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Posts Tagged ‘neurodegenerative diseases’


The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

 

On 9/25/2017, Aviva Lev-Ari, PhD, RN commissioned Dr. Larry H. Bernstein to write a short article on the following topic reported on 9/22/2017 in sciencemission.com

 

We are publishing, below the new article created by Larry H. Bernstein, MD, FCAP.

 

Background

During the period between 9/2015  and 6/2017 the Team at Leaders in Pharmaceutical Business Intelligence (LPBI)  has launched an R&D effort lead by Aviva Lev-Ari, PhD, RN in conjunction with SBH Sciences, Inc. headed by Dr. Raphael Nir.

This effort, also known as, “DrugDiscovery @LPBI Group”  has yielded several publications on EXOSOMES on this Open Access Online Scientific Journal. Among them are included the following:

 

QIAGEN – International Leader in NGS and RNA Sequencing, 10/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

cell-free DNA (cfDNA) tests could become the ultimate “Molecular Stethoscope” that opens up a whole new way of practicing Medicine, 09/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

Detecting Multiple Types of Cancer With a Single Blood Test (Human Exomes Galore), 07/02/2017

Reporter and Curator: Irina Robu, PhD

 

Exosomes: Natural Carriers for siRNA Delivery, 04/24/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

One blood sample can be tested for a comprehensive array of cancer cell biomarkers: R&D at WPI, 01/05/2017

Curator: Marzan Khan, B.Sc

 

SBI’s Exosome Research Technologies, 12/29/2016

Reporter: Aviva Lev-Ari, PhD, RN

 

A novel 5-gene pancreatic adenocarcinoma classifier: Meta-analysis of transcriptome data – Clinical Genomics Research @BIDMC, 12/28/2016

Curator: Tilda Barliya, PhD

 

Liquid Biopsy Chip detects an array of metastatic cancer cell markers in blood – R&D @Worcester Polytechnic Institute, Micro and Nanotechnology Lab, 12/28/2016

Reporters: Tilda Barliya, PhD and Aviva Lev-Ari, PhD, RN

 

Exosomes – History and Promise, 04/28/2016

Reporter: Aviva Lev-Ari, PhD, RN

 

Exosomes, 11/17/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Liquid Biopsy Assay May Predict Drug Resistance, 11/16/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Glypican-1 identifies cancer exosomes, 10/31/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Circulating Biomarkers World Congress, March 23-24, 2015, Boston: Exosomes, Microvesicles, Circulating DNA, Circulating RNA, Circulating Tumor Cells, Sample Preparation, 03/24/2015

Reporter: Aviva Lev-Ari, PhD, RN

 

Cambridge Healthtech Institute’s Second Annual Exosomes and Microvesicles as Biomarkers and Diagnostics Conference, March 16-17, 2015 in Cambridge, MA, 03/17, 2015

Reporter: Aviva Lev-Ari, PhD, RN

 

The newly created think-piece on the relationship between regulatory functions of Exosomes and Metabolic processes is developed conceptually, below.

 

The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

We have had more than a half century of research into the genetic code and transcription leading to abundant work on RNA and proteomics. However, more recent work in the last two decades has identified RNA interference in siRNA. These molecules may be found in the circulation, but it has been a challenge to find their use in therapeutics. Exosomes were first discovered in the 1980s, but only recently there has been a huge amount of research into their origin, structure and function. Exosomes are 30–120 nm endocytic membrane-bound extracellular vesicles (EVs)(1-23) , and more specifically multiple vesicle bodies (MVBs) by a budding process from invagination of the outer cell membrane that carry microRNA (miRNA), and have structures composed of protein and lipids (1,23-27 ). EVs are the membrane vesicles secreted by eukaryotic cells for intracellular communication by transferring the proteins, lipids, and RNA under various physiologic conditions as well as during the disease stage. EVs also act as a signalosomes in many biological processes. Inward budding of the plasma membrane forms small vesicles that fuse. Intraluminal vesicles (ILVs) are formed by invagination of the limiting endosomal membrane during the maturation process of early endosome.

EVs are the MVBs secreted that serve in intracellular communication by transferring a cargo consisting of proteins, lipids, and RNA under various physiologic conditions (4, 23). Exosome-mediated miRNA transfer between cells is considered to be necessary for intercellular signaling and exosome-associated miRNAs in biofluids (23). Exosomes carry various molecular constituents of their cell of origin, including proteins, lipids, mRNAs, and microRNAs (miRNAs) (. They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, hydrothoracic fluid, and ascitic fluid, as well as in culture medium of most cell types.Exosomes have also been shown to be involved in noncoding RNA surveillance machinery in generating antibody diversity (24). There are also a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) that accumulate R-loop structures upon RNA exosome ablation, thereby, resolving deleterious DNA/RNA hybrids arising from active enhancers and distal divergent eRNA-expressing elements (lncRNA-CSR) engaged in long-range DNA interactions (25). RNA exosomes are large multimeric 3′-5′ exo- and endonucleases representing the central RNA 3′-end processing factor and are implicated in processing, quality control, and turnover of both coding and noncoding RNAs. They are large macromolecular cages that channel RNA to the ribonuclease sites (29). A major interest has been developed to characterize of exosomal cargo, which includes numerous non-randomly packed proteins and nucleic acids (1). Moreover, exosomes play an active role in tumorigenesis, metastasis, and response to therapy through the transfer of oncogenes and onco-miRNAs between cancer cells and the tumor stroma. Blood cells and the vascular endothelium is also exosomal shedding, which has significance for cardiovascular,   neurologicological disorders, stroke, and antiphospholipid syndrome (1). Dysregulation of microRNAs and the affected pathways is seen in numerous pathologies their expression can reflect molecular processes of tumor onset and progression qualifying microRNAs as potential diagnostic and prognostic biomarkers (30).

Exosomes are secreted by many cells like B lymphocytes and dendritic cells of hematopoietic and non-hematopoietic origin viz. platelets, Schwann cells, neurons, mast cells, cytotoxic T cells, oligodendrocytes, intestinal epithelial cells were also found to be releasing exosomes (4). They are engaged in complex functions like persuading immune response as the exosomes secreted by antigen presenting cells activate T cells (4). They all have a common set of proteins e.g. Rab family of GTPases, Alix and ESCRT (required for transport) protein and they maintain their cytoskeleton dynamics and participate in membrane fusion. However, they are involved in retrovirus disease pathology as a result of recruitment of the host`s endosomal compartments in order to generate viral vesicles, and they can either spread or limit an infection based on the type of pathogen and its target cells (5).

Upon further consideration, it is understandable how this growing biological work on exosomes has enormous significance for laboratory diagnostics (1, 3, 5, 6, 11, 14, 15, 17-20, 23,30-41) . They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, thoracic and abdominal effusions, and ascitic fluid (1). The involvement of exosomes in disease is broad, and includes: cancer, autoimmune and infectious disease, hematologic disorders, neurodegenerative diseases, and cardiovascular disease. Proteins frequently identified in exosomes include membrane transporters and fusion proteins (e.g., GTPases, annexins, and flotillin), heat shock proteins (e.g., HSC70), tetraspanins (e.g., CD9, CD63, and CD81), MVB biogenesis proteins (e.g., alix and TSG101), and lipid-related proteins and phospholipases. The exosomal lipid composition has been thoroughly analyzed in exosomes secreted from several cell types including DCs and mast cells, reticulocytes, and B-lymphocytes (1). Dysregulation of microRNAs of pathways observed in numerous pathologies (5, 10, 12, 21, 27, 35, 37) including cancers (30), particularly, colon, pancreas, breast, liver, brain, lung (2, 6, 17-20, 30, 33-36, 38, 39). Following these considerations, it is important that we characterize the content of exosomal cargo to gain clues to their biogenesis, targeting, and cellular effects which may lead to identification of biomarkers for disease diagnosis, prognosis and response to treatment (42).

We might continue in pursuit of a particular noteworthy exosome, the NLRP3 inflammasome, which is activated by a variety of external or host-derived stimuli, thereby, initiating an inflammatory response through caspase-1 activation, resulting in inflammatory cytokine IL-1b maturation and secretion (43).
Inflammasomes are multi-protein signaling complexes that activate the inflammatory caspases and the maturation of interleukin-1b. The NLRP3 inflammasome is linked with human autoinflammatory and autoimmune diseases (44). This makes the NLRP3 inflammasome a promising target for anti-inflammatory therapies. The NLRP3 inflammasome is activated in response to a variety of signals that indicate tissue damage, metabolic stress, and infection (45). Upon activation, the NLRP3 inflammasome serves as a platform for activation of the cysteine protease caspase-1, which leads to the processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Heritable and acquired inflammatory diseases are both characterized by dysregulation of NLRP3 inflammasome activation (45).
Receptors of innate immunity recognize conserved moieties associated with either cellular damage [danger-associated molecular patterns (DAMPs)] or invading organisms [pathogen-associated molecular patterns (PAMPs)](45). Either chronic stimulation or overwhelming tissue damage is injurious and responsible for the pathology seen in a number of autoinflammatory and autoimmune disorders, such as arthritis and diabetes. The nucleotide-binding domain leucine-rich repeat (LRR)-containing receptors (NLRs) are PRRs are found intracellularly and they share a unique domain architecture. It consists of a central nucleotide binding and oligomerization domain called the NACHT domain that is located between an N-terminal effector domain and a C-terminal LRR domain (45). The NLR family members NLRP1, NLRP3, and NLRC4 are capable of forming multiprotein complexes called inflammasomes when activated.

The (NLRP3) inflammasome is important in chronic airway diseases such as asthma and chronic obstructive pulmonary disease because the activation results, in pro-IL-1β processing and the secretion of the proinflammatory cytokine IL-1β (46). It has been proposed that Activation of the NLRP3 inflammasome by invading pathogens may prove cell type-specific in exacerbations of airway inflammation in asthma (46). First, NLRP3 interacts with the adaptor protein ASC by sensing microbial pathogens and self-danger signals. Then pro-caspase-1 is recruited and the large protein complex called the NLRP3 inflammasome is formed. This is followed by autocleavage and activation of caspase-1, after which pro-IL-1β and pro-IL-18 are converted into their mature forms. Ion fluxes disrupt membrane integrity, and also mitochondrial damage both play key roles in NLRP3 inflammasome activation (47). Depletion of mitochondria as well as inhibitors that block mitochondrial respiration and ROS production prevented NLRP3 inflammasome activation. Futhermore, genetic ablation of VDAC channels (namely VDAC1 and VDAC3) that are located on the mitochondrial outer membrane and that are responsible for exchanging ions and metabolites with the cytoplasm, leads to diminished mitochondrial (mt) ROS production and inhibition of NLRP3 inflammasome activation (47). Inflammasome activation not only occurs in immune cells, primarily macrophages and dendritic cells, but also in kidney cells, specifically the renal tubular epithelium. The NLRP3 inflammasome is probably involved in the pathogenesis of acute kidney injury, chronic kidney disease, diabetic nephropathy and crystal-related nephropathy (48). The inflammasome also plays a role in autoimmune kidney disease. IL-1 blockade and two recently identified specific NLRP3 inflammasome blockers, MCC950 and β-hydroxybutyrate, may prove to have value in the treatment of inflammasome-mediated conditions.

Autophagosomes derived from tumor cells are referred to as defective ribosomal products in blebs (DRibbles). DRibbles mediate tumor regression by stimulating potent T-cell responses and, thus, have been used as therapeutic cancer vaccines in multiple preclinical cancer models (49). It has been found that DRibbles could induce a rapid differentiation of monocytes and DC precursor (pre-DC) cells into functional APCs (49). Consequently, DRibbles could potentially induce strong innate immune responses via multiple pattern recognition receptors. This explains why DRibbles might be excellent antigen carriers to induce adaptive immune responses to both tumor cells and viruses. This suggests that isolated autophagosomes (DRibbles) from antigen donor cells activate inflammasomes by providing the necessary signals required for IL-1β production.

The Hsp90 system is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function (50). The co-chaperones enable Hsp90 to chaperone structurally and functionally diverse client proteins. Sahasrabudhe et al. (50) show that the nature of the client protein dictates the contribution of a co-chaperone to its maturation. The study reveals the general importance of the cochaperone Sgt1 (50). In addition to Hsp90, we have to consider Hsp60. Adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins (21).

A new Protein Organic Solvent Precipitation (PROSPR) method efficiently isolates the EV repertoire from human biological samples. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes. In addition lipidomic characterization of enriched CNS vesicles identified previously reported EV-specific lipid families and novel lipid isoforms not previously detected in human EVs. The characterization of these structures from central nervous system (CNS) tissues is relevant to current neuroscience, especially to advance the understanding of neurodegeneration in amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD)(15). In addition, study of EVs in brain will enable characterization of the degenerative posttranslational modifications (DPMs) occurring in those proteins.
Neurodegenerative disease is characterized by dysregulation because of NLRP3 inflammasome activation. Alzheimer’s disease (AD) and Parkinson’s disease (PD), both neurodegenerative diseases are associated with the NLRP3 inflammasome. PD is characterized by accumulation of Lewy bodies (LB) formed by a-synuclein (aSyn) aggregation. A recent study revealed that aSyn induces synthesis of pro-IL-1b by an interaction with TLR2 and activates NLRP3 inflammasome resulting in caspase-1 activation and IL-1b maturation in human primary monocytes (43). In addition mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals. It is notable that in this aberrant activation mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals (43).

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Electronic Proceedings for 10th US-India BioPharma & Healthcare Summit, June 2, 2016, Marriott Cambridge, MA

 

Curator: Aviva Lev-Ari, PhD, RN

 

LIVE — 9AM-noon US-India BioPharma & Healthcare Summit, June 2, 2016, Marriott Cambridge, MA

https://pharmaceuticalintelligence.com/2016/06/02/live-9am-noon-us-india-biopharma-healthcare-summit-june-2-2016-marriott-cambridge-ma/

 

LIVE 11:45AM – 2:40PM US-India BioPharma & Healthcare Summit, June 2, 2016, Marriott Cambridge, MA

https://pharmaceuticalintelligence.com/2016/06/02/1145am-240pm-us-india-biopharma-healthcare-summit-june-2-2016-marriott-cambridge-ma/

 

LIVE 3:15PM – 5:00PM US-India BioPharma & Healthcare Summit, June 2, 2016, Marriott Cambridge, MA

https://pharmaceuticalintelligence.com/2016/06/02/315pm-500pm-us-india-biopharma-healthcare-summit-june-2-2016-marriott-cambridge-ma/

 

 

 

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LIVE — 9AM-noon US-India BioPharma & Healthcare Summit, June 2, 2016, Marriott Cambridge, MA

ANNOUNCEMENT

Leaders in Pharmaceutical Business Intelligence (LPBI) Group

will cover in Real Time using Social Media the

10th US-India BioPharma & Healthcare Summit,

June 2, 2016

Aviva Lev-Ari, PhD, RN will be streaming LIVE from the

Marriott Cambridge, MA

 

@USAIC

#USAIC2016

@pharma_BI

@AVIVA1950

SPEAKERS

http://usaindiachamber.org/current-events.shtml

9-15 AM – 9-30 AM Welcome address by Karun Rishi, President, USA-India Chamber of Commerce

Progress made in last two years, we need faster advancement. Thanks to all attendees and those who came for far away.

Opening comments
by Master of Ceremonies – Dr Andrew Plump, Chief Medical & Scientific Officer, Member of the Board of Directors, Takeda Pharmaceuticals

Biomedical field, important issues are covered today. Karun is a Force of Nature. Guests from India, welcome.

Innovation in BioTech and understanding Disease is exploding

  • Ability to attack disease is amazing
  • Pipelines synthetic, small molecule – THE Past — today new unconventional therapies

India’s role:

  • Innovation space in India – diversification in modalities
  • Partnerships
  • # of Rounds in Financing: 67 new start ups in 2015 in the US – Academic Start Ups
  • Models in India: we need to figure out which types will work best in India

Takeda and Japan

  • Strong academic science
  • rich history of productivity in R&D
  • Commercialization of R&D is not strong in Japan
  • Institute outside of Tokyo: Academia and Industry collaboration
9-30 AM – 9-55 AM India Regulatory and Clinical Research Update
K.L. Sharma, IAS, Joint Secretary, Ministry of Health and Family Welfare, Government of India

  • Ministry of Health and Family Welfare is keen to improve and progress
  • Federal funds will be distributed to 60 States for Reform, Actions
  • Good Laboratories Practices, Manufacturing Practices Processes, CMP, GMP,
  • Risk assessment based on data from Manufacturing and inspection
  • Regulatory aspects and validation – largest in the World quality effort of manufacturing, August 2016 – results will be sahred
  • Medical Devices: ISO – extensive cooperation to amend the Law and add New Lawsto provide compeling reason for rules and standards in Medical Devices and Prostesis
  • Biologics, Stem Cells
  • Collaboration between Medical Institutions, Academic Institution
  • Recruiting from industry to the Government
  • Toxicology studies
  • Acredidation process
  • Put in Public Domain: Information on products, all new regulation
  • International hamonization

Questions from the Podium

    • Academia: Collaboration
    • J-J: IF the doors are open – How we can connect to develop relations
    • How International hamonization can take place with each of the States
  • Committes from Laboratories, Central Govenrment and Industry
    • US -India collaborations: Improvements on the way
9-55 AM – 10-45 AM Panel Discussion: Neurodegenerative diseases – Matters of the mind

Moderator:
Dr. Ole Isacson, Professor of Neurology, Harvard Medical School

The most difficult field in Medicine, opportunities in Academia and in Industry — in last ten years these two groups merged in interest to solve the problem, Genetics work with Neuro to develop drugs, pathology and Neurologist discussions associations with immunology and oncology

  • 5 Millions in the US affected by Neurological Diseases
  • Role of Stem Cells
  • gene therapies

Panelists:

  • 5 – Dr. John Dunlop, Vice President & Head, Neuroscience, AstraZeneca
  1. Clinical Studies  – data on antibodies need to be sahred AZ invested in Amyloid Hypothesis
  2. ALS, MS, Parkinson
  • 3 – Dr. Douglas Feltner
  1. Antibodies made  – cause fight with inflammation
  2. MS, Parkinson, AZ – very complex diseases
  3. Longitudinal changes,
  • 4 – Philippe Lopes-Fernandes, Senior Vice-President, Merck KGaA

Adherence with medicine and treatment cause of 15 years

  • 2 – Dr. Alfred Sandrock, Executive Vice President and Chief Medical Officer, Biogen

DO belief in Beta-Amyloid hypothesis is causal hypothesis, In early patients – reduction of plaque by the drug very quickly, Early CLinical Trial, progression monitoring, TAU present and spread beyond temporal lobe, microglial cells can be protected, helpful to preserve neurons,

  1. Need to understand Pathways,
  2. know how to mitigate risk along the way, reduce risk of investment in a disease solution may not come by instead of investment on a drug for a disease curalble NEURO is more difficult
  3. How Indian Scientists can particiapte
  4. even with inheretance, protected by other factors
  5. Intracellular proteins
  • 1 – Dr. Rudolph Tanzi, Director, Genetics and Aging Research Unit, Mass General Hospital

Biology of Alzheimer’s Disease: Loss of synapses, 4 genes, Amyloid Hypothesis – debatable, head-concussions, genes for inflammation, Human models, C1-2, diet, Statin, APO-4 E2, E3 – lipid componenet – role in amyloid transport

  1. Cross -pathologies disease specifics
  2. mutations that protect us, plaque is present NO Ad, resilience
  3. Brain-Microbiome: Infections in th eBrain

Questions from the Floor

  • Etiology
  • Biomarkers
10-45 AM – 11-15 AM Fireside Chat with

  • Dr David Meeker, Head, Sanofi Genzyme

 

  1. How we build value
  2. specialty care business  – systemic growth
  3. Where is the probability the highest?
  4. Pay to Play – a start not an end game
  5. R&D effert internally must be very strong before acquision are to take place
  6. ecosystem is critically important
  7. Resource allocations remains important in strategy

 

  • Dilip Shanghvi, Managing Director, Sun Pharmaceutical Industries Limited – India based

 

  1. How innovations in India can impact positively the Cost of HealthCare
  2. 4 products at different stages
  3. Partnerships wiht International companies are needed in areas the expertise is not enough
  4. 50% of products are for international markets
  5. consistency and scalability
  6. Partnerships with Biotech are important for growth – joint value for bigger position
  7. In our strategy I am Seeking
  • partner in oncology,
  • other drugs to use out technology for drug delivery,
  • excaplulate our expertise in exosome lipidsome for other targets
  • Proof of Concept transfer to products the process id lengthy
  • BioSimilar – BioPharma — important sector for Pharma
  • Fear of failure

Moderator:
Dr. Raju Kucherlapati, Professor of Genetics, Harvard Medical School

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Neuroscience impact of synaptic pruning discovery

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Synaptic Pruning Discovery May Lead to New Therapies for Neuro Disorders

GEN 3 May, 2016    http://www.genengnews.com/gen-news-highlights/synaptic-pruning-discovery-may-lead-to-new-therapies-for-neuro-disorders/81252680/

Source: NIH      http://www.genengnews.com/Media/images/GENHighlight/thumb_May3_2016_NIH_CRANPuzzleBrain_AdolescentBrain2247219834.jpg

 

A research team led by scientists at SUNY Downstate Medical Center has identified a brain receptor that appears to initiate adolescent synaptic pruning, a process believed necessary for learning, but one that appears to go awry in both autism and schizophrenia.

Sheryl Smith, Ph.D., professor of physiology and pharmacology at SUNY Downstate, explained that “Memories are formed at structures in the brain known as dendritic spines that communicate with other brain cells through synapses. The number of brain connections decreases by half after puberty, a finding shown in many brain areas and for many species, including humans and rodents.”

This process is referred to as adolescent “synaptic pruning” and is thought to be important for normal learning in adulthood. Synaptic pruning is believed to remove unnecessary synaptic connections to make room for relevant new memories, but because it is disrupted in diseases such as autism and schizophrenia, there has recently been widespread interest in the subject.

“Our report is the first to identify the process which initiates synaptic pruning at puberty. Previous studies have shown that scavenging by the immune system cleans up the debris from these pruned connections, likely the final step in the pruning process,” added Dr. Smith. “Working with a mouse model we have shown that, at puberty, there is an increase in inhibitory GABA [gamma-aminobutyric acid] receptors, which are targets for brain chemicals that quiet down nerve cells. We now report that these GABA receptors trigger synaptic pruning at puberty in the mouse hippocampus, a brain area involved in learning and memory.”

The study (“Synaptic Pruning in the Female Hippocampus Is Triggered at Puberty by Extrasynaptic GABAA Receptors on Dendritic Spines”) is published online in eLife.

Dr. Smith noted that by reducing brain activity, these GABA receptors also reduce levels of a protein in the dendritic spine, kalirin-7, which stabilizes the scaffolding in the spine to maintain its structure. Mice that do not have these receptors maintain the same high level of brain connections throughout adolescence.

Dr. Smith pointed out that the mice with too many brain connections, which do not undergo synaptic pruning, are able to learn spatial locations, but are unable to relearn new locations after the initial learning, suggesting that too many brain connections may limit learning potential.

These findings may suggest new treatments targeting GABA receptors for “normalizing” synaptic pruning in diseases such as autism and schizophrenia, where synaptic pruning is abnormal. Research has suggested that children with autism may have an over-abundance of synapses in some parts of the brain. Other research suggests that prefrontal brain areas in persons with schizophrenia have fewer neural connections than the brains of those who do not have the condition.

 

Synaptic pruning in the female hippocampus is triggered at puberty by extrasynaptic GABAAreceptors on dendritic spines

Adolescent synaptic pruning is thought to enable optimal cognition because it is disrupted in certain neuropathologies, yet the initiator of this process is unknown. One factor not yet considered is the α4βδ GABAA receptor (GABAR), an extrasynaptic inhibitory receptor which first emerges on dendritic spines at puberty in female mice. Here we show that α4βδ GABARs trigger adolescent pruning. Spine density of CA1 hippocampal pyramidal cells decreased by half post-pubertally in female wild-type but not α4 KO mice. This effect was associated with decreased expression of kalirin-7 (Kal7), a spine protein which controls actin cytoskeleton remodeling. Kal7 decreased at puberty as a result of reduced NMDAR activation due to α4βδ-mediated inhibition. In the absence of this inhibition, Kal7 expression was unchanged at puberty. In the unpruned condition, spatial re-learning was impaired. These data suggest that pubertal pruning requires α4βδ GABARs. In their absence, pruning is prevented and cognition is not optimal.

Searches Related to Synaptic Pruning in the Female Hippocampus Is Triggered at Puberty by Extrasynaptic GABAA Receptors on Dendritic Spines

Optogenetics helps understand what causes anxiety and depression

Researchers at Ruhr University Bochum (RUB; Germany) coupled nerve cell receptors to light-sensitive retinal pigments to understand how the serotonin neurotransmitter works and, therefore, learn what causes anxiety anddepression.

Related: Optogenetics could lead to better understanding of anxiety, depression

Prof. Dr. Olivia Masseck, who led the work, researches the causes of anxiety and depression. For more than 60 years, researchers have been hypothesising that the diseases are caused by, among other factors, changes to the level of serotonin. But understanding how the serotonin system works is quite difficult, says Masseck, who became junior professor for Super-Resolution Fluorescence Microscopy at RUB in April 2016.

With a method called optogenetics, Olivia Masseck (right) creates nerve cell receptors that are controllable with light. (Copyright: RUB, Damian Gorczany)

The number of receptors for serotonin in the brain amounts to 14, occurring in different cell types. Consequently, determining the functions that different receptors fulfill in the individual cell types is a complicated task. If, however, the proteins are coupled to light-sensitive pigments, they can be switched on and off with light of a specific color at high spatial and temporal precision. Masseck used this method, known as optogenetics, to characterize, for example, the properties of different light-sensitive proteins and identified the ones that are best suited as optogenetic tools. She has analyzed several light-sensitive varieties of the serotonin receptors 5-HT1A and 5-HT2C in great detail. Together with her collaborators, she has demonstrated in several studies that both receptors can control the anxiety behavior of mice.

To investigate the serotonin system more closely, Masseck and her research team is currently developing a sensor that is going to indicate the neurotransmitter in real time. One potential approach involves the integration of a modified form of a green fluorescent protein into a serotonin receptor.

In a brain slice, Olivia Masseck measures the activity of nerve cells in which she switches on their receptors using light stimulation. Via the pipette a red dye diffuses into the cell, rendering them visible in the brain slice. (Copyright: RUB, Damian Gorczany)

This protein produces green light only if it is embedded in a specific spatial structure. If a serotonin molecule binds to a receptor, the receptor changes its three-dimensional conformation. The objective is to integrate the fluorescent protein in the receptor so that its spatial structure changes together with that of the receptor when it binds a serotonin molecule, in such a way that the protein begins to glow.

Full details of the work appear in Rubin Science Magazine; for more information, please visithttp://rubin.rub.de/en/controlling-nerve-cells-light.

Controlling nerve cells with light   

New optogenetic tools   by Julia Weiler
Anxiety and depression are two of the most frequently occurring mental disorders worldwide. Light-activated nerve cells may indicate how they are formed.

Statistically, every fifth individual suffers from depression or anxiety in the course of his or her life. The mechanisms that trigger these disorders are not yet fully understood, despite the fact that researchers have been studying the hypothesis that one of the underlying cause are changes to the level of the neurotransmitter serotonin for 60 years.

“Unfortunately, it is very difficult to understand how the serotonin system works,” says Prof Dr Olivia Masseck, who is junior professor for Super-Resolution Fluorescence Microscopy since the end of April 2016. She  intends to fathom the mysteries of the complex system. The number of receptors for the neurotransmitter in the brain amounts to 14 in total, and they occur in different cell types. Consequently, determining the functions that different receptors fulfil in the individual cell types is a complicated task.

In order to fathom the purpose of such receptors, researchers used to observe which functions were inhibited after they had been activated or blocked with the aid of pharmaceutical drugs. However, many substances affect not just one receptor, but several at the same time. Moreover, researchers cannot tell receptors in the individual cell types apart when pharmaceutical drugs have been applied. “It had been impossible to study serotonin signalling pathways at high spatial and temporal resolution,” adds Masseck. Until the development of optogenetics.

“This method has revolutionised neuroscience,” says Olivia Masseck, whose collaborator Prof Dr Stefan Herlitze was one of the pioneers in this field. Optogenetics allows precise control over the activity of specific nerve cells or receptors with light. What sounds like science fiction, is routine at RUB’s Neuroscience Research Department. Masseck: “Until now, we had been passive observers, and monitoring cell activity was all we could do; now, we are able to manipulate it precisely.”

The researcher from Bochum is mainly interested in the 5-HT1A and 5-HT1B receptors, the so-called autoreceptors of the serotonin system. They occur in serotonin-producing cells, where they regulate the amount of released neurotransmitters; that means they determine the serotonin level in the brain.

Normally, 5-HT1A and 5-HT1B are activated when a serotonin molecule bonds to the receptor. The docking triggers a chain reaction in the cell. The effects of this signalling cascade include a reduced activity of the neural cell, which releases less neurotransmitter.

By modifying certain brain cells in the brains of mice, Olivia Masseck successfully activated the 5-HT1Areceptor without the aid of serotonin. She combined it with a visual pigment – so-called opsin. More specifically, she utilised blue or red visual pigments from the cones responsible for colour vision. This is how she generated a serotonin receptor that she could switch on with red or blue light. This method enables the RUB researcher to identify the role the 5-HT1A receptor plays in anxiety and depression.

To this end, she delivered the combined protein made up of light-sensitive opsin and serotonin receptor into the brain of mice using a virus that had been rendered harmless. Like a shuttle, it transports genetic information which contains the blueprint for the combined protein. Once injected into brain tissue, the virus implants the gene for the light-activated receptor in specific nerve cells. There, it is read, and the light-activated receptor is incorporated into the cell membrane.

The researcher was now able to switch the receptors on and off in a living mouse using light. She analysed in what way this manipulation affected the animals’ behaviour in an anxiety test, i.e. Open Field Test. For the purpose of the experiment, she placed individual mice in a large, empty Plexiglas box.

Under normal circumstances, the animals avoid the centre of the brightly-lit box, because it doesn’t offer any cover. Most of the time, they stay close to the walls. When Olivia Masseck switched on the 5-HT1Areceptor using light, the behaviour of the mice changed. They were less anxious and spent more time in the middle of the Plexiglas box.

These results were confirmed in a further test. Olivia Masseck stopped the time it took the mice to eat a food pellet in the middle of a large Plexiglas box. Normal animals waited between six and seven minutes before they ventured into the centre to feed. However, mice whose serotonin receptor was switched on started to feed after one or two minutes. “This is important evidence indicating that the 5-HT1A receptor signalling pathway in the serotonin system is linked to anxiety,” concludes Masseck.

In the next step, the researcher intends to find out in what way depressive behaviour is affected by the activation of the 5-HT1A receptor. “If the animals are exposed to chronic stress, they develop symptoms similar to those in humans with depression,” describes Masseck. “They might, for example, withdraw from social interactions.”

However, just like in humans, this applies to only a certain percentage of the mice. “Not every individual who suffers from chronic stress or experiences negative situations develops depression,” points out Masseck. What happens in the serotonin system of animals that are susceptible to depression, as opposed to that of animals that do not present any depressive symptoms? This is what the researcher intends to find out by deploying the optogenetic methods described above; in addition, she is currently developing a custom-built serotonin sensor.

Olivia Masseck’s assumption is that her findings regarding the neuronal circuits and molecular mechanisms of anxiety and depression are applicable to humans. Mice have similar cell functions, and their nervous system has a similar structure. The neuroscientist expects that optogenetics will one day be deployed in human applications.

“Genetic manipulation of cells for the purpose of controlling them with light might sound like science fiction,” she says, “but I am convinced that optogenetics will be used in human applications in the next decades.” It could, for example, be utilised for deep brain stimulation in Parkinson’s patients, because it facilitates precise activation of the required signalling pathways, with fewer side effects, at that.

“In the first step, optogenetics will be used in therapy of retinal diseases,” believes Olivia Masseck. Researchers are currently conducting experiments aiming at restoring the visual function in blind mice.

Olivia Masseck is aware that her research raises ethical questions. “We have to discuss in which applications we want or don’t want to use these techniques,” she says. Her research demonstrates how easily the lines between science-fiction films and scientific research can blur.

Detect cancer hallmarks with targeted fluorescent probes.  

The smart iABP™ targeted imaging probes are based on cysteine cathepsin activity, which are highly expressed in tumor and tumor-associated cells of numerous cancers. Additionally, cathepsins are consistently expressed across multiple tumor types compared to other affinity based probes such as integrin or MMPs, which are inconsistently expressed.

http://www.vergentbio.com/hs-fs/hubfs/Banners/Activity-based-probes-cancer2.jpg

The small molecule iABP™ probes are based on smart, activatable technology:

  • Penetrates tumor tissues quickly
  • Gives you superior target localization and retention at the proteolytic site
  • only fluorescences once it’s bound to the active target giving extremely low background fluorescence and high signal to noise ratio
  • Eliminates any need to wash prior to staining cells or tissue in ex vivo analysis.

The probes are suitable for use in non-invasive small animal imaging studies, live cell imaging, fluorescence microscopy, flow cytometry and SDS-PAGE applications.

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Neuron clearing with age

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Brain Guardians Remove Dying Neurons

Salk scientists show how immune receptors clear dead and dysfunctional brain cells and how they might be targets for treating neurodegenerative diseases

By Salk Institute for Biological Studies

By adolescence, your brain already contains most of the neurons that you’ll have for the rest of your life. But a few regions continue to grow new nerve cells—and require the services of cellular sentinels, specialized immune cells that keep the brain safe by getting rid of dead or dysfunctional cells.

Now, Salk scientists have uncovered the surprising extent to which both dying and dead neurons are cleared away, and have identified specific cellular switches that are key to this process. The work was detailed in Nature on April 6, 2016.

https://youtu.be/bevh2BSuI0U

Video courtesy of the Salk Institute

“We discovered that receptors on immune cells in the brain are vital for both healthy and injured states,” says Greg Lemke, senior author of the work, a Salk professor of molecular neurobiology and the holder of the Françoise Gilot-Salk Chair. “These receptors could be potential therapeutic targets for neurodegenerative conditions or inflammation-related disorders, such as Parkinson’s disease.”

death in the brain

http://www.labmanager.com/media/Industry%20News%20Pics/April-2016/apr7-2016-salk-1-Death-in-the-brain.jpg

An accumulation of dead cells (green spots) is seen in the subventricular zone (SVZ)—a neurogenic region—of the brain in a mouse lacking the receptors Mer and Axl. (Blue staining marks all cells.) No green spots are seen in the SVZ from a normal mouse. IMAGE CREDIT: SALK INSTITUTE

Two decades ago, the Lemke lab discovered that immune cells express critical molecules called TAM receptors, which have since become a focus for autoimmune and cancer research in many laboratories. Two of the TAM receptors, dubbed Mer and Axl, help immune cells called macrophages act as garbage collectors, identifying and consuming the over 100 billion dead cells that are generated in a human body every day.

For the current study, the team asked if Mer and Axl did the same job in the brain. Specialized central nervous system macrophages called microglia make up about 10 percent of cells in the brain, where they detect, respond to and destroy pathogens. The researchers removed Axl and Mer in the microglia of otherwise healthy mice. To their surprise, they found that the absence of the two receptors resulted in a large pile-up of dead cells, but not everywhere in the brain. Cellular corpses were seen only in the small regions where the production of new neurons—neurogenesis—is observed.

Many cells die normally during adult neurogenesis, but they are immediately eaten by microglia. “It is very hard to detect even a single dead cell in a normal brain, because they are so efficiently recognized and cleared by microglia,” says Paqui G. Través, a co-first author on the paper and former Salk research associate. “But in the neurogenic regions of mice lacking Mer and Axl, we detected many such cells.”

When the researchers more closely examined this process by tagging the newly growing neurons in mice’s microglia missing Mer and Axl, they noticed something else interesting. New neurons that migrate to the olfactory bulb, or smell center, increased dramatically without Axl and Mer around. Mice lacking the TAM receptors had a 70 percent increase in newly generated cells in the olfactory bulb than normal mice.

https://youtu.be/jLAnUtCBUtU

Video courtesy of the Salk Institute

How—and to what extent—this unchecked new neural growth affects a mouse’s sense of smell is not yet known, according to Lemke, though it is an area the lab will explore. But the fact that so many more living nerve cells were able to migrate into the olfactory bulb in the absence of the receptors suggests that Mer and Axl have another role aside from clearing dead cells—they may actually also target living, but functionally compromised, cells.

“It appears as though a significant fraction of cell death in neurogenic regions is not due to intrinsic death of the cells but rather is a result of the microglia themselves, which are killing a fraction of the cells by engulfment,” says Lemke. “In other words, some of these newborn neuron progenitors are actually being eaten alive.”

This isn’t necessarily a bad thing in the healthy brain, Lemke adds. The brain produces more neurons than it can use and then prunes back the cells that aren’t needed. However, in an inflamed or diseased brain, the destruction of living cells may backfire.

Greg Lemke and Lawrence Fourgeaud

Greg Lemke and Lawrence Fourgeaud PHOTO CREDIT: SALK INSTITUTE    http://www.labmanager.com/media/Industry%20News%20Pics/April-2016/apr7-2016-salk-2-Greg-Lemke_Lawrence-Fourgeaud.jpg

The Lemke lab did one more series of experiments to understand the role of TAM receptors in disease: they looked at the activity of Axl and Mer in a mouse model of Parkinson’s disease. This model produces a human protein present in an inherited form of the disease that results in a slow degeneration of the brain. The team saw that Axl was far more active in this setting, consistent with other studies showing that increased Axl is a reliable indicator of inflammation in tissues.

the area of a brain lacking Mer and Axl

http://www.labmanager.com/media/Industry%20News%20Pics/April-2016/apr7-2016-salk-3-Trail-of-death-covercropped.jpg

In the area of a brain lacking Mer and Axl a ‘trail of death’ is apparent from the migratory pathway from the neurogenic region to the olfactory bulb (smell center of the brain). Blue staining marks all cells, and green spots are dead cells. No green spots are seen in the same section from a normal mouse. IMAGE CREDIT: SALK INSTITUTE

“It seems that we can modify the course of the disease in an animal model by manipulating Axl and Mer,” says Lawrence Fourgeaud, a co-first author on the paper and former Salk research associate. The team cautions that more research needs to be done to determine if modulating the TAM receptors could be a viable therapy for neurodegenerative disease involving microglia.

Other researchers on the paper were Yusuf Tufail, Humberto Leal-Bailey, Erin D. Lew, Patrick G. Burrola, Perri Callaway, Anna Zagórska and Axel Nimmerjahn of the Salk Institute; and Carla V. Rothlin of the Yale University School of Medicine.

The work was supported by the National Institutes of Health, the Leona M. and Harry B. Helmsley Charitable Trust, the Howard Hughes Medical Institute, and the NomisH.N. and Frances C. Berger, Fritz B. Burns, HKT, WaittRita Allen, and Hearst foundations.

Related Article: How Neurons Lose Their Connections

Related Article: Beer Compound Could Help Fend Off Alzheimer’s and Parkinson’s Diseases

 

TAM receptors regulate multiple features of microglial physiology

Lawrence FourgeaudPaqui G. TravésYusuf TufailHumberto Leal-Bailey, …., Axel Nimmerjahn Greg Lemke
Nature 532:240–244 (14 April 2016).     http://dx.doi.org:/10.1038/nature17630

Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for routine non-inflammatory clearance of dead brain cells1. Here we show that the TAM receptor tyrosine kinases Mer and Axl2 regulate these microglial functions. We find that adult mice deficient in microglial Mer and Axl exhibit a marked accumulation of apoptotic cells specifically in neurogenic regions of the CNS, and that microglial phagocytosis of the apoptotic cells generated during adult neurogenesis3, 4 is normally driven by both TAM receptor ligands Gas6 and protein S5. Using live two-photon imaging, we demonstrate that the microglial response to brain damage is also TAM-regulated, as TAM-deficient microglia display reduced process motility and delayed convergence to sites of injury. Finally, we show that microglial expression of Axl is prominently upregulated in the inflammatory environment that develops in a mouse model of Parkinson’s disease6. Together, these results establish TAM receptors as both controllers of microglial physiology and potential targets for therapeutic intervention in CNS disease.

http://www.nature.com/nature/journal/v532/n7598/carousel/nature17630-f1.jpg

http://www.nature.com/nature/journal/v532/n7598/carousel/nature17630-f2.jpg

http://www.nature.com/nature/journal/v532/n7598/carousel/nature17630-f3.jpg

http://www.nature.com/nature/journal/v532/n7598/carousel/nature17630-sf3.jpg

 

 

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Graphene Interaction with Neurons

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Graphene Shown to Safely Interact with Neurons in the Brain

University of Cambridge

(Source: University of Cambridge)

http://www.biosciencetechnology.com/sites/biosciencetechnology.com/files/bt1601_cambridge_graphene.png

 

Researchers have successfully demonstrated how it is possible to interface graphene – a two-dimensional form of carbon – with neurons, or nerve cells, while maintaining the integrity of these vital cells. The work may be used to build graphene-based electrodes that can safely be implanted in the brain, offering promise for the restoration of sensory functions for amputee or paralyzed patients, or for individuals with motor disorders such as epilepsy or Parkinson’s disease.

The research, published in the journal ACS Nano, was an interdisciplinary collaboration coordinated by the University of Trieste in Italy and the Cambridge Graphene Centre.

Previously, other groups had shown that it is possible to use treated graphene to interact with neurons. However the signal to noise ratio from this interface was very low. By developing methods of working with untreated graphene, the researchers retained the material’s electrical conductivity, making it a significantly better electrode.

“For the first time we interfaced graphene to neurons directly,” said Professor Laura Ballerini of the University of Trieste in Italy. “We then tested the ability of neurons to generate electrical signals known to represent brain activities, and found that the neurons retained their neuronal signaling properties unaltered. This is the first functional study of neuronal synaptic activity using uncoated graphene based materials.”

Our understanding of the brain has increased to such a degree that by interfacing directly between the brain and the outside world we can now harness and control some of its functions. For instance, by measuring the brain’s electrical impulses, sensory functions can be recovered. This can be used to control robotic arms for amputee patients or any number of basic processes for paralyzed patients – from speech to movement of objects in the world around them. Alternatively, by interfering with these electrical impulses, motor disorders (such as epilepsy or Parkinson’s) can start to be controlled.

Scientists have made this possible by developing electrodes that can be placed deep within the brain. These electrodes connect directly to neurons and transmit their electrical signals away from the body, allowing their meaning to be decoded.

However, the interface between neurons and electrodes has often been problematic: not only do the electrodes need to be highly sensitive to electrical impulses, but they need to be stable in the body without altering the tissue they measure.

Too often the modern electrodes used for this interface (based on tungsten or silicon) suffer from partial or complete loss of signal over time. This is often caused by the formation of scar tissue from the electrode insertion, which prevents the electrode from moving with the natural movements of the brain due to its rigid nature.

Graphene has been shown to be a promising material to solve these problems, because of its excellent conductivity, flexibility, biocompatibility and stability within the body.

Based on experiments conducted in rat brain cell cultures, the researchers found that untreated graphene electrodes interfaced well with neurons. By studying the neurons with electron microscopy and immunofluorescence the researchers found that they remained healthy, transmitting normal electric impulses and, importantly, none of the adverse reactions which lead to the damaging scar tissue were seen.

According to the researchers, this is the first step towards using pristine graphene-based materials as an electrode for a neuro-interface. In future, the researchers will investigate how different forms of graphene, from multiple layers to monolayers, are able to affect neurons, and whether tuning the material properties of graphene might alter the synapses and neuronal excitability in new and unique ways. “Hopefully this will pave the way for better deep brain implants to both harness and control the brain, with higher sensitivity and fewer unwanted side effects,” said Ballerini.

“We are currently involved in frontline research in graphene technology towards biomedical applications,” said Professor Maurizio Prato from the University of Trieste. “In this scenario, the development and translation in neurology of graphene-based high-performance biodevices requires the exploration of the interactions between graphene nano- and micro-sheets with the sophisticated signalling machinery of nerve cells. Our work is only a first step in that direction.”

“These initial results show how we are just at the tip of the iceberg when it comes to the potential of graphene and related materials in bio-applications and medicine,” said Professor Andrea Ferrari, Director of the Cambridge Graphene Centre. “The expertise developed at the Cambridge Graphene Centre allows us to produce large quantities of pristine material in solution, and this study proves the compatibility of our process with neuro-interfaces.”

The research was funded by the Graphene Flagship, a European initiative which promotes a collaborative approach to research with an aim of helping to translate graphene out of the academic laboratory, through local industry and into society.

Source: University of Cambridge

 

Remembering to Remember Supported by Two Distinct Brain Processes

http://www.biosciencetechnology.com/news/2013/08/remembering-remember-supported-two-distinct-brain-processes

To investigate how prospective memory is processed in the brain, psychological scientist Mark McDaniel of Washington University in St. Louis and colleagues had participants lie in an fMRI scanner and asked them to press one of two buttons to indicate whether a word that popped up on a screen was a member of a designated category.  In addition to this ongoing activity, participants were asked to try to remember to press a third button whenever a special target popped up. The task was designed to tap into participants’ prospective memory, or their ability to remember to take certain actions in response to specific future events.

When McDaniel and colleagues analyzed the fMRI data, they observed that two distinct brain activation patterns emerged when participants made the correct button press for a special target.

When the special target was not relevant to the ongoing activity—such as a syllable like “tor”—participants seemed to rely on top-down brain processes supported by the prefrontal cortex. In order to answer correctly when the special syllable flashed up on the screen, the participants had to sustain their attention and monitor for the special syllable throughout the entire task. In the grocery bag scenario, this would be like remembering to bring the grocery bags by constantly reminding yourself that you can’t forget them.

When the special target was integral to the ongoing activity—such as a whole word, like “table”—participants recruited a different set of brain regions, and they didn’t show sustained activation in these regions. The findings suggest that remembering what to do when the special target was a whole word didn’t require the same type of top-down monitoring. Instead, the target word seemed to act as an environmental cue that prompted participants to make the appropriate response—like reminding yourself to bring the grocery bags by leaving them near the front door.

“These findings suggest that people could make use of several different strategies to accomplish prospective memory tasks,” says McDaniel.

McDaniel and colleagues are continuing their research on prospective memory, examining how this phenomenon might change with age.

Co-authors on this research include Pamela LaMontagne, Michael Scullin, Todd Braver of Washington University in St. Louis; and Stefanie Beck of Technische Universität Dresden.

This research was funded by the National Institute on Aging, the Washington University Institute of Clinical and Translation Sciences, the National Center for Advancing Translational Sciences, and the German Science Foundation.

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