Advertisements
Feeds:
Posts
Comments

Archive for the ‘Stress Disorders’ Category


Failed pain relief drug candidate clinical trial

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

What was the drug in Clinical Trial Tragedy In France Jan 2016

by DR ANTHONY MELVIN CRASTO Ph.D

09404-notw1-BIA2

BIA 10-2474

3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide

BIA 10-2474 is an experimental fatty acid amide hydrolase inhibitor[1] developed by the Portuguese pharmaceutical company Bial-Portela & Ca. SA. The drug was developed to relieve pain,[2][3] to ease mood and anxiety problems, and to improve movement coordination linked to neurodegenerative illnesses.[4] It interacts with the humanendocannabinoid system.[5][6] It has been linked to severe adverse events affecting 5 patients in a drug trial in Rennes, France, and at least one death, in January 2016.[7]

French newspaper Le Figaro has obtained Bial study protocol documents listing the the chemical name of BIA-10-2474 as 3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide.[8] A Bial news release described BIA-10-2474 as “a long-acting inhibitor of FAAH”.[9]

Fatty acid amide hydrolase (FAAH) is an enzyme which degrades endocannabinoid neurotransmitters like anandamide,[10] which relieves pain and can affect eating and sleep patterns.[11][12] FAAH inhibitors have been proposed for a range of nervous-system disorders including anxiety, alcoholism, pain and nausea.

The Portuguese pharmaceutical company Bial holds several patents on FAAH enzyme inhibitors.[12][13][14][15]

 

No target organ was identified during toxicology studies and few adverse clinical findings were observed at the highest dose tested. For the single ascending dose part [of the clinical trial], a starting dose of 0.25 mg was judged to be safe for a first-in-human administration.[8]

The protocol defines no starting dose for the multi-dose treatment groups, noting that this will be based on the outcome of the single dose portion of the trial (an approach known as adaptive trial design). The authors note that nonetheless, the starting dose will not exceed 33% of the maximum tolerated dose (MTD) identified in the single dose groups (or 33% of the maximum administered dose if the MTD is not reached).[8]

 

In July 2015 Biotrial, a contract research organization, began testing the drug in a human phase one clinical trial for the manufacturer. The study was approved by French regulatory authority, the Agence Nationale de Sécurité du Médicament (ANSM), on June 26, 2015, and by the Brest regional ethics committee on July 3, 2015.[20] The trial commenced on July 9, 2015,[21] in the city of Rennes, and recruited 128 healthy volunteers, both men and women aged 18 to 55. According to French authorities, the study employed a three-stage design with 90 of the volunteers having received the drug during the first two stages of the trial, with no serious adverse events being reported .[17][20] Participants of the study were to receive €1,900 and, in turn, asked to stay at Biotrial’s facility for two weeks during which time they would take the drug for ten days and undergo tests.[22]

In the third stage of the trial evaluating multiple doses, six male volunteers received doses by mouth, starting on 7 January 2016. The first volunteer was hospitalized at theRennes University Hospital on January 10, became brain dead,[17][23][24][25] and died on January 17.[26] The other five men in the same dosage group were also hospitalized, in the period of January 10 through January 13[27] four of them suffering injuries including deep hemorrhagic and necrotic lesions seen on brain MRI.[7] The six men who were hospitalised were the group which received the highest dose.[26] A neurologist at the University of Rennes Hospital Center, Professor Pierre-Gilles Edan, stated in a press conference with the French Minister for Health, that 3 of the 4 men who were displaying neurological symptoms “already have a severe enough clinical picture to fear that even in the best situation there will be an irreversible handicap” and were being given corticosteroids to control the inflammation.[27] The sixth man from the group was not showing adverse effects but had been hospitalized for observation.[25][28][29] Biotrial stopped the experiment on January 11, 2016.[4]

 

Le Figaro posted a 96-page clinical study protocol for BIA 10-2474 that the French newspaper procured from an unnamed source.

According to the document, BIA 10-2474 is 3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide.

BIA 10-2474 “is designed to act as a long-active and reversible inhibitor of brain and peripheral FAAH,” notes the protocol. The compound “increases anandamide levels in the central nervous system and in peripheral tissues.”

The clinical trial protocol also notes that the company tested BIA 10-2474 on mice, rats, dogs, and monkeys for effects on the heart, kidneys, and gastrointestinal tract, among other pharmacological and toxicological evaluations.

 

The clinical trial, conducted by the company Biotrial on behalf of the Portuguese pharmaceutical firm Bial, was evaluating a pain relief drug candidate called BIA 10-2474 that inhibits fatty acid amide hydrolase (FAAH) enzymes. Blocking these enzymes prevents them from breaking down cannabinoids in the brain, a family of compounds that includes the euphoria-inducing neurotransmitter anandamide and Δ9-tetrahydrocannabinol, the major psychoactive component of marijuana.

Phase I clinical trials are conducted to check a drug candidate’s safety profile in healthy, paid volunteers. In this case, the drug caused hemorrhagic and necrotic brain lesions in five out of six men in a group who received the highest doses of the drug, said Gilles Edan, a neurologist at the University Hospital Center of Rennes.

The French health minister has stated the drug acted on natural receptors found in the body known as endocannibinoids, which regulate mood and appetite. It did not contain cannabis or anything derived from it, as was originally reported. All six trial participants were administered the doses simultaneously.

 

The trial was being performed at Biotrial, a French-based firm that was formed in 1989 and has conducted thousands of trials. A message on the company’s website stated that they are working with health authorities to understand the cause of the accident, while extending thoughts to the patients and their families. Bial has disclosed the drug was a FAAH (fatty acid amide hydrolase) inhibitor, which is an enzyme produced in the brain and elsewhere that breaks down neurotransmitters called endocannabinoids. Two scientists from the Nottingham Medical School who have worked with FAAH tried over the weekend to try and identify the drug by examining a list of drugs Bial currently has in its pipeline. They believe the culprit is one identified by the codename BIA 10-2474.

 

While safety issues like this are rare, they are not unheard of. In 2006, a clinical trial in London left six men ill. All were taking part in a study testing a drug designed to fight auto-immune disease and leukemia. Within hours of taking the drug TGN1412, all experienced a serious reaction, were admitted to intensive care, and had to be treated for organ failure.

 

The Duff Report, written in response to the TGN1412 trial, noted the medicine should have been tested in one person at a time. It also helped to put additional safety measures in place. The Medicines and Health Products Regulatory Agency (MHRA) now requires committees to look at pre-clinical data to determine the proper initial dose, and rules are in place to stop the trial if unintended reactions occur.

 

Other pharmaceutical companies, including Merck, Pfizer, Johnson & Johnson, Sanofiand Vernalis, have previously taken other FAAH inhibitors into clinical trials without experiencing such adverse events (e.g. respectively, MK-4409,[35][36] PF-04457845,JNJ-42165279,[37] SSR411298 and V158866.[38][39] Related enzyme inhibitor compounds such as URB-597 and LY-2183240 have been sold illicitly as designer drugs,[40][41] all without reports of this type of toxicity emerging, so the mechanism of the toxicity observed with BIA 10-2474 remains poorly understood.

Clinical Trial Tragedy, France, Jan 2016, PHASE 1 | Categories: Uncategorized | URL:http://wp.me/p38LX5-4ut

Advertisements

Read Full Post »


H2S-mediated protein sulfhydration in stress reveals metabolic reprogramming

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the Integrated Stress Response

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>Bo-JhihGuan, 

Ilya Bederman
Department of Pediatrics, Case Western Reserve University, Cleveland, United States
No competing interests declared

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>IlyaBederman, 

Mithu Majumder
Department of Pharmacology, Case Western Reserve University, Cleveland, United States
No competing interests declared

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>MithuMajumder, et al.
eLife 2015;10.7554/eLife.10067    

http://elifesciences.org/content/early/2015/11/23/eLife.10067http://dx.doi.org/10.7554/eLife.10067

The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the Integrated Stress Response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.
Posttranslational modification is a fundamental mechanism in the regulation of structure and function of proteins. The covalent modification of specific amino acid residues influences diverse biological processes and cell physiology across species. Reactive cysteine residues in proteins have high nucleophilicity and low pKa values and serve as a major target for oxidative modifications, which can vary depending on the subcellular environment, including the type and intensity of intracellular or environmental cues. Oxidative environments cause different post-translational cysteine modifications, including disulfide bond formation (-S-S-), sulfenylation (-S-OH), nitrosylation (-S-NO), glutathionylation (-S-SG), and sulfhydration (-S-SH) (also called persulfidation) (Finkel, 2012; Mishanina et al., 2015). In the latter, an oxidized cysteine residue included glutathionylated, 60 sulfenylated and nitrosylated on a protein reacts with the sulfide anion to form a cysteine persulfide. The reversible nature of this modification provides a mechanism to fine tune biological processes in different cellular redox states. Sulfhydration coordinates with other post-translational protein modifications such as phosphorylation and nitrosylation to regulate cellular functions (Altaany et al., 2014; Sen et al., 2012). Despite great progress in bioinformatics and advanced mass spectroscopic techniques (MS), identification of different cysteine-based protein modifications has been slow compared to other post-translational modifications. In the case of sulfhydration, a small number of proteins have been identified, among them the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, GAPDH (Mustafa et al., 2009). Sulfhydrated GAPDH at Cys150 exhibits an increase in its catalytic activity, in contrast to the inhibitory effects of nitrosylation or glutathionylation of the same cysteine residue (Mustafa et al., 2009; Paul and Snyder, 2012). The biological significance of the Cys150 modification by H2S is not well-studied, but H2S could serve as a biological switch for protein function acting via oxidative modification of specific cysteine residues in response to redox homeostasis (Paul and Snyder, 2012). Understanding the physiological significance of protein sulfhydration requires the development of genome-wide innovative experimental approaches. Current methodologies based on the modified biotin switch technique do not allow detection of a broad spectrum of sulfhydrated proteins (Finkel, 2012). Guided by a previously reported strategy (Sen et al., 2012), we developed an experimental approach that allowed us to quantitatively evaluate the sulfhydrated proteome and the physiological consequences of H2S synthesis during chronic ER stress. The new methodology allows a quantitative, close-up view of the integrated cellular response to environmental and intracellular cues, and is pertinent to our understanding of human disease development.
The ER is an organelle involved in synthesis of proteins followed by various modifications. Disruption of this process results in the accumulation of misfolded proteins, causing ER stress (Tabas and Ron, 2011; Walter and Ron, 2011), which is associated with development of many diseases ranging from metabolic dysfunction to neurodegeneration (Hetz, 2012). ER stress induces transcriptional, translational, and metabolic reprogramming, all of which are interconnected through the transcription factor Atf4. Atf4 increases expression of genes promoting adaptation to stress via their protein products. One such gene is the H2S-producing enzyme, γ-cystathionase (CTH), previously shown to be involved in the signaling pathway that negatively regulates the activity of the protein tyrosine phosphatase 1B (PTP1B) via sulfhydration (Krishnan et al., 2011). We therefore hypothesized that low or even modest levels of reactive oxygen species (ROS) during ER stress may reprogram cellular metabolism via H2S-mediated protein sulfhydration (Figure 1A).
In summary, sulfhydration of specific cysteines in proteins is a key function of H2S (Kabil and Banerjee, 2010; Paul and Snyder, 2012; Szabo et al., 2013). Thus, the development of tools that can quantitatively measure genome-wide protein sulfhydration in physiological or pathological conditions is of central importance. However, a significant challenge in studies of the biological significance of protein sulfhydration is the lack of an approach to selectively detect sulfhydrated cysteines from other modifications (disulfide bonds, glutathionylated thiols and sulfienic acids) in complex biological samples. In this study, we introduced the BTA approach that allowed the quantitative assessment of changes in the sulfhydration of specific cysteines in the proteome and in individual proteins. BTA is superior to other reported methodologies that aimed to profile cysteine modifications, such as the most commonly used, a modified biotin switch technique (BST). BST was originally designed to study protein nitrosylation and postulated to differentiate free thiols and persulfides (Mustafa et al., 2009). A key advantage of BTA over the existing methodologies, is that the experimental approach has steps to avoid false-positive and negative results, as target proteins for sulfhydration. BST is commonly generating such false targets for cysteine modifications (Forrester et al., 2009; Sen et al., 2012). Using mutiple validations, our data support the specificity and reliability of the BTA assay for analysis of protein sulfhydration both in vitro and in vivo. With this approach, we found that ATF4 is the master regulator of protein sulfhydration in pancreatic β cells during ER stress, by means of its function as a transcription factor. A large number of protein targets have been discovered to undergo sulfhydration in β cells by the BTA approach. Almost 1,000 sulfhydrated cysteine- containing peptides were present in the cells under the chronic ER stress condition of treatment with Tg for 18 h. Combined with the isotopic-labeling strategy, almost 820 peptides on more than 500 proteins were quantified in the 405 cells overexpressing ATF4. These data show the potential of the BTA method for further systematic studies of biological events. To our knowledge, the current dataset encompasses most known sulfhydrated cysteine residues in proteins in any organism. Our bioinformatics analyses revealed sulfhydrated cysteine residues located on a variety of structure-function domains, suggesting the possibility of regulatory mechanism(s) mediated by protein sulfhydration. Structure and sequence analysis revealed consensus motifs that favor sulfhydration; an arginine residue and alpha-helix dipoles are both contributing to stabilize sulfhydrated cysteine thiolates in the local environment.
Pathway analyses showed that H2S-mediated sulfhydration of cysteine residues is that part of the ISR with the highest enrichment in proteins involved in energy metabolism. The metabolic flux revealed that H2S promotes aerobic glycolysis associated with decreased oxidative phosphorylation in mitochondria during ER stress in β cells. The TCA cycle revolves by the action of the respiratory chain that requires oxygen to operate. In response to ER stress, mitochondrial function and cellular respiration are down-regulated to limit oxygen demand and to sustain mitochondria. When ATP production from the TCA cycle becomes limited and glycolytic flux increases, there is a risk of accumulation of lactate from pyruvate. One way to escape accumulation of lactate is the mitochondrial conversion of pyruvate to oxalacetic acid (OAA) by pyruvate carboxylase. This latter enzyme was found to be sulfhydrated, consistent with the notion that sulfhydration is linked to metabolic reprogramming towards glycolysis.
The switch of energy production from mitochondria to glycolysis is known as a signature of hypoxic conditions. This metabolic switch has also been observed in many cancer cells characterized as the Warburg effect, which contributes to tumor growth. The Warburg effect provides advantages to cancer cell survival via the rapid ATP production through glycolysis, as well as the increased conversion of glucose into anabolic biomolecules (amino acid, nucleic acid and lipid biosynthesis) and reducing power (NADPH) for regeneration of antioxidants. This metabolic response of tumor cells contributes to tumor growth and metastasis (Vander Heiden et al., 2009). By analogy, the aerobic glycolysis trigged by increased H2S production could give β cells the capability to acquire ATP and nutrients to adapt their cellular metabolism towards maintaining ATP levels in the ER (Vishnu et al., 2014), increasing synthesis of glycerolphospholipids, glycoproteins and protein (Krokowski et al., 2013b), all important components of the ISR. Similar to hypoxic conditions, a phenotype associated with most tumors, the decreased mitochondria function in β cells during ER stress, can also be viewed as an adaptive response by limiting mitochondria ROS and mitochondria-mediated apoptosis. We therefore view that the H2S-mediated increase in glycolysis is an adaptive mechanism for survival of β cells to chronic ER stress, along with the improved ER function and insulin production and folding, both critical factors controlling hyperglycemia in diabetes. Future work should determine which are the key proteins targeted by H2S and thus contributing to metabolic reprogramming of β cells, and if and how insulin synthesis and secretion is affected by sulfhydration of these proteins during ER stress.
Abnormal H2S metabolism has been reported to occur in various diseases, mostly through the deregulation of gene expression encoding for H2S-generating enzymes (Wallace and Wang, 2015). An increase of their levels by stimulants is expected to have similar effects on sulfhydration of proteins like the ATF4- induced CTH under conditions of ER stress. It is the levels of H2S under oxidative conditions that influence cellular functions. In the present study, ER stress in β cells induced elevated Cth levels, whereas CBS was unaffected. The deregulated oxidative modification at cysteine residues by H2S may be a major contributing factor to disease development. In this case, it would provide a rationale for the design of therapeutic agents that would modulate the activity of the involved enzymes.

Read Full Post »


Philip Seymour Hoffman’s death

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

 

Philip Seymour Hoffman, Actor of Depth, Dies at 46

Read Full Post »


Music Therapy

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

What is Music Therapy

http://www.musictherapy.org/about/listserv/

http://www.musictherapy.org/about/musictherapy/

What is Music Therapy?

Music Therapy is the clinical and evidence-based use of music interventions to accomplish individualized goals within a therapeutic relationship by a credentialed professional who has completed an approved music therapy program.

Music Therapy is an established health profession in which music is used within a therapeutic relationship to address physical, emotional, cognitive, and social needs of individuals. After assessing the strengths and needs of each client, the qualified music therapist provides the indicated treatment including creating, singing, moving to, and/or listening to music. Through musical involvement in the therapeutic context, clients’ abilities are strengthened and transferred to other areas of their lives. Music therapy also provides avenues for communication that can be helpful to those who find it difficult to express themselves in words. Research in music therapy supports its effectiveness in many areas such as: overall physical rehabilitation and facilitating movement, increasing people’s motivation to become engaged in their treatment, providing emotional support for clients and their families, and providing an outlet for expression of feelings.

https://www.youtube.com/watch?feature=player_embedded&v=UlqWw36onD4

 

Fact Sheets for Music Therapy with Individual Populations:

AMTA Strategic Priorities for Specific Populations:

References and Bibliographies:

 

History of Music Therapy

The idea of music as a healing influence which could affect health and behavior is as least as old as the writings of Aristotle and Plato. The 20th century profession formally began after World War I and World War II when community musicians of all types, both amateur and professional, went to Veterans hospitals around the country to play for the thousands of veterans suffering both physical and emotional trauma from the wars. The patients’ notable physical and emotional responses to music led the doctors and nurses to request the hiring of musicians by the hospitals. It was soon evident that the hospital musicians needed some prior training before entering the facility and so the demand grew for a college curriculum. A very brief historical glimpse of this fascinating profession follows, below.

 Earliest references

The earliest known reference to music therapy appeared in 1789 in an unsigned article in Columbian Magazine titled “Music Physically Considered.” In the early 1800s, writings on the therapeutic value of music appeared in two medical dissertations, the first published by Edwin Atlee (1804) and the second by Samuel Mathews (1806). Atlee and Mathews were both students of Dr. Benjamin Rush, a physician and psychiatrist who was a strong proponent of using music to treat medical diseases. The 1800s also saw the first recorded music therapy intervention in an institutional setting (Blackwell’s Island in New York) as well as the first recorded systematic experiment in music therapy (Corning’s use of music to alter dream states during psychotherapy).

Early Associations

Interest in music therapy continued to gain support during the early 1900s leading to the formation of several short-lived associations. In 1903, Eva Augusta Vescelius founded the National Society of Musical Therapeutics. In 1926, Isa Maud Ilsen founded the National Association for Music in Hospitals. And in 1941, Harriet Ayer Seymour founded the National Foundation of Music Therapy. Although these organizations contributed the first journals, books, and educational courses on music therapy, they unfortunately were not able to develop an organized clinical profession.

Early Educational Programs and Advocates

In the 1940s, three persons began to emerge as innovators and key players in the development of music therapy as an organized clinical profession. Psychiatrist and music therapist Ira Altshuler, MD promoted music therapy in Michigan for three decades. Willem van de Wall pioneered the use of music therapy in state-funded facilities and wrote the first “how to” music therapy text,Music in Institutions (1936). E. Thayer Gaston, known as the “father of music therapy,” was instrumental in moving the profession forward in terms of an organizational and educational standpoint. The first music therapy college training programs were also created in the 1940s. Michigan State University established the first academic program in music therapy (1944) and other universities followed suit, including the University of Kansas, Chicago Musical College, College of the Pacific, and Alverno College.

National Association for Music Therapy

The National Association for Music Therapy (NAMT) was founded at a meeting in New York City on June 2, 1950. NAMT succeeded where previous music therapy associations previously failed by creating a constitution and bylaws, developing standards for university-level educational and clinical training requirements, making research and clinical training a priority, creating a registry and, later, board-certification requirements, and publishing research and clinical journals.

Music Therapy Around the World and on the Web  

Music Therapy ENews

Music Therapy ENews is an announcement-only, electronic newsletter published by the American Music Therapy Association. It is not a discussion listserv. ENews brings timely information on music therapy to its subscribers, focusing on conferences, music therapy education and training opportunities, media alerts, on-line resources, and other information important to the profession of music therapy. Members are welcome to submit announcements and other materials for consideration by emailing the editor at ENews@musictherapy.org. AMTA reserves editorial rights on all submissions. ENews subscription is free.

The Music Therapy ListServ

The Music Therapy ListServ is a discussion forum where people can discuss the use of music to restore, maintain, and improve mental and physical health.

To join the ListServ, send an E-mail, including your first and last name, to the list owners: Alexandra Mesquita-Baer(alex.baer@comcast.net) or Lynne Hockenbury (LynneHock@aol.com) For AMTA members, please include your membership number to speed up the process.

It is a free service and you can cancel your subscription at any time. To cancel, send an email to listproc@ukans.edu without a subject or signature. In the body of your message, type:

UnsubMUSTHP-L

This ListServ is run independently of AMTA.

Music Therapy Around the World

AMTA’s International Relations Committee

The purpose of the AMTA International Relations Committee is to promote networking between music therapists around the world and to facilitate awareness of international issues among AMTA members both within the United States and abroad. Information below is provided to share information on national music therapy organizations throughout the world. If you are familiar with websites of national organizations which are not included here, feel free to send their contact us and suggest they be included.

Worldwide Music Therapy Organizations
International Music Therapy Associations
Sharing Organizations
A Music Therapy Moment   
Every music therapist has a wealth of stories about that exquisite musical/clinical moment when art and science come together to create an awareness, an accomplishment, a breakthrough. These stories – poignant, insightful, or humorous – show the power and effect of music therapy, and can help to build understanding of the benefits and applications of music therapy.  These stories – along with the latest news and research in music therapy – bring the power of music therapy to people around the world, helping to educate and inspire and ensure access to quality music therapy services for every child, teen, and adult.

The Transformative Power of Working with People Who Are Facing Death

Our work as music therapists never stops giving us powerful experiences and lessons. This seems to be magnified when spending one’s days with people who are facing death. With this experience, music therapists are sensitized to the extreme emotions surrounding death, and can empathize with these patients and their families.

I had the privilege of working with an older man, who I’ll refer to as Mr. Smith, and who dearly touched my heart. Countless patients of mine have touched me, but Mr. Smith will remain in my memory as vividly as I saw him in the very hours we spent together.

At the end of life, there is a certain amount of one’s will that determines when one dies. I have seen people hold on to their lives with extreme pain and labored breathing, for weeks, just to reconcile a broken relationship with a loved one. That being said, there is simply no substitute for the beautiful and seamless opportunity that music therapy provides for people to complete their lives with dignity.

Music allowed Mr. Smith to die peacefully. The two songs that he specifically requested conveyed the messages he needed to share before departing from this world. Music therapy provided him the crucial opportunity or medium to express what he felt.

Since Mr. Smith was in a great deal of pain at the end of his life, we never engaged in very formal lyric analysis; however, Mr. Smith naturally expressed his analysis of these songs in small, intermittent statements during our sessions.

The first song he requested was Send In The Clowns, by Stephen Sondheim. This song, to Mr. Smith, highlighted the gross irony that, in stark contrast to the beauty and potential happiness in this world, there is often great emotional and physical pain in our final hours. The grand exit and culmination of our lives is often marked “not with a bang, but a whimper,” as T.S. Elliot so poignantly writes. It is a cold reality; a cruel joke that often leaves us bitter. Send in the Clowns validated and beautifully conveyed feelings for Mr. Smith when he could not. He said “I used to be able to sing and dance, and now-” he paused and closed his eyes, wincing from a shooting pain- “well, I’m here in this place.” “This place” was where people came to die. Mr. Smith knew that, because, in addition to being fully alert and oriented, he had a sister who had passed away there just two years before.

The second song he requested was “Try to Remember,” from the Broadway musical The Fantastiks. This is a beautiful song that he particularly wanted his family to hear. There are several lines in this song that Mr. Smith highlighted by mouthing the words to his wife:

“Try to remember… and follow.”
“Without a hurt the heart is hollow.”

“Deep in December it’s nice to remember the fire of September that made us mellow.”

I’ve wondered what Mr. Smith’s room would’ve been like without music therapy. Mr. & Mrs. Smith had four children- one who’d been estranged- all of whom were quite anxious. No one’s anxiety exceeded that of his wife, however. I was able to witness the facilitation of tears, hugs, and precious family interactions by our music therapy sessions together.

I’ve also wondered how my life would be without the experience and privilege of working with Mr. Smith. It is impossible to know for sure, but I can say that I am better able to keep an eye on the big picture of my life after working with him.

My time with Mr. Smith instilled in me a powerfully transformative thought. The music of our lives remains long after our bodies pass away; the love contained therein is eternal and will last beyond our pain.

Written by Sharon Graham, MM, MT-BC

 

Read Full Post »


microglia and brain maintenance

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Mapping mosaicism: Tracing subtle mutations in our brains

Posted on January 14, 2015 by Nancy Fliesler

Posted in All PostsInformation technology

More On: brain developmentDNA sequencinggeneticsmosaicismneurosciencesomatic mutations

DNA sequences were once thought to be the same in every cell, but the story is now known to be more complicated than that. The brain is a case in point: Mutations can arise at different times in brain development and affect only a percentage of neurons, forming a mosaic pattern.

Now, thanks to new technology described last week in Neuron, these subtle “somatic” brain mutations can be mapped spatially across the brain and even have their ancestry traced.

Like my family, who lived in Eastern Europe, migrated to lower Manhattan and branched off to Boston, California and elsewhere, brain mutations can be followed from the original mutant cells as they divide and migrate to their various brain destinations, carrying their altered DNA with them.

“Some mutations may occur on one side of the brain and not the other,” says Christopher Walsh, MD, PhD, chief of Genetics and Genomics at Boston Children’s Hospital and co-senior author on the paper. “Some may be ‘clumped,’ affecting just one gyrus [fold] of the brain, disrupting just a little part of the cortex at a time.”

This tracking capability represents a significant advance for genetics research. And for neuroscientists, it provides a new way to study both the normal brain and brain disorders like epilepsy, autism and intellectual disability.

Walsh and colleagues studied normal brain tissue from a teenage boy who had passed away from other causes. Sampling in more than 30 brain locations, they used deep, highly sensitive, whole-genome sequencing of one neuron at a time—unlike usual methods, which sequence thousands or millions of cells mixed together and simply read out an average.

http://vectorblog.org/wp-content/uploads/2015/01/Walsh-figure3B-v2-1024×735.jpg

The blue and green boxes indicate different degrees of mosaicism (based on proportion of cells affected) in the left half of this teen’s normal brain. The blue shaded area indicates that retrotransposon mutation #1 (blue boxes) is limited to a focal area in the middle frontal gyrus. The empty boxes indicate areas where mutation #1 was not detected. (Courtesy Gilad Evrony, PhD, Boston Children’s Hospital)

Next, using technology developed by Alice (Eunjung) Lee in the lab of Peter Park, PhD, at Harvard Medical School’s Center for Biomedical Informatics, they zeroed in on inserted bits of DNA caused by retrotransposons, one type of mutation that can arise as the brain develops. These essentially served as markers that allowed cell lineages to be traced.

“Our findings are intriguing because they suggest that every normal brain may in fact be a mosaic patchwork of focal somatic mutations, though in normal individuals most are likely silent or harmless,” says Gilad Evrony, PhD, in the Walsh Lab.

http://vectorblog.org/wp-content/uploads/2015/01/Walsh-figure5-1024×509.jpg

This model illustrates the origins of two somatic retrotransposon mutations during prenatal development and their subsequent dissemination in the brain. Insertion #2 (in green) occurred soon after conception; #1 (in blue) happened sometime later during brain development. The ‘pie slices’ show a closeup of the layers of the cerebral cortex. Later in development, additional somatic mutations occurred inside insertions #1 and #2, creating new, smaller sublineages of cells. (Courtesy Gilad Evrony, PhD)

A parallel study from Walsh’s lab in 2014 used single-neuron sequencing to find copy number variants— a different type of mutation affecting the number of copies of chromosomes or chromosome fragments. It, too, found the mutations to be present in normal brains as well as neurologically diseased brains.

Walsh and others speculate that some somatic brain mutations might play a role in autism, epilepsy, schizophrenia and other unsolved neuropsychiatric diseases whose causes are mostly still a mystery.

“It is possible that a whole new class of brain disorders may exist that has not been previously recognized,” says Evrony. “In such disorders, a somatic mutation may subtly affect only one small part of the brain involved in a specific ability, for example language, while sparing the rest of the brain.”

Read more:

 

Tracking subtle brain mutations, systematicallyTool can trace and spatially map “mosaic” mutations in the brain

http://www.prnewswire.com/news-releases/tracking-subtle-brain-mutations-systematically-300017369.html

BOSTON, Jan. 7, 2015 /PRNewswire-USNewswire/ — DNA sequences were once thought to be identical from cell to cell, but it’s increasingly understood that mutations can arise during brain development that affect only certain groups of brain cells. A technique developed at Boston Children’s Hospital allows these subtle mutation patterns to be traced and mapped spatially for the first time. This capability is a significant advance for genetics research and provides a new way to study both the normal brain and brain disorders such as epilepsy and autism.

Described in the January 7th issue of Neuron, the technique uses “deep,” highly sensitive whole-genome sequencing of single neurons and a new technology that identifies inserted bits of DNA caused by retrotransposons, one of several kinds of so-called somatic mutations that can arise as the brain develops.

The technique picks up somatic mutations that affect just a fraction of the brain’s cells, in a “mosaic” pattern. It also allows “lineage tracing,” showing when during brain development the mutations arise and how they spread through brain tissue as the mutated cells grow, replicate and migrate, carrying the mutation with them.

“There is a lot of genetic diversity from one neuron to the other, and this work gets at how somatic mutations are distributed in the brain,” says Christopher Walsh, MD, PhD, chief of Genetics and Genomics at Boston Children’s and co-senior author on the paper. “Some mutations may occur on one side of the brain and not the other. Some may be ‘clumped,’ affecting just one gyrus [fold] of the brain, disrupting just a little part of the cortex at a time.”

The study examined brain tissue from a deceased 17-year-old who had been neurologically normal, sampling in more than 30 brain locations. It builds on work published by the Walsh lab in 2012, which developed methods to sequence the genomes of single neurons, and represents the first time single neurons have been sequenced in their entirety. The single-cell technique is better at detecting subtle mosaicism than usual DNA sequencing methods, which sequence many thousands or millions of cells mixed together and read out an average for the sample.

Somatic brain mutations, affecting just pockets of cells, can be harmful, and have been suggested as a possible cause of neurodevelopmental disorders such as autism, epilepsy or intellectual disability (see this review article for further background). But they also can be completely benign or have just a subtle effect.

“Our findings are intriguing because they suggest that every normal brain may in fact be a mosaic patchwork of focal somatic mutations, though in normal individuals most are likely silent or harmless,” says Gilad Evrony, PhD, in the Walsh Lab, co-first author on the Neuron paper. “These same technologies can now be used to study the brains of people who died from unexplained neuropsychiatric diseases to determine whether somatic mutations may be the cause.”

Finally, says Evrony, the findings provide a proof-of-principle for a systematic way of studying how brain cells disperse and migrate during development, “something that has not been possible to do before in humans,” he says.

Co-first author Alice Eunjung Lee, PhD, from the lab of Peter Park, PhD, at the Center for Biomedical Informatics at Harvard Medical School, developed the study’s retrotransposon analysis tool, which detects somatic retrotransposon mutations in single-cell sequencing data.

Mirroring these findings, study published by Walsh’s lab in 2014 used single-neuron sequencing to detect copy number variants—another type of mutation affecting the number of copies of chromosomes or chromosome fragments. The study found that these mutations can occur in both normal and neurologically diseased brains.

Evrony and Lee are first authors on the Neuron paper; Walsh and Park are senior authors. The research was supported by the National Institutes of Health (MSTP grant T32GM007753), the National Institute of Neurological Disorders and Stroke (R01 NS079277 and R01 NS032457), the Louis Lange III Scholarship in Translational Research, the Eleanor and Miles Shore Fellowship, the Research Connection and the Manton Center for Orphan Disease Research at Boston Children’s Hospital, the Paul G. Allen Family Foundation and the Howard Hughes Medical Institute.

SOURCE Boston Children’s Hospital

 

Beth Stevens: A transformative thinker in neuroscience

Posted on September 29, 2015 by Nancy Fliesler

Posted in All PostsDrug discoveryProfiles

 

More On: Alzheimer’s diseaseautismFM Kirby Neurobiology Centerglial cellsneurosciencesynapse development

http://vector.childrenshospital.org/2015/09/beth-stevens-a-transformative-thinker-in-neuroscience/

https://youtu.be/6DOYTpXkLOY

When 2015 MacArthur “genius” grant winner Beth Stevens, PhD, began studying the role of glia in the brain in the 1990s, these cells—“glue” from the Greek—weren’t given much thought. Traditionally, glia were thought to merely protect and support neurons, the brain’s real players.

But Stevens, from the Department of Neurology and the F.M. Kirby Neurobiology Center at Boston Children’s Hospital, has made the case that glia are key actors in the brain, not just caretakers. Her work—at the interface between the nervous and immune systems—is helping transform how neurologic disorders like autism, amyotrophic lateral sclerosis (ALS), Alzheimer’s disease and schizophrenia are viewed.

Soon after college graduation in 1993, without prior experience in neuroscience, she helped discoveran interplay between neurons and glial cells known as Schwann cells that controlled production of the nerve insulation known as myelin It was one of the early pieces of evidence that glia and neurons talk to each other.

In 2007, while still a postdoctoral fellow, Stevens showed how star-shaped glial cells called astrocytes influence the development of synapses, or brain connections. Studying neurons, her lab showed that a gene called C1q was markedly more active when astrocytes were present. C1q is an immune gene, one nobody had expected to see in a normal brain. In the context of disease, it initiates the complement cascade, an immunologic pathway for tagging unwanted cells and debris for clearance by other immune cells.

But in healthy developing brains, Stevens showed, C1q was concentrated at developing synapses, or brain connections, apparently marking certain synapses for pruning.

Then in 2012, the Stevens lab showed that microglia—another type of glia usually thought of as immune cells themselves—actively sculpt the brain’s wiring. They literally trim away unwanted, inappropriate synapses by eating them—in the same way they’d engulf and destroy invading bacteria.

http://19g6dy4by8jx1b5cx74fh0f2.wpengine.netdna-cdn.com/wp-content/uploads/2012/06/Microglial-cell.jpg

That paper was cited by the journal Neuron as the year’s most influential paper.

The same year, she received a Presidential Early Career Award for Scientists and Engineers, honoring her innovative research and scientific leadership.

Stevens’s current investigations are looking at synapse loss—a hallmark of neurodegenerative conditions such as Alzheimer’s—and trying to understand why it occurs. Her lab’s recent work suggests that normal pruning mechanisms that are active during early brain development get re-activated later in life. Intervening with this activation could lead to a new treatment approach, she believes.

Stevens isn’t the only brain researcher at Boston Children’s to become a MacArthur fellow. Neurosurgeon Benjamin Warf, MD, received the honor in 2012.

For more:

 

 

Immune cells “sculpt” brain circuits — by eating excess connections

Posted on June 5, 2012 by Nancy Fliesler

Posted in All PostsDrug discoveryPediatrics

More On: ALSAlzheimer’s diseaseautismbrain developmentepilepsyglaucomaHuntington’s diseaseLou Gehrig’s disease,Parkinson’s diseasesynapse development

https://youtu.be/wb8UAyf8Nhw

The above movie shows an immune cell caught in the act of tending the brain—it’s just eaten away unnecessary connections, or synapses, between neurons.

That’s not something these cells, known as microglia, were previously thought to do. As immune cells, it was thought that their job was to rid the body of unwanted pathogens and debris, by engulfing and digesting them.

The involvement of microglia in the brain’s development has started to be recognized only recently. The latest research finds that microglia tune into the brain’s cues, akin to the way they survey their environment for invading microbes, and get rid of excess synapses the same way they’d dispatch these invaders—by eating them.

It’s a whole other way of understanding how the healthy brain develops—at the hands of cells that were once thought to be merely nerve “glue” (the literal meaning of “glia” from the Greek), playing a protective role to neurons, say investigators Beth Stevens, PhD, and Dori Schafer, PhD, of the F.M. Kirby Neurobiology Center at Boston Children’s Hospital.

“In the field of neuroscience, glia have often been ignored,” says Stevens. “But glia aren’t the nerve glue, they’re actively communicating with neurons. People have gotten a new respect for glia and are hungry to know more about them.”

Such knowledge could eventually shed light on brain disorders ranging from autism to Alzheimer’s.

The “eat me” sign

We’re all born with more brain connections than we need. As we begin to encounter our world, they’re trimmed back to fine-tune our circuitry. It’s a bit of an oversimplification, but Stevens and Schafer demonstrated last week in the journal Neuron that when two neurons start talking to each other less – because their connection is no longer important to our lives– the microglia notice that and prune the synapse away.

To study microglia’s pruning activity, Stevens and Schafer used a time-honored model: the visual system. When you cover one eye soon after birth, you force the brain to rewire: Brain connections with the covered eye weaken and those synapses eventually get eliminated.

Using this model, Stevens and Schafer showed that microglia take their cues from a set of signals also used by the immune system, known as the complement cascade. Specifically, microglia carry receptors that recognize the complement protein C3—the same protein found on synapses that are destined for elimination.

“We think that weaker synapses are being tagged with C3, and that microglia are eliminating them just as macrophages would eliminate bacteria,” says Schafer.  “C3 is like an ‘eat me’ signal.”

As a postdoctoral fellow in 2007, Stevens showed that neurons are loaded with complement proteins soon after birth, just when pruning is at its peak. In the new study, she and Schafer deliberately disrupted complement signaling in mice—stripping the microglia of C3 receptors, or blocking those receptors with a drug. When they did so, pruning of irrelevant synapses didn’t occur.

Stevens thinks their findings might have relevance for brain disorders. Developmental brain disorders such as autism, epilepsy or schizophrenia are increasingly seen as disorders of synapse development, and some data suggest that microglia and/or the complement cascade are involved.

At the other end of the spectrum, scientists have noted that microglia—normally in a resting state in adults—are activated in neurodegenerative diseases like glaucoma, Alzheimer’s disease, Lou Gehrig’s disease, Huntington’s disease and Parkinson’s disease. Subtle changes have been found in synapses that might cause them to be targeted for elimination.

So could targeting microglia or the complement cascade prevent synapse loss or alter pruning in these diseases?  “All this is still very speculative,” Stevens cautions. “We first need to understand normal brain development.”

 

Beth Stevens

Neuroscientist

Assistant Professor of Neurology, F. M. Kirby Neurobiology Center, Boston Children’s Hospital

Department of Neurology, Harvard Medical School

Boston, Massachusetts

Age: 45

Published September 28, 2015

https://www.macfound.org/fellows/946/#sthash.GpHuiEC6.dpuf

Beth Stevens is a neuroscientist whose research on microglial cells is prompting a significant shift in thinking about neuron communication in the healthy brain and the origins of adult neurological diseases. Until recently, it was believed that the primary function of microglia was immunological; they protected the brain by reducing inflammation and removing foreign bodies.

Stevens identified an additional, yet critical, role: the microglia are responsible for the “pruning” or removal of synaptic cells during brain development. Synapses form the connections, or means of communication, between nerve cells, and these pathways are the basis for all functions or jobs the brain performs. Using a novel model system that allows direct visualization of synapse pruning at various stages of brain development, Stevens demonstrated that the microglia’s pruning depends on the level of activity of neural pathways. She identified immune proteins called complement that “tag” (or bind) excess synapses with an “eat me” signal in the healthy developing brain. Through a process of phagocytosis, the microglia engulf or “eat” the synapses identified for elimination. This pruning optimizes the brain’s synaptic arrangements, ensuring that it has the most efficient “wiring.”

Stevens’s discoveries indicate that our adult neural circuitry is determined not only by the nerve cells but also by the brain’s immune cells. Her work suggests that adult diseases caused by deficient neural architecture (such as autism and schizophrenia) or states of neurodegeneration (such as Alzheimer’s or Huntington’s disease) may be the result of impaired microglial function and abnormal activation of this pruning pathway. Stevens is redefining our understanding of how the wiring in the brain occurs and changes in early life and shedding new light on how the nervous and immune systems interact in the brain, both in health and disease.

Beth Stevens received B.S. (1993) from Northeastern University and a Ph.D. (2003) from the University of Maryland. She was a postdoctoral fellow (2005–2008) at Stanford University and is currently an assistant professor in the Department of Neurology at Harvard Medical School and the F. M. Kirby Neurobiology Center at Boston Children’s Hospital. She is also an Institute Member of the Broad Institute of MIT and Harvard. Her scientific papers have appeared in such journals as NeuronScienceProceedings of the National Academy of Sciences, and Nature Neuroscience, among others.

– See more at: https://www.macfound.org/fellows/946/#sthash.GpHuiEC6.dpuf

Portraits of scientists who are making a mark on autism research.

http://spectrumnews.org/news/profiles/beth-stevens-casting-immune-cells-as-brain-sculptors/

Beth Stevens: Casting immune cells as brain sculptors

BY NICHOLETTE ZELIADT  /  24 SEPTEMBER 2015

Shortly after Beth Stevens launched her lab at Boston Children’s Hospital in 2008, she invited students from the Newton Montessori School, in a nearby suburb, to come for a visit. The children peered at mouse and rat brains bobbing in fluid-filled jars. They also learned how to position delicate slices of brain tissue on glass slides and inspect them with a microscope.

This visit sparked a running relationship with the school, with a steady stream of students visiting the growing lab each year. Soon it became too complicated to bring so many children to the lab, so Stevens decided to take her neuroscience lessons on the road, visiting a number of local elementary schools each year. Last year, she dropped in on the classrooms of her 5- and 8-year-old daughters, Zoe and Riley.

“The kids got really excited,” Stevens says. “It’s become such a thing that the principal wants me to come back for the whole school.”

Stevens’ enthusiasm for science has left a lasting impression on researchers, too. Her pioneering work points to a surprise role in brain development formicroglia, a type of cell once considered to simply be the brain’s immune defense system, cleaning up cellular debris, damaged tissue and pathogens. But thanks to Stevens, researchers now appreciate that these non-neuronal cells also play a critical role in shaping brain circuits.

In a 2012 discovery that created a buzz among autism researchers, Stevens and her colleagues discovered that microglia prune neuronal connections, calledsynapses, in the developing mouse brain. The trimming of synapses is thought to go awry in autism. And indeed, emerging work from Stevens’ lab hints at a role for microglia in the disorder.

Stevens has already earned praise and several prizes for her work. In 2012, shereceived the Presidential Early Career Award for Scientists and Engineers, the most prestigious award that the U.S. government bestows on young scientists. And in October, she’ll deliver one of four presidential lectures at the world’s largest gathering of neuroscientists — the annual meeting of the Society for Neuroscience — an honor she shares with three neuroscience heavyweights, including two Nobel laureates.

“The field is probably expecting a lot from Beth,” says Jonathan Kipnis, professor of neuroscience at the University of Virginia. Stevens has put microglia at the forefront, Kipnis says. “What used to be a stepchild of neuroscience research is now getting a lot of attention, and I think in part it’s due to her research.”

Curious mind:

Stevens was born in 1970 in Brockton, Massachusetts, where her mother taught elementary school and her father was the school’s principal. As a child, she was deeply inquisitive, eager to understand how things work. She enjoyed collecting bugs and worms, and would analyze these precious specimens in makeshift labs in her backyard.

But a career in science wasn’t on her radar until high school, when she took a biology class with an inspiring teacher named Anthony Cabral. “He totally made me realize that this could be a career, that I could be a scientist,” Stevens says. “It was that one class that changed it, and I’m like, ‘Okay, I’m going to do this.’”

In 1988, she began studying biology at Northeastern University in Boston, which offered an unusual opportunity. It had a unique cooperative education program that allowed Stevens to spend several semesters working full time in medical labs after finishing her coursework.

After that experience, Stevens knew she wanted to find a job in a research lab. After graduating in 1993, she joined her then-boyfriend Rob Graham, now her husband, in Washington, D.C., where he had landed a job in the U.S. Senate. Stevens headed to the National Institutes of Health (NIH) in Rockville, Maryland, to apply for a job as a research assistant.

At around the same time, neuroscientist R. Douglas Fields was launching his lab at the NIH. He studied how neural impulses influence glia — a class of non-neuronal cells that includes microglia — and shape the structure of the developing brain. Fields readily hired Stevens despite her lack of expertise in neuroscience. “I was impressed with her work ethic, energy and drive,” he says.

Stimulating research:

In Fields’ lab, Stevens used a multi-compartment cell culture system to investigate whether stimulating neurons influences the activity of Schwann cells, glial cells that produce a fatty substance called myelin, which insulates nerves1. She discovered that patterns of neural impulses similar to those that occur during early development influence the maturation of Schwann cells and the production of myelin.

The findings added to mounting evidence that glia and neurons communicate with each other, a newly emerging concept at the time.

“What I loved about the glia research was that there were so few neuroscientists studying it; it was such a mysterious part of neuroscience,” Stevens says. “Those years in Doug’s lab were really exciting because it was a new field.”

Stevens spent five years in Fields’ lab. “She was doing extraordinary work,” Fields says. “She had the potential and the interest to do neuroscience research, and I recommended that she should consider going to graduate school.”

But Stevens didn’t want to give up her position in the lab, and at that time, the NIH did not allow its researchers to have graduate students. So she and Fields convinced the University of Maryland, College Park, just 10 miles away, to allow her to take graduate courses in neuroscience while completing the necessary research for her Ph.D. in Fields’ lab.

In 2000, less than two years after starting graduate school, Stevens published a paper in Science showing that nerves in the peripheral nervous system (located outside the brain and spinal cord) use chemical signals to communicate with Schwann cells2. Two years later, she reported in Neuron that a similar form of communication occurs in the brain, between neurons and oligodendrocytes, the myelin-producing cells in the brain3.

As she was closing in on her Ph.D., Stevens sought career advice from Story Landis, then-director of the National Institute of Neurological Disorders and Stroke. Landis turned Stevens on to the possibility of starting her own lab one day. “I convinced her that she really had the abilities and energy and intelligence to run an independent research program,” Landis says.

In 2004, Stevens sought a postdoctoral fellowship with neurobiologist Ben Barres at Stanford University. “She was already seen as a leading researcher in the glial field,” recalls Barres, who promptly hired her. “She had done all sorts of beautiful work on glia.”

In Barres’ lab, Stevens continued to explore the dialogue between neurons and glia, turning her attention to star-shaped glia called astrocytes. Barres and his team had discovered that astrocytes help neurons form synapses4. To get a better handle on this process, Stevens examined how astrocytes influence gene expression in neurons in the developing mouse brain.

To her surprise, she found that astrocytes trigger neurons to produce a ‘complement’ protein that is best known for its role in the immune system. There, the protein serves as an ‘eat me’ signal, flagging pathogens and debris for removal. She found that neurons deposit this tag around immature synapses, but not mature ones, in mouse brain tissue, and mice that lack this protein have too many immature synapses. The findings suggested that astrocytes might help eliminate synapses by triggering the complement cascade5.

 

http://spectrumnews.org/wp-content/uploads/2015/09/20150929ProfileBethStevensChild350.jpg

Young recruit: Beth Stevens’ daughter Riley inspects brain tissue during a visit to her mother’s lab. | Courtesy of Beth Stevens

But it was still unclear exactly how the tagged synapses are cleared. The prime suspects were microglia, the only cells in the brain known to have the receptor for the ‘eat me’ signal.

Stevens set out to test this hypothesis in her own lab: After four years as a postdoc, she had decided to branch out on her own. In 2008, neuroscientist Michael Greenberg — chair of the neurobiology department at Harvard — recruited her to the Harvard-affiliated Boston Children’s Hospital. Even when her lab was in its infancy, she had little trouble convincing new staff to join her.

“A lot of people might be a little hesitant to join a new lab,” says Dorothy Schafer, a former postdoctoral fellow in Stevens’ lab who is now assistant professor of neurobiology at the University of Massachusetts-Worcester. “But I was so excited by the research, and she was so energetic and extremely positive, and just seemed like a very nice person.”

One decision Stevens made early on was to continue to studying microglia in mice rather than experiment with new model systems. “You’ll never see her working on songbirds, because she has this aversion to birds,” Schafer says. “I think they think her curly blond hair is a nest or something, and she’s had really bad experiences with many types of birds dive-bombing her head.”

Just four years into her foray as an independent researcher, Stevens found the proof she had been looking for. In 2012, her team published evidence that microglia eat synapses, especially those that are weak and unused6.

The findings pinned down a new role for microglia in wiring the brain. They also helped to explain how the brain, which starts out with a surplus of neurons, trims some of the excess away. Neuron named the paper its most influential publication of 2012.

Stevens continues to study the function of microglia in the healthy brain, most recently uncovering preliminary evidence that a certain protein serves as a ‘don’t eat me’ tag that protects synapses from being engulfed by microglia. She is also exploring the role of microglia in disorders such as autism.

Several studies suggest that microglia are more active and more numerous in the brains of people with autism than in controls. Stevens and her team are looking at whether the activity of microglia is altered during brain development in mouse models of autism.

 

Immunodulatory Thalidomides in ~ conjugants unleash proteasome degradation on ~ oncoproteins with distinct mechanisms- BRD4,MYC & PIM1 & little collteral damage to 7429 other proteins!

Imagine being able to specifically target a cancer protein for immediate destruction, slipping Robert Louis Stevenson’s notorious black spot into a crevice in the secondary structure and spelling imminent death. Well, this is what Winter et al. (2015) describe in a recent drug discovery report for Science.1 Using phthalimide conjugation, the researchers not only specifically marked BRD4, a transcriptional coactivator important in MYC oncogene upregulation, for proteasomal degradation, but also achieved reduced tumor burdens in vivo.

The research team combined two drugs, thalidomide and JQ1, exploiting the properties of each to create a bifunctional compound, dBET1, that drives the proteasomal degradation of BRD4. JQ1, which in itself is anti-oncogenic, selectively binds BET bromodomains on the transcription factor, thus competitively inhibiting BRD4 activity on chromatin. Thalidomide, a phthalimide-based drug with immunomodulatory properties, binds cereblon (CRBN) in the cullin-RING ubiquitin ligase (CRL) complex, which is important in proteasomal protein degradation.

After confirming that the new phthalimide conjugate, dBET1, retained affinity for BRD4 and that this binding was specific, the team used a human acute myelocytic leukemia (AML) cell line, MV4;11, to show that treatment with the conjugate over 18 hours reduced BRD4 abundance. The researchers also found this with dBET1 treatment of other human cancer cell lines (SUM159, MOLM13). Following this, Winter et al. investigated the mechanisms by which dBET1 inhibits BRD4. By focusing primarily on proteasome function, the researchers determined that the reduction in BRD4 abundance in MV4;11 cells is proteasomal and dependent on CRBN binding activity.

Having established targeted proteasomal degradation using the dBET1 conjugate, Winter et al. then investigated the proteomic consequences of treatment in MV4;11 cells. Scientists at the Thermo Fisher Scientific Center for Multiplexed Proteomics (Harvard Medical School) used quantitative proteomics analysis with an isobaric tagging approach to compare the immediate effects of dBET1 treatment following two hours of incubation with the responses to JQ1 and vehicle control. Spectral data analysis identified 7,429 proteins with few differences in response to either treatment. JQ1 treatment reduced levels of MYC and oncoprotein PIM1 similarly to the response following dBET1 incubation. However, treatment with the latter also reduced BRD2, BRD3 and BRD4 abundance, findings that the research team confirmed with specific immunoblotting. Measuring expression of mRNA showed that both treatments reduced levels of MYC and PIM1 abundance. However, Winter et al. found no difference in BRD3 and BRD4, suggesting that dBET1 reduces the protein levels by post-transcriptional regulation.

Investigating the antiproliferative potential of the phthalimide conjugate, dBET1, Winter and coauthors examined apoptotic response in both MV4;11 and DHL4 lymphoma cells, and in primary human AML blast cultures. Compared to JQ1 treatment, dBET1 stimulated a profound and prolonged apoptotic response in both cell lines, suggesting that targeted degradation could be a more effective treatment than target inhibition.

shapes of proteins as they shift from one stable shape to a different, folded one Protein-structural-changes

shapes of proteins as they shift from one stable shape to a different, folded one Protein-structural-changes

Orchestrating the unfolded protein response in health and disease

Randal J. Kaufman Department of Biological Chemistry,
Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan, USA J. Clin. Invest. 110:1389–1398 (2002).   http://dx.doi.org:/10.1172/JCI200216886

The endoplasmic reticulum (ER), the entrance site for proteins destined to reside in the secretory pathway or the extracellular environment, is also the site of biosynthesis for steroids and for cholesterol and many lipids. Given the considerable number of resident structural proteins and biosynthetic enzymes and the high expression of many secreted proteins, the total concentration of proteins in the this organelle can reach 100 mg/ml. The ER relies on an efficient system of protein chaperones that prevent the accumulation of unfolded or aggregated proteins and correct misfolded proteins that are caught in low-energy kinetic traps (see Horwich, this Perspective series, ref. 1).

These chaperone-mediated processes expend metabolic energy to ensure high-fidelity protein folding in the lumen of the ER. For example, the most abundant ER chaperone, BiP/GRP78, uses the energy from ATP hydrolysis to promote folding and prevent aggregation of proteins within the ER. In addition, the oxidizing environment of the ER creates a constant demand for cellular protein disulfide isomerases to catalyze and monitor disulfide bond formation in a regulated and ordered manner. Operating in parallel with chaperone dependent protein folding are several “quality control” mechanisms, which ensure that, of all proteins translocated into the ER lumen, only those that are properly folded transit to the Golgi compartment. Proteins that are misfolded in the ER are retained until they reach their native conformation or are retrotranslocated back into the cytosol for degradation by the 26S proteasome. The ER has evolved highly specific signaling pathways to ensure that its protein-folding capacity is not overwhelmed. These pathways, collectively termed the unfolded protein response (UPR), are required if the cell is to survive the ER stress (see Ron, this Perspective series, ref. 2) that can result from perturbation in calcium homeostasis or redox status, elevated secretory protein synthesis, expression of misfolded proteins, sugar/glucose deprivation, or altered glycosylation. Upon accumulation of unfolded proteins in the ER lumen, the UPR is activated, reducing the amount of new protein translocated into the ER lumen, increasing retrotranslocation and degradation of ER-localized proteins, and bolstering the protein-folding capacity of the ER. The UPR is orchestrated by the coordinate transcriptional activation of multiple genes, a general decrease in translation initiation, and a concomitant shift in the mRNAs that are translated.

The recent discovery of the mechanisms of ER stress signaling, coupled with the ability to genetically engineer model organisms, has led to major new insights into the diverse cellular and physiological processes that are regulated by the UPR. Here, I summarize current discoveries that have offered insights into the complex regulation of the UPR and its relevance to human physiology and disease.

Glucose and protein folding Early studies demonstrated that both viral transformation and glucose depletion induce transcription of a set of related genes that were termed glucose-regulated proteins (GRPs) (3). Since viral transformation increases both the cellular metabolic rate and ATP utilization, it became evident that, in both cases, this signal emanates from the ER as a consequence of energy deprivation. Because proteins have different ATP requirements for protein folding prior to export, it has been proposed that the threshold for UPR activation might differ among various cell types, depending on their energy stores and the amount and nature of the secretory proteins they produce (4). Glucose not only provides the metabolic energy needed by cells but also participates directly in glycoprotein folding as a component of oligosaccharide structures.

The recognition and modification of oligosaccharide structures in the lumen of the ER is intimately coupled to polypeptide folding (5). As the growing nascent chain is translocated into the lumen of the ER, a 14-oligosaccharide core (GlcNAc2Man9Glc3) is added to consensus asparagine residues. Immediately after the addition of this core, the three terminal glucose residues are cleaved by the sequential action of glucosidases I and II to yield a GlcNAc2Man9 structure. If the polypeptide is not folded properly, a UDP-glucose:glycoprotein glucosyltransferase (UGGT) recognizes the unfolded nature of the glycoprotein and reglucosylates the core structure to re-establish the glucose-α(1, 3)–mannose glycosidic linkage. Monoglucosylated oligosaccharides containing this bond bind to the ER-resident protein chaperones calnexin and calreticulin.

This quality control process ensures that unfolded glycoproteins do not exit the ER. Treatment of cells with castanospermine, a transition-state analogue inhibitor of glucosidases I and II, inhibits this monoglucosylation cycle, prevents interaction of unfolded glycoproteins with calnexin and calreticulin, and activates the UPR. Genetic alterations that reduce the nucleotide sugar precursor pool or glycosyltransferase reactions likewise activate the UPR (6). Therefore, the recognition of altered carbohydrate structures is in some manner linked to UPR activation.

The UPR in yeast and higher eukaryotes On a cellular level, the accumulation of unfolded proteins in the ER lumen induces the transcription of a large set of genes whose products increase the ER’s volume or its capacity for protein folding or promote the degradation of misfolded proteins through the process of ER-associated protein degradation (ERAD) (7). For example, transcription of the ER protein chaperone BiP is a classical marker for UPR activation in yeast and mammalian cells (8). BiP binds hydrophobic exposed patches on the surfaces of unfolded proteins and interactive sites on unassembled protein subunits, and it releases its polypeptide substrates upon ATP binding.

In parallel, as Ron (this Perspective series, ref. 2) details in his accompanying article, translation is attenuated to decrease the protein-folding load. The complex network of physiological responses to ER stress is regulated by only a few ER transmembrane proteins: IRE1, PERK, and ATF6 (9). IRE1, PERK, and ATF6 are proximal sensors that regulate the production and/or quality of basic leucine zipper–containing (bZIP-containing) transcription factors that may form homo- and heterodimers. Combinatorial interactions of these factors generate diversity in responses for different subsets of UPRresponsive genes. In multicellular organisms, if these adaptive responses are not sufficient to relieve ER stress, the cell dies through apoptosis or necrosis.

IRE1-dependent splicing The UPR-signaling pathway was first described less than ten years ago in the budding yeast Saccharomyces cerevisiae. Elegant studies identified IRE1 as the sensor of unfolded proteins in the ER lumen. IRE1 is a type 1 transmembrane Ser/Thr protein kinase that also has a site-specific endoribonuclease (RNase) activity. The presence of unfolded proteins in the ER lumen promotes dimerization and trans-autophosphorylation, rendering IRE1 active as an RNase, and allowing it to cleave a 252-base intron from the mRNA encoding the transcription factor HAC1 (10). The 5′ and 3′ ends of HAC1 mRNA are spliced together by tRNA ligase in a process that is independent of the spliceosome and the usual intranuclear machinery for mRNA splicing. Splicing of HAC1 mRNA increases its translational efficiency and alters sequence of the encoded HAC1 protein, yielding a potent transcriptional activator (11) that can bind and activate the UPR elements (UPREs) upstream of many UPR-inducible genes. In S. cerevisiae, the UPR activates transcription of approximately 381 genes (7).

All eukaryotic cells appear to have maintained the essential and unique properties of the UPR present in S. cerevisiae, but higher eukaryotes possess additional sensors that generate diverse, coordinately regulated responses that promote stress adaptation or cell death. The mammalian genome contains two homologues of yeast IRE1 — IRE1α and IRE1β. Whereas IRE1α is expressed in most cells and tissues, with high-level expression in the pancreas and placenta (12), IRE1β expression is prominent only in intestinal epithelial cells (13). Both IRE1 molecules respond to the accumulation of unfolded proteins in the ER, which activate their kinase and, thereby, their RNase activities. The cleavage specificities of IRE1α and IRE1β are similar, if not identical, suggesting that they do not recognize different sets of substrates but rather generate temporally specific and tissue-specific expression (14, 15).

Searching for transcription factors that mediate the UPR, Yoshida et al. defined a mammalian ER stress response element [ERSEI; CCAAT(N9)CCACG] that is necessary and sufficient for UPR gene activation. Using a yeast one-hybrid screen, these authors isolated XBP1, a bZIP transcription factor X-box DNA binding protein (16). Subsequently, several groups demonstrated that XBP1 mRNA is a substrate for mammalian IRE1, much as the HAC1 mRNA in S. cerevisiae is processed by the yeast IRE1; this pathway is also conserved in Caenorhabditis elegans (17–20). On activation of the UPR, XBP1 mRNA is cleaved by IRE1 to remove a 26-nucleotide intron and generate a translational frameshift. As expected given the precedent of HAC1 regulation in yeast, the resulting processed mRNA encodes a protein with a novel carboxy-terminus that acts as a potent transcriptional activator.

Overexpression of either IRE1α or IRE1β is sufficient to activate transcription from a BiP promoter reporter construct (15). Analysis of a minimal UPRE motif (TGACGTGC/A) (21) uncovered a transcriptional defect in IRE1α-null mouse embryo fibroblasts that could be complemented by expression of spliced XBP1 mRNA (20), and Yoshida et al. (unpublished data) recently identified a UPR-inducible gene that uniquely requires IRE1α-mediated splicing of XBP1 mRNA. However, neither IRE1α nor IRE1β is necessary for transcriptional activation of the BiP gene, as judged by the phenotype of IRE1α/β–deleted murine cells (20, 22, 23). These results indicate that a subset of UPR targets require IRE1 but that at least one IRE1-independent pathway exists for UPR-mediated transcriptional induction. Deletion of IRE1α causes embryonic lethality at embryonic day 10.5 (E10.5) (20, 22, 23). Therefore, although IRE1α is not required for the UPR, it is clearly required for mammalian embryogenesis. XBP1 deletion also causes embryonic lethality, but the mutant embryos can survive up to day E14.5, consistent with the notion that XBP1 acts downstream of IRE1α. XBP1 deletion causes cardiomyopathy and liver hypoplasia (24, 25). In contrast, IRE1β-null mice develop normally but exhibit increased susceptibility to experimentally induced colitis, a phenotype that is consistent with the specific expression of this kinase in the intestinal epithelium (26).

Activation of ATF6 and PERK by ER stress The activating transcription factor ATF6 (16) has been identified as another regulatory protein that, like XBP1, can bind ERSEI elements in the promoters of UPRresponsive genes. There are two forms of ATF6, both synthesized as ER transmembrane proteins. ATF6α (90 kDa) and ATF6β (110 kDa, also known as CREB-RP) both require the presence of the transcription factor CBF (also called NF-Y) to bind ERSEI (27–30).

On activation of the UPR, both forms of ATF6 are processed to generate 50- to 60-kDa cytosolic, bZIP containing transcription factors that migrate to the nucleus (27). Processing of ATF6 by site-1 protease (S1P) and site-2 protease (S2P) occurs within the transmembrane segment and at an adjacent site exposed to the ER lumen. S1P and S2P are the processing enzymes that cleave the ER-associated transmembrane sterolresponse element–binding protein (SREBP) upon cholesterol deprivation (31). The cytosolic fragment of cleaved SREBP migrates to the nucleus to activate transcription of genes required for sterol biosynthesis. Interestingly, although the mechanism regulating ATF6 processing is similar to that regulating SREBP processing (32), the UPR only elicits ATF6 processing, whereas sterol deprivation alone induces SREBP processing. The SREBP cleavage–activating protein (SCAP) confers specificity for SREBP transport to the Golgi compartment, and consequently cleavage in response to sterol deprivation (33). It is unknown whether another cleavage-activating protein, analogous to SCAP but active only following induction of the UPR, promotes the specific cleavage and activation of ATF6 by S1P and S2P.

Transcription of UPR-responsive genes is induced when the cleaved form of ATF6 activates the XBP1 promoter. Therefore, signaling through ATF6 and IRE1 merges to induce XBP1 transcription and mRNA splicing, respectively (Figure 1, a and b). ATF6 increases XBP1 transcription to produce more substrate for IRE1- mediated splicing that generates more active XBP1, providing a positive feedback for UPR activation. However, cells that lack either IRE1α or ATF6 cleavage can induce XBP1 mRNA (20). These two pathways may thus provide parallel signaling pathways for XBP1 transcriptional induction. Alternatively, another pathway — possibly mediated by the ER-localized protein kinase PERK (see Ron, this Perspective series, ref. 2) — may also contribute to induction of XBP1 mRNA. The binding specificities of XBP1 and ATF6 are similar, although ATF6 binding requires CBF binding to an adjacent site, whereas XBP1 binds independently (17, 20, 21, 34). These binding specificities provide another avenue for complementary interaction between the IRE1-XBP1 and ATF6 pathways at the level of transcriptional activation. In addition, these transcription factors might regulate transcription from a second ERSE (ERSEII), which also contains a CCACG motif (35).

In parallel with the activation of ATF6 processing and the consequent changes in gene transcription, the accumulation of unfolded proteins in the ER also alters cellular patterns of translation. The protein kinase PERK has been implicated in this aspect of the ER stress response (see Ron, this Perspective series, ref. 2). Activated PERK phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2α) and attenuates general protein synthesis. Inactivation of the PERK-eIF2α phosphorylation pathway decreases cells’ ability to survive ER stress (36, 37). The PERK pathway promotes cell survival not only by limiting the protein-folding load on the ER, but also by inducing transcription of UPR- activated genes, one-third of which require phosphorylation of eIF2α for their induction (36). Preferential translation of the transcription factor ATF4 allows for continued activation of these genes under conditions of stress, when general protein synthesis is inhibited (36, 37).

A coordinated mechanism for activation One puzzling question about the UPR is how three independent sensors are activated by a common stimulus, the accumulation of unfolded proteins in the ER lumen. BiP, which negatively regulates the UPR, interacts with all three sensors, IRE1, PERK, and ATF6, under nonstressed conditions and may indeed be the master regulator of UPR activation.

Upon accumulation of unfolded proteins in the ER, BiP is released from IRE1, PERK, and ATF6. It is believed that the unfolded proteins bind BiP and sequester it from interacting with IRE1, PERK, and ATF6 to elicit their activation. In this manner, BiP senses both the level of unfolded proteins and the energy (ATP) level in the cell in regulating the UPR. Following release from BiP, IRE1 and PERK are each free to undergo spontaneous homodimerization mediated by their lumenal domains and to become phosphorylated by their endogenous kinase activities (38, 39). BiP interaction with ATF6 prevents trafficking of ATF6 to the Golgi compartment. For this reason, BiP release permits ATF6 transport to the Golgi compartment, where it gains access to S1P and S2P proteases (32). The regulation of signaling through the free level of BiP is an attractive hypothesis providing a direct mechanism by which all three ER stress sensors could be activated by the same stimulus. In addition, the increase in BiP during the UPR would provide a negative feedback to turn off UPR signaling. However, in certain cells, different stress conditions can selectively activate only one or two of the ER stress sensors. For example, in pancreatic β cells, glucose limitation appears to activate PERK prior to activation of IRE1 (D. Scheuner and R.J. Kaufman, unpublished results). It will be important to elucidate how general BiP repression permits the selective activation of individual components of the UPR that mediate various downstream effects.

Signaling the UPR in eukaryotes

Signaling the UPR in eukaryotes

Figure 1 Signaling the UPR in eukaryotes.

http://dm5migu4zj3pb.cloudfront.net/manuscripts/16000/16886/small/JCI0216886.f1.gif

Three proximal sensors, IRE1, PERK, and ATF6, coordinately regulate the UPR through their various signaling pathways. Whereas IRE1 and PERK are dispensable for many aspects of the response, ATF6 cleavage is required for UPR transcriptional induction and appears to be the most significant of these effectors in mammalian cells. BiP negatively regulates these pathways. BiP interacts with ATF6 to prevent its transport to the Golgi compartment (a). BiP binds to the lumenal domains of IRE1 (b) and PERK (c) to prevent their dimerization. As unfolded proteins accumulate, they bind BiP and reduce the amount of BiP available to bind and inhibit activation of IRE1, PERK, and ATF6. (a) BiP release from ATF6 permits transport to the Golgi compartment. In the Golgi, ATF6 is cleaved by S1P and S2P proteases to yield a cytosolic fragment that migrates to the nucleus to activate transcription of responsive genes, including XBP1. (b) BiP release from IRE1 permits dimerization to activate its kinase and RNase activities to initiate XBP1 mRNA splicing. XBP1 splicing removes a 26-base intron, creating a translational frameshift to yield a more potent transcriptional activator. (c) BiP release permits PERK dimerization and activation to phosphorylate Ser51 on eIF2α to reduce the frequency of AUG initiation codon recognition. As eIF2α phosphorylation reduces the functional level of eIF2, the general rate of translation initiation is reduced. However, selective mRNAs, such as ATF4 mRNA, are preferentially translated under these conditions, possibly by the presence of open reading frames within the 5′ untranslated region of the mRNA. Upon recovery from the UPR, GADD34 targets PP1 to dephosphorylate eIF2α and increase protein translation.

The UPR as a mediator of programmed cell death In contrast to UPR-signaling adaptation in response to ER stress, prolonged UPR activation leads to apoptotic cell death (Figure 2). The roles of several death-promoting signaling pathways have been shown by analysis of specific gene-deleted cells. Activated IRE1 recruits c-Jun-N-terminal inhibitory kinase (JIK) and the cytosolic adaptor TRAF2 to the ER membrane (22, 40). TRAF2 activates the apoptosis-signaling kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase (MAPKKK) (41). Activated ASK1 leads to activation of the JNK protein kinase and mitochondriadependent caspase activation (40–42).

ER insults lead to caspase activation by mitochondria/APAF-1–dependent and –independent pathways. ER stress promotes cytochrome c release from mitochondria, possibly by c-ABL kinase (43) or calcium (44). However, APAF1–/– cells are susceptible to ER stress–induced apoptosis, indicating that the mitochondrial pathway is not essential (45). Caspase-12 is an ER-associated proximal effector in the caspase activation cascade, and cells lacking this enzyme are partially resistant to inducers of ER stress (46). ER stress induces TRAF2 release from procaspase 12, allowing it to bind activated IRE1. As shown in Figure 2, release of TRAF2 permits clustering of procaspase-12 at the ER membrane, leading to its activation (40). Caspase-12 can activate caspase-9, which in turn activates caspase- 3 (47). Procaspase-12 can also be activated by m-calpain in response to calcium release from the ER, although the physiological significance of this pathway is not known (48). In addition, upon ER stress, procaspase-7 is activated and recruited to the ER membrane (49). These findings support the notion that ER stress leads to several redundant pathways for caspase activation.

A second death-signaling pathway activated by ER stress is mediated by transcriptional activation of genes encoding proapoptotic functions. Activation of UPR sensor IRE1, PERK, or ATF6 leads to transcriptional activation of CHOP/GADD153, a bZIP transcription factor that potentiates apoptosis (see Ron, this Perspective series, ref. 2).

The UPR in health and disease Primary amino acid sequence contains all the information for a protein to attain its final folded conformation. However, many folding intermediates exist along the folding pathway (see Horwich, this Perspective series, ref. 1), and some of these intermediates can become irreversibly trapped in low-energy states and activate the UPR. Clearance of such misfolded species requires a functional ER-associated degradation (ERAD) pathway, which is regulated by the UPR. Proteasomal degradation of ER-associated misfolded proteins is required to protect from UPR activation. Proteasomal inhibition is sufficient to activate the UPR, and, in turn, genes encoding several components of ERAD are transcriptionally induced by the UPR (7). Therefore, it is to be expected that UPR activation and impaired ERAD function might contribute to a variety of diseases and that polymorphisms affecting the UPR and ERAD responses could modify disease progression. The following examples provide the best available evidence linking the UPR pathway to the natural history of human diseases and animal models of these diseases.

The UPR and ERAD in genetic disease Many recessive inherited genetic diseases are due to loss  of-function mutations that disturb productive folding and that produce proteins that are either not secreted or not functional. In other cases, protein-folding mutations can interfere with cellular processes, resulting in a gain of function and a dominant pattern of inheritance. In several instances, UPR activation by the accumulation of unfolded proteins in the ER is known to contribute to disease progression. The distinction between these two classes of genetic disease is important, because gain-of-function protein-misfolding mutations will be less amenable to treatment by gene therapy to deliver a wild-type copy of the mutant gene.

One well-characterized protein-folding defect results from a mutation that leads to type 1 diabetes. The Akita mouse has a gain-of-function Cys96Tyr mutation in the proinsulin 2 (Ins2) gene; this mutation disrupts proinsulin folding. The mutant protein is retained in the ER of the pancreatic β cell and activates the UPR. Crucially, the progressive development of diabetes in this model is not solely due to the lack of insulin but is rather a consequence of the misfolded protein accumulation, UPR activation, and β cell death. When bred into a Chop–/–background, the Akita mutation causes a lesser degree of β cell death and delayed onset of diabetes (50), indicating that the loss of at least one downstream signaling component of the UPR can ameliorate pathogenesis in this setting.

Signaling UPR-mediated cell death

Signaling UPR-mediated cell death

Figure 2 Signaling UPR-mediated cell death.

http://dm5migu4zj3pb.cloudfront.net/manuscripts/16000/16886/medium/JCI0216886.f2.jpg

The activation of procaspase-12 is likely the major pathway that induces apoptosis in response to ER stress. Upon activation of the UPR, c-Jun-N-terminal inhibitory kinase (JIK) release from procaspase-12 permits clustering and activation of procaspase-12. Caspase-12 activates procaspase-9 to activate procaspase-3, the executioner of cell death. In addition, activated IRE1 binds JIK and recruits TRAF2, which signals through apoptosis-signaling kinase 1 (ASK1) and JNK to promote mitochondria-dependent apoptosis. In addition, in vitro studies suggest that localized calcium release from the ER activates m-calpain to cleave and activate procaspase-12. Upon UPR activation, procaspase-7 is activated and recruited to the ER membrane. Finally, IRE1, PERK, and ATF6 induce transcription of several genes encoding apoptotic functions, including CHOP/GADD153. CSP, caspase; pCSP, procaspase.

Deficiency in α1-proteinase inhibitor (α1-PI, also known as α1-antitrypsin) results in emphysema and destructive lung disease in one out of 1,800 births. However, a subgroup of affected individuals develop chronic liver disease and hepatocellular carcinoma as a consequence of a secretion defect in the misfolded protein at the site of synthesis, the hepatocyte. This is the most common genetic cause of liver disease in children. The Z allele of the α1 gene PI (Glu342Lys mutation) produces a protein that polymerizes and is retained in the ER for degradation by the proteasome (see Lomas and Mahadeva, this Perspective series, ref. 51; and Perlmutter, this series, ref. 52). While α1-PI Z neither binds BiP nor activates the UPR, analysis of fibroblasts obtained from these patients demonstrates that individuals susceptible to liver disease have inherited a second trait that slows degradation of the misfolded protein in the ER (53), consistent with the idea that polymorphisms that reduce ERAD function can exacerbate pathogenesis of certain diseases.

There are numerous additional genetic misfolding diseases that are also likely influenced by UPR signaling. Because BiP release from IRE1, PERK, or ATF6 can activate the UPR, the expression of any wild-type or mutant protein that binds BiP can have a similar effect. In contrast, misfolded proteins that do not bind BiP are unlikely to activate the UPR. For example, cystic fibrosis is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Approximately 70% of patients with this disease carry a common mutation, deletion of Phe508, that results in a molecule that is retained in the ER and eventually degraded by the proteosome (see Gelman and Kopito, this Perspective series, ref. 54). Although expression of ∆508 CFTR does not activate the UPR in cultured cells, the protein does interact with calnexin, as well as HSP70, and requires ERAD function for cell survival.

Osteogenesis imperfecta (OI) results from misfolding mutations in procollagen that produce molecules that bind BiP and activate the UPR (55). Interestingly, Wolcott-Rallison syndrome is due to inactivating mutations in the PERK gene. Affected individuals, as well as mice with deletions in Perk, display osteoporosis and deficient mineralization throughout the skeletal system (56, 57), the same defects that are observed in OI. Procollagen type I accumulates to high levels and mature collagen is not detected in bone and osteoblasts from PERKnull mice. Osteoblasts from PERK-null humans and mice display fragmented and distended ER that is filled with electron-dense material (56, 57). These observations suggest that procollagen type 1 uniquely requires PERK function to maintain its transport out of the ER, processing, and secretion In this case, PERK may be required to limit procollagen synthesis so that it does not saturate the ER protein-folding capacity.

The UPR and ERAD in conformational diseases Diseases caused by expansion of polyglutamine repeats and neurodegenerative diseases, such as Alzheimer disease and Parkinson disease, represent a large class of conformational diseases associated with accumulation of abnormal protein aggregates in and around affected neurons. Recent evidence indicates that the pathogenesis of these diseases is due to a defect in proteasomal function that results in UPR activation, leading to cell death. The protein aggregates in these diseases are localized to the nucleus or the cytoplasm and would not be predicted to disturb ER function directly. Nevertheless, they have been found in some cases to activate the UPR and to promote cell death. Analysis of the polyglutamine repeat associated with the spinocerebrocellular atrophy protein (SCA3) in Machado-Joseph disease suggests that cytoplasmic accumulation of the SCA3 aggregate can inhibit proteasome function, thereby interfering with ERAD to induce the UPR and elicit caspase-12 activation (41, 58). These findings support the idea that the UPR can signal the accumulation of unfolded proteins in the cytosol via proteasomal inhibition and disruption of ERAD function.

Parkinson disease is the most common movement disorder, affecting about 1% of individuals 65 years of age or older. Autosomal recessive juvenile parkinsonism (AR-JP) results from defects in the Parkin gene (59), which encodes a ubiquitin protein ligase (E3) that functions with ubiquitin-conjugating enzyme UbcH7 or UbcH8 to tag proteins for degradation. Overexpression of Parkin suppresses cell death associated with ER stress (60). Inherited Parkinson disease is associated with the accumulation in the ER of dopaminergic neurons of PAEL-R, a putative transmembrane receptor protein that is detected in an insoluble form in the brains of AR-JP patients (61). The accumulation of PAEL-R results from defective Parkin that does not maintain the proteasome-degrading activity necessary to maintain ER function (62). Other, still-unidentified substrates of the Parkin E3 ligase may also be relevant to the pathogenesis of AR-JP.

The UPR in diabetes The metabolism of glucose is tightly controlled at the levels of synthesis and utilization through hormonal regulation. The most dramatic phenotype in Wolcott-Rallison syndrome is pancreatic β cell death with infancy onset diabetes (56). A similar defect is observed in PERK-null mice; this defect also correlated with increased apoptosis of β cells (57, 63). In addition, mice with a homozygous Ser51Ala mutation at the PERK phosphorylation site in eIF2α display an even greater β cell loss that appears in utero (36). Therefore, translational control through PERK-mediated phosphorylation of eIF2α is required to maintain β cell survival (see Ron, this Perspective series, ref. 2). The more severe β cell loss in mice harboring the Ser51Ala eIF2α mutation suggests that additional eIF2α kinases partially complement the requirement for PERK in β cell function (36)

Glucose not only promotes the secretion of insulin but also stimulates insulin transcription and translation (64–66). Our group has proposed that glucose stimulated proinsulin mRNA translation is regulated by PERK-mediated phosphorylation of eIF2α in response to UPR activation 36). As blood glucose declines, energy may become limiting for protein folding in the ER and therefore activate the UPR to promote PERK-mediated phosphorylation of eIF2α. Conversely, a rise in blood glucose would turn off the UPR so that translation would accelerate, allowing entry of new preproinsulin into the ER. In this manner, PERK mediated phosphorylation of eIF2α provides a brake on protein synthesis, including proinsulin translation. Continual elevation of blood glucose may also prolong elevated proinsulin translation, eventually activating the UPR as the secretion capacity of the ER is overwhelmed. Therefore, a delicate balance between glucose levels and eIF2α phosphorylation needs to be maintained: Disturbances in either direction may lead to excessive UPR activation, with eventual β cell death.

The insulin resistance and hyperglycemia associated with type 2 diabetes is accommodated by an increase in proinsulin translation. Under these conditions the UPR is activated to compensate for the increased protein-folding requirement in the ER. Prolonged activation of the UPR could contribute to the β cell death associated with insulin resistance. Thus, the signaling mechanisms that β cells use for sensing glucose levels, triggering insulin secretion, and rapidly controlling insulin biosynthesis may have coevolved with ER signaling pathways to support these specialized functions. Pancreatic β cells are exquisitely sensitive to physiological fluctuations in blood glucose, because, in contrast to other cell types, they lack hexokinase, an enzyme with a low affinity but a high capacity for binding glucose. Therefore, in β cells, the production of glucose 6-phosphate and the production of ATP through glycolysis are controlled by glucokinase (67), and the ratio of ATP to ADP correlates directly with the blood glucose level. Periodic decreases in blood glucose level (as occurs between meals) would decrease the ATP/ADP ratio and compromise protein folding in the ER so that the UPR may be frequently activated in these cells. Hence, when glucose levels vary within the normal physiological range, the ER compartment of the β cell may be exposed to greater energy fluctuations than is the ER of other cell types, making the β cell uniquely dependent on the UPR for survival during intermittent decreases in blood glucose levels, as happens between meals. Additionally, the high-level expression of PERK and IRE1α in the pancreas may predispose these kinases to dimerization and activation in response to intermittent stress.

The UPR in organelle expansion The UPR is required for ER expansion that occurs upon differentiation of highly specialized secretory cells, but ER membrane expansion can also proceed independently of UPR activation. Overexpression of membrane proteins, such as HMG CoA reductase or the peroxisomal protein Pex15, promotes the expansion of smooth membranes without UPR activation (68, 69), as does overexpression of the p180 ribosome acceptor in the rough ER membrane (70). Conversely, protein overexpression, even under circumstances in which secretory capacity is unchanged (as occurs following the induction of high levels of cytochrome p450), can activate the UPR to induce ER chaperone levels to match the expanded membrane area (71, 72).

During the terminal differentiation of certain secretory cells, such as those in the pancreas or liver, membrane expansion is accompanied by a dramatic increase in protein secretion. Likewise, upon B cell maturation into high-level antibody-secreting plasma cells, the ER compartment expands approximately fivefold to accommodate the large increase in Ig synthesis. The requirement for the UPR in this latter process has been demonstrated in XBP1–/– cells. Since deletion of XBP1 produces an embryonic-lethal phenotype at day E14.5, the role of XBP1 in B and T cell development had to be studied in immunoincompetent RAG1–/– mice reconstituted with XBP1–/– embryonic stem cells (73). Work in these chimeric mice demonstrated that XBP1 is required for high-level Ig production. Interestingly, the induction of Ig heavy-chain and light-chain gene rearrangement and the assembly and transport of Igµ to the surface of the B cells occurred normally. However, plasma cells were not detected, suggesting a role for XBP1 in plasma cell differentiation or survival.

These findings support the hypothesis that induction of Ig synthesis activates the UPR to induce ER expansion to accommodate the high-level antibody expression. Alternatively, activation of the UPR may be part of the differentiation program that occurs prior to induction of high-level antibody synthesis. Plasma cell differentiation is stimulated in vivo by treatment with LPS or by ligation of CD40 receptors, treatments that activate the innate immune response and have been shown to induce XBP1 mRNA splicing (19). Thus, the UPR may contribute to a programmed response to signals that increase a cell’s protein-secretory demand.

The UPR in hyperhomocysteinemia. The association between high levels of serum homocysteine and the development of ischemic heart disease and stroke is supported by substantial epidemiological data. Unfortunately, it is not known whether homocysteine is the underlying cause of atherosclerosis and thrombosis. Severe hyperhomocysteinemia is caused by mutation in the cystathionine β-synthase (CBS) gene, whose product is a vitamin B6–dependent enzyme required for the conversion of homocysteine to cysteine. Elevated homocysteine is also associated with vitamin B deficiency. In cultured vascular endothelial cells, homocysteine induces protein misfolding in the ER by interfering with disulfide bond formation, and it activates the UPR to induce expression of several ER stress response proteins, such as BiP, GRP94, CHOP, and HERP (74–76). Homocysteine also activates apoptosis in a manner that requires an intact IRE1-signaling pathway (76).

These findings suggest that homocysteine acts intracellularly to disrupt ER homoeostasis. Indeed, recent studies confirm that induction of hyperhomocysteinemia elicits UPR activation in the livers of normal or Cbs+/– mice (77). In addition, hyperhomocysteinemia activates SREBP cleavage, leading to intracellular accumulation of cholesterol (77). Increased cholesterol biosynthesis may explain the hepatic steatosis and possibly the atherosclerotic lesions associated with hyperhomocysteinemia. Finally, hyperhomocysteinemia accelerates atherosclerosis in ApoE–/– mice (78, 79), although the molecular mechanisms remain to be elucidated.

Hyperhomocysteinemia is also associated with increased amyloid production and increased amyloid-mediated neuronal death in animal models of Alzheimer disease (80). These observations suggest that the UPR may link the disease etiologies of hyperhomocysteinemia and Alzheimer disease. HERP, a homocysteine-induced ER stress–responsive gene, appears to be involved in amyloid β-protein (Aβ) accumulation, including the formation of senile plaques and vascular Aβ deposits (81), and that it interacts with both presenilin-1 (PS1) and presenilin-2 (PS2), thus regulating presenilin-mediated Aβ generation. Immunohistochemical analysis of brains from patients with Alzheimer disease reveals intense HERP staining in activated microglia in senile plaques.

The UPR in cancer Hypoxia is a common feature of solid tumors that display increased malignancy, resistance to therapy, and poor prognosis. Hypoxia in the tumor results from increased demand due to dysregulated cell growth and from vascular abnormalities associated with cancerous tissue. The importance of hypoxia has been seen in the clinic, since it predicts for poor outcome of treatments, independent of treatment modality. Hypoxia activates the UPR, whose downstream signaling events can undermine the efficacy of treatment. Tumor cells need to adapt to the increasingly hypoxic environment that surrounds them as they grow, and the induction of the UPR is key to this response. Induction of the ER stress response genes, for example BiP and GRP94, in cancerous tissue correlates with malignancy, consistent with their antiapoptotic function (82). In addition, the UPR confers resistance to topoisomerase inhibitors, such as etoposide, and some UPR-induced genes directly mediate drug resistance via the multi-drug-resistance gene MDR. Therefore, approaches to prevent UPR activation in cancerous cells may significantly improve treatment outcome.

The proteasome inhibitor PS-341 is now in earlyphase clinical evaluation for the treatment of multiple myeloma, a clonal B cell tumor of differentiated plasma cells (83). The mechanism of PS-341 function is thought to be inhibition of IκB degradation, which prevents activation of the antiapoptotic transcription factor NF-κB. However, proteasomal inhibition would also prevent ERAD. As high-level heavy- or light-chain Ig production is likely associated with a certain degree of protein misfolding, it is possible that inhibition of ERAD function may be selectively toxic to B cell myelomas through activation of the UPR and apoptosis.

The UPR and viral pathogenesis The two major mediators of the IFN-induced arm of the innate immune response are evolutionarily related to IRE1 and PERK. The kinase/endoribonuclease domain of IRE1 is homologous to RNaseL, and the protein kinase domain of PERK is related to the double-stranded RNA–activated (dsRNA-activated) eIF2α protein kinase PKR. RNase L and PKR mediate the IFN induced antiviral response of the host, which is required to limit viral protein synthesis and pathogenesis. As part of the innate immune response to viral infection, RNase L and PKR are activated by dsRNAs produced as intermediates in viral replication. In contrast to activation by dsRNA, IRE1 and PERK are activated by ER stress, which can be induced by high-level viral glycoprotein expression. All enveloped viruses produce excess glycoproteins that could elicit PERK and IRE1 activation to meet the need for increased folding and secretory capacity. More studies will be required to elucidate the role of the UPR in various viral diseases.

Hepatitis C virus (HCV) is a positive-stranded RNA virus encoding a single polyprotein. Polyprotein cleavage generates at least ten polypeptides, including two glycoproteins, E1 and E2. A large amount of E1 forms disulfide–cross-linked aggregates with E2 in the ER (84). Since the accumulation of misfolded α1-PI elicits UPR activation, with subsequent hepatocyte death and hepatocellular carcinoma, it is possible that the aggregated E1/E2 complexes in the HCV-infected hepatocyte also contribute to hepatitis and hepatocellular carcinoma. Future studies should identify whether these glycoprotein aggregates activate the UPR to mediate the hepatocyte cell death and transformation associated with the pathogenesis of HCV infection.

The UPR in tissue ischemia Finally, neuronal death due to reperfusion after ischemic injury is associated with activation of the UPR (85, 86). Immediately after reperfusion, protein synthesis is inhibited, due at least in part to phosphorylation of eIF2α; this inhibition may represent a protective mechanism to prevent further neuron damage. Recent studies support the idea that eIF2α phosphorylation in response to reperfusion injury is mediated by PERK and hence that it depends on the UPR (87). If so, UPR activation prior to ischemic injury might protect the brain and other tissues from cell death during periods of reperfusion.

Summary A variety of approaches have been employed to identify the UPR signaling components, their function, and their physiological role. Yeast genetics allowed the definition of the basic ER stress–signaling pathway. The identification of homologous and parallel signaling pathways in higher eukaryotes has produced a mechanistic framework the cell uses to sense and compensate for ER over-load and stress. The high-level tissue-specific expression patterns of several ER stress–signaling molecules indicated the pancreas and intestine as organs that require UPR for physiological function. Analysis of UPR-induced gene expression established that protein degradation is required to reduce the stress of unfolded protein accumulation in the ER. Major advances in identifying UPR function and rele vance to disease were derived from mutation of UPR signaling components in model organisms and the identification of mutations in humans.

Despite tremendous progress, our knowledge of the UPR pathway remains incomplete. Further studies promise to expand our understanding of how ER stress impacts the other cellular signaling pathways. It will be very exciting and informative to understand how the UPR varies when critical components are genetically manipulated by deletion or other types of mutations. In addition, although the accumulation of unfolded protein in the ER is now known to contribute to pathogenesis in a variety of diseases, there are still few therapeutic approaches that target these events. With a greater understanding of protein-folding processes, pharmacological intervention with chemical chaperones to promote proper folding becomes feasible, as observed with sodium phenylbutyrate for ∆508 CFTR (see Gelman and Kopito, this Perspective series, ref. 53). Future intervention should consider activation of different subpathways of the UPR or overexpression of appropriate protein chaperones, as in the case of overexpression of the J domain of cytosolic HSP70, which suppresses polyglutamine toxicity in flies (88). Treatments that activate the ERAD response may also ameliorate pathogenesis in a number of the conformational diseases.

Over the past ten years, tremendous progress has been made in understanding the mechanisms and physiological significance of the UPR. The processes of protein folding and secretion, transcriptional and translational activation, and protein degradation are intimately interconnected to maintain homeostasis in the ER. A variety of environmental insults, genetic disease, and underlying genetic modifiers of UPR function contribute to the pathogenesis of different disease states. As we gain a greater understanding of the mechanisms that control UPR activation, it should be possible to discover methods to activate or inhibit the UPR as desired for therapeutic benefit.

  1. Horwich, A. 2002. Protein aggregation in disease: a role for folding intermediates forming specific multimeric interactions. J. Clin. Invest. 110:1221–1232. doi:10.1172/JCI200216781.
  2. Ron, D. 2002. Translational control in the endoplasmic reticulum stress response. J. Clin. Invest. 110:1383–1388. doi:10.1172/JCI200216784.
  3. Lee, A.S. 1992. Mammalian stress response: induction of the glucose-regulated protein family. Curr. Opin. Cell Biol. 4:267–273.
  4. Kaufman, R.J., et al. 2002. The unfolded protein response in nutrient sensing and differentiation. Nat. Rev. Mol. Cell Biol. 3:411–421.
  5. Ellgaard, L., and Helenius, A. 2001. ER quality control: towards an understanding at the molecular level. Curr. Opin. Cell Biol. 13:431–437.
  6. Jakob, C.A., Burda, P., Te Heesen, S., Aebi, M., and Roth, J. 1998. Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo. Glycobiology. 8:155–164.
  7. Travers, K.J., et al. 2000. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell. 101:249–258.
    ……

 

Citation: Cell Death and Disease (2014) 5, e1578; doi:10.1038/cddis.2014.539
Published online 18 December 2014

An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina
http://www.nature.com/cddis/journal/v5/n12/full/cddis2014539a.html

T Rana1, V M Shinde1, C R Starr1, A A Kruglov1, E R Boitet1, P Kotla1, S Zolotukhin2, A K Gross1 and M S Gorbatyuk1

  1. 1Department of Vision Sciences, University of Alabama at Birmingham, AL, USA
  2. 2Department of Pediatrics, University of Florida, FL, USA

Correspondence: M Gorbatyuk, Department of Vision Sciences, University of Alabama at Birmingham, 1670 University Boulevard, Birmingham, 35233 AL, USA. Tel: +1 205 934 6762; Fax: +1 205 934 3425; E-mail:mgortk@uab.edu

Received 20 July 2014; Revised 23 October 2014; Accepted 27 November 2014

Edited by P Ekert

Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1β, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1β was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1β. We observed ~19%reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1β.

Abbreviations: 

ERG, electroretinography; SD-OCT, spectral domain optical coherence tomography; UPR, unfolded protein response; IL-1β, Interleukin-1β; TNF-α, tumor necrosis factor-α; MCP-1, monocyte chemoattractant protein-1; NF-kB, ; nuclear factor kappa B, ; ER, endoplasmic reticulum; ADRP, autosomal dominant retinitis pigmentosa; RHO, rhodopsin; ERAI, ER stress activated indicator; Tn, tunicamycin; ONL, outer nuclear layer; H&E, hematoxylin and eosin; ONH, optic nerve head

 

ER stress and neuroinflammation: connecting the unfolded protein response to JAK/STAT signaling (P5196)

Gordon Meares,1 and Etty Benveniste1

1Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL

J Immunol May 2013 190 (Meeting Abstract Supplement) 198.5

http://www.jimmunol.org/cgi/content/meeting_abstract/190/1_MeetingAbstracts/198.5

Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. ER stress is brought on by misfolded proteins. In turn, cells respond with activation of the unfolded protein response (UPR). The UPR is a highly conserved pathway that transmits both adaptive and apoptotic signals to restore homeostasis or eliminate the irreparably damaged cell. Recent evidence indicates that ER stress and inflammation are linked. In this study, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in astrocytes. The JAK/STAT pathway mediates the biological actions of many cytokines and growth factors. We have found that ER stress leads to the activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of IL-6 and several chemokines. The activation of STAT3 signaling is dependent on the protein kinase PERK, a central component of the UPR. Knockdown of PERK abrogates ER stress-induced activation of STAT3 and overexpression of PERK is sufficient to activate STAT3. Additionally, ER stressed astrocytes, via paracrine signaling, can stimulate activation of microglia leading to production of oncostatin M (OSM). OSM can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK-JAK1-STAT3 signaling pathway that may elicit a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation.

 

Neural Plasticity
Volume 2014 (2014), Article ID 610343, 15 pages
http://dx.doi.org/10.1155/2014/610343

Review Article

Surveillance, Phagocytosis, and Inflammation: How Never-Resting Microglia Influence Adult Hippocampal Neurogenesis

Amanda Sierra,1,2,3 Sol Beccari,2,3 Irune Diaz-Aparicio,2,3 Juan M. Encinas,1,2,3 Samuel Comeau,4,5 and Marie-Ève Tremblay4,5

1Ikerbasque Foundation, 48011 Bilbao, Spain
2Achucarro Basque Center for Neuroscience, Bizkaia Science and Technology Park, 48170 Zamudio, Spain
3Department of Neurosciences, University of the Basque Country, 48940 Leioa, Spain
4Centre de Recherche du CHU de Québec, Axe Neurosciences, Canada G1P 4C7
5Département de Médecine Moléculaire, Université Laval, Canada G1V 4G2

Received 10 December 2013; Accepted 11 February 2014; Published 19 March 2014

Academic Editor: Carlos Fitzsimons

http://www.hindawi.com/journals/np/2014/610343/

Microglia cells are the major orchestrator of the brain inflammatory response. As such, they are traditionally studied in various contexts of trauma, injury, and disease, where they are well-known for regulating a wide range of physiological processes by their release of proinflammatory cytokines, reactive oxygen species, and trophic factors, among other crucial mediators. In the last few years, however, this classical view of microglia was challenged by a series of discoveries showing their active and positive contribution to normal brain functions. In light of these discoveries, surveillant microglia are now emerging as an important effector of cellular plasticity in the healthy brain, alongside astrocytes and other types of inflammatory cells. Here, we will review the roles of microglia in adult hippocampal neurogenesis and their regulation by inflammation during chronic stress, aging, and neurodegenerative diseases, with a particular emphasis on their underlying molecular mechanisms and their functional consequences for learning and memory.

  1. Microglia: The Resident Immune Cells of the Brain

Microglia were first described in 1919 by the Spanish neuroanatomist Pío del Río Hortega, a disciple of the renowned Santiago Ramón y Cajal, almost half a century later than neurons and astrocytes and just before oligodendrocytes [1]. This delayed appearance into the neuroscience arena is still apparent today, as microglia remain one of the least understood cell types of the brain. Traditionally, microglia were simply considered as “brain macrophages” controlling the inflammatory response during acute insults and neurodegenerative conditions, and only recently was their unique origin revealed. Indeed, microglia were shown to derive from primitive myeloid progenitors of the yolk sac that invade the central nervous system (CNS) during early embryonic development (reviewed in [2]). In contrast, circulating monocytes and lymphocytes, as well as most tissue macrophages, derive from hematopoietic stem cells located initially in the foetal liver and later in the bone marrow [3]. In the adult brain, the microglial population is maintained exclusively by self-renewal during normal physiological conditions [2]. As a consequence, microglia are the only immune cells which permanently reside in the CNS parenchyma, alongside neural tube-derived neurons, astrocytes, and oligodendrocytes.

These past few years, unprecedented insights were also provided into their extreme dynamism and functional behaviour, in health as much as in disease. Indeed, microglia were revealed to be exceptional sensors of their environment, responding on a time scale of minutes to even subtle variations of their milieu, by undergoing concerted changes in morphology and gene expression [45]. During pathological insults, “activated” microglia were particularly shown to thicken and retract their processes, extend filopodia, proliferate and migrate, release factors and compounds influencing neuronal survival (such as proinflammatory cytokines, trophic factors, reactive oxygen species (ROS), etc.), and phagocytose pathogens, degenerating cells and debris, thus providing better understanding of their roles in orchestrating the inflammatory response [6]. These abilities as immune cells are also recruited during normal physiological conditions, where “surveillant” microglia further participate in the remodeling of neuronal circuits by their phagocytic elimination of synapses and their regulation of glutamatergic receptors maturation and synaptic transmission, among other previously unexpected roles [79], in addition to their crucial involvement in the phagocytic elimination of newborn cells in the context of adult neurogenesis [10].

Our review will discuss the emerging roles of microglia in adult hippocampal neurogenesis and their regulation by inflammation during chronic stress, aging, and neurodegenerative diseases, with a particular emphasis on their underlying molecular mechanisms and their functional consequences for learning and memory (Figure 1).

 

http://www.hindawi.com/journals/np/2014/floats/610343/thumbnails/610343.fig.001_th.jpg

Figure 1: The effects of surveillant and inflammatory microglia on the adult hippocampal neurogenic cascade. During physiological conditions, surveillant microglia effectively phagocytose the excess of apoptotic newborn cells and may release antineurogenic factors such as TGF. This anti-inflammatory state is maintained by neuronal (tethered or released) fractalkine. Enriched environment drives microglia towards a phenotype supportive of neurogenesis, via the production of IGF-1. In contrast, inflammatory challenge triggered by LPS, irradiation, aging, or AD induces the production of proinflammatory cytokines such as IL-1, TNF, and IL-6 by microglia as well as resident astrocytes and infiltrating monocytes, neutrophils, and lymphocytes. These cytokines have profound detrimental effects on adult neurogenesis by reducing the proliferation, survival, integration, and differentiation of the newborn neurons and decreasing their recall during learning and memory paradigms.

  1. A Brief Overview of Adult Hippocampal Neurogenesis

Adult hippocampal neurogenesis is continuously maintained by the proliferation of neural stem cells located in the subgranular zone (SGZ) [1113]. These neuroprogenitors have been named “radial glia-like cells” (rNSCs), or type 1 cells, since they morphologically and functionally resemble the embryonic radial glia. They have also been defined as “quiescent neuroprogenitors” because only a small percentage of the population is actively dividing during normal physiological conditions. The lineage of these cells is frequently traced by using analogs of the nucleotide thymidine, such as bromodeoxyuridine (BrdU) which gets incorporated into the DNA of dividing cells during the S phase and can be detected by immunofluorescence. Alternatively, their lineage can be traced by labeling with fluorescent reporters which are delivered to dividing cells by retroviral vectors or expressed by specific cell type promoters via inducible transgenic mice (for a review of the methods commonly used to study adult neurogenesis, see [14]). The daughter cells of rNSCs, also called type 2 cells or amplifying neuroprogenitors (ANPs), rapidly expand their pool by proliferating before becoming postmitotic neuroblasts. Within a month, these neuroblasts differentiate and integrate as mature neurons into the hippocampal circuitry [15]. They however display unique electrophysiological characteristics during several months, being more excitable than mature neurons [16], and constitute a special cell population that is particularly inclined to undergo synaptic remodeling and activity-dependent plasticity [17].

These unique properties of the newborn neurons and the neurogenic cascade in general suggested that adult hippocampal neurogenesis could play an important role in hippocampal-dependent functions that require extensive neuroplasticity such as learning and memory. Indeed, activity-dependent plasticity and learning are long known for modulating adult neurogenesis in a complex, yet specific manner, with adult hippocampal neurogenesis being influenced by learning tasks which depend on the hippocampus [4445]. For instance, hippocampal-dependent learning paradigms were found to regulate the survival of newborn neurons, in a positive manner that depends on the timing between their birth and the phases of learning [4647]. Young (1.5–2 months old) newborn neurons were also shown to be preferentially activated during memory recall in a water maze task, compared to mature neurons, as determined by colabeling of BrdU with immediate early genes such as c-Fos and Arc, in which expression correlates with neuronal firing [48]. Nonetheless, it has only been in the last few years that loss-of-function and gain-of-function approaches with inducible transgenic mice were able to confirm that adult hippocampal neurogenesis is necessary for synaptic transmission and plasticity, including the induction of long-term potentiation (LTP) and long-term depression [49], as well as trace learning in conditioned protocols [50], memory retention in spatial learning tasks [5152], and encoding of overlapping input patterns, that is, pattern separation [53].

Adult hippocampal neurogenesis and its functional implications for learning and memory are however influenced negatively by a variety of conditions that are commonly associated with microglial activation and inflammation in the brain, such as chronic stress, aging, and neurodegenerative diseases, as we will review herein. Indeed, inflammation caused by irradiation produces a sustained inhibition of neurogenesis, notably by decreasing the proliferation and neuronal differentiation of the progenitors, and therefore, exposure to therapeutic doses of cranial irradiation has been widely used for modulating neurogenesis experimentally before the development of more specific approaches [54].

  1. Regulation of Adult Hippocampal Neurogenesis by Inflammation

Inflammation is a natural bodily response to damage or infection that is generally mediated by proinflammatory cytokines such as interleukin 1 beta (IL-1), interleukin 6 (IL-6), and tumour necrosis factor alpha (TNF), in addition to lipidic mediators such as prostaglandins and leukotrienes. Oftentimes, it is associated with an increased production of ROS, as well as nitric oxide (NO). Together, these proinflammatory mediators lead to an increase in local blood flow, adhesion, and extravasation of circulating monocytes, neutrophils, and lymphocytes [55]. In the brain, microglia are the main orchestrator of the neuroinflammatory response, but other resident cell types, including astrocytes, endothelial cells, mast cells, perivascular and meningeal macrophages, and even neurons, can produce proinflammatory mediators, though perhaps not to the same extent as microglia [56]. In addition, peripheral immune cells invading the CNS during inflammation can further produce proinflammatory mediators, but the respective contribution of microglia versus other cell types in the inflammatory response of the brain is poorly understood.

The harmful effects of inflammation are also widely determined by the actual levels of proinflammatory mediators released, rather than the occurrence or absence of an inflammatory response in itself. For instance, TNF regulates synaptic plasticity by potentiating the cell surface expression of AMPA glutamatergic receptors, thus resulting in a homeostatic scaling following prolonged blockage of neuronal activity during visual system development [57]. However, TNF also produces differential effects at higher concentrations,ranging from an inhibition of long-term potentiation to an enhancement of glutamate-mediated excitotoxicityin vitro [58]. Inflammation induced by chronic ventricular infusion of bacterial lipopolysaccharides (LPS; a main component of the outer membrane of Gram-negative bacteria), that is, the most widely used method for inducing an inflammatory challenge, also increases ex vivo the hippocampal levels of TNF and IL-1, thereby impairing novel place recognition, spatial learning, and memory formation, but all these cognitive deficits can be restored by pharmacological treatment with a TNF protein synthesis inhibitor, a novel analog of thalidomide, 3,6′-dithiothalidomide [59].

The impact of inflammation on adult hippocampal neurogenesis was originally discovered by Olle Lindvall and Theo Palmer’s groups in 2003, showing that systemic or intrahippocampal administration of LPS reduces the formation of newborn neurons in the adult hippocampus, an effect that is prevented by indomethacin, a nonsteroidal anti-inflammatory drug (NSAID) which inhibits the synthesis of proinflammatory prostaglandins [6061]. Similarly, inflammation can determine the increase in neurogenesis that is driven by seizures, a context in which neurogenesis can be prevented by LPS and increased by the anti-inflammatory antibiotic minocycline [60]. In these studies, hippocampal proliferation remained unaffected by LPS or minocycline and thus it is likely that inflammation targeted the survival of newborn cells [6061], as LPS is known to increase SGZ apoptosis [62]. Inflammation also has further downstream effects on the neurogenic cascade. For instance, LPS increases the number of thin dendritic spines and the expression of the excitatory synapses marker “postsynaptic density protein of 95kDa” (PSD95) in newborn neurons. LPS in addition increases the expression of GABAA receptors at early stages of synapse formation, leading to suggesting a possible imbalance of excitatory and inhibitory neurotransmission in these young neurons [63]. Finally, LPS also prevents the integration of newborn neurons into behaviourally relevant networks, including most notably their activation during spatial exploration, as determined by the percentage of BrdU cells colabeled with the immediate early gene Arc [64].

Importantly, none of these manipulations is specific to microglia and may directly or indirectly affect other brain cells involved in the inflammatory response of the brain. For instance, both LPS and minocycline affect astrocytic function in vitro and in vivo [6569]. Furthermore, LPS is known to drive infiltration of monocytes and neutrophils into the brain parenchyma [70]. Monocytes and neutrophils produce major proinflammatory mediators and could therefore act on the neurogenic cascade as well. The implication of microglia in LPS-induced decrease in neurogenesis is nonetheless supported in vivo by the negative correlation between the number of newborn neurons (BrdU+, NeuN+ cells) and the number of “activated” microglia (i.e., expressing ED1) [60]. ED1, also called CD68 or macrosialin, is a lysosomal protein which is overexpressed during inflammatory challenge. While the location of ED1 previously suggested its involvement in phagocytosis, its loss of function did not result in phagocytosis deficits and thus, its function still remains unknown (reviewed in [10]). The number of ED1-positive microglia also negatively correlates with neurogenesis during inflammation provoked by cranial irradiation [61]. While correlation does not involve causation, nor can pinpoint to the underlying mechanism, these experiments were the first to reveal a potential role for “activated” microglia in the regulation of adult hippocampal neurogenesis. More direct evidence of microglial mediation in LPS deleterious effects was obtained from in vitro experiments, as it was shown that conditioned media from LPS-challenged microglia contained IL-6, which in turn caused apoptosis of neuroblasts [61]. Nonetheless, astrocytes can also release IL-6 when stimulated with TNF or IL-1 [71] and chronic astrocytic release of IL-6 in transgenic mice reduced proliferation, survival, and differentiation of newborn cells, thus resulting in a net decrease in neurogenesis [72]. In summary, while the detrimental impact of inflammation on neurogenesis is well established, more work is needed to define the specific roles played by the various inflammatory cells populating the brain.

  1. Inflammation Associated with Chronic Stress

Across health and disease, the most prevalent condition that is associated with neuroinflammation is “chronic stress,” which commonly refers to the repeated or sustained inability to cope with stressful environmental, social, and psychological constraints. Chronic stress is characterized by an imbalanced secretion of glucocorticoids by the hypothalamic-pituitary-adrenal (HPA) axis (most notably cortisol in humans and corticosterone in rodents), which leads to an altered brain remodeling, massive loss of synapses, and compromised cognitive function [73]. In particular, an impairment of spatial learning, working memory, novelty seeking, and decision making has been associated with chronic stress [74]. Glucocorticoids are well known for their anti-inflammatory properties, as they interfere with NF-B-mediated cytokine transcription, ultimately delaying wound healing [75]. They are also potent anti-inflammatory mediators in vivo [76] and in purified microglia cultures [77]. Recently, repeated administration of high doses of glucocorticoids by intraperitoneal injection, to mimic their release by chronic stress, was also shown to induce a loss of dendritic spines in the motor cortex, while impairing learning of a motor task. A transcription-dependent pathway acting downstream of the glucocorticoid receptor GR was proposed [7879] but the particular cell types involved were not identified.

Microglia are considered to be a direct target of the glucocorticoids, as they were shown to express GR during normal physiological conditions in vivo [77]. In fact, transgenic mice lacking GR in microglia and macrophages show an increased production of proinflammatory mediators (including TNF and IL-1) and greater neuronal damage in response to an intraparenchymal injection of LPS, compared to wild-type mice [80]. In contrast, glucocorticoids are considered to be proinflammatory in the chronically stressed brain [81], where among other changes they can promote inflammation, oxidative stress, neurodegeneration, and microglial activation [82]. For example, repeated restraint stress induces microglial proliferation and morphological changes, including a hyperramification of their processes in the adult hippocampus following restraint stress [83], but a nearly complete loss of processes in the context of social defeat [84]. Prenatal restraint stress also causes an increase in the basal levels of TNF and IL-1, while increasing the proportion of microglia showing a reactive morphology in the adult hippocampus [85]. Similarly, social defeat leads to an enhanced response to the inflammatory challenge induced by intraperitoneal injection of LPS, including an increased production of TNF and IL-1, and expression of inducible NO synthase (iNOS) by microglia, accompanied by an increased infiltration of circulating monocytes [8486]. Therefore, microglia are a strong candidate for mediating some of the effects of stress on adult neurogenesis, as will be discussed below, in synergy with other types of inflammatory cells.

Chronic stress is well known for its negative effects on hippocampal neurogenesis (reviewed in [8788]), although not all stress paradigms are equally effective [89]. Several stress paradigms can decrease neuroprogenitors proliferation in the tree shrew [90] and in mice [9192], although this effect seems to be compensated by an increased survival of newborn neurons [92] and whether stress results in a net increase or decrease in neurogenesis remains controversial (reviewed in [8788]). The effects of stress on adult neurogenesis seem to be mediated at least partially by glucocorticoids, because mice lacking a single copy of the GR gene show behavioural symptoms of depression including learned helplessness, neuroendocrine alterations of the HPA axis, and impaired neurogenesis [93]. In parallel, chronic stress is associated with an increased inflammatory response, which may inhibit neurogenesis as well. For instance, serum levels of IL-1and IL-6 are significantly increased in depressed patients [94]. In mice, restraint stress leads to a widespread activation of NF-B in the hippocampus, including at the level of neuroprogenitors [95] and increased protein levels of IL-1 [96]. In addition to the direct role of glucocorticoids, IL-1 also seems to mediate some of the effects of mild chronic stress, because in vivo manipulations that block IL-1 (either pharmacologically or in null transgenic mice) prevent the anhedonic stress response and the antineurogenic effect of stress [9196]. Moreover, the corticoids and IL-1 pathways may regulate each other in a bidirectional manner because the administration of a GR antagonist can blunt the LPS-induced production of hippocampal IL-1 in stressed mice [97], whereas mice knockout for the IL-1 receptor (IL-1R1) fail to display the characteristic elevation of corticosterone induced by mild chronic stress [96]. Another stress-related cytokine, IL-6, induces depressive phenotypes and prevents the antidepressant actions of fluoxetine when administered to mice in vivo [98]. So far the effects of stress on neurogenesis via corticosteroids and inflammation have been assumed to be cell autonomous, as neuroprogenitors express both GR [99] and IL-1R1 [95]. The potential participation of microglia is yet to be determined, but there are some reports of a direct effect of stress on microglial activation. For instance, microglia acutely isolated from mice subjected to acute stress (by inescapable tail shock) showed a primed response to LPS challenge by producing higher levels of IL-1 mRNA ex vivo [100], and the specific loss of expression of GR in microglia leads to a blunted inflammatory response in vitro and to a decreased neuronal damage in vivo in response to LPS [80]. In stress paradigms, these enhanced responses of microglia to inflammatory challenges are similar to their age-related “priming” which has been associated with and is possibly due to an increased basal production of proinflammatory mediators. However, whether microglia express increased levels of IL-1 and other proinflammatory cytokines in response to stressful events is presently unclear [101]. It is thus possible that some of the antineurogenic effects of stress are exerted by means of microglial-dependent inflammation, but this hypothesis remains to be experimentally tested.

  1. Inflammation Associated with Aging and Neurodegenerative Diseases

Inflammation is also commonly associated with normal aging and neurodegenerative diseases and, therefore, could represent a putative underlying mechanism that explains their decrease in hippocampal neurogenesis. Nonetheless, inflammation is also associated with neurological diseases, such as epilepsy or stroke, where neurogenesis is thought to be increased, although the data from rodents and humans is somewhat conflictive [102]. Neurogenesis is well known to decline throughout adulthood and normal aging in rodents and humans [103104], but the decay is more pronounced and occurs later in life in mice than in humans [105]. The aging-associated decrease in neurogenesis has been shown to occur mainly as a consequence of exhaustion of the rNSC population which, after being recruited and activated, undergo three rounds of mitosis in average and then terminally differentiate into astrocytes [12106]. In addition, a reduced mitotic capacity of the neuroprogenitors could further contribute to decreasing neurogenesis [106], and moreover, an age-related increase in the levels of proinflammatory cytokines could also hinder neurogenesis in the aging brain. Serum levels of IL-1, IL-6, and TNF are elevated in elderly patients [107108]. Aged microglia express higher levels of these proinflammatory cytokines and show a greater response to LPS inflammatory challenge, that is, a “primed” response, than their younger counterparts [109]. The origin of this low-grade age-related inflammation (“inflamm-aging” [110]) remains unknown and may be related to both aging and damage to the surrounding neurons, as well as aging of the immune system per se.

At the cellular level, stress to the endoplasmic reticulum (ER) caused by various perturbations, such as nutrient depletion, disturbances in calcium or redox status, or increased levels of misfolded proteins, can induce a cell-autonomous inflammatory response to neurons. Stress to the ER, a multifunctional organelle which is involved in protein folding, lipid biosynthesis, and calcium storage triggers a homeostatic response mechanism named the unfolding protein response (UPR), aiming to clear the unfolded proteins in order to restore normal ER homeostasis [111]. However, if the ER stress cannot be resolved, the UPR also initiates inflammatory and apoptotic pathways via activation of the transcription factor NF-B which controls the expression of most proinflammatory cytokines [112]. In the brain, ER stress is often initiated by the formation of abnormal protein aggregates in several neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and prion-related disorders [113]. This neurodegeneration-associated ER stress is assumed to occur mostly in neurons, but there are some examples of microglial protein misfolding as well. For instance, both microglia and neurons overexpress CHOP (C/EBP homologous protein), a transcription factor which is activated during ER stress in human patients and mouse models of ALS [114]. Inflammation has been speculated to be a main negative contributor to the pathology of ALS [115], but a direct microglial involvement in mediating the inflammatory response to abnormal protein aggregation in ALS and other neurodegenerative conditions remains to be tested. Finally, ER stress has been linked to a variety of inflammatory conditions [116117], including chronic stress, diet-induced obesity, and drug abuse, as well as atherosclerosis and arthritis [118120]. During normal aging, a progressive decline in expression and activity of key ER molecular chaperones and folding enzymes could also compromise the adaptive response of the UPR, thereby contributing to the age-associated decline in cellular functions [118]. Therefore, aging is strongly associated with a chronic ER stress which leads to increased activation of NF-B [112]; however, the contribution of the different brain cell types to “inflamm-aging” is still poorly understood. The detrimental effects on neurogenesis of increased proinflammatory cytokines in the aging brain are not necessarily related to microglia, but also to stressed neurons. Furthermore, ER stress may also cause a cell-autonomous response in neural stem cells [121], although its impact on neurogenesis remains to be experimentally determined.

In addition, aging is accompanied by an increased level of mitochondrial oxidative stress, which in turn activates the “Inflammasome” [122], a group of multimeric proteins comprising the interleukin 1 converting enzyme (ICE, caspase 1) which serves to release the active form of the cytokine [123]. IL-1 may act directly on rNSCs (visualised by labeling with the Sox2 marker), as they express IL-1R1 in the adult hippocampus [91]. Treatment with IL-1 decreases hippocampal proliferation in young mice [91] and pharmacological inhibition of ICE partially restores the number of newborn neurons in aged mice without significantly affecting their differentiation rate [124]. Transgenic IL-1 overexpression results in chronic inflammation and depletion of doublecortin-labeled neuroblasts, thus mimicking the aging-associated depletion of neurogenesis [125]. The actual mechanism of action of IL-1 on neurogenesis in aged mice, including decreased proliferation of rNSCs/ANPs and survival of newborn neurons, remains undetermined. Microglia are a main source of IL-1in the aging brain, but the hypothesis that microglia-derived IL-1 is responsible for depleting neurogenesis in the aging brain remains to be directly tested.

The regulation of neurogenesis by IL-1 in the aging brain has been further linked to the activity of another cytokine, the chemokine fractalkine, or CX3CL1. Fractalkine has soluble and membrane-tethered forms and is exclusively expressed by neurons, while the fractalkine receptor (CX3CR1) is expressed in the brain by microglia alone [126]. This module forms a unique neuron-microglia signalling unit that controls the extent of microglial inflammation in several neurodegenerative conditions including PD, ALS [127], or AD [128]. In fact, CX3CR1 blocking antibodies increase the production of hippocampal IL-1 when administered to young adult rats [129]. Importantly, chronic treatment with fractalkine increases hippocampal proliferation and the number of neuroblasts in aged (22 months old) but not young (3 months old) or middle-aged rats (12 months old), whereas an antagonists of CX3CR1 has the opposite effects in young, but not in middle-aged nor old rats [129]. Since fractalkine expression is decreased during aging [129], a reduced neuron-microglia signalling might be releasing the brake on microglial contribution to inflammatory responses, although increased levels of fractalkine were instead reported in aged rat hippocampus by other studies [68]. Additional insights into the role of fractalkine signalling come from knock-in mice in which the endogenous CX3CR1 locus is replaced by the fluorescent reporter GFP [126]. The initial studies suggested that  (i.e., ) mice have no significant differences in brain development and functions [130], but more systematic investigations recently revealed a long list of hippocampal-dependent changes in young (3 months old)  and  mice compared to wild-type mice. These changes notably included decreased neuroprogenitors proliferation and neuroblasts number, impaired LTP, performance in contextual fear conditioning and water maze spatial learning and memory, and, importantly, increased IL-1 protein levels [131]. The signalling pathway of fractalkine-IL-1 is functionally relevant, because IL-1R1 antagonists rescued LTP and cognitive function in  mice [131]. In sum, even though neuronal fractalkine seems to be sufficient for restraining the inflammatory activity of microglia in young rats, its downregulation during aging could activate the microglial inflammatory response and thereby subsequently reduce the proliferation of remaining neuroprogenitors.

In AD, inflammatory cytokines such as IL-1 are overexpressed in the microglia associated with the amyloid beta (A) plaques of postmortem samples [132] and in transgenic mice modeling the disease [133]. The loss of synapses (from hippocampus to frontal cortex) is one of the main pathological substrates in this disease, but adult neurogenesis is also severely reduced in most mouse models of AD, possibly due to a decreased proliferation of neuroprogenitors and a decreased survival of newborn cells, even though the putative changes in the neurogenic cascade in postmortem samples remain controversial (reviewed in [102]). This lack of agreement is possibly explained by the fact that the vast majority of AD cases have a late onset over 65 years of age, when little neurogenesis remains. In contrast, in most transgenic AD mouse models, the Aaccumulation, cognitive deficits, and changes in neurogenesis are already detectable in young animals (2-3 months old). The study of AD is further hindered by the difficulty in comparing the time course and pathology across different mouse models. For instance, early treatment with minocycline can improve cognition and reduce A burden in mice expressing the human amyloid precursor protein (APP) [134]. In contrast, in mice expressing APP and a mutated form of presenilin 1 (PS1), which is part of the  secretase pathway that cleaves A, inflammation is reduced without any detectable changes in A plaques deposition [135]. Concomitantly with a decrease in tissue inflammatory cytokines and number of microglial cells, minocycline restores neurogenesis and hippocampus-dependent memory deficits in these APP/PS1 mice [135], indirectly suggesting that cognitive decay in AD may be at least in part related to a detrimental effect of inflammation on hippocampal neurogenesis. Direct evidence that neurogenesis is associated with the cognitive performance in AD is still lacking. Further research is also necessary to determine the neurogenic targets of AD-related inflammation. One central open question for future therapies aiming at increasing neurogenesis and cognition in AD is whether neuroprogenitors are spared or whether their age-induced loss becomes accelerated. Rather than increasing the proliferation and neurogenic output of the few rNSCs remaining in an old AD brain, it may be more relevant to develop strategies that prevent the age-related loss of neuroprogenitors in presymptomatic patients.

In summary, inflammation associated with a wide variety of experimental models of disease produces strong detrimental effects on hippocampal neurogenesis. These effects on human neurogenesis are however not so well described and, in vitro, IL-1 increases the proliferation of hippocampal embryonic neuroprogenitors but decreases their differentiation into neurons [136]. Novel methods to assess hippocampal neurogenesis in the living human brain, from metabolomics of neuroprogenitors to hippocampal blood brain volume (reviewed in [102]), will help to determine the contribution of inflammation to adult neurogenesis in the healthy and diseased human brain during aging.

  1. Normal Physiological Conditions

In the healthy mature brain, microglia are an essential component of the neurogenic SGZ niche, where they physically intermingle with neuroprogenitors, neuroblasts, and newborn neurons [62]. Here, surveillant microglia effectively and rapidly phagocytose the excess of newborn cells undergoing apoptosis [62]. Importantly, microglial phagocytosis in the adult SGZ is not disturbed by inflammation associated with aging or by LPS challenge, as the phagocytic index (i.e., the proportion of apoptotic cells completely engulfed by microglia) is maintained over 90% in these conditions [62]. Nonetheless, the consequences of microglial phagocytosis on adult hippocampal neurogenesis remain elusive. Treatment of mice with annexin V, which binds to the phosphatidylserine (PS) receptor and prevents the recognition of PS on the surface of apoptotic cells, presumably blocking phagocytosis, increases the number of apoptotic cells in the SGZ [40]. Concomitantly, annexin V reduces neurogenesis by decreasing the survival of neuroblasts without affecting neuroprogenitors proliferation [40]. Similar results were obtained in transgenic mice knock-out for ELMO1, a cytoplasm protein which promotes the internalization of apoptotic cells, although the effects on neurogenesis were ascribed to a decreased phagocytic activity of neuroblasts [40]. The actual phagocytic target of the neuroblasts remains undetermined, but the newborn apoptotic cells in the adult SGZ are exclusively phagocytosed by microglia, at least in physiological conditions [62]. Nevertheless, none of the above manipulations has specifically tested the role of microglial phagocytosis in hippocampal-dependent learning and memory and thus, the functional impact of microglial phagocytosis in adult neurogenic niches during normal physiological conditions remains to be elucidated.

Microglial phagocytosis of apoptotic cells is actively anti-inflammatory, at least in vitro, and thus it has been hypothesized that anti-inflammatory cytokines produced by phagocytic microglia may further regulate neurogenesis [10]. For instance, transforming growth factor beta (TGF), which is produced by phagocytic microglia in vitro [137], inhibits the proliferation of SGZ neuroprogenitors [138]. Microglia are further able to produce proneurogenic factors in vitro [139]. When primed with cytokines associated with T helper cells such as interleukin 4 (IL-4) or low doses of interferon gamma (IFN), cultured microglia support neurogenesis and oligodendrogenesis through decreased production of TNF and increased production of insulin-like growth factor 1 (IGF-1) [139], an inducer of neuroprogenitor proliferation [26]. A list of potential factors produced by microglia and known to act on neuroprogenitor proliferation can be found in Table 1. In addition, recent observations suggest that neuroprogenitor cells may not only regulate their own environment, but also influence microglial functions. For instance, vascular endothelial growth factor (VEGF) produced by cultured neuroprecursor cells directly affects microglial proliferation, migration, and phagocytosis [20]. More potential factors produced by neuroprogenitors shown to be influencing microglial activity and function can be found in Table 2. However, it has to be taken into account that most of these observations were obtained in culture and that further research is needed in order to elucidate whether those factors are also secreted and have the same regulatory responses in vivo.

Table 1: Summary of factors secreted by microglia and the potential effect they have on neuroprogenitors in vitro.
Microglia secreted
factors
Reference Modulation of neural progenitor cells Reference
BDNF [18] Differentiation [19]
EGF [20] Survival, expansion, proliferation, differentiation [21]
FGF [22] Survival and expansion [23]
GDNF [24] Survival, migration, and differentiation [25]
IGF-1 [21] Proliferation [26]
IL-1 [27] Reduction in migration [27]
IL-6 [28] Inhibition of neurogenesis [29]
IL-7 [20] Differentiation [30]
IL-11 [20] Differentiation [30]
NT-4 [24] Differentiation [31]
PDGF [32] Expansion and differentiation [33]
TGF [34] Inhibition of proliferation [19]

 

Table 1: Summary of factors secreted by microglia and the potential effect they have on neuroprogenitors in vitro.

http://www.hindawi.com/journals/np/2014/610343/tab1/

 

 

Table 2: Summary of factors secreted by neuroprogenitors and the potential effect they have on microglia in vitro.

NPC secreted factors Reference Modulation of microglia Reference
BDNF [18] Proliferation and induction of phagocytic activity [35]
Haptoglobin [24] Neuroprotection [36]
IL-1 [37] Intracellular Ca+2 elevation and proliferation [22]
IL-6 [37] Increase in proliferation [38]
M-CSF [20] Mitogen [39]
NGF [40] Decrease in LPS-induced NO [41]
TGF [37] Inhibition of TNF secretion [42]
TNF [37] Upregulation of IL-10 secretion [43]
VEGF [20] Induction of chemotaxis and proliferation [20]

http://www.hindawi.com/journals/np/2014/floats/610343/thumbnails/610343.tab2_th.jpg

Table 2: Summary of factors secreted by neuroprogenitors and the potential effect they have on microglia in vitro.

In addition, microglial capacity to remodel and eliminate synaptic structures during normal physiological conditions has suggested that microglia could also control the synaptic integration of the newborn neurons generated during adult hippocampal neurogenesis [140]. Three main mechanisms were proposed: (1) the phagocytic elimination of nonapoptotic axon terminals and dendritic spines, (2) the proteolytic remodeling of the perisynaptic environment, and (3) the concomitant structural remodeling of dendritic spines [7140]. Indeed, microglial contacts with synaptic elements are frequently observed in the cortex during normal physiological conditions, sometimes accompanied by their engulfment and phagocytic elimination [141143], as in the developing retinogeniculate system [144]. Microglial cells are distinctively surrounded by pockets of extracellular space, contrarily to all the other cellular elements [142], suggesting that microglia could remodel the volume and geometry of the extracellular space, and thus the concentration of various ions, neurotransmitters, and signalling molecules in the synaptic environment. Whether microglia create the pockets of extracellular space themselves or not remains unknown, but these pockets could result from microglial release of extracellular proteases such as metalloproteinases and cathepsins [145], which are well known for influencing the formation, structural remodeling, and elimination of dendritic spines in situ and also experience-dependent plasticity in vivo [7146]. More recently, microglial phagocytosis of synaptic components was also observed in the developing hippocampus, in the unique time window of synaptogenesis, a process which is notably regulated by fractalkine-CX3CR1 signalling [147]. Therefore, the attractive hypothesis that microglial sculpts the circuitry of newborn cells in the adult hippocampus deserves further attention.

Lastly, microglia were also involved in increasing adult hippocampal neurogenesis in the enriched environment (EE) experimental paradigm. EE is a paradigm mimicking some features of the normal living circumstances of wild animals, as it gives them access to social interactions, toys, running wheels, and edible treats. EE has long been known to enhance neurogenesis by acting on newborn cells survival, resulting ultimately in an enlargement of the dentate gyrus [148]. Functionally, these changes are accompanied by enhanced spatial learning and memory formation with the water maze paradigm [149]. Similar increases in neurogenesis are obtained by subjecting mice to voluntary running paradigms, although in this case the effect is mediated by increased neuroprogenitor proliferation [150]. During inflammatory conditions, EE is antiapoptotic and neuroprotective [151] and it limits the hippocampal response to LPS challenge by decreasing the expression of several cytokines and chemokines, including IL1- and TNF [152]. In fact, EE is believed to counteract the inflammatory environment and rescue the decreased number of neuroblasts in mice compared to wild-type mice [153]. The effects of EE are independent of the IL-1 signalling pathway, as it increases neurogenesis in mice that are null for IL-1R1 [154]. EE also induces microglial proliferation and expression of the proneurogenic IGF-1 [155], but the full phenotype of microglia in EE compared to standard housing and its impact on the neurogenic cascade remains to be determined.

The mechanisms behind the anti-inflammatory actions of EE are unknown, but they were suggested to involve microglial interactions with T lymphocytes through an increased expression of the major histocompatibility complex of class II (MHC-II) during EE [155]. MHC-II is responsible for presenting the phagocytosed and degraded antigens to the antibodies expressed on the surface of a subtype of T lymphocytes (T helper or CD4+ cells), thus initiating their activation and production of antigen-specific antibodies. Severe combined immunodeficient (SCID) mice lacking either T and B lymphocytes or nude mice lacking only T cells have impaired proliferation and neurogenesis in normal and EE housing compared to wild-type mice [155], as well as impaired performance in the water maze [156]. Similarly, antibody-based depletion of T helper lymphocytes impairs basal and exercise-induced proliferation and neurogenesis [157]. Furthermore, a genetic study in heterogeneous stock mice, which descend from eight inbred progenitor strains, has found a significant positive correlation between genetic loci associated to hippocampal proliferation and to the proportion of CD4+ cells among blood CD3+ lymphocytes [158]. Additional experiments are needed to fully determine the possible interactions between microglia and T cells in neurogenesis, because, at least in normal physiological conditions, (1) T cell surveillance of the brain parenchyma is minimal, (2) microglia are poor antigen presenting cells, and (3) antigen presentation by means of MHC-II family of molecules is thought to occur outside the brain, that is, in the meninges and choroid plexus [159]. In fact, during voluntary exercise, there are no significant changes in T cell surveillance of the hippocampus, nor a direct interaction between T cells and microglia, nor any changes in the gene expression profile of microglia, including that of IGF-1, IL-1, and TNF [160]. The number of microglia is also inversely correlated with the number of hippocampal proliferating cells, rNSCs, and neuroblasts in aged (8 months) mice subjected to voluntary running, as well asin vitro cocultures of microglia and neuroprogenitors, which has been interpreted as resulting from an overall inhibitory effect of microglia on adult neurogenesis [161]. Even though EE is clearly a more complex environmental factor than voluntary running, further research is necessary to disregard nonspecific or indirect effects of genetic or antibody-based T cells depletion on microglia and other brain cell populations, including rNSCs. For instance, adoptive transfer of T helper cells treated with glatiramer acetate, a synthetic analog of myelin basic protein (MBP) approved for the treatment of multiple sclerosis, produces a bystander effect on resident astrocytes and microglia by increasing their expression of anti-inflammatory cytokines such as TGF[162]. Alternatively, it has been suggested that T cells may mediate an indirect effect on adult hippocampal neurogenesis by increasing the production of brain-derived neurotrophic factor (BDNF) [157], which is involved in the proneurogenic actions of EE [163]. Whether BDNF can counteract the detrimental effects of T cell depletion on neurogenesis remains unknown. Overall, the roles of microglia in EE and running-induced neurogenesis are unclear and have to be addressed with more precise experimental designs. In summary, surveillant microglia are part of the physical niche surrounding the neural stem cells and newborn neurons of the mature hippocampus, where they continuously phagocytose the excess of newborn cells. Microglia were also linked to the proneurogenic and anti-inflammatory effects of voluntary running and EE, but direct evidence is missing. The overall contribution of microglia to neurogenesis and learning and memory in normal physiological conditions remains largely unexplored at this early stage in the field.

  1. Conclusion

In light of these observations, microglia are now emerging as important effector cells during normal brain development and functions, including adult hippocampal neurogenesis. Microglia can exert a positive or negative influence on the proliferation, survival, or differentiation of newborn cells, depending on the inflammatory context. For instance, microglia can compromise the neurogenic cascade during chronic stress, aging, and neurodegenerative diseases, by their release of proinflammatory cytokines such as IL-1, IL-6, and TNF. A reduced fractalkine signalling between neurons and microglia could also be involved during normal aging. However, microglia are not necessarily the only cell type implicated because astrocytes, endothelial cells, mast cells, perivascular and meningeal macrophages, and to a lesser extent neurons and invading peripheral immune cells could further contribute by releasing proinflammatory mediators.

Additionally, microglia were shown to phagocytose the excess of newborn neurons undergoing apoptosis in the hippocampal neurogenic niche during normal physiological conditions, while a similar role in the synaptic integration of newborn cells was also proposed in light of their capacity to phagocytose synaptic elements. Lastly, microglial interactions with T cells, leading to the release of anti-inflammatory cytokines, neurotrophic factors, and other proneurogenic mediators (notably during EE and voluntary running), could counteract the detrimental effects of inflammation on adult hippocampal neurogenesis and their functional implications for learning and memory.

However, further research is necessary to assess the relative contribution of microglia versus other types of resident and infiltrating inflammatory cells and to determine the nature of the effector cytokines and other inflammatory mediators involved, as well as their cellular and molecular targets in the neurogenic cascade. Such research will undoubtedly help to develop novel strategies aiming at protecting the neurogenic potential and ultimately its essential contribution to learning and memory.

Abbreviations

AD: Alzheimer’s disease
ANPs: Amplifying neuroprogenitors
APP: Amyloid precursor protein
A: Amyloid beta
BDNF: Brain-derived neurotrophic factor
BrdU: 5-Bromo-2′-Deoxyuridine
CX3CL1: Fractalkine
CX3CR1: Fractalkine receptor
EAE: Experimental acute encephalomyelitis
EE: Enriched environment
EGF: Epidermal growth factor
FGFb: Basic fibroblast growth factor
GDNF: Glial cell line-derived neurotrophic factor
GFAP: Glial fibrillary acidic protein
GR: Glucocorticoid receptor
HPA: Hypothalamic-pituitary-adrenal axis
ICE: Interleukin 1 converting enzyme
IL-1: Interleukin 1 beta
IL-1R1: Interleukin 1 beta receptor
IL-4: Interleukin 4
IL-6: Interleukin 6
IL-7: Interleukin 7
IL-11: Interleukin 11
IFN: Interferon gamma
IGF-1: Insulin-like growth factor 1
iNOS: Inducible nitric oxide synthase
LPS: Bacterial lipopolysaccharides
LTP: Long term potentiation
M-CSF: Macrophage colony-stimulating factor
MBP: Myelin basic protein
MHC-II: Major histocompatibility complex class II
MOG: Myelin oligodendrocyte glycoprotein
NF-B: Nuclear factor kappa-light-chain-enhancer of activated B cells
NGF: Nerve growth factor
NO: Nitric oxide
NSAID: Nonsteroidal anti-inflammatory drug
NT-4: Neurotrophin-4
PDGF: Platelet-derived growth factor
PS: Phosphatidylserine
PS1: Presenilin 1
ROS: Radical oxygen species
SCID: Severe combined immunodeficiency
SGZ: Subgranular zone
TGF: Transforming growth factor beta
TNF: Tumor necrosis factor alpha
VEGF: Vascular endothelial growth factor.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was supported by grants from the Spanish Ministry of Economy and Competitiveness to Amanda Sierra (BFU2012-32089) and Juan M. Encinas (SAF2012-40085), from Basque Government (Saiotek S-PC 12UN014) and Ikerbasque start-up funds to Juan M. Encinas and Amanda Sierra, and from The Banting Research Foundation, the Scottish Rite Charitable Foundation of Canada, and start-up funds from Université Laval and Centre de recherche du CHU de Québec to Marie-Ève Tremblay.

References

  1. P. Rezaie and D. Male, “Mesoglia and microglia—a historical review of the concept of mononuclear phagocytes within the central nervous system,” Journal of the History of the Neurosciences, vol. 11, no. 4, pp. 325–374, 2002. View at Publisher · View at Google Scholar · View at Scopus
  2. F. Ginhoux, S. Lim, G. Hoeffel, D. Low, and T. Huber, “Origin and differentiation of microglia,”Frontiers in Cellular Neuroscience, vol. 7, article 45, 2013. View at Publisher · View at Google Scholar
  3. E. Gomez Perdiguero, C. Schulz, and F. Geissmann, “Development and homeostasis of “resident” myeloid cells: the case of the microglia,” GLIA, vol. 61, no. 1, pp. 112–120, 2013. View at Publisher ·View at Google Scholar
  4. H. Kettenmann, F. Kirchhoff, and A. Verkhratsky, “Microglia: new roles for the synaptic stripper,”Neuron, vol. 77, no. 1, pp. 10–18, 2013. View at Publisher · View at Google Scholar
  5. A. Aguzzi, B. A. Barres, and M. L. Bennett, “Microglia: scapegoat, saboteur, or something else?” Science, vol. 339, no. 6116, pp. 156–161, 2013. View at Publisher · View at Google Scholar
  6. K. Helmut, U. K. Hanisch, M. Noda, and A. Verkhratsky, “Physiology of microglia,” Physiological Reviews, vol. 91, no. 2, pp. 461–553, 2011. View at Publisher · View at Google Scholar · View at Scopus
  7. M. È. Tremblay, B. Stevens, A. Sierra, H. Wake, A. Bessis, and A. Nimmerjahn, “The role of microglia in the healthy brain,” Journal of Neuroscience, vol. 31, no. 45, pp. 16064–16069, 2011. View at Publisher ·View at Google Scholar · View at Scopus
  8. A. Miyamoto, H. Wake, A. J. Moorhouse, and J. Nabekura, “Microglia and synapse interactions: fine tuaning neural circuits and candidate molecules,” Frontiers in Cellular Neuroscience, vol. 7, article 70, 2013. View at Publisher · View at Google Scholar
  9. C. Bechade, Y. Cantaut-Belarif, and A. Bessis, “Microglial control of neuronal activity,” Frontiers in Cellular Neuroscience, vol. 7, article 32, 2013. View at Publisher · View at Google Scholar
  10. A. Sierra, O. Abiega, A. Shahraz, and H. Neumann, “Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis,” Frontiers in Cellular Neuroscience, vol. 7, article 6, 2013. View at Publisher · View at Google Scholar
  11. G. Kempermann, S. Jessberger, B. Steiner, and G. Kronenberg, “Milestones of neuronal development in the adult hippocampus,” Trends in Neurosciences, vol. 27, no. 8, pp. 447–452, 2004. View at Publisher ·View at Google Scholar · View at Scopus
  12. J. M. Encinas, T. V. Michurina, N. Peunova et al., “Division-coupled astrocytic differentiation and age-related depletion of neural stem cells in the adult hippocampus,” Cell Stem Cell, vol. 8, no. 5, pp. 566–579, 2011. View at Publisher · View at Google Scholar · View at Scopus
  13. M. A. Bonaguidi, M. A. Wheeler, J. S. Shapiro et al., “In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics,” Cell, vol. 145, no. 7, pp. 1142–1155, 2011. View at Publisher · View at Google Scholar · View at Scopus
  14. J. J. Breunig, J. I. Arellano, J. D. Macklis, and P. Rakic, “Everything that glitters isn’t gold: a critical review of postnatal neural precursor analyses,” Cell Stem Cell, vol. 1, no. 6, pp. 612–627, 2007. View at Publisher · View at Google Scholar · View at Scopus
  15. L. S. Overstreet-Wadiche and G. L. Westbrook, “Functional maturation of adult-generated granule cells,” Hippocampus, vol. 16, no. 3, pp. 208–215, 2006. View at Publisher · View at Google Scholar · View at Scopus

……………….

 

Review

Nature Reviews Molecular Cell Biology 8, 519-529 (July 2007) | doi:10.1038/nrm2199

Signal integration in the endoplasmic reticulum unfolded protein response

David Ron & Peter Walter

http://www.nature.com/nrm/journal/v8/n7/full/nrm2199.html

The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways — cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate and the metabolism of proteins, amino acids and lipids. The arms of the UPR are integrated to provide a response that remodels the secretory apparatus and aligns cellular physiology to the demands imposed by ER stress.

 

Figure 1: The unfolded protein response (UPR) signalling pathways.

FromThe impact of the endoplasmic reticulum protein-folding environment on cancer development

Nature Reviews Cancer 14, 581–597 (2014)  http://dx.doi.org:/10.1038/nrc3800

(UPR) signalling pathways

(UPR) signalling pathways

http://www.nature.com/nrc/journal/v14/n9/images/nrc3800-f1.jpg

Upon endoplasmic reticulum (ER) stress, unfolded and misfolded proteins bind and sequester immunoglobulin heavy-chain binding protein (BIP), thereby activating the UPR. The UPR comprises three parallel signalling branches: PRKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α), inositol-requiring protein 1α (IRE1α)–X-box binding protein 1 (XBP1) and activating transcription factor 6α (ATF6α). The outcome of UPR activation increases protein folding, transport and ER-associated protein degradation (ERAD), while attenuating protein synthesis. If protein misfolding is not resolved, cells enter apoptosis. CHOP, C/EBP homologous protein; GADD34, growth arrest and DNA damage-inducible protein 34; JNK, JUN N-terminal kinase; P, phosphorylation; RIDD, regulated IRE1-dependent decay; ROS, reactive oxygen species; XBP1s, transcriptionally active XBP1; XBP1u, unspliced XBP1.

Figure 3: The unfolded protein response (UPR) and inflammation.

(UPR) and inflammation

(UPR) and inflammation

http://www.nature.com/nrc/journal/v14/n9/images/nrc3800-f3.jpg

The three UPR pathways augment the production of reactive oxygen species (ROS) and activate nuclear factor-κB (NF-κB) and activator protein 1 (AP1) pathways, thereby leading to inflammation. NF-κB, which is a master transcriptional regulator of pro-inflammatory pathways, can be activated through binding to the inositol-requiring protein 1α (IRE1α)–TNF receptor-associated factor 2 (TRAF2) complex in response to endoplasmic reticulum (ER) stress, leading to recruitment of the IκB kinase (IKK), IκB phosphorylation (P) and degradation, and nuclear translocation of NF-κB196. Moreover, the IRE1α–TRAF2 complex can recruit apoptosis signal-regulating kinase 1 (ASK1) and activate JUN N-terminal kinase (JNK), increasing the expression of pro-inflammatory genes through enhanced AP1 activity197. The PRKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α) and activating transcription factor 6α (ATF6α) branches of the UPR activate NF-κB through different mechanisms. Engaging PERK–eIF2α signalling halts overall protein synthesis and increases the ratio of NF-κB to IκB, owing to the short half-life of IκB, thereby freeing NF-κB for nuclear translocation198199. ATF6α activation following exposure to the bacterial subtilase cytotoxin that cleaves immunoglobulin heavy-chain binding protein (BIP) leads to AKT phosphorylation and consequent NF-κB activation109200.

 

 

Figure 4

The cancer-supporting role of the unfolded protein response (UPR).

http://www.nature.com/nrc/journal/v14/n9/images/nrc3800-f4.jpg

cancer-supporting role of the unfolded protein response

cancer-supporting role of the unfolded protein response

Read Full Post »


The relationship of stress hypermetabolism to essential protein needs

Curator: Larry H. Bernstein, MD, FCAP

 

 

The relationship of stress hypermetabolism to essential protein needs

A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Subtitle: Transthyretin and the Systemic Inflammatory Response

Larry H. Bernstein, MD, FACP, Clinical Pathologist, Biochemist, and Transfusion Physician
President, Triplex, Trumbull, CT 06611, USA

 

Brief introduction

Transthyretin  (also known as prealbumin) has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompted a review of the  benefit of using this test in acute and chronic care. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases. It is sensitive to the systemic inflammatory response syndrome (SIRS), and needs to be understood in the context of acute illness to be used effectively. There are a number of physiologic changes associated with SIRS and the injury/repair process that affect TTR. The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.  A much better understanding of the significance of this program has emerged from studies of nitrogen and sulfur in health and disease.

Transthyretin protein structure

Transthyretin protein structure (Photo credit: Wikipedia)

Age-standardised disability-adjusted life year...

Age-standardised disability-adjusted life year (DALY) rates from Protein-energy malnutrition by country (per 100,000 inhabitants). (Photo credit: Wikipedia)

_________________________________________________________________________________________________________

The systemic inflammatory response syndrome C-reactive protein and transthyretin conundrum.
Larry H Bernstein
Clin Chem Lab Med 2007; 45(11):0
ICID: 939932
Article type: Editorial

The Transthyretin Inflammatory State Conundrum
Larry H. Bernstein
Current Nutrition & Food Science, 2012, 8, 00-00

Keywords: Tranthyretin (TTR), systemic inflammatory response syndrome (SIRS), protein-energy malnutrition (PEM), C- reactive protein, cytokines, hypermetabolism, catabolism, repair.

Transthyretin has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompts a review of the actual benefit of using this test in a number of settings. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases. It is sensitive to the systemic inflammatory response syndrome (SIRS), and needs to be understood in the context of acute illness to be used effectively.

There are a number of physiologic changes associated with SIRS and the injury/repair process that affect TTR and  in the context of an ICU setting, the contribution of TTR is essential.  The only consideration is the timing of initiation since the metabolic burden is sufficiently high that a substantial elevation is expected in the first 3 days post admission, although the level of this biomarker is related to the severity of injury. Despite the complexity of the situation, TTR is not to be considered a test “for all seasons”. In the context of age, prolonged poor meal intake, chronic or acute illness, TTR needs to be viewed in a multivariable lens, along with estimated lean body mass, C-reactive protein, the absolute lymphocyte count, presence of neutrophilia, and perhaps procalcitonin if there is remaining uncertainty. Furthermore, the reduction of risk of associated complication requires a systematized approach to timely identification, communication, and implementation of a suitable treatment plan.

The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.

_________________________________________________________________________________________________________

Title: The Automated Malnutrition Assessment
Accepted 29 April 2012. http://www.nutritionjrnl.com. Nutrition (2012), doi:10.1016/j.nut.2012.04.017.
Authors: Gil David, PhD; Larry Howard Bernstein, MD; Ronald R Coifman, PhD
Article Type: Original Article

Keywords: Network Algorithm; unsupervised classification; malnutrition screening; protein energy malnutrition (PEM); malnutrition risk; characteristic metric; characteristic profile; data characterization; non-linear differential diagnosis

We have proposed an automated nutritional assessment (ANA) algorithm that provides a method for malnutrition risk prediction with high accuracy and reliability.  The problem of rapidly identifying risk and severity of malnutrition is crucial for minimizing medical and surgical complications. These are not easily performed or adequately expedited. We characterized for each patient a unique profile and mapped similar patients into a classification. We also found that the laboratory parameters were sufficient for the automated risk prediction.

_________________________________________________________________________________________________________

Title: The Increasing Role for the Laboratory in Nutritional Assessment
Article Type: Editorial
Section/Category: Clinical Investigation
Accepted 22 May 2012. http://www.elsevier.com/locate/clinbiochem.
Clin Biochem (2012), doi:10.1016/j.clinbiochem.2012.05.024
Keywords: Protein Energy Malnutrition; Nutritional Screening; Laboratory Testing
Author: Dr. Larry Howard Bernstein, MD

The laboratory role in nutritional management of the patient has seen remarkable growth while there have been dramatic changes in technology over the last 25 years, and it is bound to be transformative in the near term. This editorial is an overview of the importance of the laboratory as an active participant in nutritional care.

The discipline emerged divergently along separate paths with unrelated knowledge domains in physiological chemistry, pathology, microbiology, immunology and blood cell recognition, and then cross-linked emerging into clinical biochemistry, hematology-oncology, infectious diseases, toxicology and therapeutics, genetics, pharmacogenomics, translational genomics and clinical diagnostics.

In reality, the more we learn about nutrition, the more we uncover of metabolic diversity of individuals, the family, and societies in adapting and living in many unique environments and the basic reactions, controls, and responses to illness. This course links metabolism to genomics and individual diversity through metabolomics, which will be enlightened by chemical and bioenergetic insights into biology and translated into laboratory profiling.

Vitamin deficiencies were discovered as clinical entities with observed features as a result of industrialization (rickets and vitamin D deficiency) and mercantile trade (scurvy and vitamin C)[2].  Advances in chemistry led to the isolation of each deficient “substance”.  In some cases, a deficiency of a vitamin and what is later known as an “endocrine hormone” later have confusing distinctions (vitamin D, and islet cell insulin).

The accurate measurement and roles of trace elements, enzymes, and pharmacologic agents was to follow within the next two decades with introduction of atomic absorption, kinetic spectrophotometers, column chromatography and gel electrophoresis.  We had fully automated laboratories by the late 1960s, and over the next ten years basic organ panels became routine.   This was a game changer.

Today child malnutrition prevalence is 7 percent of children under the age of 5 in China, 28 percent in sub-Saharan African, and 43 percent in India, while under-nutrition is found mostly in rural areas with 10 percent of villages and districts accounting for 27-28 percent of all Indian underweight children. This may not be surprising, but it is associated with stunting and wasting, and it has not receded with India’s economic growth. It might go unnoticed viewed alongside a growing concurrent problem of worldwide obesity.

The post WWII images of holocaust survivors awakened sensitivity to nutritional deprivation.

In the medical literature, Studley [HO Studley.  Percentage of weight loss. Basic Indicator of surgical risk in patients with chronic peptic ulcer.  JAMA 1936; 106(6):458-460.  doi:10.1001/jama.1936.02770060032009] reported the association between weight loss and poor surgical outcomes in 1936.  Ingenbleek et al [Y Ingenbleek, M De Vissher, PH De Nayer. Measurement of prealbumin as index of protein-calorie malnutrition. Lancet 1972; 300[7768]: 106-109] first reported that prealbumin (transthyretin, TTR) is a biomarker for malnutrition after finding very low TTR levels in African children with Kwashiorkor in 1972, which went unnoticed for years.  This coincided with the demonstration by Stanley Dudrick  [JA Sanchez, JM Daly. Stanley Dudrick, MD. A Paradigm ShiftArch Surg. 2010; 145(6):512-514] that beagle puppies fed totally through a catheter inserted into the superior vena cava grew, which method was then extended to feeding children with short gut.  Soon after Bistrian and Blackburn [BR Bistrian, GL Blackburn, E Hallowell, et al. Protein status of general surgical patients. JAMA 1974; 230:858; BR Bistrian, GL Blackburn, J Vitale, et al. Prevalence of malnutrition in general medicine patients, JAMA, 1976, 235:1567] showed that malnourished hospitalized medical and surgical patients have increased length of stay, increased morbidity, such as wound dehiscence and wound infection, and increased postoperative mortality, later supported by many studies.

Michael Meguid,MD, PhD, founding editor of Nutrition [Elsevier] held a nutrition conference “Skeleton in the Closet – 20 years later” in Los Angeles in 1995, at which a Beckman Prealbumin Roundtable was held, with Thomas Baumgartner and Michael M Meguid as key participants.  A key finding was that to realize the expected benefits of a nutritional screening and monitoring program requires laboratory support. A Ross Roundtable, chaired by Dr. Lawrence Kaplan, resulted in the first Standard of Laboratory Practice Document of the National Academy of Clinical Biochemists on the use of the clinical laboratory in nutritional support and monitoring. Mears then showed a real benefit to a laboratory interactive program in nutrition screening based on TTR [E Mears. Outcomes of continuous process improvement of a nutritional care program incorporating serum prealbumin measurements. Nutrition 1996; 12 (7/8): 479-484].

A later Ross Roundtable on Quality in Nutritional Care included a study of nutrition screening and time to dietitian intervention organized by Brugler and Di Prinzio that showed a decreased length of hospital stay with $1 million savings in the first year (which repeated), which included reduced cost for dietitian evaluations and lower complication rates.

Presentations were made at the 1st International Transthyretin Congress in Strasbourg, France by Mears [E Mears.  The role of visceral protein markers in protein calorie malnutrition. Clin Chem Lab Med 2002; 40:1360-1369] on the impact of TTR in screening for PEM in a public hospital in Louisiana, and by Potter [MA Potter, G Luxton. Prealbumin measurement as a screening tool for patients with protein calorie malnutrition in emergency hospital admissions: a pilot study.  Clin Invest Med. 1999; 22(2):44-52] that indicated a 17% in-hospital mortality rate in a Canadian hospital for patients with PCM compared with 4% without PCM (p < 0.02), while only 42% of patients with PCM received nutritional supplementation. Cost analysis of screening with prealbumin level projected a saving of $414 per patient screened.  Ingenbleek and Young [Y Ingenbleek, VR Young.  Significance of transthyretin in protein metabolism.  Clin Chem Lab Med. 2002; 40(12):1281–1291.  ISSN (Print) 1434-6621, DOI: 10.1515/ CCLM.2002.222, December 2002. published online: 01/06/2005] tied the TTR to basic effects reflected in protein metabolism.

_______________________________________________________________________________________________

Transthyretin as a marker to predict outcome in critically ill patients.
Arun Devakonda, Liziamma George, Suhail Raoof, Adebayo Esan, Anthony Saleh, Larry H Bernstein
Clin Biochem 2008; 41(14-15):1126-1130
ICID: 939927
Article type: Original article

TTR levels correlate with patient outcomes and are an accurate predictor of patient recovery in non-critically ill patients, but it is uncertain whether or not TTR level correlates with level of nutrition support and outcome in critically ill patients. This issue has been addressed only in critically ill patients on total parenteral nutrition and there was no association reported with standard outcome measures. We revisit this in all patients admitted to a medical intensive care unit.

Serum TTR was measured on the day of admission, day 3 and day 7 of their ICU stay. APACHE II and SOFA score was assessed on the day of admission. A registered dietician for their entire ICU stay assessed the nutritional status and nutritional requirement. Patients were divided into three groups based on initial TTR level and the outcome analysis was performed for APACHE II score, SOFA score, ICU length of stay, hospital length of stay, and mortality.

TTR showed excellent concordance with the univariate or multivariate classification of patients with PEM or at high malnutrition risk, and followed for seven days in the ICU, it is a measure of the metabolic burden.  TTR levels decline from day 1 to day 7 in spite of providing nutritional support. Twenty-five patients had an initial TTR serum concentration more than 17 mg/dL (group 1), forty-eight patients had mild malnutrition with a concentration between 10 and 17 mg/dL (group 2), Forty-five patients had severe malnutrition with a concentration less than 10 mg/dL (group 3).  Initial TTR level had inverse correlation with ICU length of stay, hospital length of stay, and APACHE II score, SOFA score; and predicted mortality, especially in group 3.

___________________________________________________________________________________________________________

A simplified nutrition screen for hospitalized patients using readily available laboratory and patient
information.
Linda Brugler, Ana K Stankovic, Madeleine Schlefer, Larry Bernstein
Nutrition 2005; 21(6):650-658
ICID: 825623
Article type: Review article
The role of visceral protein markers in protein calorie malnutrition.
Linda Brugler, Ana Stankovic, Larry Bernstein, Frederick Scott, Julie O’Sullivan-Maillet
Clin Chem Lab Med 2002; 40(12):1360-1369
ICID: 636207
Article type: Original article

The Automated Nutrition Score is a data-driven extension of continuous quality improvement.

Larry H Bernstein
Nutrition 2009; 25(3):316-317
ICID: 939934

______________________________________________________________________________________________________
Transthyretin: its response to malnutrition and stress injury. clinical usefulness and economic implications.
LH Bernstein, Y Ingenbleek
Clin Chem Lab Med 2002; 40(12):1344-1348
ICID: 636205
Article type: Original article

_______________________________________________________________________________________________________

THE NUTRITIONALLY-DEPENDENT ADAPTIVE DICHOTOMY (NDAD) AND STRESS HYPERMETABOLISM
Yves Ingenbleek  MD  PhD  and  Larry Bernstein MD
J CLIN LIGAND ASSAY  (out of print)

The acute reaction to stress is characterized by major metabolic, endocrine and immune alterations. According to classical descriptions, these changes clinically present as a succession of 3 adaptive steps – ebb phase, catabolic flow phase, and anabolic flow phase. The ebb phase, shock and resuscitation, is immediate, lasts several hours, and is characterized by hypokinesis, hypothermia, hemodynamic instability and reduced basal metabolic rate. The catabolic flow phase, beginning within 24 hours and lasting several days, is characterized by catabolism with the flow of gluconeogenic substrates and ketone bodies in response to the acute injury. The magnitude of the response depends on the acuity and the severity of the stress. The last, a reparative anabolic flow phase, lasts weeks and is characterized by the accretion of amino acids (AAs) to rebuilding lean body mass.

The current opinion is that the body economy is reset during the course of stress at novel thresholds of metabolic priorities. This is exemplified mainly by proteolysis of muscle, by an effect on proliferating gut mucosa and lymphoid tissue as substrates are channeled to support wound healing, by altered syntheses of liver proteins with preferential production of acute phase proteins (APPs) and local repair in inflamed tissues (3). The first two stages demonstrate body protein breakdown exceeding the rate of protein synthesis, resulting in a negative nitrogen (N) balance, muscle wasting and weight loss. In contrast, the last stage displays reversed patterns, implying progressive recovery of endogenous N pools and body weight.

These adaptive alterations undergo continuing elucidation. The identification of cytokines, secreted by activated macrophages/monocytes or other reacting cells, has provided further insights into the molecular mechanisms controlling energy expenditure, redistribution of protein pools, reprioritization of syntheses and secretory processes.

The free fraction of hormones bound to specific binding-protein(s) [BP(s)] manifests biological activities, and any change in the BP blood level modifies the effect of the hormone on the end target organ.  The efficacy of these adaptive responses may be severely impaired in protein-energy malnourished (PEM) patients. This is especially critical with respect to changes of the circulating levels of transthyretin (TTR), retinol-binding protein (RBP) and corticosteroid-binding globulin (CBG) conveying thyroid hormones (TH), retinol and cortisol, respectively.  This reaction is characterized by cytokine mediated autocrine, paracrine and endocrine changes. Among the many inducing molecules identified, interleukins 1 and 6 (Il-1, Il-6) and tumor necrosis factor a (TNF) are associated with enhanced production of 3 counterregulatory hormonal families (cortisol, catecholamines and glucagon). Growth hormone (GH) and TH also have roles in these metabolic adjustments.

There is overproduction of cortisol mediated by several cytokines acting on both the adrenal cortex (10) and on the pituitary through hypothalamic CRH with loss of feedback regulation of ACTH production (11). Hypercortisolemia is a major finding observed after surgery (12), sepsis (13), and medical insults, usually correlated with severity of insult and of complications. Rising cortisol values parallel hyperglycemic trends, as an effect of both gluconeogenesis and insulin resistance. Working in concert with TNF, glucocorticoids govern the breakdown of muscle mass, which is regarded as the main factor responsible for the negative N balance.

Under normal conditions, GH exerts both lipolytic and anabolic influences in the whole body economy under the dual control of the hypothalamic hormones somatocrinin (GHRH) and somatostatin (SRIH). GH secretion is usually depressed by rising blood concentrations of glucose and free fatty acids (FFAs) but is paradoxicaly elevated despite hyperglycemia in stressed patients.

The oversecretion of counterregulatory hormones working in concert generates subtle equilibria between glycogenolytic/glycolytic/gluconeogenic adaptive processes. The net result is the neutralization of the main hypoglycemic and anabolic activities of insulin and the development of a persisting and controlled hyperglycemic tone in the stressed body. The molecular mechanisms whereby insulin resistance occurs in the course of stress refer to
cytokine-  and  hormone-induced  phosphorylation abnormalities affecting receptor signaling. The insulin-like anabolic processes of GH are mediated by IGF1 working as relay agent. The expected high IGF1 surge associated with GH oversecretion is not observed in severe stress as plasma values are usually found at the lower limit of normal or even in the subnormal range.  The end result of this dissociation between high GH and low IGF1 levels is to favor the proteolysis of muscle mass to release AAs for gluconeogenesis and the breakdown of adipose tissue to provide ketogenic substrates.

The acute stage of stress is associated with the onset of a low T3 syndrome typically delineated by the drop of both total (TT3) and free (FT3) triiodothyronine plasma levels in the subnormal range. In contrast, both total (TT4) and free (FT4) thyroxine values usually remain within normal ranges with declining trends observed for TT4 and rising tendencies for FT4 (44). This last free compound is regarded as the sensor reflecting the actual thyroid status and governing the release of TSH whereas FT3 works as the active hormonal mediator at nuclear receptor level. The maintenance of an euthyroid sick syndrome is compatible with the down-regulation of most metabolic and energetic processes in healthy tissues. These inhibitory effects , negatively affecting all functional steps of the hypothalamo-pituitary-thyroid axis concern TSH production, iodide uptake, transport and organification into iodotyrosyl residues, peroxidase coupling activity as well as thyroglobulin synthesis and TH leakage. Taken together, the above-mentioned data indicate that the development of hyperglycemia and of insulin-resistance in healthy tissues – mainly in the muscle mass – are hallmarks resulting from the coordinated activities of the counterregulatory hormones.

A growing body of recent data suggest that the stressed territory, whatever the causal agent – bacterial or viral sepsis, auto-immune disorder, traumatic or toxic shock, burns, cancer – manifest differentiated metabolic and immune reactions. The amplitude, duration and efficacy of these responses are reportedly impaired along several ways in PEM patients. These last detrimental effects are accompanied by a number of medical, social and economical consequences, such as extended length of hospital stay and increased complication / mortality rates. It is therefore mandatory to correctly identify and follow up the nutritional status of hospitalized patients. Such approaches are prerequisite to timely and scientifically grounded nutritional and pharmacological mediated interventions.

Contrary to the rest of the body, energy requirements of the inflamed territory are primarily fulfilled by anaerobic glycolysis, an effect triggered by the inhibition of key-enzymes of carbohydrate metabolism, notably pyruvate-dehydrogenase. This non-oxidative combustion of glucose reveals low conversion efficiency but offers the major advantage to maintain, in the context of hyperglycemia, fuel provision to poorly irrigated and/or edematous tissues. The depression of the 5’-monodeiodinating activity (5’-DA) plays a pivotal role in these adaptive changes, yielding inactive reverse T3 (rT3) as index of impaired T4 to T3 conversion rates, but at the same time there is an augmented supply of bioactive T3 molecules and local overstimulation of thyro-dependent processes characterized by thyroid down-regulation.  The same differentiated evolutionary pattern applies to IGF1. In spite of lowered plasma total concentrations, the proportion of IGF1 released in free form may be substantially increased owing to the proteolytic degradation of IGFBP-3 in the intravascular compartment. The digestion of  BP-3 results from the surge of several proteases occurring the course of stress, yielding biologically active IGF1 molecules available for the repair of damaged tissues. In contrast, healthy receptors oppose a strong resistance to IGF1 ligands freed in the general circulation, likely induced by an acquired phosphorylation defect very similar in nature to that for the insulin transduction pathway.

PEM is the generic denomination of a broad spectrum of nutritional disorders, commonly found in hospital settings, and whose extreme poles are identified as marasmus and kwashiorkor. The former condition is usually regarded as the result of long-lasting starvation leading to the loss of lean body mass and fat reserves but relatively well-preserved liver function and immune capacities. The latter condition is typically the consequence of (sub)acute deprivation predominantly affecting the protein content of staplefood, an imbalance causing hepatic steatosis, fall of visceral proteins, edema and increased vulnerability to most stressful factors. PEM may be hypometabolic or hypermetabolic, usually coexists with other diseased states and is frequently associated with complications. Identification of PEM calls upon a large set of clinical and analytical disciplines comprising anthropometry, immunology, hematology and biochemistry.

CBG, TTR and RBP share in common the transport of specific ligands exerting their metabolic effects at nuclear receptor level. Released from their specific BPs in free form, cortisol, FT4 and retinol immediately participe to the strenghtening of the positive and negative responses to stressful stimuli. CBG is a relatively weak responder to short-term nutritional influences (73)  although long-lasting PEM is reportedly capable of causing its significant diminution (74). The dramatic drop of CBG in the course of stress appears as the combined effect of Il-6-induced posttranscriptional blockade of its liver synthesis (75) and peripheral overconsumption by activated neutrophils (61). The divergent alterations outlined by CBG and total cortisolemia result in an increased disposal of free ligand reaching proportions considerably higher than the 4 % recorded under physiological conditions.

The appellation of negative APPs that was once given to the visceral group of carrier-proteins. The NDAD concept takes the opposite view, defending the opinion that their suppressed synthesis releases free ligands which positively contribute to strengthen all aspects of the stress reaction, justifying the ABR denomination. This implies that the role played by ABRs should no longer be interpreted in terms of concentrations but in terms of functionality.

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

THE OXIDATIVE STRESS OF HYPERHOMOCYSTEINEMIA RESULTS FROM REDUCED BIOAVAILABILITY OF SULFUR-CONTAINING REDUCTANTS.
Yves Ingenbleek. The Open Clinical Chemistry Journal, 2011, 4, 34-44.

Vegetarian subjects consuming subnormal amounts of methionine (Met) are characterized by subclinical protein malnutrition causing reduction in size of their lean body mass (LBM) best identified by the serial measurement of plasma transthyretin (TTR). As a result, the transsulfuration pathway is depressed at cystathionine-β-synthase (CβS) level triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and promoting its conversion to Met. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CβS causing in turn declining generation of hydrogen sulfide (H2S) from enzymatic sources. The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions. Combination of subclinical malnutrition and S8-deficiency thus maximizes the defective production of Cys, GSH and H2S reductants, explaining persistence of unabated oxidative burden. The clinical entity increases the risk of developing cardiovascular diseases (CVD) and stroke in underprivileged plant-eating populations regardless of Framingham criteria and vitamin-B status. Although unrecognized up to now, the nutritional disorder is one of the commonest worldwide, reaching top prevalence in populated regions of Southeastern Asia. Increased risk of hyperhomocysteinemia and oxidative stress may also affect individuals suffering from intestinal malabsorption or westernized communities having adopted vegan dietary lifestyles.

Metabolic pathways: Met molecules supplied by dietary proteins are submitted to TM processes allowing to release Hcy which may in turn either undergo Hcy – Met RM pathways or be irreversibly committed into TS decay. Impairment of CbS activity, as described in protein malnutrition, entails supranormal accumulation of Hcy in body fluids, stimulation of activity and maintenance of Met homeostasis. This last beneficial effect is counteracted by decreased concentration of most components generated downstream to CbS, explaining the depressed CbS- and CbL-mediated enzymatic production of H2S along the TS cascade. The restricted dietary intake of elemental S further operates as a limiting factor for its non-enzymatic reduction to H2S which contributes to downsizing a common body pool. Combined protein- and S-deficiencies work in concert to deplete Cys, GSH and H2S from their body reserves, hence impeding these reducing molecules to properly face the oxidative stress imposed by hyperhomocysteinemia.

see also …

McCully, K.S. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am. J. Pathol., 1996, 56, 111-128.

Cheng, Z.; Yang, X.; Wang, H. Hyperhomocysteinemia and endothelial dysfunction. Curr. Hypertens. Rev., 2009, 5,158-165.

Loscalzo, J. The oxidant stress of hyperhomocyst(e)inemia. J. Clin.Invest., 1996, 98, 5-7.

Ingenbleek, Y.; Hardillier, E.; Jung, L. Subclinical protein malnutrition is a determinant of hyperhomocysteinemia. Nutrition, 2002, 18, 40-46.

Ingenbleek, Y.; Young, V.R. The essentiality of sulfur is closely related to nitrogen metabolism: a clue to hyperhomocysteinemia. Nutr. Res. Rev., 2004, 17, 135-153.

Hosoki, R.; Matsuki, N.; Kimura, H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem. Biophys. Res. Commun., 1997, 237, 527-531.

Tang, B.; Mustafa, A.; Gupta, S.; Melnyk, S.; James S.J.; Kruger, W.D. Methionine-deficient diet induces post-transcriptional downregulation of cystathionine-􀀁-synthase. Nutrition, 2010, 26, 1170-1175.

Yves Ingenbleek. Plasma Transthyretin Reflects the Fluctuations of Lean Body Mass in Health and Disease. Chapter 20. In S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_20, # Springer‐Verlag Berlin Heidelberg 2009.

Transthyretin (TTR) is a 55-kDa protein secreted mainly by the choroid plexus and the liver. Whereas its intracerebral production appears as a stable secretory process allowing even distribution of intrathecal thyroid hormones, its hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly. Both morbid conditions are governed by distinct pathogenic mechanisms leading to the reduction in size of lean body mass (LBM). The liver production of TTR integrates the dietary and stressful components of any disease spectrum, explaining why it is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequalled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyperhomocysteinemic states, acquired metabolic disorders currently ascribed to dietary restriction in water-soluble vitamins. Sulfur (S)-deficiency is proposed as an additional causal factor in the sizeable proportion of hyperhomocysteinemic patients characterized by adequate vitamin intake but experiencing varying degrees of nitrogen (N)-depletion. Owing to the fact that N and S coexist in plant and animal tissues within tightly related concentrations, decreasing LBM as an effect of dietary shortage and/or excessive hypercatabolic losses induces proportionate S-losses. Regardless of water-soluble vitamin status, elevation of homocysteine plasma levels is negatively correlated with LBM reduction and declining TTR plasma levels. These findings occur as the result of impaired cystathionine-b-synthase activity, an enzyme initiating the transsulfuration pathway and whose suppression promotes the upstream accumulation and remethylation of homocysteine molecules. Under conditions of N- and S-deficiencies, the maintenance of methionine homeostasis indicates high metabolic priority.

Schematically, the human body may be divided into two major compartments, namely fat mass (FM) and FFM that is obtained by substracting
FM from body weight (BW). The fat cell mass sequesters about 80% of the total body lipids, is poorly hydrated and contains only small quantities of lean tissues and nonfat constituents. FFM comprises the sizeable part of lean tissues and minor mineral compounds among which are Ca, P, Na, and Cl pools totaling about 1.7 kg or 2.5% of BW in a healthy man weighing 70 kg. Subtraction of mineral mass from FFM provides LBM, a composite aggregation of organs and tissues with specific functional properties. LBM is thus nearly but not strictly equivalent to FFM. With extracellular mineral content subtracted, LBM accounts for most of total body proteins (TBP) and of TBN assuming a mean 6.25 ratio between protein and N content.

SM accounts for 45% of TBN whereas the remaining 55% is in nonmuscle lean tissues. The LBM of the reference man contains 98% of total
body potassium (TBK) and the bulk of total body sulfur (TBS). TBK and TBS reach equal intracellular amounts (140 g each) and share distribution patterns (half in SM and half in the rest of cell mass).  The body content of K and S largely exceeds that of magnesium (19 g), iron (4.2 g) and zinc (2.3 g). The average hydration level of LBM in healthy subjects of all age is 73% with the proportion of the intracellular/extracellular fluid spaces being 4:3. SM is of particular relevance in nutritional studies due to its capacity to serve as a major reservoir of amino acids (AAs) and as a dispenser of gluconeogenic substrates. An indirect estimate of SM size consists in the measurement of urinary creatinine, end-product of the nonenzymatic hydrolysis of phosphocreatine which is limited to muscle cells.

During ageing, all the protein components of the human body decrease regularly. This shrinking tendency is especially well documented for SM  whose absolute amount is preserved until the end of the fifth decade, consistent with studies showing unmodified muscle structure, intracellular K content and working capacit. TBN and TBK are highly correlated in healthy subjects and both parameters manifest an age-dependent curvilinear decline
with an accelerated decrease after 65 years.  The trend toward sarcopenia is more marked and rapid in elderly men than in elderly women decreasing strength and functional capacity. The downward SM slope may be somewhat prevented by physical training or accelerated by supranormal cytokine status as reported in apparently healthy aged persons suffering low-grade inflammation. 2002) or in critically ill patients whose muscle mass undergoes proteolysis and contractile dysfunction.

The serial measurement of plasma TTR in healthy children shows that BP values are low in the neonatal period and rise linearly with superimposable concentrations in both sexes during infant growth consistent with superimposable N accretion and protein synthesis rates. Starting from the sixties, TTR values progressively decline showing steeper slopes in elderly males. The lowering trend seems to be initiated by the attenuation of androgen influences and trophic stimuli with increasing age. The normal human TTR trajectory from birth to death has been well documented by scientists belonging to the Foundation for Blood Research. TTR is the first plasma protein to decline in response to marginal protein restricion, thus working as an early signal warning that adaptive mechanisms maintaining homeostasis are undergoing decompensation.

TTR was proposed as a marker of protein nutritional status following a clinical investigation undertaken in 1972 on protein-energy malnourished (PEM) Senegalese children (Ingenbleek et al. 1972). By comparison with ALB and transferrin (TF) plasma values, TTR revealed a much higher degree of sensitivity to changes in protein status that has been attributed to its shorter biological half-life (2 days) and to its unusual Trp richness (Ingenbleek et al. 1972, 1975a). Transcription of the TTR gene in the liver is directed by CCAAT/enhancer binding protein (C/EBP) bound to hepatocyte nuclear factor 1 (HNF1) under the control of several other HNFs. The mechanism responsible for the suppressed TTR synthesis in PEM-states is a restricted AA and energy supply working as limiting factors (Ingenbleek and Young 2002). The rapidly turning over TTR protein is highly responsive to any change in protein flux and energy supply, being clearly situated on the cutting edge of the equipoise.

LBM shrinking may be the consequence of either dietary restriction reducing protein syntheses to levels compatible with survival or that of cytokine-induced tissue proteolysis exceeding protein synthesis and resulting in a net body negative N balance. The size of LBM in turn determines plasma TTR concentrations whose liver production similarly depends on both dietary provision and inflammatory conditions. In animal cancer models, reduced TBN pools were correlated with decreasing plasma TTR values and provided the same predictive ability. In kidney patients, LBM is proposed as an excellent predictor of outcome working in the same direction as TTR plasma levels.  High N intake, supposed to preserve LBM reserves, reduces significantly the mortality rate of kidney patients and is positively correlated with the alterations of TTR plasma concentrations appearing as the sole predictor of final outcome. It is noteworthy that most SELDI or MALDI workers interested in defining protein nutritional status have chosen TTR as a biomarker, showing that there exists a large consensus considering the BP as the most reliable indicator of protein depletion in most morbid circumstances.

Total homocysteine (tHcy) is a S-containing AA not found in customary diets but endogenously produced in the body of mammals by the enzymatic transmethylation of methionine (Met), one of the eight IAAs supplied by staplefoods. tHcy may either serve as precursor substrate for the synthesis of new Met molecules along the remethylation (RM) pathway or undergo irreversible kidney leakage through a cascade of derivatives defining the transsulfuration (TS) pathway. Hcy is thus situated at the crossroad of RM and TS pathways that are regulated by three water-soluble vitamins (pyridoxine, B6; folates, B9; cobalamins, B12).

Significant positive correlations are found between tHcy and plasma urea and plasma creatinine, indicating that both visceral and muscular tissues undergo proteolytic degradation throughout the course of rampant inflammatory burden. In healthy individuals, tHcy plasma concentrations maintain positive correlations with LBM and TTR from birth until the end of adulthood. Starting from the onset of normal old age, tHcy values become disconnected from LBM control and reveal diverging trends with TTR values. Of utmost importance is the finding that, contrary to all protein
components which are downregulated in protein-depleted states, tHcy values are upregulated.  Hyperhomocysteinemia is an acquired clinical entity characterized by mild or moderate elevation in tHcy blood values found in apparently healthy individuals (McCully 1969). This distinct morbid condition appears as a public health problem of increasing importance in the general population, being regarded as an independent and graded risk factor for vascular pathogenesis unrelated to hypercholesterolemia, arterial hypertension, diabetes and smoking.

Studies grounded on stepwise multiple regression analysis have concluded that the two main watersoluble vitamins account for only 28% of tHcy variance whereas vitamins B6, B9, and B12, taken together, did not account for more than 30–40% of variance. Moreover, a number of hyperhomocysteinemic conditions are not responsive to folate and pyridoxine supplementation. This situation prompted us to search for other causal factors which might fill the gap between the public health data and the vitamin triad deficiencies currently incriminated. We suggest that S – the forgotten element – plays central roles in nutritional epidemiology (Ingenbleek and Young 2004).

Aminoacidemia studies performed in PEM children, adult patients and elderly subjects have reported that the concentrations of plasma IAAs invariably display lowering trends as the morbid condition worsens. The depressed tendency is especially pronounced in the case of tryptophan and for the so-called branched-chain AAs (BCAAs, isoleucine, leucine, valine) the decreases in which are regarded as a salient PEM feature following the direction outlined by TTR (Ingenbleek et al. 1986). Met constitutes a notable exception to the above described evolutionary profiles, showing unusual stability in chronically protein depleted states.

Maintenance of normal methioninemia is associated with supranormal tHcy blood values in PEMadults (Ingenbleek et al. 1986) and increased tHcy leakage in the urinary output of PEM children. In contrast, most plasma and urinary S-containing compounds produced along the TS pathway downstream to CbSconverting step (Fig. 20.1) display significantly diminished values. This is notably the case for cystathionine (Ingenbleek et al. 1986), glutathione, taurine, and sulfaturia. Such distorted patterns are reminiscent of abnormalities defining homocystinuria, an inborn disease of Met metabolism characterized by CbS refractoriness to pyridoxine stimuli, thereby promoting the upstream retention of tHcy in biological fluids. It
was hypothesized more than 20 years ago (Ingenbleek et al. 1986) that PEM is apparently able to similarly depress CbS activity, suggesting that the enzyme is a N-status sensitive step working as a bidirectional lockgate, overstimulated by high Met intake (Finkelstein and Martin 1986) and downregulated under N-deprivation conditions (Ingenbleek et al. 2002). Confirmation that N dietary deprivation may inhibit CbS activity has recently provided. The tHcy precursor pool is enlarged in biological fluids, boosting Met remethylation processes along the RM pathway, consistent with studies showing overstimulation of Met-synthase activity in conditions of protein restriction. In other words, high tHcy plasma concentrations observed in PEM states are the dark side of adaptive mechanisms for maintaining Met homeostasis. This is consistent with the unique role played by Met in the preservation of N body stores.

The classical interpretation that strict vegans, who consume plenty of folates in their diet and manifest nevertheless higher tHcy plasma concentrations than omnivorous counterparts, needs to be revisited. On the basis of hematological and biochemical criteria, cobalamin deficiency is one of the most prevalent vitamin-deficiencies wordwide, being often incriminated as deficient in vegan subjects. It seems, however, likely that its true causal impact on rising tHcy values is substantially overestimated in most studies owing to the modest contribution played by cobalamins on tHcy
variance analyses. In contrast, there exists a growing body of converging data indicating that the role played by the protein component is largely underscored in vegan studies. It is worth recalling that S is the main intracellular anion coexisting with N within a constant mean S:N ratio (1:14.5) in animal tissues and dietary products of animal origin (Ingenbleek 2006). The mean S:N ratio found in plant items ranges from 1:20 to 1:35, a proportion that does not optimally meet human tissue requirements (Ingenbleek 2006), paving the way for borderline S and N deficiencies.

A recent Taiwanese investigation on hyperhomocysteinemic nuns consuming traditional vegetarian regimens consisting of mainly rice, soy products,
vegetables and fruits with few or no dairy items illustrates such clinical misinterpretation (Hung et al. 2002). The authors reported that folates and cobalamins, taken together, accounted for only 28.6% of tHcy variance in the vegetarian cohort whereas pyridoxine was inoperative (Hung et al. 2002). The daily vegetable N and Met intakes were situated highly significantly (p < 0.001) below the recommended allowances for humans (FAO/WHO/United Nations University 1985), causing a stage of unrecognized PEM documented by significantly depressed BCAA plasma
concentrations. Met levels escaped the overall decline in IAAs levels, emphasizing that efficient homeostatic mechanisms operate at the expense of an acquired hyperhomocysteinemic state. The diagnosis of subclinical PEM was missed because the authors ignored the exquisitely sensitive TTR detecting power. A proper PEM identification would have allowed the authors to confirm the previously described TTR–tHcy relationship that was established in Western Africa from comparable field studies involving country dwellers living on plant products.

The concept that acute or chronic stressful conditions may exert similar inhibitory effects on CbS activity and thereby promote hyperhomocysteinemic states is founded on previous studies showing that hypercatabolic states are characterized by increased urinary N and S losses maintaining tightly correlated depletion rates (Cuthbertson 1931; Ingenbleek and Young 2004; Sherman and Hawk 1900) which reflect the S:N ratio found in tissues undergoing cytokine induced proteolysis. This has been documented in coronary infarction and in acute pancreatitis where tHcy elevation evolves too rapidly to allow for a nutritional vitamin B-deficit explanation.  tHcy is considered stable in plasma and the two investigations report unaltered folate and cobalamin plasma concentrations.

The clinical usefulness of TTR as a nutritional biomarker, described in the early seventies (Ingenbleek et al. 1972) has been substantially disregarded by the scientific community for nearly four decades. This long-lasting reluctance expressed by many investigators is largely due to the fact that protein malnutrition and stressful disorders of various causes have combined inhibitory effects on hepatic TTR synthesis. Declining TTR plasma concentrations may result from either dietary protein and energy restrictions or from cytokine-induced transcriptional blockade (Murakami et al. 1988) of its hepatic synthesis. The proposed marker was therefore seen as having high sensitivity but poor specificity. Recent advances in protein metabolism settle the controversy by throwing further light on the relationships between TTR and the N-components of body composition.

The developmental patterns of LBM and TTR exhibit striking similarities. Both parameters rise from birth to puberty, manifest gender dimorphism during full sexual maturity then decrease during ageing. Uncomplicated PEM primarily affects both visceral and structural pools of LBM with distinct kinetics, reducing protein synthesis to levels compatible with prolonged survival. In acute or chronic stressful disorders, LBM undergoes muscle proteolysis exceeding the upregulation of protein syntheses in liver and injured areas, yielding a net body negative N balance. These adaptive responses are well identified by the measurement of TTR plasma concentrations which therefore appear as a plasma marker for LBM fluctuations.
Attenuation of stress and/or introduction of nutritional rehabilitation restores both LBM and TTR to normal values following parallel slopes. TTR fulfills, therefore, a unique position in assessing actual protein nutritional status, monitoring the efficacy of dietetic support and predicting the patient’s outcome (Bernstein and Pleban 1996).

see also…

Acosta PB, Yannicelli S, Ryan AS, Arnold G, Marriage BJ, Plewinska M, Bernstein L, Fox J, Lewis V, Miller M, Velazquez A (2005) Nutritional therapy improves growth and protein status of children with a urea cycle enzyme defect. Mol Genet Metab 86:448–455.

Arroyave G, Wilson D, Be´har M, Scrimshaw NS (1961) Serum and urinary creatinine in children with severe protein malnutrition. Am J Clin Nutr 9:176–179.

Bates CJ, Mansoor MA, van der Pols J, Prentice A, Cole TJ, Finch S (1997) Plasma total homocysteine in a representative sample of 972 British men and women aged 65 and over. Eur J Clin Nutr 51:691–697.

Battezzatti A, Bertoli S, San Romerio A, Testolin G (2007) Body composition: An important determinant of homocysteine and methionine concentrations in healthy individuals. Nutr Metab Cardiovasc Dis 17:525–534.

Bernstein LH, Bachman TE, Meguid M, Ament M, Baumgartner T, Kinosian B, Martindale R, Spiekerman M (1995) Prealbumin in nutritional care Consensus Group. Measurement of visceral protein status in assessing protein and energy malnutrition: Standard of care. Nutrition 11:169–171

Bernstein LH, Ingenbleek Y (2002) Transthyretin: Its response to malnutrition and stress injury. Clinical usefulness and economical implications. Clin Chem Lab Med 40:1344–1348.

Boorsook H, Dubnoff JW (1947) The hydrolysis of phosphocreatine and the origin of creatinine. J Biol Chem 168:493–510.

Briend A, Garenne M, Maire B, Fontaine O, Dieng F (1989) Nutritional status, age and survival: The muscle mass hypothesis. Eur J Clin Nutr 43:715–726

Gray GE, Landel AM, Meguid MM (1994) Taurine-supplemented total parenteral nutrition and taurine status of malnourished cancer patients. Nutrition 10:11–15

Heymsfield SB, McManus C, Stevens V, Smith J (1982) Muscle mass: Reliable indicator of protein-energy malnutrition and outcome. Am J Clin Nutr 35:1192–1199

Ingenbleek Y (2006) The nutritional relationship linking sulfur to nitrogen in living organisms. J Nutr 136:S1641–S1651
Ingenbleek Y (2008) Plasma transthyretin indicates the direction of both nitrogen balance and retinoid status in health and disease. Open Clin Chem J 1:1–12
Ingenbleek Y, Bernstein LH (1999a) The stressful condition as a nutritionally dependent adaptive dichotomy. Nutrition 15:305–320
Ingenbleek Y, Bernstein LH (1999b) The nutritionally dependent adaptive dichotomy (NDAD) and stress hypermetabolism. J Clin Ligand Assay 22:259–267
Ingenbleek Y, Carpentier YA (1985) A prognostic inflammatory and nutritional index scoring critically ill patients. Internat J Vitam Nutr Res 55:91–101

Ingenbleek Y, Young VR (1994) Transthyretin (prealbumin) in health and disease: Nutritional implications. Annu Rev Nutr 14:495–533
Ingenbleek Y, Young VR (2002) Significance of transthyretin in protein metabolism. Clin Chem Lab Med 40:1281–1291
Ingenbleek Y, Young VR (2004) The essentiality of sulfur is closely related to nitrogen metabolism. Nutr Res Rev 17:135–151

Pharma Intell Links

Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II
Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I 
Mitochondrial dynamics and cardiovascular diseases 
“Seductive Nutrition”: Making Popular Dishes a Bit Healthier – Culinary Institute of America
Low Bioavailability of Nitric Oxide due to Misbalance in Cell Free Hemoglobin in Sickle Cell Disease – A Computational Model
Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis
Nitric Oxide and Immune Responses: Part 2
Mitochondrial Damage and Repair under Oxidative Stress
Endothelial Function and Cardiovascular Disease
Nitric Oxide and Sepsis, Hemodynamic Collapse, and the Search for Therapeutic Options
Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?
Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control
Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation
Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes
Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?
Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?

Simple representation of the toll-like recepto...

Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control

Curator and Author: Larry H Bernstein, MD, FCAP

What is Septic Shock?
Scripps Research Professor Wolfram Ruf and colleagues have identified a key connection between the signaling pathways and the immune system spiraling out of control involving the coagulation system and vascular endothelium that, if disrupted may be a target for sepsis. (Science Daily, Feb 29, 2008). It may be caused by a bacterial infection that enters the bloodstream, but we now recognize the same cascade not triggered by bacterial invasion. These invading bacteria produce endotoxins and other toxins that trigger a widespread inflammatory response of the innate immune system–a response that is necessary, as it turns out, because without the inflammation, the body cannot fight off the bacterial infection. During sepsis, the inflammation triggers widespread coagulation in the bloodstream. This coagulation can block blood vessels in vital organs, starving the organs of oxygen and damaging them. The organs can be further damaged when the blood starts to flow again because the lining of the blood vessels remain leaky due to inflammatory cytokines and damage by intravascular coagulation.
What is the Pathogenesis of Sepsis?
The acute respiratory distress syndrome (ARDS) has been defined as a severe form of acute lung injury featuring pulmonary inflammation and increased capillary leak. ARDS is associated with a high mortality rate and accounts for 100,000 deaths annually in the United States. ARDS may arise in a number of clinical situations, especially in patients with sepsis. A well-described pathophysiological model of ARDS is one form of the acute lung inflammation mediated by neutrophils, cytokines, and oxidant stress. Neutrophils are major effect cells at the frontier of innate immune responses, and they play a critical role in host defense against invading microorganisms. The tissue injury appears to be related to proteases and toxic reactive oxygen radicals released from activated neutrophils. In addition, neutrophils can produce cytokines and chemokines that enhance the acute inflammatory response. Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is mediated by a group of chemoattractants, especially CXC chemokines. Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP). Under these conditions of stimulation, activation of MAPKs (p38, p42/p44) occurs in sham neutrophils but not in CLP neutrophils, while under the same conditions phosphorylation of p38 and p42/p44 occurs in both sham and CLP alveolar macrophages. These data indicate that, under septic conditions, there is impaired signaling in neutrophils and enhanced signaling in alveolar macrophages, resulting in CXC chemokine production, and C5a appears to play a pivotal role in this process. As a result, CXC chemokines increase in lung, setting the stage for neutrophil accumulation in lung during sepsis.
Uncontrolled activation of the coagulation cascade following lung injury contributes to the development of lung inflammation and fibrosis in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and fibrotic lung disease. This article reviews our current understanding of the mechanisms leading to the activation of the coagulation cascade in response to lung injury and the evidence that excessive procoagulant activity is of pathophysiological significance in these disease settings. This is consistent with a pneumonia or lung injury preceding sepsis. On the other hand, it is not surprising that abdominal, cardiac bypass, and post cardiac revascularization may also lead to events resembling sepsis and/or cardiovascular collapse. The tissue factor-dependent extrinsic pathway is the predominant mechanism by which the coagulation cascade is locally activated in the lungs of patients with ALI/ARDS and pulmonary fibrosis. The cellular effects mediated via activation of proteinase-activated receptors (PARs) may be of particular importance in influencing inflammatory and fibroproliferative responses in experimental models involving direct injury to the lung. In this regard, studies in PAR1 knockout mice have shown that this receptor plays a major role in orchestrating the interplay between coagulation, inflammation and lung fibrosis.
The activation of the coagulation cascade is one of the earliest events initiated following tissue injury. The prime function of this complex and highly regulated proteolytic system is to generate insoluble, crosslinked fibrin strands, which bind and stabilize weak platelet hemostatic plugs, formed at sites of tissue injury. The formation of this provisional clot is critically dependent on the action of thrombin, and is generated following the stepwise activation of coagulation proteinases via the extrinsic and intrinsic systems. Under normal circumstances, blood is not exposed to tissue factor (TF). However, upon tissue injury, exposure of plasma to TF expressed on non-vascular cells or on activated endothelial cells results in the formation of the TF-activated factor VII (FVIIa) complex. The TF–FVIIa complex subsequently catalyses the initial activation of FX to activated factor X (FXa) and FIX to activated factor IX. FXa in association with activated factor V catalyses the conversion of prothrombin to thrombin. Sustained coagulation is achieved when thrombin synthesized through the initial TF–FVIIa–FXa complex catalyses the activation of FXI, FIX, FVIII and FX. In this manner, the intrinsic pathway is activated.
The systemic inflammatory response syndrome (SIRS) is the massive inflammatory reaction resulting from systemic mediator release that may lead to multiple organ dysfunction. I introduce an analysis of the roles of cytokines, cytokine production, and the relationship of cytokine production to the development of SIRS. The article postulates a three-stage development of SIRS, in which stage 1 is a local production of cytokines in response to an injury or infection. Stage 2 is the protective release of a small amount of cytokines into the body’s circulation. Stage 3 is the massive systemic reaction where cytokines turn destructive by compromising the integrity of the capillary walls and flooding end organs. While cytokines are generally viewed as a destructive development in the patient that generally leads to multiple organ dysfunction, cytokines also protect the body when localized. It will be necessary to study the positive effects of cytokines while also studying their role in causing SIRS. It will also be important to investigate the relationship between cytokines and their blockers in SIRS.
Monocyte/macrophage- and neutrophil-mediated inflammatory responses can be stimulated through a variety of receptors, including G protein-linked 7-transmembrane receptors (e.g., FPR1; MIM 136537), Fc receptors (see MIM 146790), CD14 (MIM 158120) and Toll-like receptors (e.g., TLR4; MIM 603030), and cytokine receptors (e.g., IFNGR1; MIM 107470). Engagement of these receptors can also prime myeloid cells to respond to other stimuli. Myeloid cells express receptors belonging to the Ig superfamily, such as TREM1, or to the C-type lectin superfamily. Depending on their transmembrane and cytoplasmic sequence structure, these receptors have either activating (e.g., KIR2DS1; MIM 604952) or inhibitory functions (e.g., KIR2DL1; MIM 604936).[supplied by OMIM].
TREM-1 associates with and signals via the adapter protein 12DAP12/12TYROBP, which contains an ITAM. To mediate activation, TREM-1 associates with the transmembrane adapter molecule 12DAP12. In sharp contrast to the effect by Ad-FDAP12, transgene expression in the liver of soluble form of extracellular domain of TREM-1 as an antagonist of 12DAP12 signaling, remarkably inhibited zymosan A-induced granuloma formation at every time point examined.
For signal transduction, 01TREM-1 couples to the ITAM-containing adapter DNAX activation protein of 12 kDa (23DAP12 ). MARV and EBOV activate TREM-1 on human neutrophils, resulting in 12DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. TREM-1 is the best-characterized member of a growing family of 12DAP12-associated receptors that regulate the function of myeloid cells in innate and adaptive responses. TREM-1 (triggering receptor expressed on myeloid cells), a recently discovered receptor of the immunoglobulin superfamily, activates neutrophils and monocytes/macrophages by signaling through the adapter protein 12DAP12. 522Granulocyte TREM-1 expression was high at baseline and immediately down-regulated upon LPS exposure along with an increase in soluble TREM-1.
DIC is primarily a laboratory diagnosis, based on the combination of elevated fibrin-related markers (FRM), with decreased procoagulant factors and platelets. Non-overt DIC is observed in most patients with sepsis, whereas overt DIC is less frequent. Consumption coagulopathy is a bleeding disorder caused by low levels of platelets and procoagulant factors associated with massive coagulation activation. Treatment with drotrecogin alfa (activated) improves survival and other outcome parameters in severe sepsis, including a subgroup of patients fulfilling the laboratory criteria of overt DIC. No randomized trials demonstrating effective therapies in consumption coagulopathy have been published.
Sepsis is a complex syndrome characterized by simultaneous activation of inflammation and coagulation manifested as systemic inflammatory response syndrome (SIRS)/sepsis symptoms through release of proinflammatory cytokines, procoagulants, and adhesion molecules from immune cells and/or damaged endothelium. Conventional treatments have focused on source control, antimicrobials, vasopressors, and fluid resuscitation; however, a new treatment paradigm exists: that of treating the host response to infection with adjunct therapies including early goal-directed therapy, drotrecogin alfa (activated), and immunonutrition. The drotrecogin alfa (activated) has been shown to reduce mortality in the severely septic patient when combined with traditional treatment. Therapies targeting improved oxygen and blood flow and reduction of apoptosis and free radicals are under investigation. Ultimately, intervention timing may be the most important factor in reducing severe sepsis mortality.

Cell Signaling in Sepsis
Recent data have shown stable patterns of activation among peripheral blood mononuclear cells and neutrophils in healthy human subjects. Although polymorphisms in Toll-like receptors play a contributory role in determining cellular activation, other factors are involved as well. In addition, circulating and locally released mediators of inflammation, including cytokines, complement fragments, and components of activated coagulation and fibrinolytic systems, that are generated in increased amounts during severe infection also interact with membrane-based receptors, leading to activation of intracellular path ways capable of further accelerating proinflammatory cascades. Circulating and organ-specific cell populations are activated to produce proinflammatory mediators during sepsis. Neutrophils and PBMCs bear TLR2 and TLR4, as well as other receptors, such as protein —coupled receptor, that induce increased generation of cytokines and other immunoregulatory proteins, as well as enhance release of proinflammatory mediators, including reactive oxygen species.
The expression of cytokines such as TNF-α and IL-1β is increased in sepsis, and engagement of TNF-α with type I(p55) and type II(p75) TNF receptors or IL-1β with IL-1 receptors belonging to the TLR/IL-1 receptor family produces activation of kinases (including Src, p38, extracellular signal—regulated kinase, and phosphoinositide 3–kinase) and transcriptional factors (such as nuclear factor [NF]–κB) important for further up-regulation of inflammatory proteins.
Genetic polymorphisms lead to alterations in TLR conformation (a small percentage of the variability in humans when their cells are exposed to bacterial products) that are accompanied by decreased cellular activation after exposure to bacterial products. The stable variability in cellular activation that is present among the genetically heterogeneous human population, only a limited number of studies have examined how such patterns may correlate with clinical outcome. A number of studies have examined the transcriptional factor NF-κB and kinases, including p38 and Akt, and provide insights into how heterogeneity in cell signaling may contribute to subsequent clinical course.
Increased activation of the mitogen-activated protein kinase protein 38, Akt, and nuclear factor (NF)–κB in neutrophils and other cell populations obtained at early time points in the clinical course of sepsis-induced acute lung injury or after accidental trauma is associated with a more-severe clinical course, suggesting that a proinflammatory cellular phenotype contributes to organ system dysfunction in such settings. Identification of patients with cellular phenotypes characterized by increased activation of NF-κB, Akt, and protein 38, as well as discrete patterns of gene activation, may permit identification of patients with sepsis who are likely to have a worse clinical outcome, thereby permitting early institution of therapies that modulate deleterious signaling pathways before organ system dysfunction develops, reducing morbidity and improving survival.

NF-kB

The transcriptional regulatory factor NF-κB is a central participant in modulating the expression of many immuno regulatory mediators involved in the acute inflammatory response [30–35]. NF-κB/rel transcription factors function as dimers held latently in the cytoplasm of cells by inhibitory IκB proteins. Signaling pathways initiated by engagement of TLRs, such as TLR 2 and TLR 4, by microbial products and other inflammatory mediators lead to nuclear accumulation of NF-κB and enhanced transcription of genes responsible for the expression of cytokines, chemokines, adhesion molecules, and other mediators of the inflammatory response associated with infection. Association of NF-κB with the inhibitory protein κB-α in the cytoplasm blocks the nuclear localization sequence of NF-κB, inhibiting its movement into the nucleus. Phosphorylation events, in addition to those involving IKKα/β and IκB-α, and involving NF-κB subunits (such as p 65) and nuclear coactivator proteins (such as TATA box binding protein or cAMP-responsive element—binding protein) are mediated by p 38, Akt, and other kinases and play an important role in regulating the transcriptional activity of NF-κB.

Studies have shown that greater nuclear accumulation of NF-κB is accompanied by higher mortality and worse clinical course in patients with sepsis. These clinical series demonstrated that persistent activation of NF-κB was found in nonsurvivors, with surviving patients having lower nuclear concentrations of NF-κB at early time points in their septic course than did nonsurvivors as well as more rapid return of nuclear accumulation of NF-κB.  Although studies of patients with sepsis have generally shown that nuclear concentrations of NF-κB are higher in non survivors than in survivors, an unresolved issue is whether such changes occur early and, therefore, define the subsequent course of sepsis or whether pathophysiological changes that result in poor clinical outcome also produce NF-κB activation as a secondary event, so that such changes in NF-κB are simply associated with more severe organ system dysfunction but do not contribute directly to outcome. A study of surgical patients without sepsis supports the hypothesis that neutrophil phenotypes defined by NF-κB activation patterns predict clinical outcome [54]. In that clinical series of patients undergoing repair of aortic aneurysms, higher preoperative levels of NF-κB in peripheral neutrophils were associated with death and with the development of postoperative organ dysfunction.

NF-κB

NF-κB (Photo credit: Wikipedia)

Stable high and low responder phenotypes in the healthy population, implies that the presence of a preexistent high responder neutrophil phenotype, as characterized by increased nuclear translocation of NF-κB after stimulation with TLR 2 or TLR 4 ligands, would be associated with more severe pulmonary inflammatory response and clinical course in response to infection. Conversely, persons whose neutrophils have diminished activation of NF-κB after stimulation would be expected to have less-intense neutrophil-driven inflammation, as well as organ dysfunction. In addition, Nuclear levels of nuclear factor (NF)–κB are significantly increased in neutrophils obtained within 24h of initiation of mechanical ventilation in patients whose clinical course from sepsis-induced acute lung injury is more severe (as defined by death or ventilation for >14 days—that is, ⩽14 ventilator-free days [VFD]), compared with patients with a less-severe course (as defined by mechanical ventilation for <14 days, or >14 VFD).  Baseline nuclear concentrations of NF-κB were lower in healthy volunteers than in patients with sepsis-induced acute lung injury, regardless of subsequent clinical course, demonstrating baseline activation of NF-κB in association with sepsis. *P <.05, vs. volunteers. †P< .05, vs. >14VFD.

Modulation of intracellular signaling cascades involving kinases, such as p 38 or Akt, or transcriptional factors, such as NF-κB, through specific inhibitory approaches has shown their pathophysiological importance in experimental models. However, the role of specific intra cellular pathways in contributing to clinical outcomes in patients with sepsis remains incompletely determined, primarily because such alterations in cellular activation patterns have not been examined at early time points before the onset of multiple organ dysfunction. Recent information shows that alterations in p38, Akt, and NF-κB among neutrophils and other cell populations not only precedes the development of organ system dysfunction but also has predictive value in identifying patients with a more severe subsequent clinical course.

RC Chambers. Procoagulant signalling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention? British Journal of Pharmacology 2008; 153, S367–S378; doi:10.1038/sj.bjp.0707603.

RC Bone. Toward a theory regarding the pathogenesis of the systemic inflammatory response syndrome: What we do and do not know about cytokine regulation. Crit Care Med 1996; 24:163-172.

Bouchon A, Facchetti F, Weigand MA, Colonna M. TREM-1 amplifies inflammation and is a crucial mediator of septic shock. Nature 2001; 410 (6832): 1103-7. doi:/10.1038/35074114. PMID 11323674.

Bleharski JR, Kiessler V, Buonsanti C, et al. A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response. J. Immunol. 2003; 170 (7): 3812-8. PMID 12646648.

Colonna M, Facchetti F. TREM-1 (triggering receptor expressed on myeloid cells): a new player in acute inflammatory responses. J. Infect. Dis 2003; 187 (Suppl 2): S397-401. PMID 12792857.

Dempfle CE. Coagulopathy of Sepsis. Thromb Hemost 2004; 91:213-224.

Cunneen J, Cartwright M. The Puzzle of Sepsis: Fitting the Pieces of the Inflammatory Response with Treatment. AACN Clin Issues 2004;15:18-44.

Ren-Feng Guo, NC Riedemann, Lei Sun, Hongwei Gao, KX Shi, et al. Divergent Signaling Pathways in Phagocytic Cells during Sepsis. The Journal of Immunology, 2006, 177: 1306–1313.

Abraham E.  Alterations in Cell Signaling in Sepsis. Clin Infect Dis 2005: 41 (Supplement 7): S459-S464. doi: 10.1086/431997

Yang KY, Arcaroli JJ, Abraham E. Early alterations in neutrophil activation are associated with outcome in acute lung injury. Am J Respir Crit Care Med 2003; 167:1567-74.

Abraham E. Neutrophils and Acute Lung Injury. Crit Care Med 2003; 31:195-9.

Abraham E, Carmody A, Shenkar R, Arcaroli J. Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol 2000; 279:1137-45.

Sepsis Bundles

The Institute for Healthcare Improvement (IHI) has highlighted sepsis as an area of focus and has identified several deficiencies that may cause suboptimal care of patients with severe sepsis.

These deficiencies include inconsistency in the early diagnosis of severe sepsis and septic shock, frequent inadequate volume resuscitation without defined endpoints, late or inadequate use of antibiotics, frequent failure to support the cardiac output when depressed, frequent failure to control hyperglycemia adequately, frequent failure to use low tidal volumes and pressures in acute lung injury, and frequent failure to treat adrenal inadequacy in refractory shock.

To address these deficiencies, the Surviving Sepsis Campaign and IHI have revised and added to the Surviving Sepsis Guidelines and created 2 sepsis treatment bundles (resuscitation and management) to guide therapy for patients with severe sepsis.

“Implicit in the use of the bundles is the need to adopt all the elements contained in the bundle,” the authors write. “One cannot choose to apply only selected items from the bundle and expect to achieve comparable benefit. The IHI sepsis website provides tools to screen patients for severe sepsis, as well as to measure success with adherence to implementing the bundles (http://www.ihi.org/IHI/Topics/CriticalCare/Sepsis/).” (The authors are employees of Eli Lilly and Co, the maker of drotrecogin alfa (activated). South Med J. 2007;100:594-600.

The sepsis resuscitation bundle, which should be accomplished as soon as possible and scored during the first 6 hours

Prealbumin (Transthyretin)

Discharge prealbumin and the change in prealbumin were positively correlated with protein and energy intake and inversely correlated with markers of inflammation, particularly CRP and IL-6. When all covariates were included in a multivariable regression analysis, the markers of inflammation predominantly accounted for the variance in prealbumin change (56%), whereas discharge protein intake accounted for 6%.

These authors propose an updated approach that incorporates current understanding of the systemic inflammatory response to help guide assessment, diagnosis, and treatment. An appreciation of a continuum of inflammatory response in relation to malnutrition syndromes is described. This discussion serves to highlight a research agenda to address deficiencies in diagnostics, biomarkers, and therapeutics of inflammation in relation to malnutrition.

Procalcitonin

The most frequent indication for antibiotic prescriptions in the northwestern hemisphere is lower respiratory tract infections (LRTIs),which range in severity from self-limited acute bronchitis to severe acute exacerbation of chronic obstructive pulmonary disease (COPD), and to life-threatening bacterial community-acquired pneumonia (CAP).4 Clinical signs and symptoms, as well as commonly used laboratory markers, are unreliable in distinguishing viral from bacterial LRTI. As many as 75% of patients with LRTI are treated with antibiotics, despitethe predominantly viral origin of their infection. An approach to estimate the probability of bacterial origin in LRTI is the measurement of serum procalcitonin (PCT).

In patients with LRTIs, a strategy of PCT guidance compared with standard guidelines resulted in similar rates of adverse outcomes, as well as lower rates of antibiotic exposure and antibiotic-associated adverse effects. (Trial Registration isrctn.org Identifier: ISRCTN95122877)

Neutrophil CD64

Despite improvements in the treatment of sepsis in recent years, there have been few diagnostic innovations which improve the sensitivity and specificity of diagnosis or facilitate therapeutic monitoring. The clinical reliance on the CBC and leukocyte differential with associated band count to indicate myeloid left shift of immaturity is not accurate, and it is not comparable to the measurement of the metamyeloctes and myelocytes. Only the introduction of a test which measures procalcitonin (PCT), an acute phase marker which is claimed to be more specific for bacterial infections than for viral infections, can be cited as a new diagnostic for the evaluation of patients with suspected infection. A need still persists for improved diagnostic indictors of infection or sepsis, as well as better tests to facilitate monitoring of therapy in the treatment of infection, so that use of antibiotics might be less empirical.

Studies have indicated that quantitative neutrophil CD64 expression is a sensitive and specific laboratory indicator of sepsis or the presence of a systemic acute inflammatory response.  Neutrophil CD64 is a highly sensitive marker for neonatal sepsis. Prospective studies incorporating CD64 into a sepsis scoring system are warranted. Studies have indicated that quantitative neutrophil CD64 (high affinity Fc receptor) expression is a worth­while candidate for evaluation as a more sensitive and specific laboratory indi­cator of sepsis or the presence of a systemic acute inflammatory response than available diagnostics . Neutrophil (PMN) CD64 is one of many activa­tion-related antigenic changes manifested by neutrophils during the normal pathophysiological acute inflammatory or innate immune response. PMN expression of CD64 is up-regulated under the influence of inflammatory relat­ed cytokines such as interleukin 12 (IL-12), interferon gamma (IFN-y) and granulocyte colony stimulating factor (G-CSF).

The first commercially available assay for PMN CD64, developed by Trillium Diagnostics, LLC is a fluorescence based, no wash flow cytometric assay, namely the Leuko64. The assay kit contains a cocktail of monoclonal antibodies includ­ing two monoclonal antibodies to CD64 and a monoclonal antibody to CD163, red cell lysis buffer, fluorescence quantitation beads, and a software program for automated analysis of the flow cytometric data that reports PMN CD64 as a CD64 index. The PMN CD64 index is designed so that normal inactivated PMNs yield values of < 1.00 and blood samples from individuals with docu­mented infection or sepsis typically show values > 1.50. Using clinical flow cytometers, the assay can be completed within 30 minutes. While this initial assay format was developed for multiparameter flow cytometers, a new version of the assay has been developed to give nearly identical results on the CD4000 and Sapphire (manufactured by Abbott Diagnostics, Santa Clara, CA) blood cell counters, which are equipped with laser light sources and fluorescence detection capabilities. If these blood cell counters are available in diagnostic haematology laboratories, the Leuko64 assay can be utilised on a 24 hour basis, in contrast to the more typical daytime operation hours of flow cytometric diagnostic laboratories.

Leukocare and Trillium Diagnostics entered an agreement to develop and market Leukocare’s method for detecting inflammatory activity using circulating cell-free DNA. Trillium aims to create a cf-DNA test as a “simple and cost effective” tool that healthcare professionals can use to obtain clinically relevant data on patients who are suspected of having sepsis. The companies said that they expect to finish developing the assay and market it in two years.

B Casserly, R Read, MM Levy. Multimarker Panels  in Sepsis. Crit Care Clin 27 (2011) 391–405 doi:10.1016/j.ccc.2010.12.011 criticalcare.theclinics.com

Dennis RA, Johnson LE, Roberson PK, Heif M, Bopp MM, et al.  Changes in prealbumin, nutrient intake, and systemic inflammation in elderly recuperative care patients.  J Am Geriatr Soc. 2008; 56(7):1270-5. Epub 2008 Jun 10. PMID: 18547360

Jensen GL, Bistrian B, Roubenoff R, Heimburger DC.  Malnutrition Syndromes: A Conundrum vs Continuum.

Bernstein LH. The systemic inflammatory response syndrome C-reactive protein and transthyretin conundrum. Clinical Chemistry Laboratory Medicine 2007; 45(11):1566–1567, ISSN (Online) 14374331, ISSN (Print) 14346621, DOI: 10.1515/CCLM.2007.334.

Schuetz P, Christ-Crain M, Thomann R, Falconnier C, Wolbers M, et al.  for the ProHOSP Study Group. Effect of Procalcitonin-Based Guidelines vs Standard Guidelines on Antibiotic Use in Lower Respiratory Tract Infections: The ProHOSP Randomized Controlled Trial.  JAMA  2009; 302(10): 1059

Bhandari V, Wang C, Rinder C, Rinder H. Hematologic Profile of Sepsis in Neonates: Neutrophil CD64 as a Diagnostic Marker. Pediatrics 2007; 31:4005.   (ISSN Numbers: Print, 0031-4005; Online, 1098-4275). doi:10.1542/peds.2007-1308

Davis BH.  Neutrophil CD64 expression in infection and sepsis. CLI Ocober 2006.

Chapter 1 Statement of Inferential    Second Opinion

Realtime Clinical Expert Support

Gil David and Larry Bernstein have developed, in consultation with Prof. Ronald Coifman, in the Yale University Applied Mathematics Program, a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options.

Keywords: Entropy, Maximum Likelihood Function, separatory clustering, peripheral smear, automated hemogram, Anomaly, classification by anomaly, multivariable and multisyndromic, automated second opinion

Abbreviations: Akaike Information Criterion, AIC;  Bayes Information Criterion, BIC, Systemic Inflammatory Response Syndrome, SIRS.

Background: The current design of the Electronic Medical Record (EMR) is a linear presentation of portions of the record by services, by diagnostic method, and by date, to cite examples.  This allows perusal through a graphical user interface (GUI) that partitions the information or necessary reports in a workstation entered by keying to icons.  This requires that the medical practitioner finds the history, medications, laboratory reports, cardiac imaging and EKGs, and radiology in different workspaces.  The introduction of a DASHBOARD has allowed a presentation of drug reactions, allergies, primary and secondary diagnoses, and critical information about any patient the care giver needing access to the record.  The advantage of this innovation is obvious.  The startup problem is what information is presented and how it is displayed, which is a source of variability and a key to its success.

Intent: We are proposing an innovation that supercedes the main design elements of a DASHBOARD and utilizes the conjoined syndromic features of the disparate data elements.  So the important determinant of the success of this endeavor is that it facilitates both the workflow and the decision-making process with a reduction of medical error. Continuing work is in progress in extending the capabilities with model datasets, and sufficient data because the extraction of data from disparate sources will, in the long run, further improve this process.  For instance, the finding of  both ST depression on EKG coincident with an elevated cardiac biomarker (troponin), particularly in the absence of substantially reduced renal function. The conversion of hematology based data into useful clinical information requires the establishment of problem-solving constructs based on the measured data.

The most commonly ordered test used for managing patients worldwide is the hemogram that often incorporates the review of a peripheral smear.  While the hemogram has undergone progressive modification of the measured features over time the subsequent expansion of the panel of tests has provided a window into the cellular changes in the production, release or suppression of the formed elements from the blood-forming organ to the circulation.  In the hemogram one can view data reflecting the characteristics of a broad spectrum of medical conditions.

Progressive modification of the measured features of the hemogram has delineated characteristics expressed as measurements of size, density, and concentration, resulting in many characteristic features of classification. In the diagnosis of hematological disorders proliferation of marrow precursors, the domination of a cell line, and features of suppression of hematopoiesis provide a two dimensional model.  Other dimensions are created by considering the maturity of the circulating cells.  The application of rules-based, automated problem solving should provide a valid approach to the classification and interpretation of the data used to determine a knowledge-based clinical opinion. The exponential growth of knowledge since the mapping of the human genome enabled by parallel advances in applied mathematics that have not been a part of traditional clinical problem solving.  As the complexity of statistical models has increased the dependencies have become less clear to the individual.  Contemporary statistical modeling has a primary goal of finding an underlying structure in studied data sets.  The development of an evidence-based inference engine that can substantially interpret the data at hand and convert it in real time to a “knowledge-based opinion” could improve clinical decision-making by incorporating multiple complex clinical features as well as duration of onset into the model.

An example of a difficult area for clinical problem solving is found in the diagnosis of SIRS and associated sepsis.  SIRS (and associated sepsis) is a costly diagnosis in hospitalized patients.   Failure to diagnose sepsis in a timely manner creates a potential financial and safety hazard.  The early diagnosis of SIRS/sepsis is made by the application of defined criteria (temperature, heart rate, respiratory rate and WBC count) by the clinician.   The application of those clinical criteria, however, defines the condition after it has developed and has not provided a reliable method for the early diagnosis of SIRS.  The early diagnosis of SIRS may possibly be enhanced by the measurement of proteomic biomarkers, including transthyretin, C-reactive protein and procalcitonin.  Immature granulocyte (IG) measurement has been proposed as a more readily available indicator of the presence of granulocyte precursors (left shift).  The use of such markers, obtained by automated systems in conjunction with innovative statistical modeling, provides a promising approach to enhance workflow and decision making.   Such a system utilizes the conjoined syndromic features of disparate data elements with an anticipated reduction of medical error.  This study is only an extension of our approach to repairing a longstanding problem in the construction of the many-sided electronic medical record (EMR).  In a classic study carried out at Bell Laboratories, Didner found that information technologies reflect the view of the creators, not the users, and Front-to-Back Design (R Didner) is needed.

Costs would be reduced, and accuracy improved, if the clinical data could be captured directly at the point it is generated, in a form suitable for transmission to insurers, or machine transformable into other formats.  Such data capture, could also be used to improve the form and structure of how this information is viewed by physicians, and form a basis of a more comprehensive database linking clinical protocols to outcomes, that could improve the knowledge of this relationship, hence clinical outcomes.

How we frame our expectations is so important that it determines the data we collect to examine the process.   In the absence of data to support an assumed benefit, there is no proof of validity at whatever cost.   This has meaning for hospital operations, for nonhospital laboratory operations, for companies in the diagnostic business, and for planning of health systems.

In 1983, a vision for creating the EMR was introduced by Lawrence Weed,  expressed by McGowan and Winstead-Fry (J J McGowan and P Winstead-Fry. Problem Knowledge Couplers: reengineering evidence-based medicine through interdisciplinary development, decision support, and research. Bull Med Libr Assoc. 1999 October; 87(4): 462–470.)   PMCID: PMC226622    Copyright notice

They introduce Problem Knowledge Couplers as a clinical decision support software tool that  recognizes that functionality must be predicated upon combining unique patient information, but obtained through relevant structured question sets, with the appropriate knowledge found in the world’s peer-reviewed medical literature.  The premise of this is stated by LL WEED in “Idols of the Mind” (Dec 13, 2006): “ a root cause of a major defect in the health care system is that, while we falsely admire and extol the intellectual powers of highly educated physicians, we do not search for the external aids their minds require”.  HIT use has been focused on information retrieval, leaving the unaided mind burdened with information processing.

The data presented has to be comprehended in context with vital signs, key symptoms, and an accurate medical history.  Consequently, the limits of memory and cognition are tested in medical practice on a daily basis.  We deal with problems in the interpretation of data presented to the physician, and how through better design of the software that presents this data the situation could be improved.  The computer architecture that the physician uses to view the results is more often than not presented as the designer would prefer, and not as the end-user would like.  In order to optimize the interface for physician, the system would have a “front-to-back” design, with the call up for any patient ideally consisting of a dashboard design that presents the crucial information that the physician would likely act on in an easily accessible manner.  The key point is that each item used has to be closely related to a corresponding criterion needed for a decision.  Currently, improved design is heading in that direction.  In removing this limitation the output requirements have to be defined before the database is designed to produce the required output.  The ability to see any other information, or to see a sequential visualization of the patient’s course would be steps to home in on other views.  In addition, the amount of relevant information, even when presented well, is a cognitive challenge unless it is presented in a disease- or organ-system structure.  So the interaction between the user and the electronic medical record has a significant effect on practitioner time, ability to minimize errors of interpretation, facilitate treatment, and manage costs.  The reality is that clinicians are challenged by the need to view a large amount of data, with only a few resources available to know which of these values are relevant, or the need for action on a result, or its urgency. The challenge then becomes how fundamental measurement theory can lead to the creation at the point of care of more meaningful actionable presentations of results.  WP Fisher refers to the creation of a context in which computational resources for meeting the challenges will be incorporated into the electronic medical record.  The one which he chooses is a probabilistic conjoint (Rasch) measurement model, which uses scale-free standard measures and meets data quality standards. He illustrates this by fitting a set of data provided by Bernstein (19)(27 items for the diagnosis of acute myocardial infarction (AMI) to a Rasch multiple rating scale model testing the hypothesis that items work together to delineate a unidimensional measurement continuum. The results indicated that highly improbable observations could be discarded, data volume could be reduced based on internal, and increased ability of the care provider to interpret the data.

 

Classified data a separate issue from automation

 Feature Extraction. This further breakdown in the modern era is determined by genetically characteristic gene sequences that are transcribed into what we measure.  Eugene Rypka contributed greatly to clarifying the extraction of features in a series of articles, which set the groundwork for the methods used today in clinical microbiology.  The method he describes is termed S-clustering, and will have a significant bearing on how we can view hematology data.  He describes S-clustering as extracting features from endogenous data that amplify or maximize structural information to create distinctive classes.  The method classifies by taking the number of features with sufficient variety to map into a theoretic standard. The mapping is done by a truth table, and each variable is scaled to assign values for each: message choice.  The number of messages and the number of choices forms an N-by N table.  He points out that the message choice in an antibody titer would be converted from 0 + ++ +++ to 0 1 2 3.

Even though there may be a large number of measured values, the variety is reduced by this compression, even though there is risk of loss of information.  Yet the real issue is how a combination of variables falls into a table with meaningful information.  We are concerned with accurate assignment into uniquely variable groups by information in test relationships. One determines the effectiveness of each variable by its contribution to information gain in the system.  The reference or null set is the class having no information.  Uncertainty in assigning to a classification is only relieved by providing sufficient information.  One determines the effectiveness of each variable by its contribution to information gain in the system.  The possibility for realizing a good model for approximating the effects of factors supported by data used for inference owes much to the discovery of Kullback-Liebler distance or “information”, and Akaike found a simple relationship between K-L information and Fisher’s maximized log-likelihood function. A solid foundation in this work was elaborated by Eugene Rypka.  Of course, this was made far less complicated by the genetic complement that defines its function, which made  more accessible the study of biochemical pathways.  In addition, the genetic relationships in plant genetics were accessible to Ronald Fisher for the application of the linear discriminant function.    In the last 60 years the application of entropy comparable to the entropy of physics, information, noise, and signal processing, has been fully developed by Shannon, Kullback, and others,  and has been integrated with modern statistics, as a result of the seminal work of Akaike, Leo Goodman, Magidson and Vermunt, and unrelated work by Coifman. Dr. Magidson writes about Latent Class Model evolution:

The recent increase in interest in latent class models is due to the development of extended algorithms which allow today’s computers to perform LC analyses on data containing more than just a few variables, and the recent realization that the use of such models can yield powerful improvements over traditional approaches to segmentation, as well as to cluster, factor, regression and other kinds of analysis.

Perhaps the application to medical diagnostics had been slowed by limitations of data capture and computer architecture as well as lack of clarity in definition of what are the most distinguishing features needed for diagnostic clarification.  Bernstein and colleagues had a series of studies using Kullback-Liebler Distance  (effective information) for clustering to examine the latent structure of the elements commonly used for diagnosis of myocardial infarction (CK-MB, LD and the isoenzyme-1 of LD),  protein-energy malnutrition (serum albumin, serum transthyretin, condition associated with protein malnutrition (see Jeejeebhoy and subjective global assessment), prolonged period with no oral intake), prediction of respiratory distress syndrome of the newborn (RDS), and prediction of lymph nodal involvement of prostate cancer, among other studies.   The exploration of syndromic classification has made a substantial contribution to the diagnostic literature, but has only been made useful through publication on the web of calculators and nomograms (such as Epocrates and Medcalc) accessible to physicians through an iPhone.  These are not an integral part of the EMR, and the applications require an anticipation of the need for such processing.

Gil David et al. introduced an AUTOMATED processing of the data available to the ordering physician and can anticipate an enormous impact in diagnosis and treatment of perhaps half of the top 20 most common causes of hospital admission that carry a high cost and morbidity.  For example: anemias (iron deficiency, vitamin B12 and folate deficiency, and hemolytic anemia or myelodysplastic syndrome); pneumonia; systemic inflammatory response syndrome (SIRS) with or without bacteremia; multiple organ failure and hemodynamic shock; electrolyte/acid base balance disorders; acute and chronic liver disease; acute and chronic renal disease; diabetes mellitus; protein-energy malnutrition; acute respiratory distress of the newborn; acute coronary syndrome; congestive heart failure; disordered bone mineral metabolism; hemostatic disorders; leukemia and lymphoma; malabsorption syndromes; and cancer(s)[breast, prostate, colorectal, pancreas, stomach, liver, esophagus, thyroid, and parathyroid].

Extension of conditions and presentation to the electronic medical record (EMR)

We have published on the application of an automated inference engine to the Systemic Inflammatory Response (SIRS), a serious infection, or emerging sepsis.  We can report on this without going over previous ground.  Of considerable interest is the morbidity and mortality of sepsis, and the hospital costs from a late diagnosis.  If missed early, it could be problematic, and it could be seen as a hospital complication when it is not. Improving on previous work, we have the opportunity to look at the contribution of a fluorescence labeled flow cytometric measurement of the immature granulocytes (IG), which is now widely used, but has not been adequately evaluated from the perspective of diagnostic usage.  We have done considerable work on protein-energy malnutrition (PEM), to which the automated interpretation is currently in review.  Of course, the

cholesterol, lymphocyte count, serum albumin provide the weight of evidence with the primary diagnosis (emphysema, chronic renal disease, eating disorder), and serum transthyretin would be low and remain low for a week in critical care.  This could be a modifier with age in providing discriminatory power.

Chapter  3           References

The Cost Burden of Disease: U.S. and Michigan. CHRT Brief. January 2010. @www.chrt.org

The National Hospital Bill: The Most Expensive Conditions by Payer, 2006. HCUP Brief #59.

Rudolph RA, Bernstein LH, Babb J: Information-Induction for the diagnosis of

myocardial infarction. Clin Chem 1988;34:2031-2038.

Bernstein LH (Chairman). Prealbumin in Nutritional Care Consensus Group.

Measurement of visceral protein status in assessing protein and energy malnutrition: standard of care. Nutrition 1995; 11:169-171.

Bernstein LH, Qamar A, McPherson C, Zarich S, Rudolph R. Diagnosis of myocardial infarction: integration of serum markers and clinical descriptors using information theory. Yale J Biol Med 1999; 72: 5-13.

Kaplan L.A.; Chapman J.F.; Bock J.L.; Santa Maria E.; Clejan S.; Huddleston D.J.; Reed R.G.; Bernstein L.H.; Gillen-Goldstein J. Prediction of Respiratory Distress Syndrome using the Abbott FLM-II amniotic fluid assay. The National Academy of Clinical Biochemistry (NACB) Fetal Lung Maturity Assessment Project.  Clin Chim Acta 2002; 326(8): 61-68.

Bernstein LH, Qamar A, McPherson C, Zarich S. Evaluating a new graphical ordinal logit method (GOLDminer) in the diagnosis of myocardial infarction utilizing clinical features and laboratory data. Yale J Biol Med 1999; 72:259-268.

Bernstein L, Bradley K, Zarich SA. GOLDmineR: Improving models for classifying patients with chest pain. Yale J Biol Med 2002; 75, pp. 183-198.

Ronald Raphael Coifman and Mladen Victor Wickerhauser. Adapted Waveform Analysis as a Tool for Modeling, Feature Extraction, and Denoising. Optical Engineering, 33(7):2170–2174, July 1994.

R. Coifman and N. Saito. Constructions of local orthonormal bases for classification and regression. C. R. Acad. Sci. Paris, 319 Série I:191-196, 1994.

Chapter 4           Clinical Expert System

Realtime Clinical Expert Support and validation System

We have developed a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options. The primary purpose is to gather medical information, generate metrics, analyze them in realtime and provide a differential diagnosis, meeting the highest standard of accuracy. The system builds its unique characterization and provides a list of other patients that share this unique profile, therefore utilizing the vast aggregated knowledge (diagnosis, analysis, treatment, etc.) of the medical community. The main mathematical breakthroughs are provided by accurate patient profiling and inference methodologies in which anomalous subprofiles are extracted and compared to potentially relevant cases. As the model grows and its knowledge database is extended, the diagnostic and the prognostic become more accurate and precise. We anticipate that the effect of implementing this diagnostic amplifier would result in higher physician productivity at a time of great human resource limitations, safer prescribing practices, rapid identification of unusual patients, better assignment of patients to observation, inpatient beds, intensive care, or referral to clinic, shortened length of patients ICU and bed days.

The main benefit is a real time assessment as well as diagnostic options based on comparable cases, flags for risk and potential problems as illustrated in the following case acquired on 04/21/10. The patient was diagnosed by our system with severe SIRS at a grade of 0.61 .

The patient was treated for SIRS and the blood tests were repeated during the following week. The full combined record of our system’s assessment of the patient, were derived from the further Hematology tests.  Following treatment, the SIRS risk as a major concern was eliminated and the system provides a positive feedback for the treatment of the physician.

 

Method for data organization and classification via characterization metrics.

Our database organized to enable linking a given profile to known profiles. This is achieved by associating a patient to a peer group of patients having an overall similar profile, where the similar profile is obtained through a randomized search for an appropriate weighting of variables. Given the selection of a patients’ peer group, we build a metric that measures the dissimilarity of the patient from its group. This is achieved through a local iterated statistical analysis in the peer group.

We then use this characteristic metric to locate other patients with similar unique profiles, for each of whom we repeat the procedure described above. This leads to a network of patients with similar risk condition. Then, the classification of the patient is inferred from the medical known condition of some of the patients in the linked network. Given a set of points (the database) and a newly arrived sample (point), we characterize the behavior of the newly arrived sample, according to the database. Then, we detect other points in the database that match this unique characterization. This collection of detected points defines the characteristic neighborhood of the newly arrived sample. We use the characteristic neighbor hood in order to classify the newly arrived sample. This process of differential diagnosis is repeated for every newly arrived point.   The medical colossus we have today has become a system out of control and beset by the elephant in the room – an uncharted complexity. We offer a method that addresses the complexity and enables rather than disables the practitioner.  The method identifies outliers and combines data according to commonality of features.

Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/11/09/summary-and-perspectives-impairments-in-pathological-states-endocrine-disorders-stress-hypermetabolism-cancer/

This summary is the last of a series on the impact of transcriptomics, proteomics, and metabolomics on disease investigation, and the sorting and integration of genomic signatures and metabolic signatures to explain phenotypic relationships in variability and individuality of response to disease expression and how this leads to  pharmaceutical discovery and personalized medicine.  We have unquestionably better tools at our disposal than has ever existed in the history of mankind, and an enormous knowledge-base that has to be accessed.  I shall conclude here these discussions with the powerful contribution to and current knowledge pertaining to biochemistry, metabolism, protein-interactions, signaling, and the application of the -OMICS to diseases and drug discovery at this time.

The Ever-Transcendent Cell

Deriving physiologic first principles By John S. Torday | The Scientist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41282/title/The-Ever-Transcendent-Cell/

Both the developmental and phylogenetic histories of an organism describe the evolution of physiology—the complex of metabolic pathways that govern the function of an organism as a whole. The necessity of establishing and maintaining homeostatic mechanisms began at the cellular level, with the very first cells, and homeostasis provides the underlying selection pressure fueling evolution.

While the events leading to the formation of the first functioning cell are debatable, a critical one was certainly the formation of simple lipid-enclosed vesicles, which provided a protected space for the evolution of metabolic pathways. Protocells evolved from a common ancestor that experienced environmental stresses early in the history of cellular development, such as acidic ocean conditions and low atmospheric oxygen levels, which shaped the evolution of metabolism.

The reduction of evolution to cell biology may answer the perennially unresolved question of why organisms return to their unicellular origins during the life cycle.

As primitive protocells evolved to form prokaryotes and, much later, eukaryotes, changes to the cell membrane occurred that were critical to the maintenance of chemiosmosis, the generation of bioenergy through the partitioning of ions. The incorporation of cholesterol into the plasma membrane surrounding primitive eukaryotic cells marked the beginning of their differentiation from prokaryotes. Cholesterol imparted more fluidity to eukaryotic cell membranes, enhancing functionality by increasing motility and endocytosis. Membrane deformability also allowed for increased gas exchange.

Acidification of the oceans by atmospheric carbon dioxide generated high intracellular calcium ion concentrations in primitive aquatic eukaryotes, which had to be lowered to prevent toxic effects, namely the aggregation of nucleotides, proteins, and lipids. The early cells achieved this by the evolution of calcium channels composed of cholesterol embedded within the cell’s plasma membrane, and of internal membranes, such as that of the endoplasmic reticulum, peroxisomes, and other cytoplasmic organelles, which hosted intracellular chemiosmosis and helped regulate calcium.

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.  ….

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.

Given that the unicellular toolkit is complete with all the traits necessary for forming multicellular organisms (Science, 301:361-63, 2003), it is distinctly possible that metazoans are merely permutations of the unicellular body plan. That scenario would clarify a lot of puzzling biology: molecular commonalities between the skin, lung, gut, and brain that affect physiology and pathophysiology exist because the cell membranes of unicellular organisms perform the equivalents of these tissue functions, and the existence of pleiotropy—one gene affecting many phenotypes—may be a consequence of the common unicellular source for all complex biologic traits.  …

The cell-molecular homeostatic model for evolution and stability addresses how the external environment generates homeostasis developmentally at the cellular level. It also determines homeostatic set points in adaptation to the environment through specific effectors, such as growth factors and their receptors, second messengers, inflammatory mediators, crossover mutations, and gene duplications. This is a highly mechanistic, heritable, plastic process that lends itself to understanding evolution at the cellular, tissue, organ, system, and population levels, mediated by physiologically linked mechanisms throughout, without having to invoke random, chance mechanisms to bridge different scales of evolutionary change. In other words, it is an integrated mechanism that can often be traced all the way back to its unicellular origins.

The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.

http://www.the-scientist.com/Nov2014/TE_21.jpg

A MECHANISTIC BASIS FOR LUNG DEVELOPMENT

The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.

http://www.the-scientist.com/Nov2014/TE_21.jpg

A MECHANISTIC BASIS FOR LUNG DEVELOPMENT: Stress from periodic atmospheric hypoxia (1) during vertebrate adaptation to land enhances positive selection of the stretch-regulated parathyroid hormone-related protein (PTHrP) in the pituitary and adrenal glands. In the pituitary (2), PTHrP signaling upregulates the release of adrenocorticotropic hormone (ACTH) (3), which stimulates the release of glucocorticoids (GC) by the adrenal gland (4). In the adrenal gland, PTHrP signaling also stimulates glucocorticoid production of adrenaline (5), which in turn affects the secretion of lung surfactant, the distension of alveoli, and the perfusion of alveolar capillaries (6). PTHrP signaling integrates the inflation and deflation of the alveoli with surfactant production and capillary perfusion.  THE SCIENTIST STAFF

From a cell-cell signaling perspective, two critical duplications in genes coding for cell-surface receptors occurred during this period of water-to-land transition—in the stretch-regulated parathyroid hormone-related protein (PTHrP) receptor gene and the β adrenergic (βA) receptor gene. These gene duplications can be disassembled by following their effects on vertebrate physiology backwards over phylogeny. PTHrP signaling is necessary for traits specifically relevant to land adaptation: calcification of bone, skin barrier formation, and the inflation and distention of lung alveoli. Microvascular shear stress in PTHrP-expressing organs such as bone, skin, kidney, and lung would have favored duplication of the PTHrP receptor, since sheer stress generates radical oxygen species (ROS) known to have this effect and PTHrP is a potent vasodilator, acting as an epistatic balancing selection for this constraint.

Positive selection for PTHrP signaling also evolved in the pituitary and adrenal cortex (see figure on this page), stimulating the secretion of ACTH and corticoids, respectively, in response to the stress of land adaptation. This cascade amplified adrenaline production by the adrenal medulla, since corticoids passing through it enzymatically stimulate adrenaline synthesis. Positive selection for this functional trait may have resulted from hypoxic stress that arose during global episodes of atmospheric hypoxia over geologic time. Since hypoxia is the most potent physiologic stressor, such transient oxygen deficiencies would have been acutely alleviated by increasing adrenaline levels, which would have stimulated alveolar surfactant production, increasing gas exchange by facilitating the distension of the alveoli. Over time, increased alveolar distension would have generated more alveoli by stimulating PTHrP secretion, impelling evolution of the alveolar bed of the lung.

This scenario similarly explains βA receptor gene duplication, since increased density of the βA receptor within the alveolar walls was necessary for relieving another constraint during the evolution of the lung in adaptation to land: the bottleneck created by the existence of a common mechanism for blood pressure control in both the lung alveoli and the systemic blood pressure. The pulmonary vasculature was constrained by its ability to withstand the swings in pressure caused by the systemic perfusion necessary to sustain all the other vital organs. PTHrP is a potent vasodilator, subserving the blood pressure constraint, but eventually the βA receptors evolved to coordinate blood pressure in both the lung and the periphery.

Read Full Post »


To reduce symptoms of mental illness and retrain the brain

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 5.11

Researchers have found that by specifically targeting a central signaling pathway in the brain, they can improve the innate behavioral response to stress in mice. Stress-induced behaviors in rodents reflect many of the symptoms that affect people suffering from major depression and other clinical conditions associated with stress. The findings, published July 20th online in the journalNature Neuroscience, suggest a new strategy for treating depression and other stress-associated disorders.

The study was led by James A. Bibb, Ph.D., at The University of Texas Southwestern Medical Center, who received NARSAD Young Investigator grants in 2000 and 2003, and lead authorFlorian Plattner, Ph.D. The scientific team also included Paul Greengard, Ph.D., a member of BBRF’s Scientific Council and a 1992, 2002, and 2008 Distinguished Investigator; Eric J. Nestler, M.D., Ph.D., a Scientific Council Member and a 1996 Distinguished Investigator; 2006 Young Investigator Kanehiro Hayashi, Ph.D.; 2007 Young Investigator Eunice Y. Yuen, Ph.D.; 1999 and 2004 Young Investigator Zhen Yan, Ph.D.; and 1999 Independent Investigator and 2006 Distinguished Investigator Angus C. Nairn, Ph.D.

The team’s findings are a result of a detailed investigation into a “signaling cascade,” called the cAMP/PKA pathway, which regulates a wide range of processes in the central nervous system. Disruption of the pathway has been linked with several mental disorders, including depression. Some existing antidepressant medications are known to boost cAMP signaling, but better understanding how this signaling network works could help researchers develop treatments that are more effective or cause fewer side effects.

Two new small-molecule compounds tested in mice can alleviate some symptoms of schizophrenia-like behaviors, including movement abnormalities, social avoidance, and cognitive performance. As reported in the July 1st issue of Neuropsychopharmacology, these research results may point the way toward new kinds of medications that treat specific aspects ofschizophrenia behaviors.

Current antipsychotic drugs used to treat the disorder target the dopamine D2 receptor, an important communication port for some neurons in the brain. These drugs are used primarily to treat schizophrenia “positive” symptoms such as delusions and hallucinations. They are less effective in treating “negative” symptoms such as a lack of pleasure in everyday life, or concentration and memory problems (“cognitive symptoms”), according to Duke University Medical Center scientists William C. Wetsel, Ph.D., a 1998 NARSAD Independent Investigator grantee, and Marc G. Caron, Ph.D., a Foundation Scientific Council member and 2005 NARSAD Distinguished Investigator grantee.

Current antipsychotic drugs were developed to bind to and block one specific type of communication pathway through dopamine D2 receptors, but the receptors are involved in more than one type of signaling pathway. Dr. Wetsel and colleagues decided to look for drug candidates that would block other pathways related to the dopamine D2 receptor, in the hope that this might reveal novel ways to treat a wider variety of schizophrenia symptoms.

They tested two dopamine D2 receptor-targeting compounds called UNC9975 and UNC9994 (developed by Jian Jin, Ph.D., of the University of North Carolina) that influence the beta-arrestin communication pathway, a different pathway than the one typically affected by antipsychotic drugs. The researchers showed that the compounds could normalize schizophrenia-like symptoms in mice by reducing their hyperactive movements, improving their memory for novel stimuli, and making them more social around other mice, among other improvements.

The new compounds also produced a much lower level of catalepsy—the rigid muscle, “trance-like” state that is sometimes a side effect of schizophrenic treatment—than traditional antipsychotic drugs such as haloperidol. Targeting different pathways connected to the dopamine D2 receptors, the researchers say, may increase the possibilities for treating people with schizophrenia in more individualized, fine-tuned ways that match their exact symptoms and vulnerabilities to side effects.

Effects of β-Arrestin-Biased Dopamine D2 Receptor Ligands on Schizophrenia-Like Behavior in Hypoglutamatergic Mice

Su M Park1, Meng Chen1, Claire M Schmerberg1, Russell S Dulman1, Ramona M Rodriguiz1,2, Marc G Caron3, Jian Jin4 and William C Wetsel1,2,3,5
Neuropsychopharmacology 2015; http://dx.doi.org:/10.1038/npp.2015.196

Current antipsychotic drugs (APDs) show efficacy with positive symptoms, but are limited in treating negative or cognitive features of schizophrenia. Whereas all currently FDA-approved medications target primarily the dopamine D2 receptor (D2R) to inhibit Gi/o-mediated adenylyl cyclase, a recent study has shown that many APDs affect not only Gi/o– but they can also influence β-arrestin- (βArr)-mediated signaling. The ability of ligands to differentially affect signaling through these pathways is termed functional selectivity. We have developed ligands that are devoid of D2R-mediated Gi/o protein signaling, but are simultaneously partial agonists for D2R/βArr interactions. The purpose of this study was to test the effectiveness of UNC9975 or UNC9994 on schizophrenia-like behaviors in phencyclidine-treated or NR1-knockdown hypoglutamatergic mice. We have found the UNC compounds reduce hyperlocomotion in the open field, restore PPI, improve novel object recognition memory, partially normalize social behavior, decrease conditioned avoidance responding, and elicit a much lower level of catalepsy than haloperidol. These preclinical results suggest that exploitation of functional selectivity may provide unique opportunities to develop drugs with fewer side effects, greater therapeutic selectivity, and enhanced efficacy for treating schizophrenia and related conditions than medications that are currently available.

The estrogen-related drug raloxifene can improve attention and memory in men and women with schizophrenia, according to a new study published in the journal Molecular Psychiatry.

University of New South Wales researcher Cynthia S. Weickert, Ph.D., a 1999 and 2001 NARSAD Young Investigator and 2004 Independent Investigator grantee, and her colleagues say their raloxifene findings could help improve some cognitive problems related to schizophrenia that have been the most difficult to treat with drugs. Dr. Weickert’s research team included NARSAD Young Investigator grantees Rhoshel K. Lenroot, M.D., (2003), and Ans Vercammen, Ph.D., (2010), along with Independent Investigator grantee Jayashri Kulkarni, Ph.D., (2000), and her husband and first author Tom Weickert, Ph.D.

A growing body of evidence suggests that estrogen plays a beneficial role in the brain, supporting growth and protecting neurons from damage. From work supported by her NARSAD Young Investigator awards, Dr. Weickert and her colleagues found that brain estrogen receptors are altered in some people with schizophrenia, blunting their ability to respond to estrogen’s beneficial effects. Raloxifene stimulates estrogen receptors and can help overcome a blunted estrogen response. Raloxifene is probably best known as a treatment for osteoporosis in women, where it mimics estrogen’s beneficial action in bones. The drug also stimulates estrogen receptors in the brain and may guard against memory loss in aging, making it potentially useful for cognitive problems in schizophrenia patients.

The research team examined the drug’s effect in 98 people diagnosed with schizophrenia or schizoaffective disorder (which combines symptoms of schizophrenia and depression). All of the patients received a daily dose of raloxifene along with their usual antipsychotic medications in one phase of the clinical trial and a placebo in another phase.

After the first six-week period, patients taking raloxifene had improved scores on memory and attention, compared to those taking placebo. When considering all the people in the study during both phases, raloxifene treatment significantly improved attention and thought processing speed. Raloxifene didn’t reduce the severity of schizophrenia symptoms more than the placebo did, but both groups showed less symptoms overall during the study, and none of the patients had severe side effects from the treatment.

Dr. Weickert and colleagues did detect some signs that the positive impact of raloxifene lasted more than one month after the treatment stopped. Although the researchers do not know the exact reasons for the lasting effects, they note that stimulating estrogen receptors might protect neurons, reduce inflammation, and increase connections between nerve cells in the brain over a longer time frame than drugs working on other neurotransmitter receptors. In light of their findings, they suggest future studies should replicate these results in a larger group of schizophrenia patients and also determine how long the cognitive benefits of a six-week treatment with raloxifene may last.

An injectable antipsychotic medication whose effects last for three months has successfully delayed the return of schizophrenia symptoms, researchers have found. Taking the drug in this form may help people with schizophrenia who struggle to stay on treatment by enabling them to take medication less frequently.

The research team, which included Adam J. Savitz, M.D., Ph.D., recipient of a NARSAD Young Investigator Grant in 2001, examined use of the long-acting antipsychotic medication paliperidone palmitate (Invega) in treating symptoms of schizophrenia such as hallucinations, delusions, and strong feelings of suspicion. The research was published online March 29th inJAMA Psychiatry.

When a person with schizophrenia cannot maintain a daily medication schedule, the drug level in his or her body can dip too low to combat symptoms, leading to relapse (return of symptoms) and an increased risk of being hospitalized. This study aimed to determine whether a dosage that would only have to be taken once every three months, rather than every day, would effectively hold off symptoms. After starting with a once-monthly dosage, patients took a three-month dosage to maintain symptom prevention and then were given randomly either placebo or the same three-month dosage every 12 weeks to see whether the medication’s positive effects would persist. (A placebo is a look-alike with inactive ingredients.)

Significantly fewer people who got the second three-month dosage experienced symptom relapse during the experimental phase of the study, compared to those taking a placebo. During this phase, the placebo group also reported more severe symptoms, while the paliperidone palmitate group’s symptoms remained constant.

The two groups also showed different patterns of side effects. The more serious side effects occurred in the placebo group: anxiety and return of other symptoms of schizophrenia. The paliperidone palmitate group more frequently experienced headaches, weight gain, common colds, as well as so-called extrapyramidal symptoms, which involve disruptions to movement.

This study did not include people with a history of substance dependence, major active medical problems, or other serious mental disorders. More research is needed to know whether less frequent dosage of long-acting injectable medication like paliperidone palmitate can help prevent relapse in these groups.

Efficacy and Safety of the 3-Month Formulation of Paliperidone Palmitate vs Placebo for Relapse Prevention of Schizophrenia – A Randomized Clinical Trial

Joris Berwaerts, MD1; Yanning Liu, MS2; Srihari Gopal, MD, MHS1; Isaac Nuamah, PhD1; Haiyan Xu, PhD1; Adam Savitz, MD, PhD1; Danielle Coppola, MD1; Alain Schotte, PhD3; Bart Remmerie, Chem Eng3; Nataliya Maruta, MD, PhD4; David W. Hough, MD1
JAMA Psychiatry. 2015; 72(8):830-839. http://dx.doi.org:/10.1001/jamapsychiatry.2015.0241.

Design, Setting, and Participants  This randomized, multicenter trial conducted from April 26, 2012, through April 9, 2014, in 8 countries consisted of 4 phases: 3-week screening phase, flexible-dose 17-week open-label transition phase, 12-week open-label maintenance phase, and open-ended double-blind (DB) phase. Of the 506 patients enrolled (aged 18-70 years; DSM-IV-TR diagnosis of schizophrenia), 305 were randomized to 3-month paliperidone palmitate (n = 160) or placebo (n = 145) in the DB phase.

Interventions  Patients received once-monthly doses of the 1-month formulation of paliperidone palmitate (50, 75, 100, or 150 mg eq) during the transition phase, followed by a single dose of the 3-month formulation (3.5 times the stabilized dose of once-monthly paliperidone palmitate) during the maintenance phase. Stabilized patients were randomized to receive either a fixed dose of 3-month paliperidone palmitate (175, 263, 350, or 525 mg eq) or placebo once every 3 months during the DB phase.

Main Outcomes and Measures  Time from randomization to the first relapse event (time to relapse) in the DB phase.

Results  In the interim analysis, time to first relapse was significantly different in favor of the paliperidone palmitate group vs the placebo group (hazard ratio = 3.45; 95% CI, 1.73-6.88; P < .001); median time to relapse was 274 days for placebo but not estimable for 3-month paliperidone palmitate. An independent data monitoring committee recommended early study termination due to efficacy. In the DB phase, 183 of 305 patients (62% with 3-month paliperidone palmitate; 58% with placebo) had at least 1 treatment-emergent adverse event; those noted more frequently in the group receiving paliperidone palmitate than in the placebo group were headache (9% vs 4%), weight increased (9% vs 3%), nasopharyngitis (6% vs 1%), and akathisia (4% vs 1%).

Conclusions and Relevance  Compared with placebo, the 3-month formulation of paliperidone palmitate administered 4 times yearly significantly delayed time to relapse in patients with schizophrenia. The 3-month formulation was generally tolerable and has a safety profile consistent with other marketed paliperidone formulations.

Trial Registration  clinicaltrials.gov Identifier:NCT01529515

 

Tracking Down the Causes of Alzheimer’s

University of Basel
http://www.biosciencetechnology.com/news/2015/09/tracking-down-causes-alzheimers?et_cid=4792750&et_rid=535648082&location=top

 

Researchers from the University of Basel were able to show that memory function (image shows the hippocampus highlighted) depends on calcium-regulating genes. (Source: MCN University of Basel

http://www.biosciencetechnology.com/sites/biosciencetechnology.com/files/bt1509_basel_brain.jpg

Genes are not only important for regular memory performance, but also for the development of Alzheimer’s disease. Researchers at the University of Basel now identified a specific group of genes that plays a central role in both processes. This group of molecules controls the concentration of calcium ions inside the cell. Their results appear in the current issue of the journal JAMA Psychiatry.

Intact memory capacity is crucial for everyday life. This fact becomes apparent once a memory disorder has developed. Alzheimer’s disease is the most common cause of age-associated memory disorders. Due to increasing life expectancy, the disease is on the rise in Switzerland and worldwide. Unfortunately, there is no effective treatment to cure or even slow down Alzheimer’s yet. Thus, understanding the origins of this neurodegenerative disorder is key to the development of much needed treatments.

Scientists have known for some years now, that genes do not only play a crucial role in normal memory performance, but also in the development of Alzheimer’s. However, it was so far unclear if specific genes are involved in both these processes.

Researchers at the transfaculty research platform at the Psychiatric University Clinics Basel and the Faculty of Psychology at the University of Basel were now able to show in a large scale study that a specific group of genes controls several processes that are central for regular brain functions as well as for the development of Alzheimer. First author Dr. Angela Heck collected and analyzed data of over 57,000 participants for this study.

Calcium is crucial

The study identified genes responsible for the concentration of calcium ions in the cell as central players of physiological and disease processes in the brain. Calcium genes stand in mutual relationship with memory performance of young and older healthy adults as well as with the function of the hippocampus, a brain region that is central to intact memory. Furthermore, calcium genes correlate with the risk for Alzheimer disease. The results contribute to the understanding of the complex processes that lead to memory disorders, such as Alzheimer’s.

 

 

 

Read Full Post »

« Newer Posts - Older Posts »