Posts Tagged ‘Neuroscience’

Memory Gene Goes Viral

Reporter: Irina Robu, PhD

A gene crucial for learning, called Arc can send genetic material from one neuron to another by using viruses was discovered by two independent team of scientist from University of Massachusetts Medical School and University of Utah which was published in Cell.  According to Dr. Edmund Talley, a program director at National Institute of Neurological Disorders and Stroke “this work is a great example of the importance of basic neuroscience research”.

Arc plays an important role in the brain’s ability to store new information, however little is known of how it works. According to the University of Utah scientists, research into the examination of the Arc gene began by introducing it into bacterial cells. When the cells made the Arc protein, it clumped together into a form that resembled a viral capsid, the shell that contains a virus’ genetic information. The Arc “capsids” appeared to mirror viral capsids in their physical structure in addition as their behavior and other properties.

At the same time, University of Massachusetts scientist led by Vivian Budnik, Ph. D and Travis Thomson, Ph.D. set out to scrutinize the contents of tiny sacks released by cells called extracellular vesicles. Their experiments in fruit flies revealed that motor neurons that control the flies’ muscles release vesicles containing a high concentration of the Arcgene’s messenger RNA (mRNA), the DNA-like intermediary molecule cells use to create the protein encoded by a DNA sequence.

Both groups similarly found evidence that Arc capsids contain Arc mRNA and that the capsids are released from neurons inside those vesicles. Also, both groups suggest that Arc capsids act like viruses by delivering mRNA to nearby cells. Furthermore, Dr. Shepherd’s team presented that the more active neurons are, the more of those vesicles they release. Dr. Shepherd’s group grew mouse neurons lacking the Arc gene in petri dishes filled with Arc-containing vesicles or Arc capsids alone. They revealed that the formerly Arc-less neurons took in the vesicles and capsids and used the Arc mRNA contained within to produce the Arc protein themselves. Finally, just like neurons that naturally manufacture the Arc protein, those cells made more of it when their electrical activity increased.

Both groups of scientists plan to examine why cells use this virus-like strategy to shuttle Arc mRNA between cells and which might allow the toxic proteins responsible for Alzheimer’s disease to spread through the brain.



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


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








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

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


  • 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

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

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Recent progress in neurodegenerative diseases and gliomas

Curator: Larry H. Bernstein, MD, FCAP




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

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.


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



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 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.


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.



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

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.


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.


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.


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.



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.


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.


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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Neuroscience impact of synaptic pruning discovery

Larry H. Bernstein, MD, FCAP, Curator



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.


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



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.


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


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.


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


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.







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Brain Biobank and studies of disease structure correlates

Larry H. Bernstein, MD, FCAP, Curator



Unveiling Psychiatric Diseases

Researchers create neuropsychiatric cellular biobank

Image: iStock/mstroz
Image: iStock/mstroz
Researchers from Harvard Medical School and Massachusetts General Hospital have completed the first stage of an important collaboration aimed at understanding the intricate variables of neuropsychiatric disease—something that currently eludes clinicians and scientists.

The research team, led by Isaac Kohane at HMS and Roy Perlis at Mass General, has created a neuropsychiatric cellular biobank—one of the largest in the world.

It contains induced pluripotent stem cells, or iPSCs, derived from skin cells taken from 100 people with neuropsychiatric diseases such as schizophrenia, bipolar disorder and major depression, and from 50 people without neuropsychiatric illness.

In addition, a detailed profile of each patient, obtained from hours of in-person assessment as well as from electronic medical records, is matched to each cell sample.

As a result, the scientific community can now for the first time access cells representing a broad swath of neuropsychiatric illness. This enables researchers to correlate molecular data with clinical information in areas such as variability of drug reactions between patients. The ultimate goal is to help treat, with greater precision, conditions that often elude effective management.

The cell collection and generation was led by investigators at Mass General, who in collaboration with Kohane and his team are working to characterize the cell lines at a molecular level. The cell repository, funded by the National Institutes of Health, is housed at Rutgers University.

“This biobank, in its current form, is only the beginning,” said Perlis, director of the MGH Psychiatry Center for Experimental Drugs and Diagnostics and HMS associate professor of psychiatry. “By next year we’ll have cells from a total of four hundred patients, with additional clinical detail and additional cell types that we will share with investigators.”

A current major limitation to understanding brain diseases is the inability to access brain biopsies on living patients. As a result, researchers typically study blood cells from patients or examine post-mortem tissue. This is in stark contrast with diseases such as cancer, for which there are many existing repositories of highly characterized cells from patients.

The new biobank offers a way to push beyond this limitation.


A Big Step Forward

While the biobank is already a boon to the scientific community, researchers at MGH and the HMS Department of Biomedical Informatics will be adding additional layers of molecular data to all of the cell samples. This information will include whole genome sequencing and transcriptomic and epigenetic profiling of brain cells made from the stem cell lines.

Collaborators in the HMS Department of Neurobiology, led by Michael Greenberg, department chair and Nathan Marsh Pusey Professor of Neurobiology,  will also work to examine characteristics of other types of neurons derived from these stem cells.

“This can potentially alter the entire way we look at and diagnose many neuropsychiatric conditions,” said Perlis.

One example may be to understand how the cellular responses to medication correspond to the patient’s documented responses, comparing in vitro with in vivo. “This would be a big step forward in bringing precision medicine to psychiatry,” Perlis said.

“It’s important to recall that in the field of genomics, we didn’t find interesting connections to disease until we had large enough samples to really investigate these complex conditions,” said Kohane, chair of the HMS Department of Biomedical Informatics.

“Our hypothesis is that here we will require far fewer patients,” he said. “By measuring the molecular functioning of the cells of each patient rather than only their genetic risk, and combining that all that’s known of these people in terms of treatment response and cognitive function, we will discover a great deal of valuable information about these conditions.”

Added Perlis, “In the early days of genetics, there were frequent false positives because we were studying so few people. We’re hoping to avoid the same problem in making cellular models, by ensuring that we have a sufficient number of cell lines to be confident in reporting differences between patient groups.”

The generation of stem cell lines and characterization of patients and brain cell lines is funded jointly by the the National Institute of Mental Health, the National Human Genome Research Institute and a grant from the Centers of Excellence in Genomic Science program.


On C.T.E. and Athletes, Science Remains in Its Infancy

Se Hoon ChoiYoung Hye KimMatthias Hebisch, et al.


Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles1. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau2, 3. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology4, 5. Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles6, 7, 8, 9, 10, 11. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.



Figure 2: Robust increases of extracellular amyloid-β deposits in 3D-differentiated hNPCs with FAD mutations.close

Robust increases of extracellular amyloid-[bgr] deposits in 3D-differentiated hNPCs with FAD mutations.

a, Thin-layer 3D culture protocol. HC, histochemistry; IF, immunofluorescence; IHC, immunohistochemistry. b, Amyloid-β deposits in 6-week differentiated control and FAD ReN cells in 3D Matrigel (green, GFP; blue, 3D6; scale bar, …


Stem Cell-Based Spinal Cord Repair Enables Robust Corticospinal Regeneration


Novel use of EPR spectroscopy to study in vivo protein structure



α-synuclein is a protein found abundantly throughout the brain. It is present mainly at the neuron ends where it is thought to play a role in ensuring the supply of synaptic vesicles in presynaptic terminals, which are required for the release of neurotransmitters to relay signals between neurons. It is critical for normal brain function.

However, α-synuclein is also the primary protein component of the cerebral amyloid deposits characteristic of Parkinson’s disease and its precursor is found in the amyloid plaques of Alzheimer’s disease. Although α-synuclein is present in all areas of the brain, these disease-state amyloid plaques only arise in distinct areas.

Alpha-synuclein protein. May play role in Parkinson’s and Alzheimer’s disease.  © molekuul.be / Shutterstock.com

Imaging of isolated samples of α-synuclein in vitro indicate that it does not have the precise 3D folded structure usually associated with proteins. It is therefore classed as an intrinsically disordered protein. However, it was not known whether the protein also lacked a precise structure in vivo.

There have been reports that it can form helical tetramers. Since the 3D structure of a biological protein is usually precisely matched to the specific function it performs, knowing the structure of α-synuclein within a living cell will help elucidate its role and may also improve understanding of the disease states with which it is associated.

If α-synuclein remains disordered in vivo, it may be possible for the protein to achieve different structures, and have different properties, depending on its surroundings.

Techniques for determining protein structure

It has long been known that elucidating the structure of a protein at an atomic level is fundamental for understanding its normal function and behavior. Furthermore, such knowledge can also facilitate the development of targeted drug treatments. Unfortunately, observing the atomic structure of a protein in vivo is not straightforward.

X-ray diffraction is the technique usually adopted for visualizing structures at atomic resolution, but this requires crystals of the molecule to be produced and this cannot be done without separating the molecules of interest from their natural environment. Such processes can modify the protein from its usual state and, particularly with complex structures, such effects are difficult to predict.

The development of nuclear magnetic resonance (NMR) spectroscopy improved the situation by making it possible for molecules to be analyzed under in vivo conditions, i.e. same pH, temperature and ionic concentration.

More recently, increases in the sensitivity of NMR and the use of isotope labelling have enabled determinations of the atomic level structure and dynamics of proteins to be determined within living cells1. NMR has been used to determine the structure of a bacterial protein within living cells2 but it is difficult to achieve sufficient quantities of the required protein within mammalian cells and to keep the cells alive for NMR imaging to be conducted.

Electron paramagnetic resonance (EPR) spectroscopy for determining protein structure

Recently, researchers have managed to overcome these obstacles by using in-cell NMR and electron paramagnetic resonance (EPR) spectroscopy. EPR spectroscopy is a technique that is similar to NMR spectroscopy in that it is based on the measurement and interpretation of the energy differences between excited and relaxed molecular states.

In EPR spectroscopy it is electrons that are excited, whereas in NMR signals are created through the spinning of atomic nuclei. EPR was developed to measure radicals and metal complexes, but has also been utilized to study the dynamic organization of lipids in biological membranes3.

EPR has now been used for the first time in protein structure investigations and has provided atomic-resolution information on the structure of α-synuclein in living mammalians4,5.

Bacterial forms of the α-synuclein protein labelled with 15N isotopes were introduced into five types of mammalian cell using electroporation. Concentrations of α-synuclein close to those found in vivo were achieved and the 15N isotopes allowed the protein to be clearly defined from other cellular components by NMR. The conformation of the protein was then determined using electron paramagnetic resonance (EPR).

The results showed that within living mammalian cells α-synuclein remains as a disordered and highly dynamic monomer. Different intracellular environments did not induce major conformational changes.


The novel use of EPR spectroscopy has resolved the mystery surrounding the in vivo conformation of α-synuclein. It showed that α-synuclein maintains its disordered monomeric form under physiological cell conditions. It has been demonstrated for the first time that even in crowded intracellular environments α-synuclein does not form oligomers, showing that intrinsic structural disorder can be sustained within mammalian cells.


  1. Freedberg DI and Selenko P. Live cell NMR Annu. Rev. Biophys. 2014;43:171–192.
  2. Sakakibara D, et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 2009;458:102–105.
  3. Yashroy RC. Magnetic resonance studies of dynamic organisation of lipids in chloroplast membranes. Journal of Biosciences 1990;15(4):281.
  4. Alderson TA and Bax AD. Parkinson’s Disease. Disorder in the court. Nature 2016; doi:10.1038/nature16871.
  5. Theillet FX, et al. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 2016; doi:10.1038/nature16531.


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Removing Alzheimer plaques

Larry H. Bernstein, MD, FCAP, Curator



Transdermal implant releases antibodies to trigger immune system to clear Alzheimer’s plaques            March 21, 2016

Test with mice over 39 weeks showed dramatic reduction of amyloid beta plaque load in the brain and reduced phosphorylation of the protein tau, two signs of Alzheimer’s


An implant that can prevent Alzheimer’s disease. A new capsule can be implanted under the skin to release antibodies that “tag” amyloid beta, signalling the patient’s immune system to clear it before it forms Alzheimer’s plaques. (credit: École polytechnique fédérale de Lausanne

EPFL scientists have developed an implantable capsule containing genetically engineered cells that can recruit a patient’s immune system to combat Alzheimer’s disease.

Placed under the skin, the capsule releases antibody proteins that make their way to the brain and “tag” amyloid beta proteins, signalling the patient’s own immune system to attack and clear the amyloid beta proteins, which are toxic to neurons.

To be most effective, this treatment has to be given as early as possible, before the first signs of cognitive decline. Currently, this requires repeated vaccine injections, which can cause side effects. The new implant can deliver a steady, safe flow of antibodies.

Protection from immune-system rejection

Cell encapsulation device for long-term subcutaneous therapeutic antibody delivery. (B) Macroscopic view of the encapsulation device, composed of a transparent frame supporting polymer permeable membranes and reinforced with an outer polyester mesh. (C) Dense neovascularization develops around a device containing antibody-secreting C2C12 myoblasts, 8 months after implantation in the mouse subcutaneous tissue. (D and E) Representative photomicrographs showing encapsulated antibody-secreting C2C12 myoblasts surviving at high density within the flat sheet device 39 weeks after implantation. (E) Higher magnification: note that the cells produce a collagen-rich matrix stained in blue with Masson’s trichrome protocol. Asterisk: polypropylene porous membrane. Scale bars = 750 mm (B and C),100 mm (D), 50 mm (E). (credit: Aurelien Lathuiliere et al./BRAIN)

The lab of Patrick Aebischer at EPFL designed the “macroencapsulation device” (capsule) with two permeable membranes sealed together with a polypropylene frame, containing a hydrogel that facilitates cell growth. All the materials used are biocompatible and the device is reproducible for large-scale manufacturing.

The cells of choice are taken from muscle tissue, and the permeable membranes let them interact with the surrounding tissue to get all the nutrients and molecules they need. The cells have to be compatible with the patient to avoid triggering the immune system against them, like a transplant can. To do that, the capsule’s membranes shield the cells from being identified and attacked by the immune system. This protection also means that cells from a single donor can be used on multiple patients.

The researchers tested the device mice in a genetic line commonly used to simulate Alzheimer’s disease over a course of 39 weeks, showing dramatic reduction of amyloid beta plaque load in the brain. The treatment also reduced the phosphorylation of the protein tau, another sign of Alzheimer’s observed in these mice.

“The proof-of-concept work demonstrates clearly that encapsulated cell implants can be used successfully and safely to deliver antibodies to treat Alzheimer’s disease and other neurodegenerative disorders that feature defective proteins,” according to the researchers.

The work is published in the journal BRAIN. It involved a collaboration between EPFL’s Neurodegenerative Studies Laboratory (Brain Mind Institute), the Swiss Light Source (Paul Scherrer Institute), and F. Hoffmann-La Roche. It was funded by the Swiss Commission for Technology and Innovation and F. Hoffmann-La Roche Ltd.

Abstract of A subcutaneous cellular implant for passive immunization against amyloid-β reduces brain amyloid and tau pathologies

Passive immunization against misfolded toxic proteins is a promising approach to treat neurodegenerative disorders. For effective immunotherapy against Alzheimer’s disease, recent clinical data indicate that monoclonal antibodies directed against the amyloid-β peptide should be administered before the onset of symptoms associated with irreversible brain damage. It is therefore critical to develop technologies for continuous antibody delivery applicable to disease prevention. Here, we addressed this question using a bioactive cellular implant to deliver recombinant anti-amyloid-β antibodies in the subcutaneous tissue. An encapsulating device permeable to macromolecules supports the long-term survival of myogenic cells over more than 10 months in immunocompetent allogeneic recipients. The encapsulated cells are genetically engineered to secrete high levels of anti-amyloid-β antibodies. Peripheral implantation leads to continuous antibody delivery to reach plasma levels that exceed 50 µg/ml. In a proof-of-concept study, we show that the recombinant antibodies produced by this system penetrate the brain and bind amyloid plaques in two mouse models of the Alzheimer’s pathology. When encapsulated cells are implanted before the onset of amyloid plaque deposition in TauPS2APP mice, chronic exposure to anti-amyloid-β antibodies dramatically reduces amyloid-β40 and amyloid-β42 levels in the brain, decreases amyloid plaque burden, and most notably, prevents phospho-tau pathology in the hippocampus. These results support the use of encapsulated cell implants for passive immunotherapy against the misfolded proteins, which accumulate in Alzheimer’s disease and other neurodegenerative disorders.


A subcutaneous cellular implant for passive immunization against amyloid-β reduces brain amyloid and tau pathologies


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Passive immunization using monoclonal antibodies has recently emerged for the treatment of neurological diseases. In particular, monoclonal antibodies can be administered to target the misfolded proteins that progressively aggregate and propagate in the CNS and contribute to the histopathological signature of neurodegenerative diseases. Alzheimer’s disease is the most prevalent proteinopathy, characterized by the deposition of amyloid plaques and neurofibrillary tangles. According to the ‘amyloid cascade hypothesis’, which is supported by strong genetic evidence (Goate and Hardy, 2012), the primary pathogenic event in Alzheimer’s disease is the accumulation and aggregation of amyloid-β into insoluble extracellular plaques in addition to cerebral amyloid angiopathy (Hardy and Selkoe, 2002). High levels of amyloid-β may cause a cascade of deleterious events, including neurofibrillary tangle formation, neuronal dysfunction and death. Anti-amyloid-β antibodies have been developed to interfere with the amyloid-β cascade. Promising data obtained in preclinical studies have validated immunotherapy against Alzheimer’s disease, prompting a series of clinical trials (Bard et al., 2000;Bacskai et al., 2002; Oddo et al., 2004; Wilcock et al., 2004a; Bohrmann et al., 2012). Phase III trials using monoclonal antibodies directed against soluble amyloid-β (bapineuzumab and solanezumab) in patients with mild-to-moderate Alzheimer’s disease showed some effects on biomarkers that are indicative of target engagement. These trials, however, missed the primary endpoints, and it is therefore believed that anti-amyloid-β immunotherapy should be administered at the early presymptomatic stage (secondary prevention) to better potentiate therapeutic effects (Doody et al., 2014; Salloway et al., 2014). For the treatment of Alzheimer’s disease, it is likely that long-term treatment using a high dose of monoclonal antibody will be required. However, bolus administration of anti-amyloid-β antibodies may aggravate dose-dependent adverse effects such as amyloid-related imaging abnormalities (ARIA) (Sperling et al., 2012). In addition, the cost of recombinant antibody production and medical burden associated with repeated subcutaneous or intravenous bolus injections may represent significant constraints, especially in the case of preventive immunotherapy initiated years before the onset of clinical symptoms in patients predisposed to develop Alzheimer’s disease.

Therefore, alternative methods need to be developed for the continuous, long-term administration of antibodies. Here, we used an implant based on a high-capacity encapsulated cell technology (ECT) (Lathuiliere et al., 2014b). The ECT device contains myogenic cells genetically engineered for antibody production. Macromolecules can be exchanged between the implanted cells and the host tissue through a permeable polymer membrane. As the membrane shields the implanted cells from immune rejection in allogeneic conditions, it is possible to use a single donor cell source for multiple recipients. We demonstrate that anti-amyloid immunotherapy using an ECT device implanted in the subcutaneous tissue can achieve therapeutic effects inside the brain. Chronic exposure to anti-amyloid-β monoclonal antibodies produced in vivo using the ECT technology leads to a significant reduction of the amyloid brain pathology in two mouse models of Alzheimer’s disease.


Microglial phagocytosis study

The measurement of antibody-mediated amyloid-β phagocytosis was performed as proposed previously (Webster et al., 2001). In this study, we used either purified preparations of full mAb-11 IgG2a antibody, or a purified Fab antibody fragment. A suspension of 530 µM fluorescent fibrillar amyloid-β42 was prepared in 10 mM HEPES (pH 7.4) by stirring overnight at room temperature. The resulting suspension contained 30 µM fluorescein-conjugated amyloid-β42 and 500 µM unconjugated amyloid-β42 (Bachem). IgG-fibrillar amyloid-β42 immune complexes were obtained by preincubating fluorescent fibrillar amyloid-β42 at a concentration of 50 µM in phosphate-buffered saline (PBS) with various concentrations of purified mAb-11 IgG2a or Fab antibody fragment for 30 min at 37 °C. The immune complexes were washed twice by centrifugation for 5 min at 14 000g and resuspended in the initial volume to obtain a fluorescent fibrillar amyloid-β42 solution (total amyloid-β42 concentration: 530 µM). The day before the experiment, 8 × 104 C8-B4 cells were plated in 24-well plates. The medium was replaced with serum-free DMEM before the addition of the peptides. The cells were incubated for 30 min with fibrillar amyloid-β42 or IgG-fibrillar amyloid-β42 added to the culture medium. Next, the cells were washed twice with Hank’s Balanced Salt Solution (HBSS) and subsequently detached by trypsinization, which also eliminates surface-bound fibrillar amyloid-β42. The cells were fixed for 10 min in 4% paraformaldehyde and finally resuspended in PBS. The cell fluorescence was determined with a flow cytometer (Accuri C6; BD Biosciences), and the data were analysed using the FlowJo software (TreeStar Inc.). To determine the effect of the anti-amyloid-β antibodies on amyloid-β phagocytosis, the concentration of fluorescent fibrillar amyloid-β42 was set at 1.5 µM, which is in the linear region of the dose-response curve depicting fibrillar amyloid-β42 phagocytosis in C8-B4 cells (Fig. 2C). All experiments were performed in duplicate.


The implantation of genetically engineered cells within a retrievable subcutaneous device leads to the continuous production of monoclonal antibodies in vivo. This technology achieves steady therapeutic monoclonal antibody levels in the plasma, offering an effective alternative to bolus injections for passive immunization against chronic diseases. Peripheral delivery of anti-amyloid-β monoclonal antibody by ECT leads to a significant reduction of amyloid burden in two mouse models of Alzheimer’s disease. The effect of the ECT treatment is more pronounced when passive immunization is preventively administered in TauPS2APP mice, most notably decreasing the phospho-tau pathology.

With the recent development of biomarkers to monitor Alzheimer’s pathology, it is recognized that a steady increase in cerebral amyloid over the course of decades precedes the appearance of the first cognitive symptoms (reviewed in Sperling et al., 2011). The current consensus therefore suggests applying anti-amyloid-β immunotherapy during this long asymptomatic phase to avoid the downstream consequences of amyloid deposition and to leverage neuroprotective effects. Several preventive clinical trials have been recently initiated for Alzheimer’s disease. The Alzheimer’s Prevention Initiative (API) and the Dominantly Inherited Alzheimer Network (DIAN) will test antibody candidates in presymptomatic dominant mutation carriers, while the Anti-Amyloid treatment in the Asymptomatic Alzheimer’s disease (A4) trial enrols asymptomatic subjects after risk stratification. If individuals with a high risk of developing Alzheimer’s disease can be identified using current biomarker candidates, these patients are the most likely to benefit from chronic long-term anti-amyloid-β immunotherapy. However, such a treatment may pose a challenge to healthcare systems, as the production capacity of the antibody and its related cost would become a challenging issue (Skoldunger et al., 2012). Therefore, the development of alternative technologies to chronically administer anti-amyloid-β antibody is an important aspect for therapeutic interventions at preclinical disease stages.

Here, we show that the ECT technology for the peripheral delivery of anti-amyloid-β monoclonal antibodies can significantly reduce cerebral amyloid pathology in two mouse models of Alzheimer’s disease. The subcutaneous tissue is a site of implantation easily accessible and therefore well adapted to preventive treatment. It is, however, challenging to reach therapeutic efficacy, as only a small fraction of the produced anti-amyloid-β monoclonal antibodies are expected to cross the blood–brain barrier, although they can next persist in the brain for several months (Wang et al., 2011; Bohrmann et al., 2012). Our results are consistent with previous reports, which have shown that the systemic administration of anti-amyloid-β antibodies can decrease brain amyloid burden in preclinical Alzheimer’s disease models (Bard et al., 2000, 2003; DeMattos et al., 2001; Wilcock et al., 2004a, b; Buttini et al., 2005; Adolfsson et al., 2012).

Remarkably, striking differences exist among therapeutic anti-amyloid-β antibodies in their ability to clear already existing plaques. Soluble amyloid-β species can saturate the small fraction of pan-amyloid-β antibodies entering the CNS and inhibit further target engagement (Demattos et al., 2012). Therefore, antibodies recognizing soluble amyloid-β may fail to bind and clear insoluble amyloid deposits (Das et al., 2001; Racke et al., 2005; Levites et al., 2006; Bohrmann et al., 2012). Furthermore, antibody-amyloid-β complexes are drained towards blood vessels, promoting cerebral amyloid angiopathy (CAA) and subsequent microhaemorrhages. The mAb-11 antibody used in the present study is similar to gantenerumab, which is highly specific for amyloid plaques and reduces amyloid burden in patients with Alzheimer’s disease (Bohrmann et al., 2012; Demattos et al., 2012; Ostrowitzki et al., 2012). We find that the murine IgG2a mAb-11 antibody efficiently enhances the phagocytosis of amyloid-β fibrils by microglial cells. In addition, ECT administration of the mAb-11 F(ab’)2 fragment lacking the Fc region fails to recruit microglial cells, and leads only to a trend towards clearance of the amyloid plaques. Therefore, our results suggest a pivotal role for microglial cells in the clearance of amyloid plaques following mAb-11 delivery by ECT. Importantly, we do not find any evidence that this treatment may cause microhaemorrhages in the mouse models used in this study. It remains entirely possible that direct binding to amyloid plaques of a F(ab’)2 fragment lacking effector functionality can contribute to therapeutic efficacy, as suggested by previous studies using antibody fragments (Bacskai et al., 2002;Tamura et al., 2005; Wang et al., 2010; Cattepoel et al., 2011). However, compared to IgG2a, the lower plasma levels achieved with F(ab’)2 are likely to limit the efficacy of peripheral ECT-mediated immunization. The exact role of the effector domain and its interaction with immune cells expressing Fc receptors, could be determined by comparing the therapeutic effects of a control antibody carrying a mutated Fc portion, similar to a previous study which addressed this question using deglycosylated anti-amyloid-β antibodies (Wilcock et al., 2006a; Fuller et al., 2014).

Remarkably, continuous administration of mAb-11 initiated before plaque deposition had a dramatic effect on the amyloid pathology in TauPS2APP mice, underlining the efficacy of preventive anti-amyloid-β treatments. In this mouse model, where tau hyperphosphorylation is enhanced by amyloid-β (Grueninger et al., 2010), the treatment decreases the number of AT8- and phospho-S422-positive neurons in the hippocampus. Furthermore, the number of MC1-positive hippocampal neurons is significantly reduced, which also indicates an effect of anti-amyloid-β immunotherapy on the accumulation of misfolded tau. These results highlight the effect of amyloid-β clearance on other manifestations of the Alzheimer’s pathology. In line with these findings, previous studies have shown evidence for a decrease in tau hyperphosphorylation following immunization against amyloid-β, both in animal models and in patients with Alzheimer’s disease (Oddo et al., 2004; Wilcock et al., 2009;Boche et al., 2010; Serrano-Pozo et al., 2010; Salloway et al., 2014).

Similar to the subcutaneous injection of recombinant proteins (Schellekens, 2005), ECT implants can elicit significant immune responses against the secreted recombinant antibody. An anti-drug antibody response was detected in half of the mice treated with the mAb-11-releasing devices, in the absence of any anti-CD4 treatment. The glycosylation profile of the mAb-11 synthesized in C2C12 myoblasts is comparable to standard material produced by myeloma or HEK293 cells (Lathuiliere et al., 2014a). Although we cannot exclude that local release by ECT leads to antibody aggregation and denaturation, it is unlikely that this mode of administration further contributes to compound immunogenicity. Because the Fab regions of the chimeric recombinant mAb-11 IgG2a contain human CDRs, it remains to be determined whether the ECT-mediated delivery of antibodies fully matched with the host species would trigger an anti-drug antibody response.

Further developments will be needed to scale up this delivery system to humans. The possibility of using a single allogeneic cell source for all intended recipients is a crucial advantage of the ECT technology to standardize monoclonal antibody delivery. However, the development of renewable cell sources of human origin will be essential to ECT application in the clinic. Although the ARPE-19 cell line has been successfully adapted to ECT and used in clinical trials (Dunn et al., 1996; Zhang et al., 2011), the development of human myogenic cells (Negroni et al., 2009) is an attractive alternative that is worth exploring. Based on the PK analysis of recombinant mAb-11 antibody subcutaneously injected in mice (Supplementary material), we estimate that the flat sheet devices chronically release mAb-11 at a rate of 6.8 and 11.8 µg/h, to reach a plasma level of 50 µg/ml in the implanted animals. In humans, injected IgG1 has a longer half-life (21–25 days), with a volume of distribution of ∼100 ml/kg and an estimated clearance of 0.2 ml/h/kg. These values indicate that the predicted antibody exposure in humans, based on the rate of mAb-11 secretion achieved by ECT in mice, would be only 10 to 20-fold lower than the typical regimens based on monthly bolus injection of 1 mg/kg anti-amyloid-β monoclonal antibody. Hence, it is realistic to consider ECT for therapeutic monoclonal antibody delivery in humans, as the flat sheet device could be scaled up to contain higher amounts of cells. Furthermore, recent progress to engineer antibodies for increased penetration into the brain will enable lowering dosing of biotherapeutics to achieve therapeutic efficacy (Bien-Ly et al., 2014; Niewoehner et al., 2014). For some applications, intrathecal implantation could be preferred to chronically deliver monoclonal antibodies directly inside the CNS (Aebischer et al., 1996; Marroquin Belaunzaran et al., 2011).

Overall, ECT provides a novel approach for the local and systemic delivery of recombinant monoclonal antibodies in the CNS. It will expand the possible therapeutic options for immunotherapy against neurodegenerative disorders associated with the accumulation of misfolded proteins, including Alzheimer’s and Parkinson’s diseases, dementia with Lewy bodies, frontotemporal lobar dementia and amyotrophic lateral sclerosis (Gros-Louis et al., 2010; Bae et al., 2012; Rosenmann, 2013).

encapsulated cell technology
monoclonal antibody

The most powerful part of the article is the methodology of the transdermal patch of genetically engineered cells which appear to give a constant stream of antibody therapy. This would be better for the authors to have highlighted because the protein delivery system is great work. However it is a shame that the Alzheimer’s field is still relying on these mouse models which are appearing to lead the filed down a path which leads to failed clinical trials. Lilly has just dropped their Alzheimer’s program and unless the filed shifts to a different hypothesis of disease etilology I feel the whole field will be stuck where it is.



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Brain, learning and memory

Larry H. Bernstein, MD, FCAP, Curator



March 23, 2016   Exploring long-range communications in the brain

Red and green dots reveal a region in the brain that that is very dense with synapses. A optically activated fluorescent protein allows Ofer Yizhar, PhD, and his group to record the activity of the synapses. (credit: Weizmann Institute of Science)

Weizmann Institute of Science researchers have devised a new way to track long-distance communications between nerve cells in different areas of the brain. They used optogenetic techniques (using genetic engineering of neurons and laser light in thin optical fibers to temporarily silence long-range axons, effectively leading to a sustained “disconnect” between two distant brain nodes.

By observing what happens when crucial connections are disabled, the researchers could begin to determine the axons’ role in the brain. Mental and neurological diseases are often thought to result from changes in long-range brain connectivity, so these studies could contribute to a better understanding of the mechanisms behind health and disease in the brain.

The study, published in Nature Neuroscience, “led us to a deeper understanding of the unique properties of the axons and synapses that form the connections between neurons,” said Ofer Yizhar, PhD, in the Weizmann Institute of Science’s Neurobiology Department. “We were able to uncover the responses of axons to various optogenetic manipulations. Understanding these differences will be crucial to unraveling the mechanisms for long-distance communication in the brain.”

Abstract of Biophysical constraints of optogenetic inhibition at presynaptic terminals

We investigated the efficacy of optogenetic inhibition at presynaptic terminals using halorhodopsin, archaerhodopsin and chloride-conducting channelrhodopsins. Precisely timed activation of both archaerhodopsin and halorhodpsin at presynaptic terminals attenuated evoked release. However, sustained archaerhodopsin activation was paradoxically associated with increased spontaneous release. Activation of chloride-conducting channelrhodopsins triggered neurotransmitter release upon light onset. Thus, the biophysical properties of presynaptic terminals dictate unique boundary conditions for optogenetic manipulation.


DARPA’s ‘Targeted Neuroplasticity Training’ program aims to accelerate learning ‘beyond normal levels’

The transhumanism-inspired goal: train superspy agents to rapidly master foreign languages and cryptography
New DARPA “TNT” technology will be designed to safely and precisely modulate peripheral nerves to control synaptic plasticity during cognitive skill training. (No mention of NZT.) (credit: DARPA)

DARPA has announced a new program called Targeted Neuroplasticity Training (TNT) aimed at exploring how to use peripheral nerve stimulation and other methods to enhance learning.

DARPA already has research programs underway to use targeted stimulation of the peripheral nervous system as a substitute for drugs to treat diseases and accelerate healing*, to control advanced prosthetic limbs**, and to restore tactile sensation.

But now DARPA plans to to take an even more ambitious step: It aims to enlist the body’s peripheral nerves to achieve something that has long been considered the brain’s domain alone: facilitating learning — specifically, training in a wide range of cognitive skills.

The goal is to reduce the cost and duration of the Defense Department’s extensive training regimen, while improving outcomes. If successful, TNT could accelerate learning and reduce the time needed to train foreign language specialists, intelligence analysts, cryptographers, and others.

“Many of these skills, such as understanding and speaking a new foreign language, can be challenging to learn,” says the DARPA statement. “Current training programs are time consuming, require intensive study, and usually require evidence of a more-than-minimal aptitude for eligibility. Thus, improving cognitive skill learning in healthy adults is of great interest to our national security.”

Going beyond normal levels of learning

The program is also notable because it will not just train; it will advance capabilities beyond normal levels — a transhumanist approach.

“Recent research has shown that stimulation of certain peripheral nerves, easily and painlessly achieved through the skin, can activate regions of the brain involved with learning,” by releasing neurochemicals in the brain that reorganize neural connections in response to specific experiences, explained TNT Program Manager Doug Weber,

“This natural process of synaptic plasticity is pivotal for learning, but much is unknown about the physiological mechanisms that link peripheral nerve stimulation to improved plasticity and learning,” Weber said. “You can think of peripheral nerve stimulation as a way to reopen the so-called ‘Critical Period’ when the brain is more facile and adaptive. TNT technology will be designed to safely and precisely modulate peripheral nerves to control plasticity at optimal points in the learning process.”

The goal is to optimize training protocols that expedite the pace of learning and maximize long-term retention of even the most complicated cognitive skills. DARPA intends to take a layered approach to exploring this new terrain:

  • Fundamental research will focus on gaining a clearer and more complete understanding of how nerve stimulation influences synaptic plasticity, how cognitive skill learning processes are regulated in the brain, and how to boost these processes to safely accelerate skill acquisition while avoiding potential side effects.
  • The engineering side of the program will target development of a non-invasive device that delivers peripheral nerve stimulation to enhance plasticity in brain regions responsible for cognitive functions.

Proposers Day

TNT expects to attract multidisciplinary teams spanning backgrounds such as cognitive neuroscience, neural plasticity, electrophysiology, systems neurophysiology, biomedical engineering, human performance, and computational modeling.

To familiarize potential participants with the technical objectives of TNT, DARPA will host a Proposers Day on Friday, April 8, 2016, at the Westin Arlington Gateway in Arlington, Va. (registration closes on Thursday, March 31, 2016). ADARPA Special Notice announces the Proposers Day and describes the specific capabilities sought. A Broad Agency Announcement with full technical details on TNT will be forthcoming. For more information, please email DARPA-SN-16-20@darpa.mil.

* DARPA’s ElectRx program is looking for “demonstrations of feedback-controlled neuromodulation strategies to establish healthy physiological states,” along with “disruptive biological-interface technologies required to monitor biomarkers and peripheral nerve activity … [and] deliver therapeutic signals to peripheral nerve targets, using in vivo, real-time biosensors and novel neural interfaces using optical, acoustic, electromagnetic, or engineered biology strategies to achieve precise targeting with potentially single-axon resolution.”

** DARPA’s HAPTIX (Hand Proprioception and Touch Interfaces) program “seeks to create a prosthetic hand system that moves and provides sensation like a natural hand. … HAPTIX technologies aim to tap in to the motor and sensory signals of the arm, allowing users to control and sense the prosthesis via the same neural signaling pathways used for intact hands and arms. … The system will include electrodes for measuring prosthesis control signals from muscles and motor nerves, and sensory feedback will be delivered through electrodes placed in sensory nerves.”


Fading of Epigenetic Memories across Generations Is Regulated
Neurons involved in working memory fire in bursts, not continuously

  • Epigenetic “remembering” is better understood than epigenetic “forgetting,” and so it is an open question whether epigenetic forgetting is, like epigenetic remembering, active—a distinct biomolecular process—or passive—a matter of dilution or decay. New research, however, suggests that epigenetic forgetting is an active process, one in which a feedback mechanism determines the duration of transgenerational epigenetic memories.

    The new research comes out of Tel Aviv University, where researchers have been working with the nematode worm Caenorhabditis elegans to elucidate epigenetic mechanisms. In particular, the researchers, led by Oded Rechavi, Ph.D., have been preoccupied with how the effects of stress, trauma, and other environmental exposures are passed from one generation to the next.

    In previous work, Dr. Rechavi’s team enhanced the state of knowledge of small RNA molecules, short sequences of RNA that regulate the expression of genes. The team identified a “small RNA inheritance” mechanism through which RNA molecules produced a response to the needs of specific cells and how they were regulated between generations.

    “We previously showed that worms inherited small RNAs following the starvation and viral infections of their parents. These small RNAs helped prepare their offspring for similar hardships,” Dr. Rechavi explained. “We also identified a mechanism that amplified heritable small RNAs across generations, so the response was not diluted. We found that enzymes called RdRPs [RNA-dependent RNA polymerases] are required for re-creating new small RNAs to keep the response going in subsequent generations.”

    Most inheritable epigenetic responses in C. elegans were found to persist for only a few generations. This created the assumption that epigenetic effects simply “petered out” over time, through a process of dilution or decay. “But this assumption,” said Dr. Rechavi, “ignored the possibility that this process doesn’t simply die out but is regulated instead.”

    This possibility was explored in the current study, in which C. elegans were treated with small RNAs that target the GFP (green fluorescent protein) gene, a reporter gene commonly used in experiments. “By following heritable small RNAs that regulated GFP—that ‘silenced’ its expression—we revealed an active, tunable inheritance mechanism that can be turned ‘on’ or ‘off,'” declared Dr. Rechavi.

    Details of the work appeared March 24 in the journal Cell, in an article entitled, “A Tunable Mechanism Determines the Duration of the Transgenerational Small RNA Inheritance in C. elegans.” The article shows that exposure to double-stranded RNA (dsRNA) activates a feedback loop whereby gene-specific RNA interference (RNAi) responses “dictate the transgenerational duration of RNAi responses mounted against unrelated genes, elicited separately in previous generations.”

    Essentially, amplification of heritable exo-siRNAs occurs at the expense of endo-siRNAs. Also, a feedback between siRNAs and RNAi genes determines heritable silencing duration.

    “RNA-sequencing analysis reveals that, aside from silencing of genes with complementary sequences, dsRNA-induced RNAi affects the production of heritable endogenous small RNAs, which regulate the expression of RNAi factors,” wrote the authors of the Cell paper. “Manipulating genes in this feedback pathway changes the duration of heritable silencing.”

    The scientists also indicated that specific genes, which they named MOTEK (Modified Transgenerational Epigenetic Kinetics), were involved in turning on and off epigenetic transmissions.

    “We discovered how to manipulate the transgenerational duration of epigenetic inheritance in worms by switching ‘on’ and ‘off’ the small RNAs that worms use to regulate genes,” said Dr. Rechavi. “These switches are controlled by a feedback interaction between gene-regulating small RNAs, which are inheritable, and the MOTEK genes that are required to produce and transmit these small RNAs across generations.

    “The feedback determines whether epigenetic memory will continue to the progeny or not, and how long each epigenetic response will last.”

    Although its research was conducted on worms, the team believes that understanding the principles that control the inheritance of epigenetic information is crucial for constructing a comprehensive theory of heredity for all organisms, humans included.

    “We are now planning to study the MOTEK genes to know exactly how these genes affect the duration of epigenetic effects,” said Leah Houri-Ze’evi, a Ph.D. student in Dr. Rechavi’s lab and first author of the paper. “Moreover, we are planning to examine whether similar mechanisms exist in humans.”

    The current study notes that the active control of transgenerational effects could be adaptive, because ancestral responses would be detrimental if the environments of the progeny and the ancestors were different.

    A Tunable Mechanism Determines the Duration of the Transgenerational Small RNA Inheritance in C. elegans

    Leah Houri-Ze’ev, Yael Korem, Hila Sheftel,…, Luba Degani, Uri Alon, Oded Rechavi
    Cell 24 March 2016; Volume 165, Issue 1, p88–99.  http://dx.doi.org/10.1016/j.cell.2016.02.057
  • New RNAi episodes extend the duration of heritable epigenetic effects
  • Amplification of heritable exo-siRNAs occurs at the expense of endo-siRNAs
  • A feedback between siRNAs and RNAi genes determines heritable silencing duration
  • Modified transgenerational epigenetic kinetics (MOTEK) mutants are identified

Figure thumbnail fx1

In C. elegans, small RNAs enable transmission of epigenetic responses across multiple generations. While RNAi inheritance mechanisms that enable “memorization” of ancestral responses are being elucidated, the mechanisms that determine the duration of inherited silencing and the ability to forget the inherited epigenetic effects are not known. We now show that exposure to dsRNA activates a feedback loop whereby gene-specific RNAi responses dictate the transgenerational duration of RNAi responses mounted against unrelated genes, elicited separately in previous generations. RNA-sequencing analysis reveals that, aside from silencing of genes with complementary sequences, dsRNA-induced RNAi affects the production of heritable endogenous small RNAs, which regulate the expression of RNAi factors. Manipulating genes in this feedback pathway changes the duration of heritable silencing. Such active control of transgenerational effects could be adaptive, since ancestral responses would be detrimental if the environments of the progeny and the ancestors were different.

How we are able to keep several things simultaneously in working memory
Pictured is an artist’s interpretation of neurons firing in sporadic, coordinated bursts. “By having these different bursts coming at different moments in time, you can keep different items in memory separate from one another,” Earl Miller says. (credit: Jose-Luis Olivares/MIT)

Think of a sentence you just read. Like that one. You’re now using your working memory, a critical brain system that’s roughly analogous to RAM memory in a computer.

Neuroscientists have believed that as information is held in working memory, brain cells associated with that information must be firing continuously. Not so — they fire in sporadic, coordinated bursts, says Earl Miller, the Picower Professor in MIT’s Picower Institute for Learning and Memory and the Department of Brain and Cognitive Sciences.

That makes sense. These different bursts could help the brain hold multiple items in working memory at the same time, according to the researchers. “By having these different bursts coming at different moments in time, you can keep different items in memory separate from one another,” says Miller, the senior author of a study that appears in the March 17 issue of Neuron.

Bursts of activity, not averaged activity

So why hasn’t anyone noticed this before? Because previous studies averaged the brain’s activity over seconds or even minutes of performing the task, Miller says. “We looked more closely at this activity, not by averaging across time, but from looking from moment to moment. That revealed that something way more complex is going on.”

To do that, Miller and his colleagues recorded neuron activity in animals as they were shown a sequence of three colored squares, each in a different location. Then, the squares were shown again, but one of them had changed color. The animals were trained to respond when they noticed the square that had changed color — a task requiring them to hold all three squares in working memory for about two seconds.

The researchers found that as items were held in working memory, ensembles of neurons in the prefrontal cortex were active in brief bursts, and these bursts only occurred in recording sites in which information about the squares was stored. The bursting was most frequent at the beginning of the task, when the information was encoded, and at the end, when the memories were read out.

The findings fit well with a model that Lundqvist had developed as an alternative to the model of sustained activity as the neural basis of working memory. According to the new model, information is stored in rapid changes in the synaptic strength of the neurons. The brief bursts serve to “imprint” information in the synapses of these neurons, and the bursts reoccur periodically to reinforce the information as long as it is needed.

The bursts create waves of coordinated activity at the gamma frequency (45 to 100 hertz), like the ones that were observed in the data. These waves occur sporadically, with gaps between them, and each ensemble of neurons, encoding a specific item, produces a different burst of gamma waves, like a fingerprint.

Implications for other cognitive functions

The findings suggest that it would be worthwhile to look for this kind of cyclical activity in other cognitive functions such as attention, the researchers say. Oscillations like those seen in this study may help the brain to package information and keep it separate so that different pieces of information don’t interfere with each other.

Robert Knight, a professor of psychology and neuroscience at the University of California at Berkeley, says the new study “provides compelling evidence that nonlinear oscillatory dynamics underlie prefrontal dependent working memory capacity.”

“The work calls for a new view of the computational processes supporting goal-directed behavior,” adds Knight, who was not involved in the research. “The control processes supporting nonlinear dynamics are not understood, but this work provides a critical guidepost for future work aimed at understanding how the brain enables fluid cognition.”

editor’s comments: I’m curious how this relates to forgetting things to make space to learn new things. (Turns out the hippocampus works closely with the prefrontal cortex in working memory, as this open-access Nature paper explains.) Also, what’s the latest on how many things we can keep in working memory (it used to be around five)? Is that number limited by forgetting or by the capacity to differentiate different spike trains? Any tricks for keeping more things in working memory?

Abstract of Gamma and Beta Bursts Underlie Working Memory

Working memory is thought to result from sustained neuron spiking. However, computational models suggest complex dynamics with discrete oscillatory bursts. We analyzed local field potential (LFP) and spiking from the prefrontal cortex (PFC) of monkeys performing a working memory task. There were brief bursts of narrow-band gamma oscillations (45–100 Hz), varied in time and frequency, accompanying encoding and re-activation of sensory information. They appeared at a minority of recording sites associated with spiking reflecting the to-be-remembered items. Beta oscillations (20–35 Hz) also occurred in brief, variable bursts but reflected a default state interrupted by encoding and decoding. Only activity of neurons reflecting encoding/decoding correlated with changes in gamma burst rate. Thus, gamma bursts could gate access to, and prevent sensory interference with, working memory. This supports the hypothesis that working memory is manifested by discrete oscillatory dynamics and spiking, not sustained activity.

Gamma and Beta Bursts Underlie Working Memory

Mikael Lundqvist5, Jonas Rose5, Pawel Herman, Scott L. Brincat, Timothy J. Buschman, Earl K. Miller

  • Working memory information in neuronal spiking is linked to brief gamma bursts
  • The narrow-band gamma bursts increase during encoding, decoding, and with WM load
  • Beta bursting reflects a default network state interrupted by gamma
  • Support for a model of WM is based on discrete dynamics and not sustained activity


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Amit, D.J. and Brunel, N.Model of global spontaneous activity and local structured activity during delay periods in the cerebral cortex.

Cereb. Cortex. 1997; 7: 237–252

Asaad, W.F. and Eskandar, E.N.A flexible software tool for temporally-precise behavioral control in Matlab.

J. Neurosci. Methods. 2008;174: 245–258

Axmacher, N., Henseler, M.M., Jensen, O., Weinreich, I., Elger, C.E., and Fell, J.Cross-frequency coupling supports multi-item working memory in the human hippocampus.

Proc. Natl. Acad. Sci. USA.2010; 107: 3228–3233

Brunel, N. and Wang, X.J.What determines the frequency of fast network oscillations with irregular neural discharges? I. Synaptic dynamics and excitation-inhibition balance.

J. Neurophysiol. 2003; 90:415–430

Buschman, T.J., Siegel, M., Roy, J.E., and Miller, E.K.Neural substrates of cognitive capacity limitations.

Proc. Natl. Acad. Sci. USA.2011; 108: 11252–11255

Johan Eriksson, Edward K. Vogel, Anders Lansner, Fredrik Bergström, Lars Nyberg
Neuron, Vol. 88, Issue 1, p33–46
Published in issue: October 07, 2015
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Current Biology, Vol. 26, Issue 3, p351–355
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Open Archive

Synaptic Amplifier

Gene discovery reveals mechanism behind how we think

By ELIZABETH COONEY   March 16, 2016

Skyler Jackman and colleagues studied the phenomenon known as synaptic facilitation by using light to turn neuronal connections on and off. The optogenetic protein used in this technique appears yellow. Image: Regehr lab

Our brains are marvels of connectivity, packed with cells that continually communicate with one another. This communication occurs across synapses, the transit points where chemicals called neurotransmitters leap from one neuron to another, allowing us to think, to learn and to remember.
Now Harvard Medical School researchers have discovered a gene that provides that boost by increasing neurotransmitter release in a phenomenon known as synaptic facilitation. And they did so by turning on a light or two.
Image: Jasmine Vazquez
image: Jasmine Vazquez

The gene is synaptotagmin 7 (syt7 for short), a calcium sensor that dynamically increases neurotransmitter release; each release serves to strengthen communication between neurons for about a second. These swift releases are thought to be critical for the brain’s ability to perform computations involved in short-term memory, spatial navigation and sensory perception.

A team of researchers who made this discovery was led by Skyler Jackman, a postdoctoral researcher in the lab of Wade Regehr, professor of neurobiology at HMS. They recently reported their findings in Nature.

 The calcium sensor synaptotagmin 7 is required for synaptic facilitationSkyler L. JackmanJosef TurecekJustine E. Belinsky & Wade G. Regehr
Nature 529, 88–91 (07 January 2016)
It has been known for more than 70 years that synaptic strength is dynamically regulated in a use-dependent manner1. At synapses with a low initial release probability, closely spaced presynaptic action potentials can result in facilitation, a short-term form of enhancement in which each subsequent action potential evokes greater neurotransmitter release2. Facilitation can enhance neurotransmitter release considerably and can profoundly influence information transfer across synapses3, but the underlying mechanism remains a mystery. One proposed mechanism is that a specialized calcium sensor for facilitation transiently increases the probability of release24, and this sensor is distinct from the fast sensors that mediate rapid neurotransmitter release. Yet such a sensor has never been identified, and its very existence has been disputed56. Here we show that synaptotagmin 7 (Syt7) is a calcium sensor that is required for facilitation at several central synapses. In Syt7-knockout mice, facilitation is eliminated even though the initial probability of release and the presynaptic residual calcium signals are unaltered. Expression of wild-type Syt7 in presynaptic neurons restored facilitation, whereas expression of a mutated Syt7 with a calcium-insensitive C2A domain did not. By revealing the role of Syt7 in synaptic facilitation, these results resolve a longstanding debate about a widespread form of short-term plasticity, and will enable future studies that may lead to a deeper understanding of the functional importance of facilitation.

“We really think one of the most important things the brain can do is change the strength of connections between neurons,” Jackman said. “Now that we have a tool to selectively turn off facilitation, we can test some long-held beliefs about its importance for thinking and working memory.”

Although synaptic facilitation was first described 70 years ago by Te-Pei Feng, known as the father of Chinese physiology, Jackman and colleagues were able to identify the mechanism behind synaptic strengthening by taking advantage of advanced laboratory techniques unavailable to previous generations of scientists.

A dozen years ago, Regehr suspected that syt7 might drive this synaptic strengthening process: The gene turns on slowly and then ramps up in speed, which would fit gradual release of neurotransmitters.

About eight years ago scientists in another lab engineered “knockout” mice that lack the syt7 gene, setting the stage for experiments to test Regehr’s speculations. But when grown in a lab dish, neurons from these knockout mice behaved no differently than other neurons; results that, at the time, dashed hopes that syt7 could explain the synaptic boost.

A year ago Jackman took another tack. He tested synaptic connections in brain tissue taken from the knockout mice but still having intact brain circuits, an experiment more reflective of how neurons and synapses might work in a living animal.

“It was striking. It was amazing,” Jackman said. “As soon as we probed these connections we saw there was a huge deficit, a complete lack of synaptic facilitation in the knockout mice, completely different from their wild-type brothers and sisters.”

To be certain that knocking out syt7 was responsible for this change, Jackman had to find a way to reinsert syt 7 and restore its function. He did that by using optogenetics, a genetic manipulation tool that allows neuronal connections to be turned on and off with light. He augmented this technique with bicistronic expression, a method that packages one optogenetic protein and one syt7protein into a single virus that infects all neurons equally. Using these two techniques, Jackman could selectively study what happened when syt7 was reinserted into a neuron and measure its effects reliably.


We need to forget things to make space to learn new things, scientists discover

Mice study, if confirmed in people, might help forget traumatic experiences

The three routes into the hippocampus seem to be linked to different aspects of learning: forming memories (green), recalling them (yellow) and forgetting them (red) (credit: John Wood)

While you’re reading this (and learning about this new study), your brain is actively trying to forget something.

We apologize, but that’s what scientists at the European Molecular Biology Laboratory (EMBL) and the University Pablo Olavide in Sevilla, Spain, found in a new study published Friday (March 18) in an open-access paper in Nature Communications.

“This is the first time that a pathway in the brain has been linked to forgetting — to actively erasing memories,” says Cornelius Gross, who led the work at EMBL.

Working with mice, Gross and colleagues studied the hippocampus, a region of the brain known to help form memories. Information enters this part of the brain through three different routes. As memories are formed, connections between neurons along the “main” route become stronger.

When they blocked this main route (dentate gyrus granule cells), the scientists found that the mice were no longer capable of learning (in this case, a specific Pavlovian response).* But surprisingly, blocking that main route  also resulted in its connections weakening, meaning the memory was actually being erased.

Limited space in the brain

Gross proposes that one explanation: “There is limited space in the brain, so when you’re learning, you have to weaken some connections to make room for others,” says Gross.

Interestingly, this active push for forgetting only happens in learning situations. When the scientists blocked the main route into the hippocampus under other circumstances, the strength of its connections remained unaltered.

The findings were made using genetically engineered mice, but the scientists demonstrated that it is possible to produce a drug that activates this “forgetting” route in the brain without the need for genetic engineering. This approach, they say, might help people forget traumatic experiences.

* But if the mice had learned that association before the scientists stopped information flow in that main route, they could still retrieve that memory. This confirmed that this route is involved in forming memories, but isn’t essential for recalling those memories. The latter probably involves the second route into the hippocampus, the scientists surmise.

Abstract of Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells

The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories.


Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells

Noelia MadroñalJosé M. Delgado-GarcíaAzahara Fernández-GuizánJayanta ChatterjeeMaja KöhnCamilla Mattucci, et al.

Nature Communications7,Article number:10923    http://www.nature.com/ncomms/2016/160318/ncomms10923/full/ncomms10923.html

The hippocampus is an evolutionarily ancient part of the cortex that makes reciprocal excitatory connections with neocortical association areas and is critical for the acquisition and retrieval of episodic and contextual memories. The hippocampus has been the subject of extensive investigation over the last 50 years as the site of plasticity thought to be critical for memory encoding. Models of hippocampal function propose that sensory information reaching the hippocampus from the entorhinal cortex via dentate gyrus (DG) granule cells is encoded in CA3 auto-association circuits and can in turn be retrieved via Schaffer collateral (SC) projections linking CA3 and CA1 (refs 1, 2, 3, 4; Fig. 1a). Learning-associated plasticity in CA3–CA3 auto-associative networks encodes the memory trace, and plasticity in SC connections is necessary for the efficient retrieval of this trace2, 5, 6, 7, 8, 9, 10. In addition, both CA3 and CA1 regions receive direct, monosynaptic inputs from entorhinal cortex that are thought to convey information about ongoing sensory inputs that could modulate CA3 memory trace acquisition and/or retrieval via SC (refs 11,12, 13; Fig. 1a). In DG granule cells, sensory information is thought to undergo pattern separation into orthogonal cell ensembles before encoding (or reactivating, in the case of retrieval) memories in CA3 (ref. 14). However, how the hippocampus executes both the acquisition and recall of memories stored in CA3 remains a question of debate with some models attributing a role for DG inputs in memory acquisition, but not retrieval2, 15, 16, 17.

Rapid and selective inhibition of DG neurotransmission in vivo.

(a) The hippocampal tri-synaptic circuit receives PP inputs from entorhinal cortex to DG, CA3 and CA1. (b) A stimulating electrode was implanted in the PP and a recording electrode in CA3 pyramidal layer. (c) Strength of CA3 pyramidal layer fEPSPs evoked in anaesthetized mice by electrical stimulation of PP inputs showed fast and slow latency population spike components corresponding to direct PP-CA3 and indirect PP–DG-CA3 inputs, respectively. Systemic administration of the selective Htr1a agonist, 8-OH-DPAT (0.3mgkg−1, subcutaneous), to Htr1aDG (Tg) mice caused a rapid and selective decrease in the long-latency component that persisted for several hours. Quantification indicated a significant decrease in DG neurotransmission following agonist treatment of Htr1aDG, but not Htr1aKO (KO) littermates or vehicle treated wild-type mice that reached 80% suppression and persisted for >2h (mean±s.e.m.; n=10;*P<0.05; two-way analysis of variance followed by Holm–Sidak post hoc test). (d) Representative fEPSPs evoked at CA3 pyramidal layer after stimulation of PP inputs before and after agonist treatment. The fast and the slow latency population spike components are indicated (black arrow, short; grey arrow, long).


Figure 2

Inhibition of DG induces rapid and persistent loss of hippocampal memory and plasticity.

Figure 4

Loss of plasticity depends on entorhinal cortex inputs and local adenosine signalling.

In the present study we examined the contribution of DG granule cells to learning and recall and its associated synaptic plasticity in animals that had previously acquired a hippocampal memory. We found that transient pharmacogenetic inhibition of DG granule cells did not impair conditioned responding to CS presentation nor alter SC synaptic plasticity demonstrating that DG is not required for memory recall (Fig. 3c,d). However, when DG inhibition occurred during paired presentation of CS and US, we observed a rapid loss of SC synaptic plasticity and conditioned responding to CS (Fig. 2d,e and Supplementary Fig. 3). Strikingly, the synaptic plasticity and behavioural impairment persisted in the absence of further stimulus presentation and later relearning occurred at a rate indistinguishable from initial learning, suggesting a loss of the memory trace (Fig. 2f,g).

One possible explanation for the memory loss seen on DG inhibition is that presentation of paired CS–US has a dual effect on CA1 plasticity, on the one hand strengthening SC synapses via a DG-dependent mechanism (indirect inputs to CA1 via the tri-synaptic circuit) and on the other hand weakening SC synapses in a non-DG-dependent manner (direct PP-CA1 inputs). This explanation is consistent with several studies in the literature reporting mechanistic and functional differences between the direct and the indirect inputs to CA1 (refs 12, 13, 30, 31, 32). Furthermore, earlier in vitro12, 23 and in vivo33 electrophysiology studies found that stimulation of PP-CA1 inputs to the hippocampus could depotentiate synaptic plasticity that had been previously acquired at SC synapses suggesting that the direct PP pathway might promote depotentiation during hippocampal learning. To test this possibility, we used dual, orthogonal pharmacogenetic inhibition of DG and entorhinal cortex to show that the memory loss phenomenon we observed depended on PP inputs (Fig. 4e). Furthermore, one of the earlier studies23 had shown that PP stimulation-induced SC depotentiation could be inhibited by blockade of adenosine A1 receptors, but not several other receptors, and we found that bilateral administration of DPCPX to the CA1 region of the hippocampus blocked synaptic depotentiation in our model (Fig. 4g).

Our data lead us to propose a novel function for PP-CA1 inputs to the hippocampus. During CS–US presentation, but not during presentation of unpaired CS–US or CS alone, information arriving via this pathway actively promotes depotentiation of SC synapses, while information arriving via the DG pathway opposes this depotentiation. Thus, in an animal that has successfully acquired a hippocampal-dependent memory, and in which the direct and indirect pathways are intact, SC synaptic strength is stable and memories can be retrieved. However, when the DG pathway is blocked, as we have done artificially in our study, depotentiation is favoured and memory is lost (see scheme, Fig. 6). The precise function of PP-dependent SC depotentiation remains unclear at this point, but we speculate that it may play a role in weakening previously acquired associations to facilitate the encoding of new memories. Existing data show that selective blockade of synaptic activity in entorhinal cortex neurons projecting to CA1 impairs the acquisition of trace fear conditioning34 and support our hypothesis of a positive role for this pathway in learning13, 30, 32, 33. Moreover, our DPCPX experiments suggest that blockade of the depotentiation mechanism promotes SC synaptic plasticity during CS–US presentation in otherwise intact animals (Fig. 4g). However, further loss and gain-of-function manipulations of this pathway coupled with in vivoelectrophysiology and learning behaviour are needed to directly test a role of PP-CA1 inputs in memory clearing.

Figure 6: Model for function of PP-CA1 inputs to the hippocampus.

Figure 6

Model for function of PP-CA1 inputs to the hippocampus.

Model for function of PP-CA1 inputs to the hippocampus.

Area CA1 of the hippocampus receives information directly from the entorhinal cortex (direct PP-CA1 pathway) and also indirectly via the tri-synaptic circuit. (a) Presentation of paired CS–US promotes potentiation of SC synapses (+) via the indirect pathway depotentiation of SC synapses (–) via the PP-CA1 pathway. In an animal having successfully undergone learning, potentiation and depotentiation are balanced, SC synaptic strength is stable and memories can be retrieved. (b) Inhibition of DG during CS–US presentation suppresses potentiation via the indirect pathway, unmasking depotentiation of SC synapses and promoting memory loss.

Our finding that DG granule cells are not required for retrieval of hippocampal memory is consistent with previous data arguing that retrieval of associative information encoded in CA3–CA3 and SC plasticity is achieved via direct PP projections to CA3 (refs 1, 2, 3, 4, 35, 36, 37, 38). However, our data appear to contradict at least one recent study demonstrating a role for DG granule cells in retrieval during contextual fear conditioning39. We believe this discrepancy is due to a requirement for DG granule cells in the processing of the contextual CS (ref. 40). However, to rule out the possibility that other methodological differences between the studies underlie the discrepancy, it would be important to determine whether the cell-type specific optogenetic inhibition method used in their study left intact the recall of hippocampal-dependent memories for discrete cues.

Our study raises several questions. First, while we show SC depotentiation is adenosine receptor dependent, the location of adenosine signalling is not clear. Adenosine A1 receptors are expressed highly in CA3 pyramidal cells as well as more modestly in CA1 (ref. 28), and a study in which this receptor was selectively knocked out in one or the other of these structures demonstrated a role for presynaptic CA3, but not postsynaptic CA1 receptors in dampening SC neurotransmission41suggesting a presynaptic mechanism for our effect. The source of adenosine, on the other hand, could involve pre- and/or postsynaptic release as well as release from non-neuronal cells such as astrocytes27, 42. Second, although our DPCPX experiment pointed to a role for PP-CA1 projections in SC depotentiation, our entorhinal cortex pharmacogenetic inhibition experiment did not allow us to distinguish between contributions of PP-CA1 and PP-CA3 inputs. Although we cannot rule out a contribution of PP-CA3 projections to SC depotentiation, earlier in vitro and in vivo electrophysiology studies clearly demonstrate a role for PP-CA1 in SC depotentiation12, 22, 33. Third, the method we used to assess SC postsynaptic strength, namely electrical stimulation evoked field potentials does not allow us to rule out that changes in synaptic plasticity at non-SC inputs underlie our plasticity effects. Experiments using targeted optogenetic stimulation of CA3 efferents could be used to more selectively measure SC synaptic strength. Fourth, our observation that SC depotentiation and memory loss occurred only during paired, but not unpaired CS–US presentation (Fig. 2d,e) suggests that the memory loss phenomenon we describe is distinct from other well-described avenues for memory degradation, including enhancement of extinction43 and blockade of reconsolidation44. Finally, our findings demonstrating generalization of DG inhibition-induced memory loss across tasks coupled with our identification of an endogenous pharmacological target that can induce similar memory loss raise the possibility that the novel memory mechanism we have uncovered may be useful for erasing unwanted memories in a clinical setting.

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Sleep and memory

Larry H. Bernstein, MD, FCAP, Curator



Learning with the Lights Out

Researchers are uncovering the link between sleep and learning and how it changes throughout our lives.

By Jenny Rood | March 1, 2016    http://www.the-scientist.com/?articles.view/articleNo/45335/title/Learning-with-the-Lights-Out

NIGHTY NIGHT: Goffredina Spanò from Jamie Edgin’s University of Arizona lab uses polysomnography to measure sleep in a toddler with Down syndrome.PAMELA SPEDER

By the early 2000s, scientists had found that sleep helps young adults consolidate memory by reinforcing and filing away daytime experiences. But the older adults that Rebecca Spencer was studying at the University of Massachusetts Amherst didn’t seem to experience the same benefit. Spencer wondered if developmental stage altered the relationship between sleep and memory, and chose nearby preschool children as subjects. She found that habitual nappers benefitted the most from daytime rest, largely because their memories decayed the most without a nap. “By staying awake, they have more interference from daytime experiences,” Spencer explains.

Until recently, most of the research into the relationship between memory and sleep has been conducted using young adults or animal models. These studies have suggested that dampened sensory inputs during sleep allow the brain to replay the day’s events during a period relatively free of distracting information, helping to solidify connections and transfer daytime hippocampal memories into long-term storage in the cortex. But how sleep and memory interact at different ages has been an open question.

In children younger than 18 months, learning is thought to occur in the cortex because the hippocampus isn’t yet fully developed. As a result, researchers hypothesize that infants don’t replay memories during sleep, the way adults do. Instead, sleep merely seems to prevent infants from forgetting as much as they would if they were awake. “The net effect is that sleep permits infants to retain more of the redundant details of a learning experience,” says experimental psychologist Rebecca Gómez of the University of Arizona. By the time they are two years old, “we think that children have the brain development that supports an active process of consolidation,” she adds.

At that age, adequate nighttime sleep becomes critical for learning. Toddlers who sleep less than 10 hours display lasting cognitive deficits, even if they catch up on sleep later in their development (Sleep, 30:1213-19, 2007). The effects are particularly strong in children with developmental disorders, who often suffer from sleep disruptions. “Kids with Down syndrome that are sleep-impaired look like they have very large differences in language,” says Jamie Edgin of the University of Arizona who studies sleep and cognition in such children. When comparing Down syndrome children who are sleep deprived with those who sleep normally, she has observed a vocabulary difference of more than 190 words on language tests, even after controlling for behavioral differences.

Understanding the impact of sleep on memory could also help another at-risk group of learners at the other end of the age spectrum. Previous research has suggested that older adults don’t remember newly acquired motor skills as well as young adults do, perhaps because the posttraining stages of the learning process appear diminished. But neuroscientist Maria Korman and her colleagues at the University of Haifa in Israel recently demonstrated that a nap soon after learning can allow the elderly to retain procedural memories just as well as younger people (Neurosci Lett, 606:173-76, 2015). Korman hypothesizes that by shortening the interval between learning and consolidation, the nap prevents intervening experiences from weakening the memory before it solidifies. Overnight sleep might be even better, if the motor skills—in this case a complex sequence of finger and thumb movements on the nondominant hand—are taught late enough in the day, something she is testing now.

Optimizing the timing of sleep and training in the elderly takes advantage of something Korman sees as a positive side of growing old. “As we age, our neural system becomes more aware of the relevance of the task,” Korman says. Unlike young adults who solidify all the information they acquire throughout the day, older people consolidate “those experiences that were tagged by the brain as very important.”

Tests for older adults’ memory acuity are generating new findings about the relationship between sleep and memory at other ages as well. After learning at a conference about a memory test for cognitive impairment and dementia in older adults, neuroscientist Jeanne Duffy of Brigham and Women’s Hospital in Boston wondered if sleep could help strengthen the connection between names and faces. She and her colleagues found that young adults who slept overnight after learning a list of 20 names and faces showed a 12 percent increase in retention when tested 12 hours later compared with subjects who didn’t sleep between training and testing (Neurobiol Learn Mem, 126:31-38, 2015). The findings have “an immediate real-world application,” Duffy says, as they address a common memory concern among people of all ages.

A poll by the National Sleep Foundation found that adolescents have a deficit of nearly two hours of sleep per night during the school week compared with the weekend, suggesting the potential for serious learning impairments, according to Jared Saletin, a postdoctoral sleep researcher at Brown University. In fact, one study found that restricting 13- to 17-year-olds to six and a half hours of sleep a night for five nights reduced the information they absorbed in a school-like setting (J Adolesc Health, 47:523-25, 2010). However, other studies have suggested that four nights of just five hours of sleep didn’t impair 14- to 16-year-olds’ performance on tests of skills and vocabulary (Sleep Med, 12:170-78, 2011). A lack of consistency in study design and the ages of the subjects makes these conflicting results difficult to interpret, Gómez writes in a review, and much remains to be discovered about the true impact of sleep deficits on teenagers’ learning (Trans Issues in Psych Sci, 1:116-25, 2015).

Developing a fuller picture of what happens to memories during sleep—and how best to tweak sleep habits to aid the recall process—could benefit some of society’s most sleep-deprived members of every age. “We need to understand this role of sleep in memory because there is such potential for intervention,” Spencer says. “Now that we have a well-founded concept of what sleep can do for memory, it’s time to put it to the test.”


Associations Between Sleep Duration Patterns and Behavioral/Cognitive Functioning at School Entry

Évelyne Touchette, MPs,1,2 Dominique Petit, PhD,1 Jean R. Séguin, PhD,3,4,5 Michel Boivin, PhD,6,7 Richard E. Tremblay, PhD,2,3,4,5,8 and Jacques Y. Montplaisir, MD, CRCP(c), PhD1,5
Sleep. 2007 Sep 1; 30(9): 1213–1219.     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1978413/
See commentary “Sleep and the Developing Brain

The aim of the study was to investigate the associations between longitudinal sleep duration patterns and behavioral/cognitive functioning at school entry.
Design, Setting, and Participants:   Hyperactivity-impulsivity (HI), inattention, and daytime sleepiness scores were measured by questionnaire at 6 years of age in a sample of births from 1997 to 1998 in a Canadian province (N=1492). The Peabody Picture Vocabulary Test – Revised (PPVT-R) was administered at 5 years of age and the Block Design subtest (WISC-III) was administered at 6 years of age. Sleep duration was reported yearly by the children’s mothers from age 2.5 to 6 years. A group-based semiparametric mixture model was used to estimate developmental patterns of sleep duration. The relationships between sleep duration patterns and both behavioral items and neurodevelopmental tasks were tested using weighted multivariate logistic regression models to control for potentially confounding psychosocial factors.  Results:   Four sleep duration patterns were identified: short persistent (6.0%), short increasing (4.8%),10-hour persistent (50.3%), and 11-hour persistent (38.9%). The association of short sleep duration patterns with high HI scores (P=0.001), low PPVT-R performance (P=0.002), and low Block Design subtest performance (P=0.004) remained significant after adjusting for potentially confounding variables.   Conclusions:   Shortened sleep duration, especially before the age of 41 months, is associated with externalizing problems such as HI and lower cognitive performance on neurodevelopmental tests. Results highlight the importance of giving a child the opportunity to sleep at least 10 hours per night throughout early childhood.

Citation: Touchette E; Petit D; Séguin JR; Boivin M; Tremblay RE; Montplaisir JY. Associations between sleep duration patterns and behavioral/cognitive functioning at school entry. SLEEP 2007;30(9):1213-1219.

Nap it or leave it in the elderly: A nap after practice relaxes age-related limitations in procedural memory consolidation

M. Kormana, , Y. DaganbA. Karnib   


•   Elderly individuals gain in practicing a new motor task as do young adults.
•   But elderly individuals fail to show delayed (offline) memory related gains.
•   A post-training nap uncovered robust offline skill consolidation in the elderly.
•   Consolidation processes are preserved in aging but are more stringently controlled.
•   Sleep scheduling can relax age related constraints on mnemonic processes.       
Using a training protocol that effectively induces procedural memory consolidation (PMC) in young adults, we show that older adults are good learners, robustly improving their motor performance during training. However, performance declined over the day, and overnight ‘offline’ consolidation phase performance gains were under-expressed. A post-training nap countered these deficits. PMC processes are preserved but under-engaged in the elderly; sleep can relax some of the age-related constraints on long-term plasticity.


A new face of sleep: The impact of post-learning sleep on recognition memory for face-name associations

Leonie Maurera, c, Kirsi-Marja Zittinga, b, Kieran Elliotta, Charles A. Czeislera, b, Joseph M. Rondaa, b, Jeanne F. Duffya, b, ,


•   We tested whether sleep influences the accuracy of remembering face-name associations.
•   Presentation and recall were 12 h apart, one time with 8 h sleep and once without.
•   More correct face-name pairs were recalled when there was a sleep opportunity.
•   Sleep duration or sleep stage was not associated with improvement between conditions.     

Sleep has been demonstrated to improve consolidation of many types of new memories. However, few prior studies have examined how sleep impacts learning of face-name associations. The recognition of a new face along with the associated name is an important human cognitive skill. Here we investigated whether post-presentation sleep impacts recognition memory of new face-name associations in healthy adults.

Fourteen participants were tested twice. Each time, they were presented 20 photos of faces with a corresponding name. Twelve hours later, they were shown each face twice, once with the correct and once with an incorrect name, and asked if each face-name combination was correct and to rate their confidence. In one condition the 12-h interval between presentation and recall included an 8-h nighttime sleep opportunity (“Sleep”), while in the other condition they remained awake (“Wake”).

There were more correct and highly confident correct responses when the interval between presentation and recall included a sleep opportunity, although improvement between the “Wake” and “Sleep” conditions was not related to duration of sleep or any sleep stage.

These data suggest that a nighttime sleep opportunity improves the ability to correctly recognize face-name associations. Further studies investigating the mechanism of this improvement are important, as this finding has implications for individuals with sleep disturbances and/or memory impairments.

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Brain Matters from iBiology

Larry H. Bernstein, MD, FCAP, Curator




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


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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