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Posts Tagged ‘blood brain barrier’


Healing traumatic brain injuries with self-assembling peptide hydrogels

Reporter : Irina Robu, PhD

In 2014, TBIs resulted in about 2.53 million emergency department visits in the U.S., according to the Centers for Disease Control and Prevention. A traumatic brain injury (TBI) can range from a mild concussion to a severe head injury. It is caused by a blow to the head or body, a wound that breaks through the skull or another injury that jars or shakes the brain. Individuals with traumatic brain injuries can develop secondary disorders after the initial blow. Researchers, Biplab Sarkar and Vivek Kumar from New Jersey Institute of Technology are hoping to prevent secondary disorders by injecting a self-assembling peptide hydrogel into the brains of rats with traumatic brain injury and see what happens. They observed that the hydrogel helped blood vessels regrow in addition to neuronal survival.

The researchers explained that after traumatic brain injury, the brain can amass glutamate which kills some neurons which is marked by overactive oxygen-containing molecules (oxidative stress), inflammation and disruption of the blood-brain barrier. Furthermore, TBI survivors can experience impaired motor control and depression. Within the experiment, the researchers showed that a week after injecting the gel in rats, the neurons have twice as many neurons at the injury site than the control animals did.

The NJIT researchers distinguished that they needed to inject the hydrogel directly in a rat’s brain just seconds after a TBI, which is not ideal, because it would be impossible to give a patient the treatment within that short period of time. The next step in showing that the self-assembling peptide hydrogel works is to combine their previous blood vessel-growing peptide and the new version to see whether it could enhance recovery. And the researchers plan to inspect whether the hydrogels work for more diffuse brain injuries such as concussions.

SOURCE

https://www.fiercebiotech.com/research/healing-traumatic-brain-injuries-self-assembling-peptide-hydrogels

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Recent progress in neurodegenerative diseases and gliomas

Curator: Larry H. Bernstein, MD, FCAP

LPBI

 

 

Alzheimer’s Protein Not All Bad, Says MassGen Study

A controversial idea—that amyloid-beta (Aβ) protein fights bacterial infections in the brain—has gained additional support from a new study. Previously, the idea seemed worthy of investigation, if a bit of a stretch, on the basis of cell culture results. Now, thanks to the efforts of a scientific team lead by researchers based at Massachusetts General Hospital, it has been reinforced by observations of how the Aβ protein functions in animals’ brains.

Details of the new study appeared May 25 in the journal Science Translational Medicine, in an article entitled, “Amyloid-β Peptide Protects against Microbial Infection in Mouse and Worm Models of Alzheimer’s Disease.” The article suggests that the tendency of Aβ protein to form insoluble aggregates is not, as has been widely assumed, intrinsically abnormal, even though the aggregates are recognized as a hallmark of Alzheimer’s disease. Rather, Aβ protein appears to be a natural antibiotic that can trap and imprison bacterial pathogens that manage to pass the blood–brain barrier, which becomes increasingly “leaky” with age.

“We present in vivo data showing that Aβ expression protects against fungal and bacterial infections in mouse, nematode, and cell culture models of AD,” wrote the article’s authors. “We show that Aβ oligomerization, a behavior traditionally viewed as intrinsically pathological, may be necessary for the antimicrobial activities of the peptide.”

The MassGen scientists and their colleagues found that transgenic mice expressing human Aβ survived significantly longer after the induction of Salmonella infection in their brains than did mice with no genetic alteration. Mice lacking the amyloid precursor protein died even more rapidly. Transgenic Aβ expression also appeared to protect C. elegans roundworms from either Candida orSalmonella infection. Similarly, human Aβ expression protected cultured neuronal cells from Candida. In fact, human Aβ expressed by living cells appears to be 1000 times more potent against infection than does the synthetic Aβ used in previous studies.

That superiority appears to relate to properties of Aβ that have been considered part of Alzheimer’s disease pathology—the propensity of small molecules to form oligomers and then aggregate into Aβ plaques. This propensity, suggests the MassGen-led team, may indicate that Aβ acts like an antimicrobial peptide (AMP).

While AMPs fight infection through several mechanisms, a fundamental process involves forming oligomers that bind to microbial surfaces and then clump together into aggregates that both prevent the pathogens from attaching to host cells and allow the AMPs to kill microbes by disrupting their cellular membranes. The synthetic Aβ preparations used in earlier studies did not include oligomers. In the current study, however, oligomeric human Aβ not only showed an even stronger antimicrobial activity, its aggregation into the sorts of fibrils that form Aβ plaques was also seen to entrap microbes in both mouse and roundworm models.

“Our findings raise the intriguing possibility that β-amyloid may play a protective role in innate immunity and infectious or sterile inflammatory stimuli may drive amyloidosis,” the study’s authors concluded. “These data suggest a dual protective/damaging role for Aβ, as has been described for other antimicrobial peptides.”

One of the study’s co-corresponding authors, Rudolph Tanzi, Ph.D., director of the Genetics and Aging Research Unit in the MassGeneral Institute for Neurodegenerative Disease (MGH-MIND), pointed out that AMPs are known to play a role in the pathologies of a broad range of major and minor inflammatory disease. “For example, LL-37, which has been our model for Aβ’s antimicrobial activities, has been implicated in several late-life diseases, including rheumatoid arthritis, lupus, and atherosclerosis,” he elaborated. “The sort of dysregulation of AMP activity that can cause sustained inflammation in those conditions could contribute to the neurodegenerative actions of Aβ in Alzheimer’s disease.”

The study’s other co-corresponding author, Robert Moir, M.D., also of the MGH-MIND Genetics and Aging unit, noted that the study’s findings may lead to potential new therapeutic strategies. He also indicated that therapies designed to eliminate amyloid plaques from patient’s brains may have their limitations.

“It does appear likely that the inflammatory pathways of the innate immune system could be potential treatment targets, Dr. Moir explained. “If validated, our data also warrant the need for caution with therapies aimed at totally removing Aβ plaques. Amyloid-based therapies aimed at dialing down but not wiping out Aβ in the brain might be a better strategy.”

It remains to be determined, however, whether Aβ typically fights real infections or is apt to behave errantly, forming aggregates as though microbes are present, even if they are, in fact, not. “Our findings raise the intriguing possibility that Alzheimer’s pathology may arise when the brain perceives itself to be under attack from invading pathogens,” said Dr. Moir. “Further study will be required to determine whether or not a bona fide infection is involved.”Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer’s disease

Deepak Kumar, Vijaya Kumar, Se Hoon Choi, Kevin J. Washicosky, et al.
Science Translational Medicine  25 May 2016;  8 (340): 340ra72
http://dx.doi.org:/10.1126/scitranslmed.aaf1059

Rehabilitation of a β-amyloid bad boy

A protein called Aβ is thought to cause neuronal death in Alzheimer’s disease (AD). Aβ forms insoluble aggregates in the brains of patients with AD, which are a hallmark of the disease. Aβ and its propensity for aggregation are widely viewed as intrinsically abnormal. However, in new work, Kumar et al. show that Aβ is a natural antibiotic that protects the brain from infection. Most surprisingly, Aβ aggregates trap and imprison bacterial pathogens. It remains unclear whether Aβ is fighting a real or falsely perceived infection in AD. However, in any case, these findings identify inflammatory pathways as potential new drug targets for treating AD.

Abstract

The amyloid-β peptide (Aβ) is a key protein in Alzheimer’s disease (AD) pathology. We previously reported in vitro evidence suggesting that Aβ is an antimicrobial peptide. We present in vivo data showing that Aβ expression protects against fungal and bacterial infections in mouse, nematode, and cell culture models of AD. We show that Aβ oligomerization, a behavior traditionally viewed as intrinsically pathological, may be necessary for the antimicrobial activities of the peptide. Collectively, our data are consistent with a model in which soluble Aβ oligomers first bind to microbial cell wall carbohydrates via a heparin-binding domain. Developing protofibrils inhibited pathogen adhesion to host cells. Propagating β-amyloid fibrils mediate agglutination and eventual entrapment of unatttached microbes. Consistent with our model, Salmonella Typhimurium bacterial infection of the brains of transgenic 5XFAD mice resulted in rapid seeding and accelerated β-amyloid deposition, which closely colocalized with the invading bacteria. Our findings raise the intriguing possibility that β-amyloid may play a protective role in innate immunity and infectious or sterile inflammatory stimuli may drive amyloidosis. These data suggest a dual protective/damaging role for Aβ, as has been described for other antimicrobial peptides.

 

CRISPR Crossing New Barriers

Researchers Are Developing Ways to Edit Some of the Most Difficult-to-Edit DNA-Neuronal DNA

http://www.genengnews.com/insight-and-intelligence/crispr-crossing-new-barriers/77900666/

 

Confocal microscopic image of the hippocampus showing immunoreactivities for mEGFP (magenta) and the HA tag (green) fused to ß-Actin.

Ryohei Yasuda, Ph.D., scientific director, and his team at the Max Planck Florida Institute of Neuroscience (MPFI) are working to understand the way individual cells in our brains change as we learn and form memories. One of their main goals is to understand how different proteins behave and impact the structure and function of an individual cell, but, much like the field of genetics was once limited by the inability to visualize the structure of DNA, their research has been limited by their ability to locate and visualize the many different types of proteins within a single cell. Current imaging methods do not provide contrast and specificity high enough to see distinct proteins. Plus, the best methods are time-consuming and expensive; it can take a year or more to develop engineered models.

Over the past few years, the development of CRISPR technology has helped scientists overcome countless genetic engineering challenges, and allowed them to edit genes with unmatched precision and speed, massively increasing clarity and cutting the cost of research requiring genetic engineering. The technique has been used in myriad ways to increase understanding and treatment of diseases and disorders, but some cells are more difficult to edit than others. Brain cells have proven especially difficult to manipulate using CRISPR.

Recently, MPFI researchers Takayasu Mikuni, Ph.D., M.D., and Jun Nishiyama, Ph.D., M.D., and Dr. Yasuda were able to harness the power of the CRISPR/Cas9 system in order to create a quick, scalable, and high-resolution technique to edit neuronal DNA, which they called “SLENDR,” (single-cell labeling of endogenous proteins by CRISPR/Cas9-mediated homology-directed repair.) Using the technique, the researchers labeled several distinct proteins with fluorescence, and were able to observe protein localization in the brain that was previously invisible. That’s just the start of what researchers may be able to accomplish using this reliable, new technique for inserting genes into neurons.

CRISPR/Cas9 and Neurons

CRISPR is a tool built into bacterial DNA that the organisms use to fight infections. When a virus invades and attempts to insert its infectious DNA into that of a bacterial cell, a special section of the bacterial DNA, called CRISPR, cuts the viral DNA and renders it unable to wreak havoc on the bacteria. The organism then inserts a copy of the viral DNA into its own DNA to work as a type of adaptive immune system, to better recognize and defeat the invader in the future. As scientists have begun to understand how this system works, they have manipulated it to target and damage specific, functional genes in a variety of organisms, and in some cases, insert a new gene in its place.

Once the section of DNA is damaged, the technique relies on the cell to naturally repair its own DNA. There are two methods that the cell might use to accomplish this. One is homology-directed repair (HDR), the other is non-homologous end joining (NHEJ). HDR rebuilds or replaces the damaged locus of the genome, whereas NHEJ reattaches the damaged ends. When the reattachment occurs following the degradation of the ends, it often leads to the deletion of function of the gene (“knock-out” the gene). If a cell uses HDR to repair itself, scientists can include a desired gene in the CRISPR system that will be inserted into the DNA to replace the damaged gene.

Despite the impressive power of CRISPR system, its use in brain cells has been limited because by the time the brain has developed, its cells are no longer dividing. Most mature brain cells will repair themselves using NHEJ. The researcher can’t give the cell a gene to insert if it’s not going to insert one to begin with. While scientists can use CRISPR relatively easily to damage and knock out certain genes through NHEJ in the brain, the lack of cell division has made it very difficult for them to knock indesired sequences to genes, through HDR, with reliable precision. That’s where the SLENDR technique comes in.

  • SLENDR

SLENDR combines the power of the CRISPR/Cas9 system with the specificity and timing of in utero electroporation. Electroporation is a well-known technique used for introducing new material into cells and creating genetic knock-outs and knock-ins. Using in utero electroporation allows researchers to insert the CRISPR/CAS9 system into prenatal models, where brain cells are still developing and dividing. Thus, the broken DNA is still being repaired via HDR, giving researchers the opportunity to precisely modify a gene. This is a big deal. “I believe that SLENDR will be a standard tool for molecular and cellular neurobiology,” said Dr. Yasuda. “SLENDR provides a valuable means to determine subcellular localization of proteins, and will help researchers to determine the function of the proteins.”

In the recent study, the researchers at MPFI inserted a gene that made proteins of interest fluoresce under the microscope. They were even able to reliably label two different proteins with distinct colors at the same time in the same cell. The researchers were able to use the technique to visualize the proteins both in vivo and in vitro. And they were able to do it in a matter of days rather than years.

With existing knowledge of how brains develop, researchers can adjust the timing and position of the electroporation in utero to accurately target cells that will go on to populate particular cortical layers of the brain, even if they haven’t differentiated and moved to that layer yet.

The recent study used the technique primarily to tag certain proteins within brain cells and observe their behavior. But, with continued optimization, the method has the potential to elucidate immeasurable brain activities in both normal and diseased brains, and lead to a deeper understanding of brain function. “The most important part is that precise genome editing is possible in the brain. That’s what’s important,” said Dr.  Nishiyama, post-doctoral researcher who worked on the study. “That’s the biggest thing.” Neuroscientists would be remiss to ignore its worth and not explore its potential.

Emma Yasinski is a scientific writer at Max Planck Florida Institute for Neuroscience. Correspondence should be directed to Ryohei Yasuda, Ph.D. (ryohei.yasuda@mpfi.org), scientific director, Max Planck Florida Institute for Neuroscience.

 

Altered Metabolism of Four Compounds Drives Glioblastoma Growth

Findings suggest new ways to treat the malignancy, slow its progression and reveal its extent more precisely.

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=190732

The altered metabolism of two essential amino acids helps drive the development of the most common and lethal form of brain cancer, according to a new study led by researchers at The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James).

The study shows that in glioblastoma (GBM), the essential amino acids methionine and tryptophan are abnormally metabolized due to the loss of key enzymes in GBM cells.

The altered methionine metabolism leads to activation of oncogenes, while the changes in tryptophan metabolism shield GBM cells from detection by immune cells. Together, the changes promote tumor progress and cancer-cell survival.

“Our findings suggest that restricting dietary intake of methionine and tryptophan might help slow tumor progression and improve treatment outcomes,” says first author and OSUCCC – James researcher Kamalakannan Palanichamy, PhD, research assistant professor in Radiation Oncology.

“While we need to better understand how these abnormally regulated metabolites activate oncogenic proteins, our intriguing discovery suggests novel therapeutic targets for this disease,” says principal investigator and study leader Arnab Chakravarti, MD, chair and professor of Radiation Oncology and co-director of the Brain Tumor Program.

“For example, restoring the lost enzymes in the two metabolic pathways might slow tumor progression and reduce aggressiveness by inactivating oncogenic kinases and activating immune responses,” says Chakravarti, who holds the Max Morehouse Chair in Cancer Research.

Chakravarti further notes that because GBM cells take up methionine much faster than normal glioma cells, positron emission tomography that uses methionine as a tracer (MET-PET) might help map GBM tumors more accurately, allowing more precise surgical removal and radiation therapy planning. (MET-PET is currently an experimental imaging method.)

More than 11,880 new cases of GBM were estimated to occur in 2015, with overall survival averaging 12 to 15 months, so there is an urgent need for more effective therapies.

Amino acids are the building blocks of proteins. Tryptophan and methionine are essential amino acids – the diet must provide them because cells cannot make them. Normally, the lack of an essential amino acid in the diet can lead to serious diseases and even death. Foods rich in tryptophan and methionine include cheese, lamb, beef, pork, chicken, turkey, fish, eggs, nuts and soybeans.

Palanichamy, Chakravarti and their colleagues conducted this study using 13 primary GBM cell lines derived from patient tumors, four commercially available GBM cell lines and normal human astrocyte cells. Metabolite analyses were done using liquid chromatography coupled with mass spectrometry.

http://www.oncology-central.com/2016/04/01/study-highlights-altered-amino-acid-metabolism-in-glioblastoma/

AUTHORS: EMILY BROWN, FUTURE SCIENCE GROUP

An investigation carried out at The Ohio State University Comprehensive Cancer Center (OH, USA) has uncovered abnormal metabolism of the essential amino acids methionine and tryptophan in glioblastoma.

The study suggests that this abnormal amino acid metabolism aids in the development of the disease. Furthermore, the findings, published recently in Clinical Cancer Research, hint at novel methods to potentially treat the malignancy, slow its progression and reveal its extent more precisely.

According to the study, it is the loss of key enzymes within glioblastoma cells that results in this abnormal metabolism. Modified methionine metabolism is described as promoting the activation of oncogenes, and the changes in tryptophan aid in masking the malignant cells from the immune system.

“While we need to better understand how these abnormally regulated metabolites activate oncogenic proteins, our intriguing discovery suggests novel therapeutic targets for this disease,” commented principal investigator and study leader Arnab Chakravarti (The Ohio State University Comprehensive Cancer Center).

 

Rapid eye movement sleep (dreaming) shown necessary for memory formation


Rapid eye movement sleep (dreaming) shown necessary for memory formation
A study published in the journal Science by researchers at the Douglas Mental Health University Institute at McGill University and the University of Bern provides the first evidence that rapid eye movement (REM) sleep — the phase where dreams appear — is directly involved in memory formation (at least in mice). “We already knew that … more…

May 16, 2016

Inhibition of  media septum GABA neurons during rapid eye movement (REM) sleep reduces theta rhythm (a characteristic of REM sleep). Schematic of the in vivo recording configuration: an optic fiber delivered orange laser light to the media septum part of the brain, allowing for optogenetic inhibition of media septum GABA neurons while recording the local field potential signal from electrodes implanted in hippocampus area CA1. (credit: Richard Boyce et al./Science)

A study published in the journal Science by researchers at the Douglas Mental Health University Institute at McGill University and the University of Bern provides the first evidence that rapid eye movement (REM) sleep — the phase where dreams appear — is directly involved in memory formation (at least in mice).

“We already knew that newly acquired information is stored into different types of memories, spatial or emotional, before being consolidated or integrated,” says Sylvain Williams, a researcher and professor of psychiatry at McGill*. “How the brain performs this process has remained unclear until now. We were able to prove for the first time that REM sleep (dreaming) is indeed critical for normal spatial memory formation in mice,” said Williams.

Dream quest

Hundreds of previous studies have tried unsuccessfully to isolate neural activity during REM sleep using traditional experimental methods. In this new study, the researchers instead used optogenetics, which enables scientists to precisely target a population of neurons and control its activity by light.

“We chose to target [GABA neurons in the media septum] that regulate the activity of the hippocampus, a structure that is critical for memory formation during wakefulness and is known as the ‘GPS system’ of the brain,” Williams says.

To test the long-term spatial memory of mice, the scientists trained the rodents to spot a new object placed in a controlled environment where two objects of similar shape and volume stand. Spontaneously, mice spend more time exploring a novel object than a familiar one, showing their use of learning and recall.

Shining orange laser light on media septum (MS) GABA neurons during REM sleep reduces frequency and power (purple section) of neuron signals in dorsal CA1 area of hippocampus (credit: Richard Boyce et al./Science)

When these mice were in REM sleep, however, the researchers used light pulses to turn off their memory-associated neurons to determine if it affects their memory consolidation. The next day, the same rodents did not succeed the spatial memory task learned on the previous day. Compared to the control group, their memory seemed erased, or at least impaired.

“Silencing the same neurons for similar durations outside of REM episodes had no effect on memory. This indicates that neuronal activity specifically during REM sleep is required for normal memory consolidation,” says the study’s lead author, Richard Boyce, a PhD student.

Implications for brain disease

REM sleep is understood to be a critical component of sleep in all mammals, including humans. Poor sleep quality is increasingly associated with the onset of various brain disorders such as Alzheimer’s and Parkinson’s disease.

In particular, REM sleep is often significantly perturbed in Alzheimer’s diseases (AD), and results from this study suggest that disruption of REM sleep may contribute directly to memory impairments observed in AD, the researchers say.

This work was partly funded by the Canadian Institutes of Health Research (CIHR), the Natural Science and Engineering Research Council of Canada (NSERC), a postdoctoral fellowship from Fonds de la recherche en Santé du Québec (FRSQ) and an Alexander Graham Bell Canada Graduate scholarship (NSERC).

* Williams’ team is also part of the CIUSSS de l’Ouest-de-l’Île-de-Montréal research network. Williams co-authored the study with Antoine Adamantidis, a researcher at the University of Bern’s Department of Clinical Research and at the Sleep Wake Epilepsy Center of the Bern University Hospital.

Abstract of Causal evidence for the role of REM sleep theta rhythm in contextual memory consolidation

Rapid eye movement sleep (REMS) has been linked with spatial and emotional memory consolidation. However, establishing direct causality between neural activity during REMS and memory consolidation has proven difficult because of the transient nature of REMS and significant caveats associated with REMS deprivation techniques. In mice, we optogenetically silenced medial septum γ-aminobutyric acid–releasing (MSGABA) neurons, allowing for temporally precise attenuation of the memory-associated theta rhythm during REMS without disturbing sleeping behavior. REMS-specific optogenetic silencing of MSGABA neurons selectively during a REMS critical window after learning erased subsequent novel object place recognition and impaired fear-conditioned contextual memory. Silencing MSGABA neurons for similar durations outside REMS episodes had no effect on memory. These results demonstrate that MSGABA neuronal activity specifically during REMS is required for normal memory consolidation.

 

Quantifying Consciousness

By Tanya Lewis

Overall brain metabolic rate can distinguish between pathological states of human consciousness, a study shows.

 


Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics
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Abedini A, Plesner A, Cao P, Ridgway Z, et al.
eLife May 23, 2016; 10.7554/eLife.12977. http://dx.doi.org/10.7554/eLife.12977

Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.

 

NIH study visualizes proteins involved in cancer cell metabolism

Cryo-EM methods can determine structures of small proteins bound to potential drug candidates.

https://www.nih.gov/news-events/news-releases/nih-study-visualizes-proteins-involved-cancer-cell-metabolism

Scientists using a technology called cryo-EM (cryo-electron microscopy) have broken through a technological barrier in visualizing proteins with an approach that may have an impact on drug discovery and development. They were able to capture images of glutamate dehydrogenase, an enzyme found in cells, at a resolution of 1.8 angstroms, a level of detail at which the structure of the central parts of the enzyme could be visualized in atomic detail. The scientists from the National Cancer Institute (NCI), part of the National Institutes of Health, and their colleagues also reported achieving another major milestone, by showing that the shapes of cancer target proteins too small to be considered within the reach of current cryo-EM capabilities can now be determined at high resolution.

The research team was led by NCI’s Sriram Subramaniam, Ph.D., with contributions from scientists at the National Center for Advancing Translational Sciences (NCATS), also part of NIH. The findings appeared online May 26, 2016, in Cell.

“These advances demonstrate a real-life scenario in which drug developers now could potentially use cryo-EM to tweak drugs by actually observing the effects of varying drug structure — much like an explorer mapping the shoreline to find the best place to dock a boat — and alter its activity for a therapeutic effect,” said Doug Lowy, M.D., acting director, NCI.

Both discoveries have the potential to have an impact on drug discovery and development. Cryo-EM imaging enables analysis of structures of target proteins bound to drug candidates without first needing a step to coax the proteins to form ordered arrays. These arrays were needed for the traditional method of structure determination using X-ray crystallography, a powerful technique that has served researchers well for more than a half century. However, not all proteins can be crystallized easily, and those that do crystallize may not display the same shape that is present in their natural environment, either since the protein shape can be modified by crystallization additives or by the contacts that form between neighboring proteins within the crystal lattice.

“It is exciting to be able to use cryo-EM to visualize structures of complexes of potential drug candidates at such a high level of detail.”

Sriram Subramaniam, Ph.D.,National Caner Institute

“It is exciting to be able to use cryo-EM to visualize structures of complexes of potential drug candidates at such a high level of detail,” said Subramaniam. “The fact that we can obtain structures of small cancer target proteins bound to drug candidates without needing to form 3D crystals could revolutionize and accelerate the drug discovery process.”

Two of the small proteins the researchers imaged in this new study, isocitrate dehydrogenase (IDH1) and lactate dehydrogenase (LDH), are active targets for cancer drug development. Mutations in the genes that code for these proteins are common in several types of cancer. Thus, imaging the surfaces of these proteins in detail can help scientists identify molecules that will bind to them and aid in turning the protein activity off.

In publications in the journal Science last year and this year, Subramaniam and his team reported resolutions of 2.2 angstroms and 2.3 angstroms in cryo-EM with larger proteins, including a complex of a cancer target protein with a small molecule inhibitor. Of note, the journal Nature Methods deemed cryo-EM as the “Method of the Year” in January 2016. “Our earlier work showed what was technically possible,” Subramaniam said. “This latest advance is a delivery of that promise for small cancer target proteins.” For more information on cryo-EM, go to http://electron.nci.nih.gov.

 

Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Abedini A, Plesner A, Cao P, Ridgway Z, et al.
eLife May 23, 2016; 10.7554/eLife.12977. http://dx.doi.org/10.7554/eLife.12977

Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.

 

Single domain antibodies (sdAbs) aid in x-ray crystallography of mammalian serotonin 5-HT3 receptor

Serotonin 5-HT3 is part of the cys-loop receptor family, the mechanism of this family is not well understood due to difficulties in obtaining high resolution crystal structures. Serotonin 5-HT3 receptor is an important druggable target in alleviating nausea and vomiting induced by chemotherapy or anesthesia, as well as psychiatric disorders. It’s structure is critical in discovering new drugs to modulate its activity.

Previously, electron microscopy imaging of non-mammalian homologs of Cys-loop receptors provided basic understanding of extracellular ligand binding sites and pore forming domains. Little was known about intracellular domains and the way they interact with cellular scaffolding proteins, as they are absent in non-mammalian homologs. A recent publication in Nature extends our understanding behind the mechanism of serotonin 5-HT3 receptors, by resolving a 3.5A crystal structure.

Mouse 5-HT3 exists as a homopentamer and is difficult to express, purify and crystallize. To overcome this challenge, researchers split the receptor by proteolyzing each subunit into two fragments. In addition, an sdAb chaperone, which acts as an inhibitor locking the channel into a non-conducting conformation, was used to stabilized the pentameric structure, enabling resolution of a 3.5A crystal structure. Most importantly the split receptor displays an intracellular domain that is tightly coupled to the membrane domain, which provides important structural information that will lead to further understanding of the physiological conformation of 5-HT3 and Cys-loop receptors.

Hassaine G. et al. X-ray structure of the mouse serotonin 5-HT3 receptor Nature. Aug 2014. 512(7514):276-281

 

UCLA animal study shows how brain connects memories across time

Wednesday, May 25, 2016

Using a miniature microscope that opens a window into the brain, UCLA neuroscientists have identified in mice how the brain links different memories over time–and this may help develop new drugs in the future for memory-robbing diseases such as Alzheimer’s.

 

FDA approves new antibody drug for treating pediatric neuroblastoma

Pediatric neuroblastoma is a rare and difficult to treat cancer that forms from immature nerve cells. This form of cancer occurs in 1 in 100,000 children, with 650 new cases each year in the United States. Current therapies, which are non-specific, only provide 40-50% long term survival rate to patients suffering from high-risk neuroblastoma, making this form of cancer an area of high medical unmet need.

A new drug, called dinutuxumab was granted priority review and orphan drug designation by the FDA. It is the first drug of its kind to be approved that specifically treats pediatric neuroblastoma. In addition to the approval, the FDA also issued a rare pediatric review priority voucher to the makers of the drug, for future groundbreaking therapies in pediatric neuroblastoma.

Dinutuxumab (formerly called ch14.18) is a disialoganglioside (GD2) binding chimeric monoclonal antibody that works in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and 13-cis-retinoic acid (RA) for treating high-risk pediatric neuroblastoma.

Antibody therapeutics are highly efficacious and specific towards rare and difficult-to-treat cancers and discovery of new antibody therapeutics will help address critical needs. Antibody drug discovery may be challenging, but working with an experienced partner can help.

FDA approves first therapy for high-risk neuroblastoma

 

Electronic Biosensor Detects Molecules Linked to Cancer, Alzheimer’s, and Parkinson’s

5/20/2016  by Fundação de Amparo À Pesquisa Do Estado de São Paulo

A biosensor developed by researchers at the National Nanotechnology Laboratory (LNNano) in Campinas, São Paulo State, Brazil, has been proven capable of detecting molecules associated with neurodegenerative diseases and some types of cancer.

The device is basically a single-layer organic nanometer-scale transistor on a glass slide. It contains the reduced form of the peptide glutathione (GSH), which reacts in a specific way when it comes into contact with the enzyme glutathione S-transferase (GST), linked to Parkinson’s, Alzheimer’s and breast cancer, among other diseases. The GSH-GST reaction is detected by the transistor, which can be used for diagnostic purposes.

An inexpensive portable biosensor has been developed by researchers at Brazil’s National Nanotechnology Laboratory with FAPESP’s support. (Credit: LNNano)

The project focuses on the development of point-of-care devices by researchers in a range of knowledge areas, using functional materials to produce simple sensors and microfluidic systems for rapid diagnosis.

“Platforms like this one can be deployed to diagnose complex diseases quickly, safely and relatively cheaply, using nanometer-scale systems to identify molecules of interest in the material analyzed,” explained Carlos Cesar Bof Bufon, Head of LNNano’s Functional Devices & Systems Lab (DSF) and a member of the research team for the project, whose principal investigator is Lauro Kubota, a professor at the University of Campinas’s Chemistry Institute (IQ-UNICAMP).

In addition to portability and low cost, the advantages of the nanometric biosensor include its sensitivity in detecting molecules, according to Bufon.

“This is the first time organic transistor technology has been used in detecting the pair GSH-GST, which is important in diagnosing degenerative diseases, for example,” he explained. “The device can detect such molecules even when they’re present at very low levels in the examined material, thanks to its nanometric sensitivity.” A nanometer (nm) is one billionth of a meter (10-9 meter), or one millionth of a millimeter.

The system can be adapted to detect other substances, such as molecules linked to different diseases and elements present in contaminated material, among other applications. This requires replacing the molecules in the sensor with others that react with the chemicals targeted by the test, which are known as analytes.

The team is working on paper-based biosensors to lower the cost even further and to improve portability and facilitate fabrication as well as disposal.

The challenge is that paper is an insulator in its usual form. Bufon has developed a technique to make paper conductive and capable of transporting sensing data by impregnating cellulose fibers with polymers that have conductive properties.

The technique is based on in situ synthesis of conductive polymers. For the polymers not to remain trapped on the surface of the paper, they have to be synthesized inside and between the pores of the cellulose fibers. This is done by gas-phase chemical polymerization: a liquid oxidant is infiltrated into the paper, which is then exposed to monomers in the gas phase. A monomer is a molecule of low molecular weight capable of reacting with identical or different molecules of low molecular weight to form a polymer.

The monomers evaporate under the paper and penetrate the pores of the fibers at the submicrometer scale. Inside the pores, they blend with the oxidant and begin the polymerization process right there, impregnating the entire material.

The polymerized paper acquires the conductive properties of the polymers. This conductivity can be adjusted by manipulating the element embedded in the cellulose fibers, depending on the application for which the paper is designed. Thus, the device can be electrically conductive, allowing current to flow without significant losses, or semiconductive, interacting with specific molecules and functioning as a physical, chemical or electrochemical sensor.

 

Protein Oxidation in Aging: Not All Proteins Are Created Equal

Cancer, Alzheimer’s disease and other age-related diseases develop over the course of aging, and certain proteins are shown to play critical roles this process. Those proteins are subject to destabilization as a result of oxidation, which further leads to features of aging cells. It is estimated that almost 50% of proteins are damaged due to oxidation for people at their 80s. The oxidative damage mediated by free radicals occurs when converting food to energy in the presence of oxygen. Cellular structures, such as proteins, DNA, and lipids, are prone to these oxidation damages, which further contribute to the development of age-related diseases.

Using computational models with physics principles incorporated, de Graff el al. from Stony Brook University unfolded the molecular mechanism that how natural chemical process affects the aging of proteins. First, the authors revealed the major factor to explain stability loss in aging cells and organisms is likely to be random modification of the protein sidechains. Furthermore, through the evaluation and analysis on the protein electrostatics, the authors suggested that highly charged proteins are in particular subject to the oxidation induced destabilization. Even one single oxidation could lead to unfold the whole structure for these highly charged proteins. Old cells are enriched in those highly charged proteins, thus the destabilization effects are elevated in the aging cells. In addition, 20 proteins associated with aging are further identified to be at high risk of oxidation. The list includes telomerase proteins and histones, both of which play critical roles in the aging of cells and cancer development. The team is currently working on analyzing more proteins, with the hope to provide key information to aid targeted treatments against age-related diseases.

Further Reading: Emerging Opportunity for Treating Alzheimer Disease by Immunotherapy

Adam M.R. de Graff, Michael J. Hazoglou, Ken A. Dill. Highly Charged Proteins: The Achilles’ Heel of Aging Proteomes.Structure, 24, 285-292 (2016)

Baruch, K. et al. PD-1 Immune Checkpoint Blockade Reduces Pathology and Improves Memory in Mouse Models of Alzheimer’s Disease. Nat. Med. 22, 135-137 (2016)

 

Single domain antibodies shown to cross blood brain barrier and offers enhanced delivery of therapeutics to CNS targets

A major challenge in developing both small molecule and antibody therapeutics for CNS disorders including brain cancer and neurodegenerative diseases, is penetrating the blood brain barrier (BBB). A study published in FASEB demonstrated that monomeric variable heavy-chain domain of camel homodimeric antibodies (mVHH), can cross the BBB in-vivo, and recognize its intracellular target: glial fibrillary acidic protein (GFAP). The ability of mVHH to cross the BBB of normal animals and those undergoing pathological stress makes it a promising modality for treating CNS diseases as well as for brain imaging.

The investigators of this study expressed a recombinant fusion protein, VHH-GFP, which was able to cross the BBB in-vivo and specifically label astrocytes. GenScript is fully engaged in single-domain antibody lead generation and optimization. With our one-stop services, we are determined to be your best partner in antibody drug discovery from gene synthesis to in-vivo characterization of candidate antibodies. All you need to provide is the Genbank accession number of the antigen protein!

Li T. et al. Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging. FASEB. Oct 2012. 26:3969-79

 

New insight behind the success of fighting cancer by targeting immune checkpoint proteins

Immune checkpoint blockade has proven to be highly successful in the clinic at treating aggressive and difficult-to-treat forms of cancer. The mechanism of the blockade, targeting CTLA-4 and PD-1 receptors which act as on/off switches in T cell-mediated tumor rejection, is well understood. However, little is known about the tumor antigen recognition profile of these affected T-cells, once the checkpoint blockade is initiated.

In a recent published study, the authors used genomics and bioinformatics approaches to identify critical epitopes on 3-methylcholanthrene induced sarcoma cell lines, d42m1-T3 and F244. CD8+ T cells in anti-PD-1 treated tumor bearing mice were isolated and fluorescently labeled with tetramers loaded with predicted mutant epitopes. Out of 66 predicted mutants, mLama4 and mAlg8 were among the highest in tetramer-positive infiltrating T-cells. To determine whether targeting these epitopes alone would yield similar results as anti-PD-1 treatment, vaccines against these two epitopes were developed and tested in mice. Prophylactic administration of the combined vaccine against mLama4 and mAlg8 yielded an 88% survival in tumor bearing mice, thus demonstrating that these two epitopes are the major antigenic targets from checkpoint-blockade and therapies against these two targets are similarly efficacious.

In addition to understanding the mechanism, identification of these tumor-specific mutant antigens is the first step in discovering the next wave of cancer immunotherapies via vaccines or antibody therapeutics. Choosing the right antibody platform can speed the discovery of a new therapeutics against these new targets. Single domain antibodies have the advantage of expedited optimization, flexibility of incorporating multiple specificity and functions, superior stability, and low COG over standard antibody approaches.

Gubin MM. et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature. Nov 2014. 515:577-584

 

Anti-PD-1 is poised to be a blockbuster, which other immune-checkpoint targeting drugs are on the horizon?

Clinical studies of anti-immune-checkpoint protein therapeutics have shown not only an improved overall survival, but also a long-term durable response, compared to chemotherapy and genomically-targeted therapy. To expand the success of immune-checkpoint therapeutics into more tumor types and improving efficacy in difficult-to-treat tumors, additional targets involved in checkpoint-blockade need to be explored, as well as testing the synergy between combining approaches.

Currently, CTLA-4 and PD-1/PD-L1 are furthest along in development, and have shown very promising results in metastatic melanoma patients. This is just a fraction of targets involved in the checkpoint-blockade pathway. Several notable targets include:

  • LAG-3 – Furthest along in clinical development with both a fusion protein and antibody approach, antibody apporach being tested in combination with anti-PD-1
  • TIM-3 – Also in clinical development. Pre-clinical studies indicate that it co-expresses with PD-1 on tumor-infiltrating lymphocytes. Combination with anti-PD-improves anti-tumor response
  • VISTA – Antibody targeting VISTA was shown to improve anti-tumor immune response in mice

In addition, there are also co-stimulatory factors that are also being explored as viable therapeutic targets

  • OX40 – Both OX40 and 4-1BB are part of the TNF-receptor superfamily. Phase I data shows acceptable safety profile, and evidence of anti-tumor response in some patients
  • 4-1BB – Phase I/II data on an antibody therapeutic targeting OX40 shows promising clinical response for melanoma, renal cell carcinoma and ovarian cancer.
  • Inducible co-stimulator (ICOS) – Member of the CD28/B7 family. Its expression was found to increase upon T-cell activation. Anti-CTLA-4 therapy increases ICOS-positive effector T-cells, indicating that it may work in synergy with anti-CTLA-4. Clinical trials of anti-ICOS antibody are planned for 2015.

Sharma P and Allison JP. Immune Checkpoint Targeting in Cancer Therapy: Toward Combination Strategies with Curative Potential. Cell. April 2015;161:205-214

 

CTLA-4 found in dendritic cells suggests New cancer treatment possibilities

Both dendritic cells and T cells are important in triggering the immune response, whereas antigen presenting dendritic cells act as the “general” leading T cells “soldiers” to chase and eliminate enemies in the battle against cancer. The well-known immune checkpoint break, CTLA-4, is believed to be present only in T cells (and cells of the same lineage). However, a new study published in Stem Cells and Development suggests that CTLA-4 also presents in dendritic cells. It further explores the mechanism on how turning off the dendritic cells in the immune response against tumors.

Matthew Halpert, et al. Dendritic Cell Secreted CTLA-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7. Stem Cells and Development, 2016; DOI: 10.1089/scd.2016.0009

 

With a wide range of animal models to choose from, what are the crucial factors to consider?

A recent perspective published in Nature Medicine addresses these gaps by comparing the strengths and limitations of different tumor models, as well as best models to use for answering different biological questions and best practices for preclinical modeling.

Below is a summary of the authors’ key considerations:

  • It is important to choose a model based on the biology of the target. Several diverse tumor models may be required to address complex biology
  • If the biology of the target includes signaling between the tumor and the stroma, then it is crucial to understand drug efficacy in the presence of an appropriate tumor microenvironment with orthotopic models
  • Avoid overuse of models that are highly sensitive to the drug, unless there is clinically relevant biomarker data to support the findings
  • For studying agents that reduce pre-existing tumors, make sure that the tumors are established in the model prior to treatment
  • Understanding the pharmacokinetics of a drug in the model prior to studies is important to ensure that the dosing is within range, and that off-target and toxic side effects are not skewing anti-tumor activity.

Gould SE, Junttila MR and de Sauvage FJ. Translational value of mouse models in oncology drug development. Nat Med. May 2015. 21(5):431-439


Revolutionary Impact of Nanodrug Delivery on Neuroscience

Reza Khanbabaie1,2,3 and Mohsen Jahanshahi
Curr Neuropharmacol. 2012 Dec; 10(4): 370–392.   doi:  10.2174/157015912804143513

Brain research is the most expanding interdisciplinary research that is using the state of the art techniques to overcome limitations in order to conduct more accurate and effective experiments. Drug delivery to the target site in the central nervous system (CNS) is one of the most difficult steps in neuroscience researches and therapies. Taking advantage of the nanoscale structure of neural cells (both neurons and glia); nanodrug delivery (second generation of biotechnological products) has a potential revolutionary impact into the basic understanding, visualization and therapeutic applications of neuroscience. Current review article firstly provides an overview of preparation and characterization, purification and separation, loading and delivering of nanodrugs. Different types of nanoparticle bioproducts and a number of methods for their fabrication and delivery systems including (carbon) nanotubes are explained. In the second part, neuroscience and nervous system drugs are deeply investigated. Different mechanisms in which nanoparticles enhance the uptake and clearance of molecules form cerebrospinal fluid (CSF) are discussed. The focus is on nanodrugs that are being used or have potential to improve neural researches, diagnosis and therapy of neurodegenerative disorders.

Keywords: Nanodrug, Nanofabrication and purification, Neuroscience, Nervous system, Nano-nervous drugs.

1. INTRODUCTION

The delivery of drugs to the nervous system is mainly limited by the presence of two anatomical and biochemical dynamic barriers: the blood–brain barrier (BBB) and blood–cerebrospinal fluid barrier (BCSFB) separating the blood from the cerebral parenchyma [1]. These barriers tightly seal the central nervous system (CNS) from the changeable milieu of blood. With the advancement of electron microscopy it is found that the ultrastructural localization of the blood–brain barrier is correlated with the capillary endothelial cells within the brain [2]. The BBB inhibits the free paracellular diffusion of water-soluble molecules by an elaborate network of complex tight junctions (TJs) that interconnects the endothelial cells. Similar to the endothelial barrier, the morphological correlate of the BCSFB is found at the level of unique apical tight junctions between the choroid plexus epithelial cells inhibiting paracellular diffusion of water-soluble molecules across this barrier [1, 3]. Beside its barrier function, it allows the directed transport of ions and nutrients into the cerebrospinal fluid (CSF) and removal of toxic agents out of the CSF using numerous transport systems.

One of the most challenging steps in neuroscience researches and therapy is the availability of techniques to penetrate these permeability barriers and delivering drugs to the CNS. Several strategies have been used to circumvent the barriers inhibiting CNS penetration. These strategies generally fall into one or more of the following three categories: manipulating drugs, disrupting the BBB (BBBD) and finding alternative routes for drug delivery. Drug manipulation methods include: Lipophilic Analogs, prodrugs, chemical drug delivery systems (CDDS), Carrier-mediated transport (CMT) and Receptor-mediated drug delivery. The drug manipulating strategy has been frequently employed, but the results have often been disappointing [46]. All of these methods have major limitations: they are invasive procedures, have toxic side effects and low efficiency, and are not sufficiently safe [7]. Two methods for disrupting the BBB have been reported: osmotic blood-brain barrier disruption and biochemical blood-brain barrier disruption. However, these procedures also break down the self-defense mechanism of the brain and make it vulnerable to damage or infection from all circulating chemicals or toxins. Since the above techniques aim to enhance the penetration of drugs to the CNS via circulatory system, they will increase the penetration of drugs throughout the entire body. This will frequently result in unwanted systemic side effects. In the other hand, systemically administered agents must penetrate the BBB to enter the CNS, which is a difficult task. Despite advances in rational CNS drug design and BBBD, many potentially efficacious drug molecules still cannot penetrate into the brain parenchyma at therapeutic concentrations. The alternative strategy to enhance CNS penetration of drug molecules is based on methodology that does not rely on the cardiovascular system. These strategies circumvent the BBB altogether and do not need drug manipulation to enhance BBB permeability and/or BBBD. Using alternative routes to deliver drugs to the CNS, e.g. intraventricular/intrathecal route and olfactory pathway, is one of these strategies.

One strategy for bypassing the BBB that has been studied extensively both in laboratory and in clinical trials is the intralumbar injection or intreventricular infusion of drugs directly into the CSF. Compared to vascular drug delivery, intra-CSF drug administration theoretically has several advantages. Intra-CSF administration bypasses the BCB and results in immediate high CSF drug concentrations. Due to the fact that the drug is somewhat contained within the CNS, a smaller dose can be used, potentially minimizing systemic toxicity. Furthermore, drugs in the CSF encounter minimize protein binding and decrease enzymatic activity relative to drugs in plasma, leading to longer drug half-life in the CSF. Finally, since the CSF freely exchanges molecules with the extracellular fluid of the brain parenchyma, delivering drugs into the CSF could theoretically result in therapeutic CNS drug concentrations [7, 8]. However, for several reasons this delivery was not as successful as predicted. These include a slow rate of drug distribution within the CSF and increase in intracranial pressure associated with fluid injection or infusion into small ventricular volumes.

Another CNS drug delivery route is the intranasal route. In this method drugs are transported intranasally along olfactory sensory neurons to yield significant concentrations in the CSF and olfactory bulb. An obvious advantage of this method is that it is noninvasive relative to other strategies. This method has received relatively little attention, since there are difficulties that have to be overcome. Among these obstacles is an enzymatically active, low pH nasal epithelium, the possibility of mucosal irritation or the possibility of large variability caused by nasal pathology, such as common cold.

Based on the advantages and disadvantages of aforementioned strategies, researchers are still looking for novel and better methods of CNS drug deliveries. The most direct way of circumventing the BBB is to deliver drugs directly to the brain interstitium which mainly includes the use of small colloidal particles like liposomes and nanoparticles [8]. By directing agents uniquely to an intracranial target, interstitial drug delivery can theoretically yield high CNS drug concentrations with minimal systemic exposure and toxicity. Furthermore, with this strategy, intracranial drug concentrations can be sustained, which is crucial in treatment with many chemotherapeutic agents. The basic reason of common acceptance of these carriers is due to their controlled profile or drug release nature as well as due to their selected targeting mechanism. Targeting action maybe due to the steric hindrance created by nano-vectors for achieving targeting ability. These carriers are usually administered through parenteral route and due to their steric phenomenon they conceal themselves from opsonisation event induced by tissue macrophages. By this way they achieve targeting ability to brain and other reticuloendothelial system (RES) organs like liver, spleen, etc.

Several approaches have been developed for delivering drugs directly to the brain interstitium like injections, catheters, and pumps. One such methodology is the Ommaya reservoir or implantable pump which achieves truly continuous drug delivery. Though interstitial drug delivery to the CNS has had only modest clinical impact, its therapeutic potential may soon be realized using new advances in polymer technologies to modify the aforementioned techniques. Polymeric or lipidbased devices that can deliver drug molecules at defined rates for specific periods of time are now making a tremendous impact in clinical medicine.

Among the strategies of direct drug delivery to the CNS, nanoparticles have attracted considerable interest from the last few decades. It has been shown that nano delivery systems have great potential to facilitate the movement of drugs across barriers (e.g., BBB). Nanosystems employed for the development of nano drug delivery systems in the treatment of CNS disorders include polymeric nanoparticles, nanospheres, nanosuspensions, nanoemulsions, nanogels, nano-micelles and nano-liposomes, carbon nanotubes, nanofibers and nanorobots, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and lipid drug conjugates (LDC). Although the exact mechanism of barrier opening by nanoparticles is not known, the novel properties such as tiny size, tailored surface, better solubility, and multi-functionality of nanoparticles present the capability to interact with composite cellular functions in new ways. In fact, nanotechnology has now emerged as an area of research for invention of newer approaches for the CNS drug delivery and a revolutionary method to improve diagnosis and therapy of neurodegenerative disorders.

In this line, an overview of preparation and characterization, purification and separation, loading and delivering of nanodrugs is the first subject of this review. Different types of nanoparticle bioproducts including carbon nanotubes as a drug delivery system and also as a novel tool in neuroscience research are explored. For instance, nanodrug delivery systems like human serum albumin (HSA) nanoparticles, bovine serum albumin (BSA)-Gum Arabic (Acacia) nanoparticles and α-lactalbumin nanoparticles are explained.

The impact of nanotechnology on neuroscience and drug delivery to the central nervous system (CNS) is the subject of the second part of this review. Different mechanisms in which nanoparticles enhance the uptake of molecules both hydrophilic and hydrophobic across the BBB and the impact of various physiochemical parameters of nanoparticles on its uptake and clearance form CSF are discussed. Also nanodrugs that are being used or have potential to improve neural researches, diagnosis and therapy of neurodegenerative disorders are investigated.

2. FROM NANOTECHNOLOGY TO NEUROPHARMACOLOGY

Nanotechnology started by the suggestion of a famous physicist, Richard Feynman, that it should be possible, in principle, to make nanoscale machines that “arrange the atoms the way we want”, and do chemical synthesis by mechanical manipulation [9, 10]. Nanotechnologies exploit materials and devices with a functional organization that has been engineered at the nanometer scale. In a broad sense, they can be defined as the science and engineering involved in the design, syntheses, characterization, and application of materials and devices whose smallest functional organization in at least one dimension is on the nanometer scale, ranging from a few to several hundred nanometers. A nanometer is roughly the size of a molecule itself (e.g., a DNA molecule is about 2.5 nm long while a sodium atom is about 0.2 nm) [10]. Nanotechnology is not in itself a single emerging scientific discipline but rather a meeting of traditional sciences such as chemistry, physics, materials science, and biology to bring together the required collective expertise needed to develop these novel technologies.

The application of nanotechnology in cell biology and physiology enables targeted interactions at a fundamental molecular level. Nanotechnology, in the context of nanomedicine, can be defined as the technologies for making nanocarriers of therapeutics and imaging agents, nanoelectronic biosensors, nanodevices, and microdevices with nanostructures. It also covers possible future applications of molecular nanotechnology (MNT) and nanovaccinology. Unlike the definition in core nanotechnology field, which restricts the “nano” to at least 1–100 nm in one dimension, nanocarriers in the biomedical field are often referred to as particles with a dimension a few nanometers to 1000 nm [8, 11]. Although, the initial properties of nanomaterials studied were for its physical, mechanical, electrical, magnetic, chemical and biological applications, recently, attention has been geared towards its pharmaceutical application, especially in the area of drug delivery [8]. There are a few challenges in use of large size materials in drug deliveries. Some of these challenges are poor bioavailability, in vivo stability, solubility, intestinal absorption, sustained and targeted delivery to site of action, therapeutic effectiveness, generalized side effects, and plasma fluctuations of drugs (see Table 11).

The most important innovations are taking place in nanopharmocology and drug delivery which involves developing nanoscale particles or molecules to improve bioavailability. These pharmacological applications of nanotechnology include: the formation of novel nanoscopic entities [11, 27], exploring and matching specific compounds to particular patients for maximum effectiveness; and advanced pharmaceutical delivery systems and discovery of new pharmacological molecular entities; selection of pharmaceuticals for specific individuals to maximize effectiveness and minimize side effects, and delivery of pharmaceuticals to targeted locations or tissues within the body. Examples of nanomaterials include nanotubes and nanofibers, liposomes, nanoparticles, polymeric micelles, block ionomer complexes, nanogels, and dendrimers.

Nanotubes [28, 29] and nanofibers mimic tubular structures that appear in nature, such as rod shaped bacteria or viruses, microtubules, ion channels, as well as axons and dendrites. They are low-dimensional nanostructures, having a very large axial ratio. Properties of a molecule in a nanotube or nanofiber structure can be different from those in the bulk or in other nanomaterials, such as spherical nanoparticles. These materials have a large surface–volume ratio, which results in a high exposure of the material components to the surrounding environment [30]. This makes nanotubes and nanofibers promising structures for biosensing and molecular recognition [31]. However, it provides a way to control drug release through the nanotubes wall, while the large hollow area inside nanotubes provides an excellent storage for drugs and other agents [32]. Furthermore, nanotubes can be synthesized to be open-ended, which can be exploited for certain biological applications.

Carbon nanotubes (CNTs) was discovered by Iijima [33] which are composed of carbon atoms arranged in hexagonal ring structures similar to graphite, with some five-membered or seven-membered rings providing the structure curvature [29, 34,35]. CNTs are compatible with biological tissues for scaffolding purposes and the charge carried by the nanotubes can be manipulated to control neurite outgrowth [36, 37]. It has also been suggested that CNTs functionalized with growth factors, such as nerve growth factor or brain-derived neurotrophic factor, can stimulate growth of neurons on the nanotube scaffold [3840]. In such application the toxicity of CNTs remains an issue that must be overcome [41, 42]. It has been reported that conductive polymer coatings for living neural cells has been generated using poly (3,4-ethylenedioxythiophene) PEDOT nanotubes [43]. The electric conductivity of PEDOT was used to enhance the electrical activity of the tissue with a long range aim of treating CNS disorders, which show sensory and motor impairments. These observations suggested that nanotube and nanofiber scaffolds have potential for neuroregeneration as well as treatment of CNS trauma [27, 44]. Nanomaterials suggest a promising strategy for neuroprotection [45]. Neuroprotection is an effect that may result in salvage, recovery, or regeneration of the nervous system.

The role of nanotechnology in targeted drug delivery and imaging was discussed in many reviews and papers [46, 47]. As a step towards a realistic system, a brief overview of preparation, characterization, delivery, loading, purification and separation of nanoparticles and nanodrugs are presented herein. In next two sections the fabrication methods of nanoparticle bioproducts and also the delivery systems of nanodrugs are explained. Subsequently we go back to the CNS nanodrugs for research and therapy and the delivery systems of nanodrugs for nervous system.

……

3. NANODRUG DELIVERY SYSTEMS

The major goals in designing nanoparticles as a delivery system are to control particle size, surface properties [85] and release of pharmacologically active agents in order to achieve the site-specific action of the drug at the therapeutically optimal rate and dose regimen [86]. If nanoparticles are considered to be used as drug delivery vehicles, it depends on many factors including: (a) size of nanoparticles required; (b) inherent properties of the drug, e.g., aqueous solubility; (c) surface characteristics such as charge and permeability; (d) degree of biodegradability, biocompatibility and toxicity; (e) drug release profile desired; and (f) antigenicity of the final product. The advantages of using nanoparticles as a drug delivery system might be summarized as follow [87]:

  1. Particle size and surface characteristics of nanoparticles can be easily manipulated to achieve both passive and active drug targeting after parenteral administration.
  2. They control and sustain release of the drug during the transportation and at the site of localization, altering organ distribution of the drug and subsequent clearance of the drug so as to achieve increase in drug therapeutic efficacy and reduction in side effects.
  3. Controlled release and particle degradation characteristics can be readily modulated by the choice of matrix constituents. Drug loading is relatively high and drugs can be incorporated into the systems without any chemical reaction; this is an important factor for preserving the drug activity.
  4. Site-specific targeting can be achieved by attaching targeting ligands to surface of particles or use of magnetic guidance.
  5. The system can be used for various routes of administration including oral, nasal, parenteral, intraocular etc.

NANODRUG DELIVERY SYSTEMS

The major goals in designing nanoparticles as a delivery system are to control particle size, surface properties [85] and release of pharmacologically active agents in order to achieve the site-specific action of the drug at the therapeutically optimal rate and dose regimen [86]. If nanoparticles are considered to be used as drug delivery vehicles, it depends on many factors including: (a) size of nanoparticles required; (b) inherent properties of the drug, e.g., aqueous solubility; (c) surface characteristics such as charge and permeability; (d) degree of biodegradability, biocompatibility and toxicity; (e) drug release profile desired; and (f) antigenicity of the final product. The advantages of using nanoparticles as a drug delivery system might be summarized as follow [87]:

  1. Particle size and surface characteristics of nanoparticles can be easily manipulated to achieve both passive and active drug targeting after parenteral administration.
  2. They control and sustain release of the drug during the transportation and at the site of localization, altering organ distribution of the drug and subsequent clearance of the drug so as to achieve increase in drug therapeutic efficacy and reduction in side effects.
  3. Controlled release and particle degradation characteristics can be readily modulated by the choice of matrix constituents. Drug loading is relatively high and drugs can be incorporated into the systems without any chemical reaction; this is an important factor for preserving the drug activity.
  4. Site-specific targeting can be achieved by attaching targeting ligands to surface of particles or use of magnetic guidance.
  5. The system can be used for various routes of administration including oral, nasal, parenteral, intraocular etc.

NERVOUS SYSTEM NANODRUGS

Nanomaterials and nanoparticles can interact with biological systems at fundamental and molecular levels [100, 101]. In neuroscience, the application of nanotechnologies entails specific interactions with neurons and glial cells. Nanodevices can target the cells and glia with a high degree of specificity. This unique molecular specificity enables the nanodrugs to stimulate and interact with tissues and neurons in controlled ways, while minimizing undesirable effects. There are two main types of nervous system drugs (neurodrugs): behavioural and molecular. Behavioural neurodrugs are for the study of how different drugs affect human behaviour and human brain. These drugs are usually used for diagnosis and therapy of neurodegeneration disorders [47, 102]. Molecular neurodrugs are used for the study of neurons and their neurochemical interactions. Since for the most part, neurons in the human brain communicate with one another by releasing chemical messengers called neurotransmitters, these drugs have to target specific transmitters and receptors to have beneficial effect on neurological functions. The preparation of these two types of drugs is closely connected. Researchers are studying the interactions of different neurotransmitters [103], neurohormones [104], neuromodulators [105], enzymes [106], second messengers [107], co-transporters [108, 109], ion channels [110], and receptor proteins [111] in the central and peripheral nervous systems to develop drugs to treat many different neurological disorders, including pain [112], neurodegenerative diseases such as Parkinson’s disease [113] and Alzheimer’s disease [114], psychological disorders [115], addiction [116], and many others.

The blood–brain barrier significantly hinders the passage of systemically delivered therapeutics and the brain extracellular matrix limits the distribution and longevity of locally delivered agents. Nanoparticles represent a promising solution to these problems. They can cross blood-brain barrier and enter the CNS. Although the applications of nanotechnology in basic and clinical neuroscience are only in the early stages of development, partly because of the complexities associated with interacting with neural cells and the mammalian nervous system, however the early results show an impressive potential of nanotechnologies to contribute to neuroscience research [117]. One area in which nanotechnology may have a significant clinical impact in neuroscience is the selective transport and delivery of drugs and other small molecules across the blood brain barrier that cannot cross otherwise.

Examples of current research include technologies that are designed to better interact with neural cells, advanced molecular imaging technologies [118, 119], materials and hybrid molecules used in neural regeneration [120], neuroprotection [121], and targeted delivery of drugs and small molecules across the blood–brain barrier [122, 123]. Among all these modern methods of drug delivery to the central nervous system (CNS), the design and application of bionanotechnologies aimed at the CNS provide revolutionary new approaches for studying cell and molecular biology and physiology. The successful and meaningful development of bionanotechnologies designed to interact with the CNS as research or clinical tools require an understanding of the relevant neurophysiology and neuropathology, an appreciation of the inherent ‘nanoscale’ structure of the CNS, and an understanding of the relevant chemistry and materials science and engineering. At nanoscale, consideration of individual molecules and interacting groups of molecules in relation to the bulk macroscopic properties of the material or device becomes important, since it is control over the fundamental molecular structure that allows control over the macroscopic chemical and physical properties [124]. Applications to neuroscience and physiology imply materials and devices designed to interact with the body at subcellular (i.e., molecular) scales with a high degree of specificity. This can potentially translate into targeted cellular and tissue-specific clinical applications designed to achieve maximal therapeutic affects with minimal side effects.

It started with controlled release strategy and the development of miniaturized delivery systems [125] and continued by the application of albumin nanoparticles for the first time in the Johns Hopkins Medical Institution in Baltimore [126]. Other nanoconstructs such as drug-polymer conjugates were first proposed in the 1970s [127] and developed pre-clinically in the 1980s [128]. Prof. Kreuter [129] proposed a definition of polymeric nanoparticles for pharmaceutical purposes for the first time that later was adopted by the Encyclopaedia of Pharmaceutical Technology [130] and the Encyclopedia of nanotechnology [131]. Today, more than 25 nanomedicines have already been approved for human use [102]. Usually the application of nanodrugs to neuroscience is divided into two parts: application in basic neuroscience [124], and in clinical neuroscience [27].

The development of nanotechnology products may play an important role in adding a new group of therapeutics to the products of pharmaceutical companies [132]. Nanotechnology enhances (1) delivery of poorly water-soluble drugs; (2) delivery of large macromolecule drugs to intracellular sites of action; (3) targeted delivery of drugs in a cell- or tissue-specific manner; (4) transcytosis of drugs across tight epithelial and endothelial barriers; (5) co-delivery of two or more drugs or therapeutic modality for combination therapy; (6) visualization of sites of drug delivery by combining therapeutic agents with imaging modalities; and (7) real-time read on the in vivo efficacy of a therapeutic agent [133]. Additionally, the manufacturing complexity of nanotechnology therapeutics may also create a significant hurdle for generic drug companies to develop equivalent therapeutics readily [132].

…….

Safe, site-specific, and efficient delivery of compounds to CNS disease sites remains a singular goal in achieving optimal therapeutic outcomes to combat neurodegenerative diseases. Treatment of CNS disorders by systemic administration or local delivery of drugs is currently inefficient in many cases. Furthermore, clinical neuroscience faces great challenges due to the extremely heterogeneous cellular and molecular environment and the complexities of the brain’s anatomical and functional “wiring” and associated information processing [224]. However, the emergence of nanotechnology provides hope that it will revolutionize diagnosis and treatment of CNS disorders. Neurodegenerative diseases are usually linked to a loss of brain and spinal cord cells. For example, the neuronal damage in AD and PD is associated with abnormal protein processing and accumulation and results in gradual cognitive and motor deterioration [225].

 

 

 

 

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Insight into Blood Brain Barrier

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Gateway to The Brain

This image shows the structural model of critical transporter, Mfsd2a. Source: Duke-NUS Medical School
This image shows the structural model of critical transporter, Mfsd2a. Source: Duke-NUS Medical School.  http://www.dddmag.com/sites/dddmag.com/files/rd1604_brain.jpg

Scientists from Duke-NUS Medical School (Duke-NUS) have derived a structural model of a transporter at the blood-brain barrier called Mfsd2a. This is the first molecular model of this critical transporter, and could prove important for the development of therapeutic agents that need to be delivered to the brain — across the blood-brain barrier. In future, this could help treat neurological disorders such as glioblastoma.

Currently, there are limitations to drug delivery to the brain as it is tightly protected by the blood-brain barrier. The blood-brain barrier is a protective barrier that separates the circulating blood from the central nervous system which can prevent the entry of certain toxins and drugs to the brain. This restricts the treatment of many brain diseases. However, as a transporter at the blood-brain barrier, Mfsd2a is a potential conduit for drug delivery directly to the brain, thus bypassing the barrier.

In this study, recently published in the Journal of Biological Chemistry, first author Duke-NUS MD/PhD student Debra Quek and senior author Professor David Silver used molecular modeling and biochemical analyses of altered Mfsd2a transporters to derive a structural model of human Mfsd2a. Importantly, the work identifies new binding features of the transporter, providing insight into the transport mechanism of Mfsd2a.

“Our study provides the first glimpse into what Mfsd2a looks like and how it might transport essential lipids across the blood-brain barrier,” said Ms Quek. “It also facilitates a structure-guided search and design of scaffolds for drug delivery to the brain via Mfsd2a, or of drugs that can be directly transported by Mfsd2a.”

Currently this information is being used by Duke-NUS researchers to design novel therapeutic agents for direct drug delivery across the blood brain barrier for the treatment of neurological diseases. This initiative by the Centre for Technology and Development (CTeD) at Duke-NUS, is one of many collaborative research efforts aimed at translating Duke-NUS’ research findings into tangible commercial and therapeutic applications for patients.

Ms Quek plans to further validate her findings by purifying the Mfsd2a protein in order to further dissect how it functions as a transporter.

 

J Biol Chem. 2016 Mar 4. pii: jbc.M116.721035. [Epub ahead of print]
Structural insights into the transport mechanism of the human sodium-dependent lysophosphatidylcholine transporter Mfsd2a.

Major Facilitator Superfamily Domain containing 2A (Mfsd2a) was recently characterized as a sodium-dependent lysophosphatidylcholine (LPC) transporter expressed at the blood-brain barrier endothelium. It is the primary route for importation of docosohexaenoic acid and other long-chain fatty acids into foetal and adult brain, and is essential for mouse and human brain growth and function. Remarkably, Mfsd2a is the first identified MFS family member that uniquely transports lipids, implying that Mfsd2a harbours unique structural features and transport mechanism. Here, we present three 3D structural models of human Mfsd2a derived by homology modelling using MelB- and LacY-based crystal structures, and refined by biochemical analysis. All models revealed 12 transmembrane helices and connecting loops, and represented the partially outward-open, outward-partially occluded, and inward-open states of the transport cycle. In addition to a conserved sodium-binding site, three unique structural features were identified: A phosphate headgroup binding site, a hydrophobic cleft to accommodate a hydrophobic hydrocarbon tail, and three sets of ionic locks that stabilize the outward-open conformation. Ligand docking studies and biochemical assays identified Lys436 as a key residue for transport. It is seen forming a salt bridge with the negative charge on the phosphate headgroup. Importantly, Mfsd2a transported structurally related acylcarnitines but not a lysolipid without a negative charge, demonstrating the necessity of a negative charged headgroup interaction with Lys436 for transport. These findings support a novel transport mechanism by which LPCs are flipped within the transporter cavity by pivoting about Lys436 leading to net transport from the outer to the inner leaflet of the plasma membrane.

 

Brain and eye contain membrane phospholipids that are enriched in the omega-3 fatty acid docosohexaenoic acid (DHA). It is widely accepted that DHA is important for brain and eye function and brain development (1,2), although mechanisms for DHA function in these tissues are not well defined.   The mechanism by which DHA and other conditionally essential and essential fatty acids cross the blood-brain barrier (BBB) has been a long-standing mystery. Recently, we identified Major Facilitator Superfamily Domain containing 2a (Mfsd2a, aka NLS1) as the primary transporter by which the brain obtains DHA. Importantly, Mfsd2a does not transport unesterified DHA, but transports DHA in the chemical form of lysophosphatidylcholine (LPC) that are synthesized by the liver and circulate largely on albumin (3). This is consistent with biochemical evidence that the brain does not transport unesterified fatty acids (4) and that LPC is the preferred carrier of DHA to the brain (5,6).   Mfsd2a is a sodium-dependent transporter that is part of the Major Facilitator Superfamily (MFS) of proteins. Members of this family with elucidated structures have 12 transmembrane domains composed of two evolutionarily duplicated 6 transmembrane units (7). Transporting an LPC is a unique feature of Mfsd2a, since most members of this family transport water-soluble and minimally polar substrates such as sugars (GLUT, MelB, LacY), and amino acids (TAT1). Mfsd2a transport is not limited to LPCs containing DHA, as it can transport LPCs containing a variety of fatty acyl chains, with higher specificity for LPCs with unsaturated fatty acyl chains with a minimum chain length of 14 carbons (6,8). Crystal structures have been solved for more than a dozen members of the MFS family, with more than 19 structures, including that of Melibiose permease (MelB) of S. typhimurium (9), Lactose permease (LacY) of Escherichia coli (10), glycerol-3-phosphate transporter of E. coli (11) and the mammalian glucose transporters 1, 3, and 5 (GLUT1, GLUT3, GLUT5) (12-14). A common transport mechanism has emerged from both biochemical and structural analyses of MFSs, in which they transport via a rocker-switch, alternating access mechanism (7,15). In the rocker-switch model, rigid-body relative motion of the N- and C-termini domains renders the substrate-binding site alternatively accessible from either side of the membrane.

Mfsd2a is highly expressed at the bloodbrain barrier in both mouse and human (6,16). Mfsd2a deficient mice (KO) have significantly reduced brain DHA as a result of a 90% reduction in brain uptake of LPC containing DHA as well as other LPCs. The most prominent phenotype of Mfsd2a KO mice is microcephaly, and KO mice additionally exhibit motor dysfunction, and behavioral disorders including anxiety and memory and learning deficits (6). In line with the mouse KO phenotypes, human patients with partially or completely inactivating mutations in Mfsd2a presented with severe microcephaly, intellectual disability, and motor dysfunction (8,16). Plasma LPCs are significantly elevated in both KO mice and human patients with Mfsd2a mutations, consistent with reduced uptake at the blood-brain barrier. Taken together, these findings demonstrate that LPCs are essential for normal brain development and function in mouse and humans.

The fact that Mfsd2a transports a lysolipid, a non-canonical substrate for an MFS protein, might indicate unique structure features and a novel transport mechanism. However, no structural information or mechanism of transport of Mfsd2a is known. Human Mfsd2a is composed of 530 amino acids, with two glycosylation sites at Asn217 and Asn227. Mfsd2a is evolutionarily conserved from teleost fish to humans. Although not a functional ortholog of bacterial MFS transporters, Mfsd2a shares 25% and 26% amino acid sequence identity with S. typhimurium MelB (9,17), and LacY from E. coli (10), respectively. Given the high conservation of the MFS fold, the use of homology modeling to gain insight into the structure of S. typhimurium MelB, for example, has proven to be highly accurate and largely consistent with subsequent X-ray crystal data (9,18). Here, we take advantage of two recently derived high resolution X-ray crystal structures of S. typhimurium MelB (9), and a high resolution X-ray crystal structure of LacY (10) to generate three predictive structural models of human Mfsd2a. These models reveal three unique regions critical for function – an LPC headgroup binding site, a hydrophobic cleft occupied by the LPC fatty acyl tail, and three sets of ionic locks. These structural features indicate a novel mechanism of transport for LPCs.

Mfsd2a is a sodium-dependent lysophosphatidylcholine transporter essential for human brain growth and function (40). Mfsd2a is the only known MFS member or secondary transporter that transports a lipid. In line with its unique function, the current study has identified three unique structural features based on a combination of homology structural modeling and biochemical analysis – (1) a unique headgroup binding site and (2) a hydrophobic cleft for acyl chain binding, and (4) 3 sets of ionic locks that stabilize the outward open conformation. Drawing together these findings with studies of the mechanism of transport of other MFS family members, we propose the following alternatingaccess mechanism for LPC transport (Fig. 6). In the first steps, LPC inserts itself into the outer leaflet of the membrane and diffuses laterally into the transporter’s hydrophobic cleft. As Mfsd2a undergoes conformational changes from the outward open to the inward open conformation, the zwitterionic headgroup is inverted from the outer membrane leaflet to the inner membrane leaflet along a translocation pathway within the transporter, interacting with specific polar and charged residues lining the path. Since LPCs are hydrophobic phospholipids, it is unlikely that they will partition out of the transporter into the aqueous environment of the cytoplasm. We propose that the “flipped” LPC exits the transporter laterally into the membrane environment of the inner leaflet. This model of LPC flipping requires further biochemical proof. Of particular interest is the visualization of the interaction of the negatively charged phosphate headgroup of LPC with Lys436 that is maintained in both outward and inward open conformations. The sidechain of Lys436 is seen to be pointing in the upward direction in the outward open conformation, but pointing downward into the translocation cleft in the inward open conformation. These findings suggest that the Lys436 acts as a tether to push or pivot the headgroup down into the translocation cavity while the N- and C-termini of Mfsd2a rock and switch from outward to inward open.

Interestingly, Lys436 is orthologous to the residue Lys377 in the melibiose transporter of S. typhimurium. Based on the S. typhimurium MelB crystal structure, Lys377 has been predicted to be involved in binding melibiose, and in forming a hydrogen bond with Tyr120, likely separating the sodium binding site from the central hydrophilic cavity (9). In a recent molecular dynamic simulation of E. coli MelB, Lys377 was noted to interact differently with residues involved in the sodium binding site (Asp55, Asp59, and Asp124) in the presence or absence of a sodium ion, and thought to be critical for the spatial organization of the sodium binding site (41). Similarly, in our refined models of Mfsd2a, Lys436 is localized in close proximity to the sodium-binding site residue, Asp93, and the central translocation pathway where it has been identified by docking studies to interact with the charged headgroup of LPC. We hypothesize that Lys436 may shuttle between the two binding sites, communicating and coordinating the occupancy status of the two sites. Interestingly, there is a distinct mobility shift in Mfsd2a bands on SDS-PAGE between wild-type Mfsd2a and the L-3 mutant (R498E, R499E, R500E, K503E, K504E) (Fig. 5I) that is not seen when each of the residues are mutated individually (Fig. S1). These findings are consistent with a conformational change in the L-3 mutant. Given that the L-3 ionic lock is visualized in the outward partially occluded model, we hypothesize that the loss of the L-3 ionic lock results in Mfsd2a being trapped in an energetically more favorable inward open conformation, resulting in the loss of transport function (Fig. 5H).

Patients with the partially inactivating mutation p.(S399L) exhibited significant increases specifically in plasma LPCs having monounsaturated (18:1 – 92%, p=0.004) and polyunsaturated LPCs (18:2, 20:4, 20:3 – 254%, p=0.002; 117%, p=0.007, and 238%, p=0.002), but not in the most abundant LPCs – saturated LPCs (C16:0, C18:0) (8). This is consistent with a greater specificity of Mfsd2a for LPCs with unsaturated fatty acyl chains (6)…A possible explanation for this acyl chain specificity is related to the mobility of the acyl tail in the membrane. It is known that phospholipids with unsaturated fatty acyl chains disrupt the packing of the bilayer, resulting in greater lateral membrane fluidity (42). Therefore, one possible mechanism for LPC specificity is that LPCs with unsaturated fatty acyl chains have greater lateral mobility in the membrane, increasing the Ka for interacting with the transport cleft of Mfsd2a.

Another important structural feature of the physiological ligand, LPC, is a minimum acyl chain length of 14 carbons is required for transport by Mfsd2a. A possible explanation for this requirement is that the hydrocarbon chain must extend beyond the cleft, protruding into the hydrophobic milieu of the phospholipid bilayer core. This interaction of the fatty acyl tail with the acyl chains of the membrane bilayer may provide a hydrophobic force strong enough to pull the molecule through and out of the transporter as the LPC headgroup partitions into the inner leaflet of the membrane. A similar scenario is seen in the Sec translocon where a hydrophobic transmembrane domain of a protein partitions laterally from the Sec61p complex channel into the lipid bilayer (43,44). This proposal that the omega carbon of the fatty acyl chain sticks out of the Mfsd2a pocket is consistent with the observation that Mfsd2a can transport nitrobenzoxadiazole (NBD) or Topfluor when these moieties are attached to the omega carbon of the LPC fatty acyl tail [1].

Other known transmembrane phospholipid transporters include flippases, floppases, and scramblases. Flippases and floppases utilize ATP to drive the uphill transport of aminophospholipids from the outer to the inner leaflet, and specific substrates from the inner to the outer leaflet, respectively (45-47). Scramblases are less well understood, facilitating transport of substrates in either direction down concentration gradients upon activation. While the substrates are similar, several differences make comparisons between Mfsd2a and phospholipid transporters of limited relevance. First, the shapes of the substrates differ in shape and size – lysophospholipids are smaller and conical while phospholipids are cylindrical. Second, unlike flippases and floppases, Mfsd2a is a secondary transporter, utilizing a sodium electrochemical gradient to drive the transport of lysophospholipids from one leaflet to the other. Third, the overall structure of MFS members is different from P4- ATPases and ABC transporters. Consequently, the mechanism of action between Mfsd2a and flippases such as P4-ATPases and ABC transporters, or floppases is expected to differ.

Being expressed at the blood-brain barrier, Mfsd2a is a potential conduit for drug delivery to the brain. The blood-brain barrier is highly impermeable, protecting the brain from bloodderived molecules, pathogens, and toxins. However, its impermeability poses a challenge for pharmacological treatment of brain diseases. It has been predicted that 98% of small molecule drugs are excluded from the brain by the blood-brain barrier (48). Currently, most drugs used to treat brain diseases are lipid soluble small molecules with a molecular weight of less than 400 Da (49). A small number of drugs traverse the blood-brain barrier by carrier-mediated transport. An example of this is Levodopa, a treatment for Parkinson’s Disease, which is a precursor of the neurotransmitter dopamine. Levodopa is transported across the blood-brain barrier by the large neutral amino acid transporter, LAT1 (50). Our findings here provide a further refinement of understanding of the structure-activity relationship of LPCs to their transport, and educates the search and design of drugs that can be transported by Mfsd2a. Candidates for transport, whether as a drug itself or as a LPC scaffold, must have a zwitterionic headgroup, but not necessarily a phosphate, and a minimal threshold of hydrophobic character. As the binding pocket is several times larger than LPC, it is sterically feasible to attach a small molecule drug onto LPC or LPC-like scaffolds for delivery across the blood-brain barrier.

In summary, these studies represent a first structural model of human Mfsd2a based on homology modeling and biochemical interrogation. We expect that this model will serve as a foundation for the future development of X-ray crystal structures of the protein, which would provide further insight into the structure and function of this physiologically important transporter required for human brain growth and function.

REFERENCES

1. Salem, N., Jr., Litman, B., Kim, H. Y., and Gawrisch, K. (2001) Mechanisms of action of docosahexaenoic acid in the nervous system. Lipids 36, 945-959

2. Bazan, N. G. (2009) Neuroprotectin D1-mediated anti-inflammatory and survival signaling in stroke, retinal degenerations, and Alzheimer’s disease. Journal of lipid research 50 Suppl, S400- 405

3. Baisted, D. J., Robinson, B. S., and Vance, D. E. (1988) Albumin stimulates the release of lysophosphatidylcholine from cultured rat hepatocytes. The Biochemical journal 253, 693-701

4. Edmond, J., Higa, T. A., Korsak, R. A., Bergner, E. A., and Lee, W. N. (1998) Fatty acid transport and utilization for the developing brain. Journal of neurochemistry 70, 1227-1234

5. Lagarde, M., Bernoud, N., Brossard, N., Lemaitre-Delaunay, D., Thies, F., Croset, M., and Lecerf, J. (2001) Lysophosphatidylcholine as a preferred carrier form of docosahexaenoic acid to the brain. Journal of molecular neuroscience : MN 16, 201-204; discussion 215-221

6. Nguyen, L. N., Ma, D., Shui, G., Wong, P., Cazenave-Gassiot, A., Zhang, X., Wenk, M. R., Goh, E. L., and Silver, D. L. (2014) Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid. Nature 509, 503-506

7. Law, C. J., Maloney, P. C., and Wang, D. N. (2008) Ins and outs of major facilitator superfamily antiporters. Annual review of microbiology 62, 289-305

8. Alakbarzade, V., Hameed, A., Quek, D. Q. Y., Chioza, B. A., Baple, E. L., Cazenave-Gassiot, A., Nguyen, L. N., Wenk, M. R., Ahmad, A. Q., Sreekantan-Nair, A., Weedon, M. N., Rich, P., Patton, M. A., Warner, T. T., Silver, D. L., and Crosby, A. H. (2015) A partially inactivating mutation in the sodium-dependent lysophosphatidylcholine transporter MFSD2A causes a non-lethal microcephaly syndrome. Nat Genet 47, 814-817

9. Ethayathulla, A. S., Yousef, M. S., Amin, A., Leblanc, G., Kaback, H. R., and Guan, L. (2014) Structure-based mechanism for Na(+)/melibiose symport by MelB. Nature communications 5, 3009

10. Guan, L., Mirza, O., Verner, G., Iwata, S., and Kaback, H. R. (2007) Structural determination of wild-type lactose permease. Proceedings of the National Academy of Sciences of the United States of America 104, 15294-15298

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Brain Matters from iBiology

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

ADAM COHEN: VISUALIZING ACTIVITY IN THE BRAIN

The pattern of electrical signals propagated through neuronal networks determines brain function. Adam Cohen examines the possibility of visualizing these signals inside an intact brain using fluorescent transmembrane proteins that are sensitive to voltage. Cohen discusses the barriers to this approach, something he predicts scientists from many disciplines will eventually overcome.

https://youtu.be/Zw8lWmGuXLU      Download: High ResLow Res     Recorded: 2014

Adam Cohen is Professor in the Departments of Chemistry and Physics at Harvard University and Investigator of the Howard Hughes Medical Institute. He develops biological tools and analytical approaches to investigate the behaviors of molecules and cells in vitro and in vivo. His lab merges protein engineering, optics, and physics, among other disciplines, on a variety of projects. For example, they have developed a fluorescent transmembrane protein that detects membrane voltage, which is useful in visualizing electrical activity in cells, such as cultured neurons.

Related —

 

The Evolution of Neural Circuits and Behaviors​
Melina Hale (University of Chicago)

Evolution can be defined as a change in heritable characteristics. In her fist talk, Hale does a excellent job of explaining how these changes occur. She uses examples, such as the variable color of the pepper moth, to explain selection of characteristics and she describes how geographic isolation can lead to the evolution of new species. In her second lecture, Hale describes work from her lab on the startle response, a highly conserved behavior found in fish and other vertebrates. Comparisons of the neurons which control the startle response, across many species of fish, have allowed Hale and her colleagues to determine how this neuronal circuit, and this behavior, have evolved over hundreds of millions of years.

Part 1 is an outstanding video for high school or undergraduate educators looking for material to teach evolution.

Watch Melina Hale’s iBioSeminar:

Part 1: Introduction to Evolution

Part 2: Neural Circuits and How They Evolve: A Startling Example!

 

Discovery of a ‘Neuronal Big Bang’

University of Geneva   http://www.biosciencetechnology.com/news/2016/03/discovery-neuronal-big-bang

 

This is an expression of all the genes of a neuron during the first hours after its birth. Each circle represents a development stage (6h, 12h, 24h), and the colored points within each circle represent the level of gene expression. (Credit: Jabaudon Lab/ UNIGE)

This is an expression of all the genes of a neuron during the first hours after its birth. Each circle represents a development stage (6h, 12h, 24h), and the colored points within each circle represent the level of gene expression. (Credit: Jabaudon Lab/ UNIGE)

 

Our brain is home to different types of neurons, each with their own genetic signature that defines their function. These neurons are derived from progenitor cells, which are specialized stem cells that have the ability to divide to give rise to neurons. Neuroscientists from the Faculty of Medicine at the University of Geneva (UNIGE) shed light on the mechanisms that allow progenitors to generate neurons. By developing a novel technology called FlashTag that enables them to isolate and visualize neurons at the very moment they are born, they have deciphered the basic genetic code allowing the construction of a neuron. This discovery, which is published in Science, allows not only to understand how our brain develops, but also how to use this code to reconstruct neurons from stem cells. Researchers will now be able to better understand the mechanisms underlying neurological diseases such as autism and schizophrenia.

Directed by Denis Jabaudon, a neuroscientist and neuroscientist at the Department of Basic Neurosciences at UNIGE Faculty of Medicine and neurologist at the University Hospitals Geneva (HUG), the researchers developed a technology termed FlashTag, which visualizes neurons as they are being born. Using this approach, at the very moment where a progenitor divides, it is tagged with a fluorescent marker that persists in its progeny. Scientists can then visualize and isolate newborn neurons in order to dynamically observe which genes are expressed in the first few hours of their existence. Over time, they can then study their evolution and changes in gene expression. “Previously, we only had a few photos to reconstruct the history of neurons, which left a lot of room for speculation. Thanks to FlashTag, there is now a full genetic movie unfolding before our eyes. Every instant becomes visible from the very beginning, which allows us to understand the developmental scenario at play, identify the main characters, their interactions and their incentives”, notes Jabaudon. Working in the cerebral cortex of the mouse, the scientists have thus identified the key genesto neuronal development, and demonstrated that their expression dynamics is essential for the brain to develop normally.

A very precise primordial choreography

This discovery, by giving access to the primordial code of the formation of neurons, helps us to understand how neurons function in the adult brain. And it appears that several of these original genes are also involved in neurodevelopmental and neurodegenerative diseases, which can occur many years later. This suggests that a predisposition may be present from the very first moments in the existence of neurons, and that environmental factors can then impact on how diseases may develop later on. By understanding the genetic choreography of neurons, the researchers can therefore observe how these genes behave from the start, and identify potential anomalies predicting diseases.

After successfully reading this genetic code, the scientists we able to rewrite it in newborn neurons. By altering the expression of certain genes, they were able to accelerate neuronal growth, thus altering the developmental script. With FlashTag, it is now possible to isolate newborn neurons and recreate cerebral circuits in vitro, which enables scientists to test their function as well as to develop new treatments.

A website open to all

The UNIGE team posted a website where it is possible to enter the name of a gene and observe how it is expressed, and how it interacts with other genes. “Each research team can only focus on a handful of genes at a time, while our genome is made up of close to 20,000 genes. We therefore made our tool available for other researchers to use it, in a fully open way,” highlights Jabaudon.

Chronic Stress Causes Brain Inflammation, Memory Loss

A new study suggests that long-term stress can hurt short-term memory, in part due to inflammation brought on by an immune response.

Bevin Fletcher, Associate Editor    http://www.biosciencetechnology.com/news/2016/03/chronic-stress-causes-brain-inflammation-memory-loss

A new study suggests that long-term stress can hurt short-term memory, in part due to inflammation brought on by an immune response.

Researchers from Ohio State University performed experiments where mice were exposed to repeated social defeat by exposure to an aggressive, larger, alpha mouse.  The mice that were under chronic stress, had difficulty remembering where the escape hole was in a maze they had previously mastered before the stressful period.

The findings were published in The Journal of Neuroscience.

“The stressed mice didn’t recall it. The mice that weren’t stressed really remembered it,” lead researcher Johnathan Godbout, associate professor of neuroscience at Ohio State, said in statement.

The researchers noted that this kind of stress isn’t the once-in-a-while, acute stress someone might feel before a big meeting or presentation, but prolonged, continued stress.

The mice also displayed depressive-like behavior through social avoidance that continued after four weeks of observation.

Brain changes were also observed in the stressed mice, including inflammation associated with the presence of immune cells, known as macrophages, in the brain.  The researchers also recorded shortfalls in the development of new neurons at 10 days and 28 days after the chronic stress ended.

John Sheridan, associate director of Ohio State’s Institute for Behavioral Medicine said in a statement that there might be ways to interrupt the inflammation that occurs in the brain.

When the mice were given a chemical that inhibited inflammation, both memory loss and the inflammatory macrophages disappeared, leading researchers to conclude that post-stress memory deficits is directly tied to inflammation and the immune system. The depressive symptoms and the brain-cell problem did not go away.

“Stress releases immune cells from the bone marrow and those cells can traffic to brain areas associated with neuronal activation in response to stress,” Sheridan said. “They’re being called to the brain, to the center of memory.”

The team aims to understand the underpinnings of stress and responses that could one day lead to treatments for people that suffer from anxiety, depression, or post-traumatic stress disorder.

New information from this study could lead to immune-based treatments, Godbout said.

 

 A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources Ethan S. Lippmann, Abraham Al-Ahmad, Samira M. Azarin, Sean P. Palecek &Eric V. Shusta

Scientific Reports 4, Article number: 4160 (2014)   http://dx.doi.org:/10.1038/srep04160

Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge, we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons derived from renewable cell sources. First, retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs, particularly through adherens junction, tight junction, and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (~5,000 Ωxcm2). Overall, this scalable human BBB model may enable a wide range of neuroscience studies.

The blood-brain barrier (BBB) is composed of brain microvascular endothelial cells (BMECs) which line brain capillaries and control molecular and cellular trafficking between the bloodstream and neural tissue. These properties are tightly regulated by the surrounding neurovascular microenvironment throughout BBB development and into adulthood. While this barrier is essential for preserving healthy brain activity, its dysfunction has been implicated in a number of neurological diseases1. Moreover, an intact BBB serves as a major bottleneck for brain drug delivery2. Studies regarding BBB development and regulation can be difficult and time-consuming to conduct in vivo and testing brain penetration of therapeutics in vivo is a low throughput endeavor. As such, in vitro BBB models have been widely implemented to study interactions between BMECs and other cells of the neurovascular unit and to conduct screens for prospective BBB-permeant drugs.

In vitro BBB models are typically constructed using primary BMECs isolated from animal brain tissue, including bovine, porcine, rat, and mouse (reviewed extensively in ref. 3). These BMECs are then co-cultured with combinations of other cells of the neurovascular unit, such as neurons, pericytes, and astrocytes, to upregulate BBB properties4,5,6,7. Models derived from animal tissue have proven extremely useful in studying various aspects of the BBB, such as developmental and regulatory mechanisms8,9,10,11,12 and assaying drug permeability, but it is generally well-accepted that owing to species differences, a robust human BBB model is vital to achieve a detailed understanding of human developmental pathways and to conduct relevant drug discovery and design studies13. Human BMEC sources for BBB models have previously consisted of either primary biopsied brain tissue14,15 or immortalized cell lines16. Primary human BMECs typically possess moderate barrier properties but are of limited scale14,15, and immortalized BMECs are clonal and readily scalable but often suffer from suboptimal barrier properties16,17. From a co-culture perspective, human neurons, astrocytes, and pericytes can also be difficult to obtain from primary tissue sources in sufficient quantities for modeling purposes. These collective issues have hindered the development of in vitro human BBB models that are both high fidelity and scalable3.

We have recently demonstrated that stem cells may be attractive candidates to replace primary cells in human BBB models. Human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), can be differentiated into cells possessing both endothelial and BBB properties (coined hPSC-derived BMECs) via co-differentiation of neural and endothelial progenitors, followed by selection and subsequent culture of the endothelial cells18. The iPSC-derived BMECs co-cultured with rat astrocytes possessed reasonable barrier tightness as measured by TEER (860 ± 260 Ωxcm2)18, but the TEER remained below some primary bovine19 and porcine20,21 models (800–2,000 Ωxcm2) and substantially lower than in vivo TEER (measured up to 5,900 Ωxcm2)22. In searching for candidates to improve the BBB phenotype, we identified all-trans retinoic acid (RA). BMECs in vivo have been shown to express retinol-binding protein and its membrane receptor STRA623,STRA6 expression has been detected in brain endothelium but not peripheral endothelium in adult mice24, and STRA6 expression was increased during the differentiation of hPSC-derived BMECs in our previous work18. Moreover, RA has been shown to upregulate certain BBB properties in immortalized rodent25,26 and human27 BMEC lines. In this manuscript, we demonstrate maturation of hPSC-derived BMEC phenotypes following retinoic acid (RA) addition during the differentiation process, including enhanced adherens junction protein expression, barrier function, and multidrug resistance protein (MRP) efflux activity. We also demonstrated in previous work that primary human neural progenitor cells (NPCs) could be differentiated to a defined mixture of neurons and astrocytes capable of inducing BBB properties in rat BMECs in co-culture7. In this manuscript, it is shown that under optimized culture conditions, RA-treated hPSC-derived BMECs sequentially co-cultured with primary human brain pericytes and NPC-derived astrocytes and neurons can achieve physiologic TEER values, forming a scalable, fully human BBB model.

.…….

The purpose of this work was to construct a renewable, robust human BBB multicellular co-culture model employing hPSCs, NPCs, and pericytes. Using previous studies as guides25,26,31, RA was identified as a significant modulator of BMEC properties during hPSC differentiation that greatly enhanced physical barrier characteristics as demonstrated by elevated TEER in BMECs cultured alone or with neurovascular cell co-culture. In recent work, RA treatment on the hCMEC/D3 human brain endothelial cell line served to increase occludin and VE-cadherin expression, and the authors suggested that RA secreted by radial glia may be involved in BBB development27. In our study, when RA was added during the endothelial progenitor expansion phase of hPSC-derived BMEC differentiation, similar results were observed including an earlier onset of VE-cadherin expression and increased occludin expression. Moreover, the BMEC yield was increased 2-fold and the tightness of the hPSC-derived BMEC monolayers as measured by elevated TEER was significantly enhanced for three different hPSC lines. Somewhat unexpectedly, RA treatment resulted in decreased claudin-5 expression. However, the Western blotting analysis was conducted using whole-cell lysates and does not take into account the substantially improved intercellular claudin-5 junctional continuity upon RA treatment (Fig. 2C). We and others have previously observed a strong correlation between such junctional continuity and resultant barrier phenotype6,29,32. In addition, previous work has demonstrated claudin-5 expression is relatively constant across peripheral and BBB endothelium while occludin expression is increased at the BBB relative to other vascular beds31. Thus, a combination of claudin-5 localization and elevated occludin expression may be the key phenotypic indicators of increased barrier function31,33. RA treatment of hPSC-derived BMECs also selectively increased MRP efflux activity, which agrees with reports demonstrating that signaling via nuclear receptors can regulate efflux transporter expression and activity at the blood-brain barrier in vitro and in vivo34,35,36,37. RA influences many aspects of brain development, such as anterior/posterior axis patterning in the hindbrain and anterior spinal cord38,39,40 and regulation of neurogenesis41,42,43. During BMEC differentiation, RA could trigger several modes of action. RA may act directly on the developing endothelial cells to upregulate BBB properties, it could induce changes in the neural cells to indirectly promote BBB differentiation, or it could act by a combination of these mechanisms. Future work will be necessary to deconvolute the RA signaling mechanisms affecting the hPSC-derived BMEC differentiation scheme.

 

In Your Dreams

Understanding the sleeping brain may be the key to unlocking the secrets of the human mind.

By David Gelernter | March 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/45357/title/In-Your-Dreams

Many scientists who study the mind live in fantasyland. They ought to move back to reality: neuroscientists, psychologists, computer scientists pursuing artificial intelligence, and the philosophers of mind who are, in many cases, the sharpest thinkers in the room.

The mind makes us rational. That mind is the one we choose to study. When we study sleep or dreaming, we isolate them first—as the specialized topics they are. But, as I argue in my new book The Tides of Mind, we will never reach a deep understanding of mind unless we start with an integrated view, stretching from rational, methodical thought to nightmares.

Integrating dreaming with the rest of mind is something like being asked to assemble a car from a large pile of metal, plastic, rubber, glass, and an ocelot. Dreaming is hallucination, centering on a radically different self from our waking selves, within unreal settings and stories. Dreams can please or scare us far more vividly than our ordinary thoughts. And they are so slippery, so hard to grasp, that we start losing them the moment we wake up.

But dreaming fits easily into the big picture of mind; and we will make no basic progress on understanding the mind until we see how. Dreaming is the endpoint of the spectrum of consciousness, the smooth progression from one type of consciousness to the next, that we each experience daily.

The simplest approach to the spectrum centers on mental focus. The quality of our attention goes from concentrated to diffuse over the course of a normal day; from a state in which we can concentrate—we can think and remember in a relatively disciplined way—to one in which, with our minds wandering and memory growing increasingly vibrant and distracting, we approach sleep. Then our thinking becomes hallucinatory (as we pass through “sleep-onset thought”); and finally, we are asleep and dreaming. Usually, we oscillate down and up more than once during the day. We move partway down, come partway back, then finally slide slowly to the bottom, when we sleep and dream.

We can also describe the spectrum as a steady shift from a mind dominated by action to one dominated by passive mental experience; from mental doing to mental being. In the upper spectrum, we tend to ignore emotion as we pursue some mental object by means of reasoning or analysis. But the daydreams and fantasies that occupy us as we move down-spectrum are often emotional. And in dreaming we encounter the most saturated emotions, good and bad, that the mind can generate.

The spectrum clarifies important aspects of the mind. “Intentionality,” the quality of aboutness (“I believe that bird is a sparrow” is about “that bird”), is sometimes called “the mark of the mental”—the distinguishing attribute of mental states. But intentionality belongs strictly to the upper spectrum, and disappears gradually as we descend. At the bottom, our minds are dominated by experience, pure being. Happiness or pain or “the experience of seeing purple” are states that have causes but are about nothing.

Software simulations of the upper spectrum, of thinking-about, have grown steadily stronger over the years. That trend will continue. Being, however, is not computable. Software can no more reproduce “being happy” than it can reproduce “being rusty.” Such states depend on physical properties of particular objects. A digital computer resembles only the upper-spectrum mind. Software will never come close to reproducing the mind as a whole.
Leaving sleep outside our investigation is a good way not to see any of this. Arbitrarily hacking off one end of any natural spectrum is an invitation to conceptual chaos. There has been plenty of that in the science of mind. We must start by understanding sleep and dreaming, and go from there.

David Gelernter is a professor of computer science at Yale University. Read an excerpt from his latest book, The Tides of Mind: Uncovering the Spectrum of Consciousness at the-scientist.com.

Out in the Cold

Serotonin’s long-debated role in sleep promotion is temperature-dependent.

By Karen Zusi | March 1, 2016     http://www.the-scientist.com/?articles.view/articleNo/45346/title/Out-in-the-Cold

N.M. Murray et al., “Insomnia caused by serotonin depletion is due to hypothermia,” Sleep, 38:1985-93, 2015.

Sleepless nights
Early research into serotonin’s functions suggested that the neurotransmitter promotes sleep: lab animals deprived of the chemical often developed insomnia. More recent evidence indicated that serotonin plays a part in wakefulness instead, a theory that has gained significant traction. But explanations of the initial experimental data were scarce—so Nick Murray, then a research fellow at the University of Iowa Carver College of Medicine, started digging.

Faulty furnace?
“Over the past 5 or 10 years, we’ve found that serotonin is a key neurotransmitter for generating body heat,” says Murray. To investigate whether this role was related to serotonin’s impact on sleep, he and his colleagues injected para-chlorophenylalanine into mice to inhibit serotonin synthesis.

On ice
When kept at room temperature (20 °C), the mice with depleted serotonin slept less and developed a lower body temperature compared with their control counterparts. However, when housed at 33 °C—a thermoneutral temperature for mice—the sleep and body temperature of the treated mice stayed normal. “Serotonin isn’t a sleep-promoting neurotransmitter,” concludes Murray, now a resident at California Pacific Medical Center. He suggests that mice lacking serotonin had a tough time sleeping under early experimental conditions simply because the animals were cold, and that at higher temperatures other neurotransmitter systems in the brain would function to allow them a normal sleep-wake cycle.

Case closed
The study “solves a long-standing mystery” in the field, says Clifford Saper of Harvard University. “Not very many labs measure sleep and body temperature at the same time,” he adds. “It just basically escaped everybody’s notice for all these years.”

 

 

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Laser Therapy Opens Blood-Brain Barrier

Curator: Larry H. Bernstein, MD, FCAP

 

Laser Surgery Opens Blood-Brain Barrier to Chemotherapy

http://www.photonics.com/Article.aspx?AID=58445

ST. LOUIS, March 11, 2016 — A laser probe has been used to open the brain’s protective cover, enabling delivery of chemotherapy drugs to patients with glioblastoma — the most common and aggressive form of brain cancer.

In a pilot study conducted by the Washington University School of Medicine in St. Louis, Mo., 14 patients with glioblastoma underwent minimally invasive laser surgery to treat a recurrence of their tumors. Heat from the laser was already known to kill brain tumor cells but, unexpectedly, the researchers found that the technology penetrated the blood-brain barrier.

“The laser treatment kept the blood-brain barrier open for four to six weeks, providing us with a therapeutic window of opportunity to deliver chemotherapy drugs to the patients,” said neurosurgery professor Eric Leuthardt, MD, who also treats patients at Barnes-Jewish Hospital. “This is crucial because most chemotherapy drugs can’t get past the protective barrier, greatly limiting treatment options for patients with brain tumors.

The team is still closely following the patients, though early results indicate they are doing better on average, in terms of survival and clinical outcomes, than what the researchers would expect with other treatment methods.

Glioblastomas are one of the most difficult cancers to treat. Most patients diagnosed with this type of brain tumor survive just 15 months, according to the American Cancer Society.

The research is part of a larger phase II clinical trial that will involve 40 patients. Twenty patients were enrolled in the pilot study, 14 of whom were found to be suitable candidates for the minimally invasive laser surgery, a technology that Leuthardt helped pioneer.

The laser technology was approved by the FDA in 2009 as a surgical tool to treat brain tumors. The Washington team’s research marks the first time the laser has been shown to disrupt the blood-brain barrier, which shields the brain from harmful toxins but inadvertently blocks potentially helpful drugs, such as chemotherapy.

As part of the trial, doxorubicin, a widely used chemotherapy, was delivered intravenously to 13 patients in the weeks following the laser surgery. Preliminary data indicate that 12 patients showed no evidence of tumor progression during the short, 10-week time frame of the study. One patient experienced tumor growth before chemotherapy was delivered; the tumor in another patient progressed after chemotherapy was administered, the researcher reported.

The laser surgery was well-tolerated by the patients in the trial; most went home one to two days afterward, and none experienced severe complications. The surgery was performed while a patient lies in an MRI scanner, providing the neurosurgical team with a real-time look at the tumor. Using an incision of only 3 mm, a neurosurgeon robotically inserted the laser to heat up and kill brain tumor cells at a temperature of about 150 °F.

“The laser kills tumor cells, which we anticipated,” said Leuthardt. “But, surprisingly, while reviewing MRI scans of our patients, we noticed changes near the former tumor site that looked consistent with the breakdown of the blood-brain barrier.”

Leuthardt confirmed and further studied these imaging findings with study co-author Dr. Joshua Shimony, a professor of radiology at Washington University.

The researchers, including co-corresponding author Dr. David Tran, a neuro-oncologist now at the University of Florida, performed follow-up testing, which showed that the degree of permeability through the blood-brain barrier peaked one to two weeks after surgery but that the barrier remained open for up to six weeks.

Other successful attempts to breach the barrier have left it open for only a short time — about 24 hours — not long enough for chemotherapy to be consistently delivered, or have resulted in only modest benefits, the researchers said. The laser technology leaves the barrier open for weeks — long enough for patients to receive multiple treatments with chemotherapy. Further, the laser only opens the barrier near the tumor, leaving the protective cover in place in other areas of the brain. This has the potential to limit the harmful effects of chemotherapy drugs in other areas of the brain, the researchers said.

The findings also suggest that other approaches, such as cancer immunotherapy — which harnesses cells of the immune system to seek out and destroy cancer — could also be useful for patients with glioblastomas.

The researchers are planning another clinical trial that combines the laser technology with chemotherapy and immunotherapy, as well as trials to test targeted cancer drugs that normally can’t breach the blood-brain barrier.

The research was published in Plos One (doi: 10.1371/journal.pone.0148613).

 

Hyperthermic Laser Ablation of Recurrent Glioblastoma Leads to Temporary Disruption of the Peritumoral Blood Brain Barrier

Poor central nervous system penetration of cytotoxic drugs due to the blood brain barrier (BBB) is a major limiting factor in the treatment of brain tumors. Most recurrent glioblastomas (GBM) occur within the peritumoral region. In this study, we describe a hyperthemic method to induce temporary disruption of the peritumoral BBB that can potentially be used to enhance drug delivery.

 Methods

Twenty patients with probable recurrent GBM were enrolled in this study. Fourteen patients were evaluable. MRI-guided laser interstitial thermal therapy was applied to achieve both tumor cytoreduction and disruption of the peritumoral BBB. To determine the degree and timing of peritumoral BBB disruption, dynamic contrast-enhancement brain MRI was used to calculate the vascular transfer constant (Ktrans) in the peritumoral region as direct measures of BBB permeability before and after laser ablation. Serum levels of brain-specific enolase, also known as neuron-specific enolase, were also measured and used as an independent quantification of BBB disruption.

Results

In all 14 evaluable patients, Ktrans levels peaked immediately post laser ablation, followed by a gradual decline over the following 4 weeks. Serum BSE concentrations increased shortly after laser ablation and peaked in 1–3 weeks before decreasing to baseline by 6 weeks.

Conclusions   

The data from our pilot research support that disruption of the peritumoral BBB was induced by hyperthemia with the peak of high permeability occurring within 1–2 weeks after laser ablation and resolving by 4–6 weeks. This provides a therapeutic window of opportunity during which delivery of BBB-impermeant therapeutic agents may be enhanced.

Trial Registration  

ClinicalTrials.gov NCT01851733

Citation: Leuthardt EC, Duan C, Kim MJ, Campian JL, Kim AH, Miller-Thomas MM, et al. (2016) Hyperthermic Laser Ablation of Recurrent Glioblastoma Leads to Temporary Disruption of the Peritumoral Blood Brain Barrier. PLoS ONE 11(2): e0148613.  http://dx.doi.org:/10.1371/journal.pone.0148613

Glioblastoma (GBM) is the most common and lethal malignant brain tumor in adults [1]. Despite advanced treatment, median survival is less than 15 months, and fewer than 5% of patients survive past 5 years [2, 3]. Effective treatment options for recurrent GBM remain very limited and much of research and development efforts in recent years have focused on this area of greatly unmet needs. Up to 90% of recurrent tumors develop within the 2–3 cm margin of the primary site and are thought to arise from microscopic glioma cells that infiltrate the peritumoral brain region prior to resection of the primary tumor [4, 5]. Therefore elimination of infiltrative GBM cells in this region likely will improve long-term disease control.

Inadequate CNS delivery of therapeutic drugs due to the blood brain barrier (BBB) has been a major limiting factor in the treatment of brain tumors. The presence of contrast enhancement on standard brain MRI qualitatively reflects a disrupted state of the BBB. For this reason, drug access to the viable contrast enhanced tumor rim is likely significantly higher than to the peritumoral region, which usually does not have contrast enhancement [6, 7]. Evidence supporting this hypothesis came from studies in which drug levels of cytotoxic agents were sampled in tumors and the surrounding brain tissue at the time of surgery or autopsy. Drug concentrations were at the highest in the enhancing portion of tumors, and then rapidly decreased up to 40 fold lower by 2–3 cm distance from the viable tumor edge [810]. Overall, these observations suggest that the BBB and its integrity negatively correlate with delivery and potentially therapeutic effects of BBB impermeant drugs.

To circumvent the BBB problem in local drug delivery, recent approaches have focused on bypassing it. A previously described method is the use of Gliadel, a polymer wafer impregnated with the chemotherapeutic agent carmustine (BCNU) and placed intra-operatively in the resection cavity to bypass the BBB. This approach resulted in a statistically significant but modest survival advantage in both newly diagnosed and recurrent GBM [1113]. The modest benefit of Gliadel could be due to the short duration of drug delivery as nearly 80% of BCNU is released from the wafer over a period of only 5 days [14]. This observation further supports the notion that the BBB is critical to chemotherapy effect. However, Gliadel is not widely utilized as it requires an open craniotomy and can impair wound healing. Another approach of bypassing the BBB is the convection-enhanced delivery system in which a catheter is surgically inserted into the tumor to deliver chemotherapy [15]. This procedure requires prolonged hospitalization to maintain the external catheter to prevent serious complications and as a result has not been used extensively.

The role of hyperthermia in inducing BBB disruption has been previously described in animal models of CNS hyperthermia. In a rodent model of glioma, the global heating of the mouse’s head to 42°C for 30 minutes in a warm water bath significantly increased the brain concentration of a thermosensitive liposome encapsulated with adriamycin chemotherapy [16]. To effect more locoregional hyperthermia, retrograde infusion of a saline solution at 43°C into the left external carotid artery in the Wistar rat reversibly increased BBB permeability to Evans-blue albumin in the left cerebral hemisphere [17]. In another approach, neodymium-doped yttrium aluminum garnet (Nd:YAG) laser-induced thermotherapy to the left forebrain of Fischer rats resulted in loco-regional BBB disruption as evidenced by passage of Evans blue dye, serum proteins (e.g. fibrinogen & IgM), and the chemotherapeutic drug paclitaxel for up to several days after thermotherapy [18]. The effect of hyperthermia on the BBB of human brain has not been examined.

Here we describe an approach to induce sustained, local disruption of the peritumoral BBB using MRI-guided laser interstitial thermal therapy, or LITT. The biologic effects and correlation with MRI findings of LITT have been studied in both animal and human models since the development of LITT over twenty years ago. A well-described zonal distribution of histopathological changes with corresponding characteristic MR imaging findings centered on the light-guide track replace the lesion targeted for thermal therapy. The central treatment zone shows development of coagulative necrosis with complete loss of normal neurons or supporting structures immediately following therapy, corresponding to hyperintense T1-weighted signal intensity relative to normal brain [1922]. The peripheral zone of the post-treatment lesion is characterized by avid enhancement with intravenous gadolinium contrast agents, which peaks several days following thermal therapy and persists for many weeks after the procedure. Gadolinium contrast enhancement in the brain following LITT is due to leakage of gadolinium contrast into the extravascular space across a disrupted BBB [2023]. The perilesional zone of hyperintense signal intensity of FLAIR-weighted images develops within 1–3 days of thermal treatment and persists for 15–45 days [22].

We demonstrate that in addition to cytoreductive ablation of the main recurrent tumor, hyperthermic exposure of the peritumoral region resulted in localized, lasting disruption of the BBB as quantified by dynamic contrast-enhanced MRI (DCE-MRI) and serum levels of brain-specific enolase (BSE), thus providing a therapeutic window of opportunity for enhanced delivery of therapeutic agents.

Table 1. Patient Baseline Demographics and Characteristics.
TMZ/RT: Stupp protocol of 60 Gy radiotherapy plus concurrent 75mg/m2 daily temozolomide. Doxorubicin treatment: Timing of 20mg/m2 IV weekly doxobubicin treatment after LITT. Early = Starting within 1 week after LITT; Late = Starting at 6 weeks after LITT.  http://dx.doi.org:/10.1371/journal.pone.0148613.t001
……
Quantitative measurement of LITT-induced peritumoral BBB disruption by DCE-MRI

Brain MRI obtained within 48 hours following LITT showed the targeted tumor replaced by a post-treatment lesion corresponding to the volume of treated tissue on intraoperative thermometry maps. The post-treatment lesion lost the original rim of tumor-associated contrast enhancement and instead demonstrated central hyperintense T1-weighted signal compared to the pre-treated tumor and normal brain and a faint, newly developed discontinuous rim of peripheral contrast enhancement extending beyond the original tumor-associated enhancing rim (Fig 2A). These findings are consistent with a loss of viable tumor tissue caused by LITT, thus achieving an effective cytoreduction similar to open surgical resection. Of note, the rim of new peripheral contrast enhancement persisted for at least the next 28 days (Fig 2B–2E). Perilesional edema qualitatively evaluated on FLAIR-weighted images increased from pretreatment imaging at week 2 and persisted at week 4 following LITT (Fig 2F–2I). Perilesional edema decreased on subsequent MRI examinations. These findings qualitatively indicate that peritumoral BBB is disrupted by LITT and that the disruption peaks within approximately 2 weeks after the procedure.

……

Fig 3 demonstrates the Ktrans time curves for our cohort of patients. In all subjects the Ktrans in the ROIs within the enhancing ring around the ablated tumor is highly elevated in the first few days after the procedure and then progressively decreases at approximately the 4-week time point. The bottom right subplot in Fig 3 is an average of the Ktrans time courses from all the subjects with adjacent curves indicating the plus and minus one standard error of the mean curves. This figure demonstrates the peak Ktrans value immediately after the LITT procedure with persistent elevation out to about 4 weeks. Radiographically, persistent contrast enhancement and FLAIR hyperintensity were observed well past 6 weeks and in many cases more than 10 weeks later. Several patients had recurrent tumor by radiographic criteria (increasing size of the edema and enhancing area around the tumor site) and these patients also demonstrated a corresponding increase in the Ktrans value. These recurrences occurred after the 10-week mark and thus were not included in Fig 3. Importantly no difference in the pattern of Ktrans tracing was consistently observed between the 10 patients receiving late doxorubicin treatment and the 4 patients receiving early doxorubicin treatment. In summary, these results indicate that the peritumoral BBB disruption as measured by Ktrans peaked immediately after LITT and persisted above baseline for an additional 4 weeks.

……

To optimize the ELISA assay for BSE, we collected sera from 3 patients with a newly diagnosed low-grade (WHO grade 2) glioma before and after their planned craniotomy and surgical resection, and determined serum concentrations of BSE. WHO grade 2 gliomas were chosen for the optimization because as they are generally non-contrast enhanced tumors on brain MRI, tumor-associated BBB is relatively intact and consequently, serum concentrations of brain-specific factors are predicted to be low pre-operatively and to then rise post-operatively due to the BBB compromise from the surgery. Serum BSE concentrations were low prior to surgery and then, as predicted, consistently increased after open craniotomy and tumor resection, thus indicating that this method had adequate sensitivity in detecting changes in serum levels of BSE due to disruption of the BBB (Fig 4).

Fig 4. Optimization of the BSE ELISA assay for measuring BBB disruption.

Serum concentrations of BSE before and after open craniotomy for surgical debulking in 3 subjects (A, B, and C) with a low-grade glioma, WHO grade II. *p<0.05.  http://dx.doi.org:/10.1371/journal.pone.0148613.g004

……

Fig 5. BBB disruption induced by LITT as measured by serum biomarkers
Serum concentrations of BSE for each of the 14 evaluable subjects in the study (A-N) and as the mean + SEM (O) as a function of time in days from the LITT procedure. In 7/14 subjects, serum BSE levels slightly decreased immediately after LITT, then in 13/14 subjects, serum BSE levels rose shortly after LITT, peaked between 1–3 weeks after LITT, and then decreased by the 6-week time point. In Patient #12, serum BSE concentration increased at week 10 coincident with an increased Ktrans at the same time point, consistent with a recurrent tumor as demonstrated on diagnostic MR imaging. Patient #15’s serum BSE concentration began to rise by week 4, consistent with early multifocal recurrent disease as demonstrated on diagnostic MR imaging.  http://dx.doi.org:/10.1371/journal.pone.0148613.g005
…….

LITT is a minimally invasive neurosurgical technique that achieves effective tumor cytoreduction of brain tumors using a laser to deliver hyperthermic ablation. Here we have demonstrated that an unexpected, potentially useful effect of LITT is its ability to also disrupt the BBB in the peritumoral region that extends outwards 1–2 cm from the viable tumor rim. Importantly, the disruption persists in all 14 evaluable, treated patients for up to 4 weeks after LITT as measured quantitatively by DCE-MRI and up to 6 weeks as measured by serum levels of the brain-specific factor BSE. These observations indicate that after LITT there is a window during which enhanced local delivery of therapeutic agents into the desired location (i.e. peritumoral region) can potentially be achieved.

In all of the patients in this series, the peaks of serum concentrations of BSE showed wider variations and were delayed from several days to 1–2 weeks following the peak of BBB disruption as measured by Ktrans. The wider variations and delay of BSE concentrations lead to relatively low correlation coefficients between the two parameters and could be explained by: 1) the higher data point resolution for the serum values versus DCE-MRI values (weekly versus biweekly, respectively); 2) interval physiologic breakdown of thermally ablated tissue coupled with subsequent diffusion and equilibration between the intracranial and peripheral compartments; and 3) high inter-tumor heterogeneity among patients resulting in a wide variation in the rates at which ablated tissues of different compositions are broken down and released into the circulation. Whether these differences may be in part due to tumor-related factors such as IDH1/2 mutations and MGMT promoter methylation is unclear due to the small number of subjects. More importantly, both methods showed that the peritumoral BBB disruption induced by LITT was temporary, decreasing soon after peaking and being resolved by 4–6 weeks in most patients. In addition, although no significant difference in all the BBB measurement parameters was observed between the early and late doxorubicin treatment arms, the number of evaluable subjects was too small to allow generalization at this time.

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Accessing the Blood Brain Barrier for Chemotherapy

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Blood-Brain Barrier Opened Noninvasively with Focused Ultrasound for the First Time

Mon, 11/09/2015 – 9:26amby Focused Ultrasound Foundation
http://www.mdtmag.com/news/2015/11/blood-brain-barrier-opened-noninvasively-focused-ultrasound-first-time

The blood-brain barrier has been non-invasively opened in a patient for the first time. A team at Sunnybrook Health Sciences Centre in Toronto used focused ultrasound to enable temporary and targeted opening of the blood-brain barrier (BBB), allowing the more effective delivery of chemotherapy into a patient’s malignant brain tumor.

The team, led by neurosurgeon Todd Mainprize, MD, and physicist Kullervo Hynynen, PhD, infused the chemotherapy agent doxorubicin, along with tiny gas-filled bubbles, into the bloodstream of a patient with a brain tumor. They then applied focused ultrasound to areas in the tumor and surrounding brain, causing the bubbles to vibrate, loosening the tight junctions of the cells comprising the blood-brain barrier and allowing high concentrations of the chemotherapy to enter targeted tissues.

“The blood-brain barrier has been a persistent impediment to delivering valuable therapies to treat tumors,” said Dr. Mainprize. “We are encouraged that we were able to open this barrier to deliver chemotherapy directly into the brain, and we look forward to more opportunities to apply this revolutionary approach.”

This patient treatment is part of a pilot study of up to 10 patients to establish the feasibility, safety and preliminary efficacy of focused ultrasound to temporarily open the blood-brain barrier to deliver chemotherapy to brain tumors. The Focused Ultrasound Foundation is currently funding this trial through their Cornelia Flagg Keller Memorial Fund for Brain Research.

“Breaching this barrier opens up a new frontier in treating brain disorders,” said Neal Kassell, MD, Chairman of the Focused Ultrasound Foundation. “We are encouraged by the momentum building for the use of focused ultrasound to non-invasively deliver therapies for a number of brain disorders.”

Opening the blood-brain barrier in a localized region to deliver chemotherapy to a tumor is a predicate for utilizing focused ultrasound for the delivery of other drugs, DNA-loaded nanoparticles, viral vectors, and antibodies to the brain to treat a range of neurological conditions, including various types of brain tumors, Parkinson’s, Alzheimer’s and some psychiatric diseases.

The procedure was conducted using Insightec’s ExAblate Neuro system. “This first patient treatment is a technological breakthrough that may lead to many clinical applications,” said Eyal Zadicario, Vice President for R&D and Director of Neuro Programs, Insightec.

While the current trial is a first-in-human achievement, Dr. Kullervo Hynynen, senior scientist at the Sunnybrook Research Institute, has been performing similar pre-clinical studies for about a decade. His research has shown that the combination of focused ultrasound and microbubbles may not only enable drug delivery, but might also stimulate the brain’s natural responses to fight disease. For example, the temporary opening of the blood-brain barrier appears to facilitate the brain’s clearance of a key pathologic protein related to Alzheimer’s and improves cognitive function.

recent study by Gerhard Leinenga and Jürgen Götz from the Queensland Brain Institute in Australia further corroborated Hynynen’s research, demonstrating opening the blood-brain barrier with focused ultrasound reduced brain plaques and improved memory in a mouse model of Alzheimer’s disease.

Based on these two pre-clinical studies, a pilot clinical trial using focused ultrasound to treat Alzheimer’s is being organized.

Blood-brain Barrier Opened Non-invasively for the First Time

http://www.biosciencetechnology.com/news/2015/11/blood-brain-barrier-opened-non-invasively-first-time

Scientists, for the first time, have non-invasively opened the blood-brain barrier (BBB) in a patient.

A team at Sunnybrook Health Sciences Centre in Toronto, led by neurosurgeon Todd Mainprize, M.D., used focused ultrasound technology to more effectively introduce chemotherapy drugs into a patient’s malignant brain tumor.  The results were verified with a post procedure MRI scan, Mainprize said at a press conference Tuesday.

The blood-brain barrier is a protective layer that keeps harmful substances such as toxins from entering from the blood vessels into the brain.  Unfortunately, it also prevents many drugs from reaching the brain in adequate doses.

At the press conference, Mainprize stressed that this is a phase one safety and concept study to show that they could pass through the BBB. He noted the operation went smoothly and the patient, a 56-year-old women, who is the first of 10 to undergo the procedure for the study, is doing well.

To breach the BBB, doctors infused a chemotherapy drug, along with tiny gas-filled bubbles, into the blood stream. Then focused ultrasound was applied to the tumor and surrounding brain, causing the bubbles to vibrate, and open the BBB so high concentrations of the chemotherapy could enter targeted tissues.

The team is actively analyzing brain tissue samples to see how much of the drug was able to enter.  The findings have not been published yet, but were presented at the Focused Ultrasound Surgery Foundation meeting, according to Mainprize.

Mainprize described the device: It has 1,024 transducers that are arranged in a helmet shape that goes around the head and the forehead, and corrects for aberrations in the skull.

While the BBB has been non-invasively opened in animals, this was the first instance in humans.

“There have been hundreds and hundreds of animal models opening the blood-brain barrier, in mice, dogs, pigs, and primates, all of which have shown a very good safety profile with no changes in function behavior or hemorrhage,” Mainprize said at the press conference.

He noted that this is a reversible procedure, and the barrier is restored back to its normal function in 24 hours.

Nathan McDannold, Ph.D., associate professor of radiology at Brigham and Women’s Hospital said, “If you compare this to alternative methods, whatever risks there are, there much less than if you were invasively injecting drugs.”

The scientists believe the technology has applications beyond brain tumors, such as in Alzheimer’s and Parkinson’s diseases.

McDannold said that groups are in the process of planning a protocol that would deliver antibodies to clear amyloid proteins, associated with Alzheimer’s, and for Parkinson’s they are looking at neuroprotectives and potential gene therapies.

The trial is being funded by the Focused Ultrasound Foundation.

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impairment of cognitive function and neurogenesis

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

β2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis

Lucas K SmithYingbo HeJeong-Soo ParkGregor BieriCedric E SnethlageKarin LinGeraldine GontierRafael Wabl, et al.
Nature Medicine 21,932–937(2015)   http://dx.doi.org:/10.1038/nm.3898

Aging drives cognitive and regenerative impairments in the adult brain, increasing susceptibility to neurodegenerative disorders in healthy individuals1, 2, 3, 4. Experiments using heterochronic parabiosis, in which the circulatory systems of young and old animals are joined, indicate that circulating pro-aging factors in old blood drive aging phenotypes in the brain5, 6. Here we identify β2-microglobulin (B2M), a component of major histocompatibility complex class 1 (MHC I) molecules, as a circulating factor that negatively regulates cognitive and regenerative function in the adult hippocampus in an age-dependent manner. B2M is elevated in the blood of aging humans and mice, and it is increased within the hippocampus of aged mice and young heterochronic parabionts. Exogenous B2M injected systemically, or locally in the hippocampus, impairs hippocampal-dependent cognitive function and neurogenesis in young mice. The negative effects of B2M and heterochronic parabiosis are, in part, mitigated in the hippocampus of young transporter associated with antigen processing 1 (Tap1)-deficient mice with reduced cell surface expression of MHC I. The absence of endogenous B2M expression abrogates age-related cognitive decline and enhances neurogenesis in aged mice. Our data indicate that systemic B2M accumulation in aging blood promotes age-related cognitive dysfunction and impairs neurogenesis, in part via MHC I, suggesting that B2M may be targeted therapeutically in old age.

Figure 1: Systemic B2M increases with age and impairs hippocampal-dependent cognitive function and neurogenesis

Systemic B2M increases with age and impairs hippocampal-dependent cognitive function and neurogenesis.

http://www.nature.com/nm/journal/v21/n8/carousel/nm.3898-F1.jpg

(a,b) Schematics of unpaired young versus aged mice (a), and young isochronic versus heterochronic parabionts (b). (a,b) Changes in plasma concentration of B2M with age at 3, 6, 12, 18 and 24 months (a) and between young isochronic and…

 

Figure 2: B2M expression increases in the aging hippocampus and impairs hippocampal-dependent cognitive function and neurogenesis.close

B2M expression increases in the aging hippocampus and impairs hippocampal-dependent cognitive function and neurogenesis.

(a,b) Western blot and quantification of hippocampal lysates probed with B2M- and actin-specific antibodies from young (3 months) and aged (18 months) unpaired animals (a), or young isochronic and young heterochronic parabionts five wee…

Figure 3: Reducing MHC I surface expression mitigates the negative effects of heterochronic parabiosis on neurogenesis.close

Reducing MHC I surface expression mitigates the negative effects of heterochronic parabiosis on neurogenesis.

http://www.nature.com/nm/journal/v21/n8/carousel/nm.3898-F3.jpg

(a) Schematic of young (3 months) WT and Tap1−/− isochronic parabionts and young WT and Tap1−/− heterochronic parabionts. (b,c) Representative (of six sections per mouse) images of the DG (b) and quantification of DCX immunostaining (c)…

 

Figure 4: Absence of B2M enhances hippocampal-dependent cognitive function and neurogenesis in aged animals.

Absence of B2M enhances hippocampal-dependent cognitive function and neurogenesis in aged animals.

http://www.nature.com/nm/journal/v21/n8/carousel/nm.3898-F4.jpg

(ad) Learning and memory in young (3 months) and aged (17 months) WT and B2m-knockout (B2m−/−) mice by RAWM (a,c) and contextual fear conditioning (b,d). Data are from 10 young WT, 10 young B2m−/−, 8 aged WT, and 12 aged B2m−/− mice. (…

 

Neuroscience. 2015 Nov 12;308:75-94. doi: 10.1016/j.neuroscience.2015.09.012. Epub 2015 Sep 10.
Synergistic neuroprotection by epicatechin and quercetin: Activation of convergent mitochondrial signaling pathways.
In view of evidence that increased consumption of epicatechin (E) and quercetin (Q) may reduce the risk of stroke, we have measured the effects of combining E and Q on mitochondrial function and neuronal survival following oxygen-glucose deprivation (OGD). Relative to mouse cortical neuron cultures pretreated (24h) with either E or Q (0.1-10μM), E+Q synergistically attenuated OGD-induced neuronal cell death. E, Q and E+Q (0.3μM) increased spare respiratory capacity but only E+Q (0.3μM) preserved this crucial parameter of neuronal mitochondrial function after OGD. These improvements were accompanied by corresponding increases in cyclic AMP response element binding protein (CREB) phosphorylation and the expression of CREB-target genes that promote neuronal survival (Bcl-2) and mitochondrial biogenesis (PGC-1α). Consistent with these findings, E+Q (0.1 and 1.0μM) elevated mitochondrial gene expression (MT-ND2 and MT-ATP6) to a greater extent than E or Q after OGD. Q (0.3-3.0μM), but not E (3.0μM), elevated cytosolic calcium (Ca(2+)) spikes and the mitochondrial membrane potential. Conversely, E and E+Q (0.1 and 0.3μM), but not Q (0.1 and 0.3μM), activated protein kinase B (Akt). Nitric oxide synthase (NOS) inhibition with L-N(G)-nitroarginine methyl ester (1.0μM) blocked neuroprotection by E (0.3μM) or Q (1.0μM). Oral administration of E+Q (75mg/kg; once daily for 5days) reduced hypoxic-ischemic brain injury. These findings suggest E and Q activate Akt- and Ca(2+)-mediated signaling pathways that converge on NOS and CREB resulting in synergistic improvements in neuronal mitochondrial performance which confer profound protection against ischemic injury.
MiR-34a regulates blood–brain barrier permeability and mitochondrial function by targeting cytochrome c

 

 

The blood–brain barrier is composed of cerebrovascular endothelial cells and tight junctions, and maintaining its integrity is crucial for the homeostasis of the neuronal environment. Recently, we discovered that mitochondria play a critical role in maintaining blood–brain barrier integrity. We report for the first time a novel mechanism underlying blood–brain barrier integrity: miR-34a mediated regulation of blood–brain barrier through a mitochondrial mechanism. Bioinformatics analysis suggests miR-34a targets several mitochondria-associated gene candidates. We demonstrated that miR-34a triggers the breakdown of blood–brain barrier in cerebrovascular endothelial cell monolayer in vitro, paralleled by reduction of mitochondrial oxidative phosphorylation and adenosine triphosphate production, and decreased cytochrome c levels.

 

The blood–brain barrier (BBB) is composed of highly specialized cerebrovascular endothelial cells (CECs), separates brain tissue from the circulating blood, and maintains homeostasis of the neuronal environment.1 The CECs are interconnected by tight junctions including cytoplasmic zonula occludens (ZO) proteins, and various transmembrane proteins such as occludin and claudins.2 Disruption of BBB tight junctions has been well documented in cerebrovascular diseases and neurodegenerative disorders and is considered to be a pathological condition of the diseases and plays a key role in disease progression as well.2

A recent study demonstrates that the mitochondrial mechanisms regulate BBB integrity and permeability using oxygen–glucose deprivation and reoxygenation (OGD-R), anin vitro model of ischemic reperfusion injury.3 Our work demonstrates that compromised mitochondria lead to the disruption of tight junctions, opening of the BBB, and exacerbation of stroke outcomes.4 As such, regulation of mitochondrial function may affect BBB openings and could be critical in limiting the pathological progression of cerebrovascular diseases and neurodegenerative disorders.

MicroRNAs (miRNAs) are short non-coding functional RNAs that target certain messenger RNAs (mRNAs) through complementary base-pairing between the miRNAs and its mRNA targets, resulting in the inhibition of mRNA translation or degradation of mRNA.5 It has been documented that miRNAs are involved in mitochondrial structure and function, such as miR-181c which regulates mitochondrial morphology,6 miR-1 which affects mitochondrial mRNA translation,7 and miR-378 which targets mitochondrial enzymes involved in oxidative energy metabolism.8 Additionally, several miRNAs have recently been found to regulate BBB permeability. MiR-155, miR-181c, and miR-29c negatively affect BBB function by targeting tight junction protein genes directly or affecting related signal pathways.911 The miR-34 family members were discovered computationally and later verified experimentally as a part of the p53 tumor suppressor network. Recent work demonstrates that miR-34a modulates the expression of synaptic targets and neuronal morphology and function.12 However, little is known regarding the role of miR-34a in mitochondrial function and BBB permeability.

In the present study, we report that the overexpression of miR-34a breaks down the BBB through inhibition of mitochondrial function. Furthermore, cytochrome c (CYC) is experimentally verified as a target of miR-34a in vitro.

 

Overexpression of miR-34a affects BBB permeability and disrupts tight junctions in CECs

To determine whether miR-34a functionally affected the BBB, we transfected CECs with miR34a plasmid versus vector control in 24-well plates, cultured the cells for 48 h, conducted a BBB permeability assay in a CEC monolayer transwell system in vitro with an additional culture of 48 h, and measured the fluorescent dye FD-4 permeability of each well (Figure 1(a)). As shown in Figure 1(a), FD-4 permeability was significantly increased in wells containing miR-34a overexpression CEC monolayer. Papp, the permeability coefficient, was also significantly higher in CECs overexpressed with miR-34a in comparison to vector controls (Figure 1(a)). Furthermore, immunohis-tochemistry staining of tight junction-related proteins revealed that ZO-1 was continuously distributed in the control, but a discontinuous distribution of ZO-1 was observed in miR-34a overexpressed CEC monolayer (Figure 1(b)). Disruption of tight junctions was not associated with cell viability in CECs transfected with plasmids for 48 h or 96 h (Supplementary Figure 2). Altogether, these data suggest that overexpression of miR-34a increases BBB permeability and compromises BBB tight junctions.

Figure 1.

View larger version:

Figure 1.

Overexpression of miR-34a increases BBB permeability in vitro. (a) A schematic protocol using fluorescein isothiocyanate–dextran-4 (FD-4) to detect BBB permeability in vitro. FD-4 permeability in CECs that overexpressed miR-34a plasmid (0.017 ng) versus control was presented as real-time rate of FD-4 mean fluorescent intensity (2-way ANOVA followed by post hoc Dunnett’s test; n = 3; **, P < 0.01; ****, P < 0.0001). Calculated apparent permeability coefficient Papp(Student’s t-test; ****, P < 0.0001) is expressed as mean ± SD. (b) Confocal fluorescence images of CECs confluent monolayers confirmed microscopically after transfection with miR-34a plasmid versus control. Fluorescent staining: tight junctions ZO-1 (red), cell nuclei (DAPI, blue). Overexpression of miR-34a apparently disrupted tight junctions and resulted in gaps between cells (white arrows). Results are representative of three independent experiments.

MiR-34a affects mitochondrial function by targeting CYC in CECs

Our recent work demonstrated that mitochondria play a pivotal role in the maintenance of BBB integrity. BBB tight junctions are rapidly disrupted if oxidative phosphorylation is reduced by mitochondrial inhibitors.4 To investigate whether the miR-34a regulates BBB openings via affecting mitochondrial function in CECs, we examined cellular energetic OCRs in CECs transfected with miR-34a plasmid versus vector control. Interestingly, overexpression of miR-34a significantly impaired mitochondrial function in CECs (Figure 2(a) and Supplementary Figure 3). Basal respiration, ATP production, maximal respiration, and spare capacity were all significantly reduced in CECs overexpressing miR-34a for 48 and 72 h (Figure 2(a)). ATP level was also substantially reduced in CECs following overexpression of miR-34a in a dose dependent manner at 72 h (Figure 2(b)).

Figure 2.

View larger version:

Figure 2.

Overexpression of mir-34a reduces mitochondrial function and decreases CYC level in cerebrovascular endothelial cells. (a) Basal respiration, ATP production, maximal respiration, and spare capacity were calculated from the bioenergetics functional assay at post-transfection 48 and 72 h (raw data in Supplementary Figure 3). Data are expressed as mean ± SD (n = 5). 1-way ANOVA followed by post hoc Tukey’s test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (b) ATP level was measured at 72 h post-transfection. Data are expressed as mean ± SD (n = 5). 1-way ANOVA followed by post hoc Tukey’s test. (****, P < 0.0001). (c) Bioinfomatic analysis of miR-34a-targeting candidates related to mitochondria. (d) Flow cytometry analysis of mitochondrial specific proteins for complex I proteins (NDUFAF1, NDUFC2 and NDUFS2), complex II protein (SDHC), complex III protein (CYB), complex IV protein (CYC oxidase, Cox IV), cytochrome c (CYCS), pyruvate dehydrogenase kinase (PDK), and voltage-dependent anion channel protein (VDAC) at 72 h post-transfection. CYC level was significantly lower in the cells that were transfected with the miR-34a plasmid. Data are presented as mean ± SD (n = 3) and analyzed by Student’s t-test, *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Results are representative of three independent experiments.

To further determine miR-34a targets and uncover the mechanism that is used to affect mitochondria, we performed a bioinformatics analysis of the miR-34a database (miRbase and TargetScan). MiR-34a potentially targets several mitochondria-associated gene candidates including succinate dehydrogenase subunit c (SDHC), cytochrome B reductase 1 (CYBRD1), cytochrome B5 reductase 3 (CYBRD5), cytochrome c (CYCS), pyruvate dehydrogenase kinase isozyme 1 and 2 (PDK1 and PDK2) (Figure 2(c). However, CECs transfected with the miR-34a plasmid had robustly decreased CYCS levels measured by flow cytometry, suggesting that CYCS is one of the miR-34a targets among the potential candidates (Figure 2(d)). Moreover, overexpression of miR-34a slightly increased potential target SDHC but did not change the protein level of CYB and PKD (Figure 2(d)). Off-target genes, NDUFAF1, and VDAC showed no significant change in protein level, but NDUFC2, NDUFS2, and Cox IV were all increased in parallel with overexpression of miR-34a (Figure 2(d)). Taken together, these results experimentally verified CYCS as a miR-34a target, which is associated with the reduction of mitochondrial oxidative phosphorylation in CECs.

Discussion

In the present study, we demonstrated that the overexpression of miR-34a results in an increased BBB permeability and the disruption of tight junctions ZO-1 in CECs. Consistently, overexpression of miR-34a impaired mitochondrial oxidative phosphorylation and reduced ATP production in CECs. Bioinformatics analysis revealed series of potential miR-34a-targeting candidates related to mitochondrial function. We elucidated that CYCS is a miR-34a target, and the overexpression of miR-34a inhibited the CYCS expression and increased with the expression of other mitochondria-associated genes.

The overexpression of miR-34a disrupted tight junction protein ZO-1 (Figure 1). However, bioinformatics analysis indicated that miR-34a did not target the ZO-1 gene or other tight junction related genes, which suggests that the increased BBB permeability is not directly caused by the targeting of tight junction protein genes. The compromised mitochondrial function by overexpression of miR-34a may influence cellular metabolism in a way that is critical to maintain BBB tight junctions. Among several potential mitochondria-associated gene targets (Figure 2(c)), miR-34a initiated the reduction of CYCS level. Interestingly, potential target SDHC and other off-target gene proteins (NDUFC2, NDUFS2, and Cox IV) were concurrently upregulated (Figure 2(d)), which might be due to the compensation for the reduced target gene protein CYCS, or the disturbance of the coordinated gene translation in mitochondria. We therefore concluded that CYCS is a miR-34a target and is responsible for the miR-34a-induced reduction of mitochondrial oxidative phosphorylation.

Protein kinase C (PKC) signaling has also been shown to affect BBB or other endothelial barriers in vitro and in vivo. A recent study reported that miR-34a regulated blood–tumor barrier by targeting PKCɛ using glioma endothelial cells.13 In this study, we did not assess the PKC pathways that could contain additional targets of miR-34a. However, our data do support that miR-34a affects BBB via a mitochondrial mechanism, which is novel and may lead a new direction for designing BBB-related therapeutics.

We have noted several limitations in our study. First, we did not examine the effects of knockdown or knockout miR-34a on BBB function, which might fully establish the role of miR-34a in the BBB and mitochondria. Second, this work was conducted in cell culture models, which adequately address the mechanism of effect that miR-34a exerts on the BBB and mitochondria but do not provide evidence of its involvement in cerebrovascular or neurodegenerative conditions. Further studies in relevant experimental models are warranted.

Mitochondria play a pivotal role in cellular bioenergetics and cell survival, participating in a variety of cellular processes, including the generation of ATP, and the regulation of apoptotic signaling and other signaling pathways.14 MiR-34a targets and represses multiple genes involved in cell proliferation, apoptosis, cell cycle, migration, etc.,15 but it is not known if these effects are modulated by the observed mitochondrial effects as well. The present study provides the first description of miR-34a affecting mitochondrial activity, which could lead to a revision of current miR-34a targets and may lead to discovery of new mechanisms. The elucidation of the miR-34a’s role in mitochondrial oxidative phosphorylation and the BBB integrity offers a novel therapeutic strategy for targeting miR-34a to treat cerebrovascular and neurodegenerative diseases such as stroke and Alzheimer’s disease. These neuropathological diseases are known to involve a host of conditions that lead to mitochondrial impairment and BBB disruption. Finally, transient opening of the BBB could prove to be useful for CNS drug delivery.

 

Long-term aerobic exercise prevents age-related brain deterioration
http://www.kurzweilai.net/long-term-aerobic-exercise-prevents-age-related-brain-deterioration

October 30, 2015

A study of the brains of mice shows that structural deterioration associated with old age can be prevented by long-term aerobic exercise starting in mid-life, according to the authors of an open-access paper in the journal PLOS Biologyyesterday (October 29).

Old age is the major risk factor for Alzheimer’s disease, like many other diseases, as the authors at The Jackson Laboratory in Bar Harbor, Maine, note. Age-related cognitive deficits are due partly to changes in neuronal function, but also correlate with deficiencies in the blood supply to the brain and with low-level inflammation.

“Collectively, our data suggests that normal aging causes significant dysfunction to the cortical neurovascular unit, including basement membrane reduction and pericyte (cells that wrap around blood capillaries) loss. These changes correlate strongly with an increase in microglia/monocytes in the aged cortex,” said Ileana Soto, lead author on the study.*

Benefits of aerobic exercise

However, the researchers found that if they let the mice run freely, the structural changes that make the blood-brain barrier leaky and result in inflammation of brain tissues in old mice can be mitigated. That suggests that there are also beneficial effects of exercise on dementia in humans.**

Further work will be required to establish the mechanism(s): what is the role of the complement-producing microglia/macrophages, how does Apoe decline contribute to age-related neurovascular decline, does the leaky blood-brain barrier allow the passage of damaging factors from the circulation into the brain?

This work was funded in part by The Jackson Laboratory Nathan Shock Center, the Fraternal Order of the Eagle, the Jane B Cook Foundation and NIH.

* The authors investigated the changes in the brains of normal young and aged laboratory mice by comparing by their gene expression profiles using a technique called RNA sequencing, and by comparing their structures at high-resolution by using fluorescence microscopy and electron microscopy. The gene expression analysis indicated age-related changes in the expression of genes relevant to vascular function (including focal adhesion, vascular smooth muscle and ECM-receptor interactions), and inflammation (especially related to the complement system, which clears foreign particles) in the brain cortex.

These changes were accompanied by a decline in the function of astrocytes (key support cells in the brain) and loss of pericytes (the contractile cells that surround small capillaries and venules and maintain the blood-brain barrier). There were also effects on the basement membrane, which forms an integral part of the blood-brain barrier, as well as an increase in the density and functional activation of the immune cells known as microglia/monocytes, which scavenge the brain for infectious agents and damaged cells.

** To investigate the impact of long-term physical exercise on the brain changes seen in the aging mice, the researchers provided the animals with a running wheel from 12 months old (equivalent to middle aged in humans) and assessed their brains at 18 months (equivalent to ~60yrs old in humans, when the risk of Alzheimer’s disease is greatly increased). Young and old mice alike ran about two miles per night, and this physical activity improved the ability and motivation of the old mice to engage in the typical spontaneous behaviors that seem to be affected by aging.

This exercise significantly reduced age-related pericyte loss in the brain cortex and improved other indicators of dysfunction of the vascular system and blood-brain barrier. Exercise also decreased the numbers of microglia/monocytes expressing a crucial initiating component of the complement pathway that others have shown previously to play are role in age-related cognitive decline. Interestingly, these beneficial effects of exercise were not seen in mice deficient in a gene called Apoe, variants of which are a major genetic risk factor for Alzheimer’s disease. The authors also report that Apoe expression in the brain cortex declines in aged mice and this decline can also be prevented by exercise.


Abstract of APOE Stabilization by Exercise Prevents Aging Neurovascular Dysfunction and Complement Induction

Aging is the major risk factor for neurodegenerative diseases such as Alzheimer’s disease, but little is known about the processes that lead to age-related decline of brain structures and function. Here we use RNA-seq in combination with high resolution histological analyses to show that aging leads to a significant deterioration of neurovascular structures including basement membrane reduction, pericyte loss, and astrocyte dysfunction. Neurovascular decline was sufficient to cause vascular leakage and correlated strongly with an increase in neuroinflammation including up-regulation of complement component C1QA in microglia/monocytes. Importantly, long-term aerobic exercise from midlife to old age prevented this age-related neurovascular decline, reduced C1QA+ microglia/monocytes, and increased synaptic plasticity and overall behavioral capabilities of aged mice. Concomitant with age-related neurovascular decline and complement activation, astrocytic Apoe dramatically decreased in aged mice, a decrease that was prevented by exercise. Given the role of APOE in maintaining the neurovascular unit and as an anti-inflammatory molecule, this suggests a possible link between astrocytic Apoe, age-related neurovascular dysfunction and microglia/monocyte activation. To test this, Apoe-deficient mice were exercised from midlife to old age and in contrast to wild-type (Apoe-sufficient) mice, exercise had little to no effect on age-related neurovascular decline or microglia/monocyte activation in the absence of APOE. Collectively, our data shows that neurovascular structures decline with age, a process that we propose to be intimately linked to complement activation in microglia/monocytes. Exercise prevents these changes, but not in the absence of APOE, opening up new avenues for understanding the complex interactions between neurovascular and neuroinflammatory responses in aging and neurodegenerative diseases such as Alzheimer’s disease.

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