Cellular stress in the brain may contribute to non-alcoholic fatty liver disease

Disruptions in a protein folding process occurring in the brain, known as endoplasmic reticulum (ER) stress, may cause non-alcoholic fatty liver disease, independent of other factors. A research team at the George Washington University (GW) published their results in the Journal of Clinical Investigation Insight.

“Nearly 75 percent of obese adults experience non-alcoholic fatty liver disease. However, its underlying causes are unclear,” said Colin Young, Ph.D., senior author and assistant professor of pharmacology and physiology at the GW School of Medicine and Health Sciences. “Recent findings have pointed to ER stress as central to its development. What our research shows is that ER stress in the brain is a key contributor.”

As the primary site of cellular protein folding, the ER plays a critical role in maintaining cellular function. When there is nutritional excess, the protein load exceeds the ER folding capacity and a collection of conserved signaling pathways, termed the unfolded protein response (UPR), are activated to preserve ER function. While beneficial in the short-term, chronic UPR activation, known as ER stress, is a major pathological mechanism in metabolic disease, such as obesity.

Young’s research team demonstrated that UPR activation in the brain, specifically in the forebrain, is causally linked to non-alcoholic fatty liver disease. Also known as hepatic steatosis, the research shows that brain ER stress can cause the disease independent of changes in body weight, food intake, and other factors.

Non-alcoholic fatty liver disease impairs normal liver function and is linked to other diseases such as diabetes and cardiovascular disease. The next step is to determine how and why ER stress occurs in the brain and how it causes fat build up in the liver.

“Further research may give us another possible avenue for targeting fatty liver disease,” said Young. “The field has been focused on how we can improve the liver, for example, by developing drugs that target the liver. Our research suggests that we may also need to think about targeting the brain to treat non-alcoholic fatty liver disease.”

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Protein ‘spy’ gains new abilities

Rice University scientists have learned to spy on cells with a divide-and-conquer strategy to label proteins.

Graduate student Emily Thomas, synthetic biologist Jonathan Silberg and their colleagues built upon established techniques that attach bio-orthogonal (noninterfering), artificial amino acids to transfer RNA (tRNA), which are used by ribosomes to synthesize proteins.

Because the amino acids are “noncanonical,” they are effective tags that help researchers identify proteins being synthesized in a cell. The Rice lab’s breakthrough was the discovery of a tRNA synthetase that only adds the amino acid to the tRNA when it binds a chemical. When prompted, the tRNA synthetase charges a tRNA with the bio-orthogonal amino acid, which is then used by ribosomes to insert the tag into proteins made in the cell.

The study appears in the American Chemical Society journal ACS Synthetic Biology.

These bio-orthogonal tags give researchers a snapshot of total protein synthesis in the cell. “Instead of physically separating a cell from a mixture to find the proteins being made, we can use this engineered switch to put what amounts to a fishhook on every protein in a specific cell,” Silberg said. “This approach will allow us to increase spatial and temporal control over the tagging of proteins synthesized in a given cell.”

Since many proteins appear and disappear during the development of an organism or the spread of a disease, the technique could be helpful to identify cellular changes that underlie disease. Thomas characterized her technique as a “protein spy.”

“It spies on what proteins are being made inside the cell,” she said. “Current technologies just spy on everything, but I want to be more specific. I want more control over when I turn my spy on or off, so I can track only the cells I’m interested in.”

The researchers used an azidonorleucine (Anl) amino acid to tag proteins in Escherichia coli bacteria cells. Thomas’ engineered switch is controlled like a computer program’s AND gate. The switch only charges tRNA with Anl efficiently when the switch is synthesized and a chemical is present in the cell to flip the switch.

Silberg said the technique will provide new control over protein transcription and tagging to researchers. “In human biology, a lot of the control comes at the DNA level, but over the past 20 years it’s become apparent that a lot of control comes at the protein level as well,” he said. “We have fewer genes in our genome than people originally expected because there’s this other layer of complexity in the proteome, the collection of proteins expressed by the genome.

“Proteins are the business side of the cell,” he said. “They provide structure and do a lot of the signaling within a cell. They give rise to a lot of the complexity we observe. In the future, our technique could help people understand the details of a disease by providing snapshots of proteins synthesized in specific cells at different times during development and allowing comparisons of healthy and diseased cells.

“The prospect of doing this in humans is the genetic technology equivalent of going to Mars right now,” Silberg said. “It’s far out.”

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Immune system, brain structure and memory linked

The body’s immune system performs essential functions, such as defending against bacteria and cancer cells. However, the human brain is separated from immune cells in the bloodstream by the so-called blood-brain barrier. This barrier protects the brain from pathogens and toxins circulating in the blood, while also dividing the immune cells of the human body into those that fulfill their function in the blood and those that work specifically in the brain. Until recently, it was thought that brain function was largely unaffected by the peripheral immune system.

However, in the past few years, evidence has accumulated to indicate that the blood’s immune system could in fact have an impact on the brain. Scientists from the University of Basel’s Transfaculty Research Platform Molecular and Cognitive Neurosciences (MCN) have now carried out two independent studies that demonstrate that this link between the immune system and brain is more significant than previously believed.

Search for regulatory patterns

In the first study, the researchers searched for epigenetic profiles, i.e. regulatory patterns, in the blood of 533 young, healthy people. In their genome-wide search, they identified an epigenetic profile that is strongly correlated with the thickness of the cerebral cortex, in particular in a region of the brain that is important for memory functions. This finding was confirmed in an independent examination of a further 596 people. It also showed that it is specifically those genes that are responsible for the regulation of important immune functions in the blood that explain the link between the epigenetic profile and the properties of the brain.

Gene variant intensifies traumatic memories

In the second study, the researchers investigated the genomes of healthy participants who remembered negative images particularly well or particularly poorly. A variant of the TROVE2 gene, whose role in immunological diseases is currently being investigated, was linked to participants’ ability to remember a particularly high number of negative images, while their general memory remained unaffected.

This gene variant also led to increased activity in specific regions of the brain that are important for the memory of emotional experiences. The researchers also discovered that the gene is linked to the strength of traumatic memories in people who have experienced traumatic events.

The results of the two studies show that both brain structure and memory are linked to the activity of genes that also perform important immune regulatory functions in the blood. “Although the precise mechanisms behind the links we discovered still need to be clarified, we hope that this will ultimately lead to new treatment possibilities,” says Professor Andreas Papassotiropoulos, Co-Director of the University of Basel’s MCN research platform. The immune system can be precisely affected by certain medications, and such medications could also have a positive effect on impaired brain functions.

Innovative research methods

These groundbreaking findings were made possible thanks to cutting edge neuroscientific and genetic methods at the University of Basel’s MCN research platform. Under the leadership of Professor Andreas Papassotiropoulos and Professor Dominique de Quervain, the research platform aims to help us better understand human brain functions and to develop new treatments for psychiatric disorders.

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Skin stem cells used to generate new brain cells

Using human skin cells, University of California, Irvine neurobiologists and their colleagues have created a method to generate one of the principle cell types of the brain called microglia, which play a key role in preserving the function of neural networks and responding to injury and disease.

The finding marks an important step in the use of induced pluripotent stem (iPS) cells for targeted approaches to better understand and potentially treat neurological diseases such as Alzheimer’s. These iPS cells are derived from existing adult skin cells and show increasing utility as a promising approach for studying human disease and developing new therapies.

Skin cells were donated from patients at the UCI Alzheimer’s Disease Research Center. The study, led by Edsel Abud, Wayne Poon and Mathew Blurton Jones of UCI, used a genetic process to reprogram these cells into a pluripotent state capable of developing into any type of cell or tissue of the body.

The researchers then guided these pluripotent cells to a new state by exposing the cells to a series of differentiation factors which mimicked the developmental origin of microglia. The resulting cells act very much like human microglial cells. Their study appears in the current issue of Neuron.

In the brain, microglia mediate inflammation and the removal of dead cells and debris. These cells make up 10- to 15-percent of brain cells and are needed for the development and maintenance of neural networks.

“Microglia play an important role in Alzheimer’s and other diseases of the central nervous system. Recent research has revealed that newly discovered Alzheimer’s-risk genes influence microglia behavior. Using these cells, we can understand the biology of these genes and test potential new therapies,” said Blurton-Jones, an assistant professor of the Department of Neurobiology & Behavior and Director of the ADRC iPS Core.

“Scientists have had to rely on mouse microglia to study the immunology of AD. This discovery provides a powerful new approach to better model human disease and develop new therapies,” added Poon, a UCI MIND associate researcher.

Along those lines, the researchers examined the genetic and physical interactions between Alzheimer’s disease pathology and iPS-microglia. They are now using these cells in three-dimensional brain models to understand how microglia interact with other brain cells and influence AD and the development of other neurological diseases.

“Our findings provide a renewable and high-throughput method for understanding the role of inflammation in Alzheimer’s disease using human cells,” said Abud, an M.D./Ph.D. student. “These translational studies will better inform disease-modulating therapeutic strategies.”

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Your brain, not your white blood cells, keeps you warm, new study suggests

A new study from the Icahn School of Medicine at Mount Sinai provides important insights into how the body regulates its production of heat, a process known as thermogenesis that is currently intensely studied as a target of diabetes and obesity treatment in humans.

While researchers had previously hypothesized that macrophages, a class of white blood cells, played a major role in thermogenesis, the new study suggests that the main driver of thermogenesis is the sympathetic nervous system, which is chiefly controlled by the brain. The results were published online in Nature Medicine.

The Mount Sinai research team led by Christoph Buettner, MD, PhD, senior author of the study and Professor of Medicine (Endocrinology, Diabetes, and Bone Disease) at the Icahn School of Medicine at Mount Sinai, focused on catecholamines, hormones released by the sympathetic nervous system to activate brown fat tissue. Brown adipose tissue is a type of fat tissue that burns energy to produce heat and keep us warm. Catecholamines can also convert white fat tissue, the more familiar kind of fat tissue that stores lipids, into a tissue that resembles brown fat. The researchers tested whether macrophages could provide an alternative source of catecholamines, as had been proposed in recent years.

“Thermogenesis is a metabolic process that receives a lot of interest as a target of drugs that allow you to burn energy and hence reduce obesity and improve diabetes. It turns out that macrophages are not that important, as they are unable to make catecholamines, but clearly the brain through the sympathetic nervous system is,” says Dr. Buettner. “Therefore, it is very important to study the role of the brain and the sympathetic nervous system when it comes to understanding metabolism.”

The ability to generate heat is critical for the survival of warm-blooded animals, including humans, as it prevents death by hypothermia. “This evolutionary pressure shaped the biology of humans and that of other warm-blooded animals, and may in part explain why humans are susceptible to developing diabetes in the environment in which we live,” says Dr. Buettner.

According to Dr. Buettner, while a lot of effort has been invested in targeting the immune system to cure diabetes and insulin resistance, as of yet there are no anti-inflammatory drugs that have been shown to work well in humans with metabolic disease. “Our study suggests that perhaps the key to combating the devastating effects of diabetes and obesity in humans is to restore the control of thermogenesis and metabolism by the brain and the autonomic nervous system,” says Dr. Buettner.

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Brain tissue from a petri dish

The most complex organ in humans is the brain. Due to its complexity and, of course, for ethical reasons, it is extremely difficult to do scientific experiments on it — ones that could help us to understand neurodegenerative diseases like Parkinson’s, for example. Scientists at the Luxembourg Centre for Systems Biomedicine (LCSB) of the University of Luxembourg have now succeeded in turning human stem cells derived from skin samples into tiny, three-dimensional, brain-like cultures that behave very similarly to cells in the human midbrain. In the researchers’ petri dishes, different cell types develop, connect into a network, exchange signals and produce metabolic products typical of the active brain. “Our cell cultures open new doors to brain research,” says Prof. Dr. Jens Schwamborn, in whose LCSB research group Developmental & Cellular Biology the research work was done. “We can now use them to study the causes of Parkinson’s disease and how it could possibly be effectively treated.” The team publishes its results today in the scientific journal Stem Cell Reports.

The human midbrain is of particular interest to Parkinson’s researchers: it is the seat of the tissue structure known medically as the substantia nigra. Here, nerve cells — specifically dopaminergic neurons — produce the messenger dopamine. Dopamine is needed to maintain smooth body movements. If the dopaminergic neurons die off, then the person affected develops tremors and muscle rigidity, the distinctive symptoms of Parkinson’s disease. For ethical reasons, researchers cannot take cells from the substantia nigra to study them. Research groups around the world are therefore working on cultivating three-dimensional structures of the midbrain in petri dishes. The LCSB team led by stem cell researcher Jens Schwamborn is one such group.

The LCSB scientists worked with so-called induced pluripotent stem cells — stem cells that cannot produce a complete organism, but which can be transformed into all cell types of the human body. The procedures required for converting the stem cells into brain cells were developed by Anna Monzel as part of her doctoral thesis, which she is doing in Schwamborn’s group. “I had to develop a special, precisely defined cocktail of growth factors and a certain treatment method for the stem cells, so that they would differentiate in the desired direction,” Monzel describes her approach. To do this, she was able to draw on extensive preparatory work that had been done in Schwamborn’s team the years before. The pluripotent stem cells in the petri dishes multiplied and spread out into a three-dimensional supporting structure — producing tissue-like cell cultures.

“Our subsequent examination of these artificial tissue samples revealed that various cell types characteristic of the midbrain had developed,” says Jens Schwamborn. “The cells can transmit and process signals. We were even able to detect dopaminergic cells — just like in the midbrain.” This fact makes the LCSB scientists’ results of extraordinary interest to Parkinson’s researchers worldwide, as Schwamborn stresses: “On our new cell cultures, we can study the mechanisms that lead to Parkinson’s much better than was ever the case before. We can test what effects environmental impacts such as pollutants have on the onset of the disease, whether there are new active agents that could possibly relieve the symptoms of Parkinson’s — or whether the disease could even be cured from its very cause. We will be performing such investigations next.”

The development of the brain-like tissue cultures not only opens doors to new research approaches. It can also help to reduce the amount of animal testing in brain research. The cell cultures in the petri dishes are of human origin, and in some aspects resemble human brains more than the brains of lab animals such as rats or mice do. Therefore, the structures of human brains and its modes of function can be modelled in different ways than it is possible in animals. “There are also attractive economic opportunities in our approach,” Jens Schwamborn explains: “The production of tissue cultures is highly elaborate. In the scope of our spin-off Braingineering Technologies Sarl, we will be developing technologies by which we can provide the cultures for a fee to other labs or the pharmaceutical industry for their research.”

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Transcription factor expression tied to medial amygdala neuronal ID, sex-specific response

Neurons derived from two different types of precursor cells that later develop into neurons in the medial amygdala — one of the interconnected structures in the brain involved in emotion, motivation and memory — help to program innate reproductive and aggressive behaviors into the brain, research led by Children’s National Health System indicates.

The finding, published April 7, 2017 in the journal eLife, helps to explain how events that occur when the fetal brain is developing may program instinctual behaviors relied on by creatures big and small, such as avoiding predators, mating and protecting their territory. One precursor cell type expresses a developmentally regulated transcription factor, a protein known as Dbx1; the other cell type expresses Foxp2, the forkhead transcription factor previously identified in humans as required for appropriate production of speech. When cells derived from these distinct cell subpopulations are activated during certain encounters, they show differing patterns of activation in male, versus female brains.

“Because they’re hard wired, we reasoned there would be a process that occurs in the fetal brain to lay down these circuits,” says Joshua G. Corbin, Ph.D., principal investigator in the Center for Neuroscience Research at Children’s National and senior study author. “By going back in time, we were able to determine where these neurons came from and how they developed. What’s most surprising is the same population of neurons exists in the male brain as the female brain, yet they respond differently to mating cues,” Corbin adds.

The brain’s limbic system weaves together environmental information and social cues and balances them against our overwhelming drive to survive in order to generate an appropriate behavioral response. One brain region where this critical activity occurs is in the medial subnucleus of the amygdala, which receives input directly from the olfactory system. Across a variety of species, chemosensory information from the olfactory bulb is processed to regulate innate behaviors.

To test their hypotheses, the study authors carried out tests tied to a trifecta of instinctual behaviors — aggression, mating and avoiding predator odor — in male and female experimental models. For instance, in males they gauged territorial aggression by placing an intruder into the cage. And for females, they removed offspring from a nursing female and introduced a male intruder into the cage and also had a second control group whose offspring were removed but no intruder was added.

Then, they examined the patterns of activation of Dbx1- derived and Foxp2+ cells. The most striking sex-specific difference in activation of Dbx1-derived and Foxp2+ cells in the medial subnucleus of the amygdala occurred during mating, Corbin and co-authors write.

“These populations of neurons may act as a toggle switch, informing how the male brain interprets mating information versus how the female brain does so,” Corbin adds.

Now that the research team has identified specific neuronal populations of interest, the next challenge will be manipulating them. For that step, they will shine light of a certain wavelength on them to turn on or switch off neural activity.

“To understand how a certain part of the brain regulates behavior, we can silence a few neurons to see what those specific neurons can do. Or we can activate them for a short period of time to see which behavior arises due to that activation,” he explains. “We also are in the process of understanding which genes are associated with development of these neurons. So far, it appears that many of the genes that we hypothesize to be a part of this process also turn out to be autism spectrum susceptibility genes. That makes sense as we think about brain development. In autism, the limbic system is dysfunctional.”

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Synthetic biologists engineer inflammation-sensing gut bacteria

Synthetic biologists at Rice University have engineered gut bacteria capable of sensing colitis, an inflammation of the colon, in mice. The research points the way to new experiments for studying how gut bacteria and human hosts interact at a molecular level and could eventually lead to orally ingestible bacteria for monitoring gut health and disease.

The research, published in a new study in Molecular Systems Biology, involved a series of breakthroughs in the lab of Jeffrey Tabor, assistant professor of bioengineering and of biosciences at Rice, and key contributions from collaborators Robert Britton and Noah Shroyer at Baylor College of Medicine. Tabor’s team, including lead co-author and postdoctoral researcher Kristina Daeffler, identified the first genetically encoded sensor of a novel biomarker linked to inflammation, inserted the genes for the sensor into a well-studied gut bacterium and collaborated with Shroyer and Britton to use the engineered bacteria to detect colon inflammation in mice.

“The gut harbors trillions of microorganisms that play key roles in health and disease,” Tabor said. “However, it is a dark and relatively inaccessible place, and few technologies have been developed to study these processes in detail. On the other hand, bacteria have evolved tens of thousands of genetically encoded sensors, many of which sense gut-linked molecules. Thus, genetically engineered sensor bacteria have tremendous potential for studying gut pathways and diagnosing gut diseases.”

Synthetic biologists like Tabor specialize in programming single-celled organisms like bacteria in much the same way an engineer might program a robot. In particular, Tabor’s team is working to develop bacterial sensors that can detect disease signals in the gut. Like electrical engineers who build circuits from wires and electronic components, Tabor’s team uses genetic circuits to program single-celled creatures to carry out complex information processing.

Previous work has suggested that alterations to the gut microbiota, genetic predisposition and other environmental factors may play key roles in inflammatory bowel disease, a condition that includes Crohn’s disease and ulcerative colitis and which affects as many as 1.6 million Americans.

“Based on a number of previous studies, we hypothesized that the molecule thiosulfate may be elevated during colitis,” Daeffler said. “It has been difficult for scientists to study this link because there aren’t tools for reliably measuring thiosulfate in living animals. Our first goal in this project was to engineer such a tool.”

From the outset of the project in 2015, Daeffler said, the idea was to use sensor bacteria, in this case an engineered form of Escherichia coli, to sense thiosulfate and related sulfur-containing compounds that may also be biomarkers of colitis. There were well-understood methods for programming E. coli to produce a fluorescent green protein in response to specific stimuli, but there were no known genes — in any organism — that were used to sense thiosulfate, and few for the other compounds.

“There’s a link between gut sulfur metabolism and inflammation, and we knew that we needed to be able to measure sulfur metabolites accurately to diagnose colon inflammation,” she said.

Tabor said study co-author Ravi Sheth, an undergraduate researcher in the group in 2015, used a computer program to identify potential sensors of thiosulfate and other sulfur compounds in the genome of Shewanella, a type of bacteria that live in marine sediment. Tabor’s group believes that Shewanella likely breathe these molecules and use the sensors to turn on the proper enzymes in their presence.

Daeffler spent one year engineering E. coli to express the sensor genes, validate their function and optimize them to respond to the potential biomarkers by producing a green fluorescent protein signal. It took another year to prove that the system worked and detected colon inflammation in mice.

The researchers administered orally two drops containing about a billion sensor bacteria to both healthy mice and to mice with colitis. They measured the activity of the sensor bacteria in each group six hours later. The tell-tale green fluorescent protein showed up in the feces of the mice. Though it was not visible to the unaided eye, it could easily be measured with a standard laboratory instrument called a flow cytometer.

The team found that the thiosulfate sensor was activated in the mice with inflammation, and was not activated in the healthy mice. Furthermore, the researchers found that the more inflammation the mouse had, the more the sensor was activated.

Tabor said the study shows that gut bacteria can be outfitted with engineered sensors and used to noninvasively measure specific metabolites and that this result could open the door to many new studies that could help elucidate a wide range of gut processes.

Though it would likely take several additional years of development, and it remains unknown if thiosulfate is a biomarker of human colitis, the sensor bacteria might eventually be re-engineered to function as a diagnostic of human colitis, Tabor said. In particular, the green fluorescent protein could be replaced with an enzyme that makes a colored pigment.

“We’d like to develop a home inflammation test where a person prone to colitis flare-ups would eat yogurt that contained the engineered bacteria and see blue pigment in the toilet if they were sick,” he said.

Tabor said such a test could reduce unneeded and costly trips to the doctor and unneeded colonoscopy procedures, which are both expensive and invasive. He said his team has begun collaborations with gastroenterologists at Baylor to achieve this goal.

 

New function discovered for compound that may help slow aging

Researchers at Oregon State University have found that a compound called rapamycin has unusual properties that may help address neurologic damage such as Alzheimer’s disease.

A study just published in Aging Cell outlines a new understanding of how this compound works.

“It’s possible this could provide a new therapeutic approach to neurologic disease,” said Viviana Perez, an assistant professor in the Department of Biochemistry and Biophysics in OSU’s College of Science, expert on the biological processes of aging and principal investigator in the Linus Pauling Institute.

Scientists have now identified two mechanisms of action of rapamycin. One was already known. The newly-discovered mechanism is what researchers say might help prevent neurologic damage and some related diseases.

“The value of rapamycin is clearly linked to the issue of cellular senescence, a stage cells reach where they get old, stop proliferating and begin to secrete damaging substances that lead to inflammation,” Perez said. “Rapamycin appears to help stop that process.”

This secretion of damaging compounds, researchers say, creates a toxic environment called senescence-associated secretory phenotype, or SASP. It’s believed this disrupts the cellular microenvironment and alters the ability of adjacent cells to function properly, compromising their tissue structure and function.

This broad process is ultimately linked to aging

“The increase in cellular senescence associated with aging, and the inflammation associated with that, can help set the stage for a wide variety of degenerative disease, including cancer, heart disease, diabetes and neurologic disease, such as dementia or Alzheimer’s,” Perez said. “In laboratory animals when we clear out senescent cells, they live longer and have fewer diseases. And rapamycin can have similar effects.”

Prior to this research, it had only been observed that there was one mechanism of action for rapamycin in this process. Scientists believed it helped to increase the action of Nrf2, a master regulator that can “turn on” up to 200 genes responsible for cell repair, detoxification of carcinogens, protein and lipid metabolism, antioxidant protection and other factors. In the process, it helped reduce levels of SASP.

The new study concluded that rapamycin could also affect levels of SASP directly, separately from the Nrf2 pathway and in a way that would have impacts on neurons as well as other types of cells.

“Any new approach to help protect neurons from damage could be valuable,” Perez said. “Other studies, for instance, have shown that astrocyte cells that help protect neuron function and health can be damaged by SASP. This may be one of the causes of some neurologic diseases, including Alzheimer’s disease.”

Through its ability to help prevent SASP-related cellular damage through two pathways — one involving Nrf2 and the other more directly — rapamycin will continue to generate significant interest in addressing issues related to aging, Perez said.

Rapamycin is a natural compound first discovered from the soils of Easter Island in the South Pacific Ocean. It has already been intensively studied because it can mimic the valuable effects of dietary restriction, which in some animals has been proven to extend their lifespan.

Laboratory mice that have received rapamycin have demonstrated more fitness, less decline in activity with age, improved cognition and cardiovascular health, less cancer, and a longer life.

The use of rapamycin for that purpose in humans has so far been constrained by an important side effect, an increase in insulin resistance that may raise the risk of diabetes. That concern still exists, limiting the use of rapamycin to help address degenerative disease until ways can be found to address that problem.

This may be possible. Scientists are searching for rapamycin analogs that may have similar biological impacts but don’t cause that unwanted side effect.

This research was supported by the American Federation for Aging Research.

Mini brains from the petri dish

A new method could push research into developmental brain disorders an important step forward. This is shown by a recent study at the University of Bonn in which the researchers investigated the development of a rare congenital brain defect. To do so, they converted skin cells from patients into so called induced pluripotent stem cells. From these ‘jack-of-all-trades’ cells, they generated brain organoids — small three-dimensional tissues which resemble the structure and organization of the developing human brain. The work has now been published in the journal Cell Reports.

Investigations into human brain development using human cells in the culture dish have so far been very limited: the cells in the dish grow flat, so they do not display any three-dimensional structure. Model organisms are available as an alternative, such as mice. The human brain has, however, a much more complex structure. Developmental disorders of the human brain can thus only be resembled to a limited degree in the animal model.

Scientists at the Institute of Reconstructive Neurobiology at the University of Bonn applied a recent development in stem cell research to tackle this limitation: they grew three-dimensional organoids in the cell culture dish, the structure of which is incredibly similar to that of the human brain. These “mini brains” offer insight into the processes with which individual nerve cells organize themselves into our highly complex tissues. “The method thus opens up completely new opportunities for investigating disorders in the architecture of the developing human brain,” explains Dr. Julia Ladewig, who leads a working group on brain development.

Rare brain deformity investigated

In their work, the scientists investigated the Miller-Dieker syndrome. This hereditary disorder is attributed to a chromosome defect. As a consequence, patients present malformations of important parts of their brain. “In patients, the surface of the brain is hardly grooved but instead more or less smooth,” explains Vira Iefremova, PhD student and lead author of the study. What causes these changes has so far only been known in part.

The researchers produced induced pluripotent stem cells from skin cells from Miller-Dieker patients, from which they then grew brain organoids. In organoids, the brain cells organize themselves — very similar to the process in the brain of an embryo: the stem cells divide; a proportion of the daughter cells develops into nerve cells; these move to wherever they are needed. These processes resemble a complicated orchestral piece in which the genetic material waves the baton.

In Miller-Dieker patients, this process is fundamentally disrupted. “We were able to show that the stem cells divide differently in these patients,” explains associate professor Dr. Philipp Koch, who led the study together with Dr. Julia Ladewig. “In healthy people, the stem cells initially extensively multiply and form organized, densely packed layers. Only a small proportion of them becomes differentiated and develops into nerve cells.”

Certain proteins are responsible for the dense and even packing of the stem cells. The formation of these molecules is disrupted in Miller-Dieker patients. The stem cells are thus not so tightly packed and, at the same time, do not have such a regular arrangement. This poor organization leads, among other things, to the stem cells becoming differentiated at an earlier stage. “The change in the three-dimensional tissue structure thus causes altered division behavior,” says Ladewig. “This connection cannot be identified in animals or in two-dimensional cell culture models.”

The scientist emphasizes that no new treatment options are in sight as a result of this. “We are undertaking fundamental research here. Nevertheless, our results show that organoids have what it takes to herald a new era in brain research. And if we better understand the development of our brain, new treatment options for disorders of the brain can presumably arise from this over the long term.”

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Making a ‘beeline’ past the blood-brain barrier for drug delivery

Most medicines can’t get through the blood-brain barrier (BBB), a highly selective membrane that separates the circulatory system from the fluid bathing the brain. Certain peptides in animal venoms, however, can navigate across it to inflict damage. Now, researchers are capitalizing on venomous sneak attacks by developing a strategy based on a bee-venom peptide, apamin, to deliver medications to the brain.

The researchers will present their work today at the 253rd National Meeting & Exposition of the American Chemical Society (ACS). ACS, the world’s largest scientific society, is holding the meeting here through Thursday. It features more than 14,000 presentations on a wide range of science topics.

“We thought that because the venoms of some animals are able to attack the central nervous system, they should be able to go through the blood-brain barrier and possibly shuttle drugs across it,” Ernest Giralt, Ph.D., says. Apamin is known to accumulate in the central nervous system of people who’ve been stung by bees.

But the idea of using the apamin peptide itself had some drawbacks. “We knew we could not use apamin directly because it’s toxic,” he says. “But the good news is that the origin of the toxicity is well-known. We thought we could probably modify apamin in such a way that the toxicity would be eliminated, but it would still keep its ability to act as a transporter.”

Apamin’s toxicity stems from its interactions with a potassium channel in neurons. A positively charged group in the apamin molecule mimics the potassium ion and blocks the potassium channel when it binds. To eliminate the toxicity, Giralt’s group at the Institute for Research in Biomedicine (IRB Barcelona, Spain) removed the positively charged chemical anchor that attaches apamin to the channel. Then, the researchers checked to make sure the molecule could still cross the BBB. “This modification made apamin much less toxic, and its ability to cross the BBB was intact,” Giralt says. “This was very good news.”

As a next step, the researchers started tinkering with the molecule to make it smaller and also to make it invisible to the immune system to reduce potential side effects. Several versions of apamin later, they ended up with a promising version called Mini-Ap4. “It surprised us that this molecule crossed the blood-brain barrier much better than apamin itself — it was pure serendipity,” Giralt says. Mini-Ap4 also did not trigger a strong immune system response in animal models, an important factor in drug design.

Other BBB shuttles are in development, but many of them are based on linear peptides, which can be degraded by proteases before a medicine makes it to the brain. “Our niche is that our peptides are cyclic, or in a ring structure, making them completely resistant to proteases,” Giralt explains.

After these initial studies, the team will then put Mini-Ap4 to work, testing two different shuttling strategies. The first will be to simply attach Mini-Ap4 to a protein with a chemical bond and see if it can carry the cargo across the BBB. The second approach will involve filling a nanoparticle with medication and coating the nanoparticle with a forest of Mini-Ap4 molecules to facilitate the transfer across the BBB. The researchers will investigate these strategies in human cells and in mice.

In other preliminary work, the researchers discovered that their version of apamin actually has two conformations, or shapes, and the team is using nuclear magnetic resonance spectroscopy to figure out which one is biologically active. “With that knowledge, we could design even better analogs,” Giralt says. He adds that a person who is allergic to bees probably wouldn’t be allergic to Mini-Ap4, but more work is needed to fully address this issue.

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Hair testing shows high prevalence of new psychoactive substance use

In the last decade hundreds of new psychoactive substances (NPS) have emerged in the drug market, taking advantage of the delay occurring between their introduction into the market and their legal ban. According to the Drug Enforcement Agency (DEA) NPS describes a recently emerged drug that may pose a public health threat. The DEA issues a quarterly Emerging Threat Report, which catalogues the newest identified NPS.

NPS tend to mimic the psychotropic effects of traditional drugs of abuse, but their acute and chronic toxicity, and side-effects are largely unknown. While seizure data from the DEA is often used to indicate what new drugs are being sold in the US, there is a lack of research examining and confirming who has been using such drugs.

Joseph J. Palamar, PhD, MPH, a New York University researcher, has been researching incidental and intentional use of NPS by young adults. His current line of inquiry has focused on survey methods, qualitative interviews, and hair sampling to ascertain frequency and type of NPS use by nightclub-goers — a demographic which traditionally has a relaxed view towards recreational drug experimentation and use.

NPS are common adulterants in drugs such as ecstasy (MDMA), which has seen an increase in popularity since it became marketed as “Molly.” Ironically, “Molly” connotes a product that is pure MDMA. In a related study, Palamar and his team found that four out of ten nightclub/festival attendees who used ecstasy or “Molly” tested positive for “bath salts” despite reporting no use.

In their current study, “Hair Testing for Drugs of Abuse and New Psychoactive Substances in a High-Risk Population,” Dr. Alberto Salomone, an affiliated researcher at the Centro Regionale Antidoping e di Tossicologia “A. Bertinaria,” Orbassano, Turin, Italy and Dr. Palamar, affiliated with NYU’s Center for Drug Use and HIV Research (CDUHR), collected hair samples from 80 young adults outside of New York City nightclubs and dance festivals, from July through September of 2015. Hair samples from high-risk nightclub and dance music attendees were tested for 82 drugs and metabolites (including NPS) using ultra-high performance liquid chromatography-tandem mass spectrometry.

“Hair analysis represents a reliable and well-established means of clinical and forensic investigations to evaluate drug exposure, said Dr. Salomone. “Hair is the most helpful specimen when either long-time retrospective information on drug consumption is of interest.” “Most NPS can no longer be detected in urine, blood, or saliva within hours or days after consumption, but hair is particularly beneficial because many drugs can be detected months after use.”

Of the eighty samples, twenty-six tested positive for at least one NPS — the most common being a “bath salt” (synthetic cathinone) called butylone (present in twenty-five samples). The “bath salts” methylone and even alpha-PVP (a.k.a.: “Flakka”) were also detected. The researchers find the presence of Flakka alarming as this drug has been associated with many episodes of erratic behavior and even death in Florida. Other new drugs detected included new stimulants called 4-FA and 5/6-APB.

“We found that many people in the nightclub and festival scene have been using new drugs and our previous research has found that many of these people have been using unknowingly,” said Dr. Palamar, also an assistant professor of Population Health at NYU Langone Medical Center (NYULMC).

Hair analysis proved a powerful tool to Drs. Salomone and Palamar and their team, allowing them to gain objective biological drug-prevalence information, free from possible biases of unintentional or unknown intake and untruthful reporting of use.

“Such testing can be used actively or retrospectively to validate survey responses and inform research on consumption patterns,” notes Dr. Palamar.

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Molecular therapy set to protect at-risk patients against heart attack and stroke

Even a single dose of a specific ribonucleic acid molecule, known as a small interfering RNA (siRNA), offers patients at high risk of cardiovascular disease long-lasting protection against high LDL cholesterol — one of the main risk factors for heart attack and stroke. This is the result of a clinical study that researchers from Charité and Imperial College London have published as leading authors in the current edition of New England Journal of Medicine*.

As a component of cell walls and a building block of numerous hormones, cholesterol plays an important role in the cell’s lipid metabolism. However, too much LDL cholesterol in the blood results in an increased risk of atherosclerosis (hardening of the arteries) and problems such as heart attack and stroke. Patients suffering from a genetic disorder which causes very high levels of LDL cholesterol are at a particularly high risk. In these patients, a protein known as PCSK9 (proprotein convertase subtilisin/kexin type 9) prevents the liver from removing LDL cholesterol from the blood.

In their study, Prof. Ulf Landmesser, Head of Charité’s Department of Cardiology (Campus Benjamin Franklin), and Prof. Kausik Ray from Imperial College London, used the principle of RNA interference. The process, which was discovered a few years ago, uses RNA molecules (small interfering RNA) to inhibit the synthesis of harmful proteins. When double-stranded siRNA is introduced into a cell, it will bind to a molecule known as the RNA-induced silencing complex (RISC complex). This allows the process to be used in a targeted manner to silence specific genes.

In their study, the researchers investigated how effective and efficient a specific siRNA was at targeting the PCSK9 protein. A total of 501 high-risk patients with high LDL cholesterol levels received varying subcutaneous doses of either inclisiran or placebo. Results showed that inclisiran led to a significant reduction in levels of both the protein and LDL cholesterol, with LDL cholesterol levels being reduced by up to 41.9 percent after a single dose, and up to 52.6 percent after two doses.

“It was particularly interesting to see just how sustained the effect of treatment was, with the effect of a single dose remaining apparent for a duration of over nine months,” explains Prof. Ulf Landmesser. He adds: “The next step will be to further develop this treatment by conducting a large clinical outcome trial. We are hoping to test what might become a new type of therapy for the prevention of heart attack and stroke in high-risk patients.”

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Stem cells help explain varied genetics behind rare neurologic disease

Researchers at Case Western Reserve University School of Medicine have successfully grown stem cells from children with a devastating neurological disease to help explain how different genetic backgrounds can cause common symptoms. The work sheds light on how certain brain disorders develop, and provides a framework for developing and testing new therapeutics. Medications that appear promising when exposed to the new cells could be precisely tailored to individual patients based on their genetic background.

In the new study, published in The American Journal of Human Genetics, researchers used stem cells in their laboratory to simultaneously model different genetic scenarios that underlie neurologic disease. They identified individual and shared defects in the cells that could inform treatment efforts.

The researchers developed programmable stem cells, called induced pluripotent stem cells, from 12 children with various forms of Pelizaeus-Merzbacher Disease, or PMD. The rare but often fatal genetic disease can be caused by one of hundreds of mutations in a gene critical to the proper production of nerve cell insulation, or myelin. Some children with PMD have missing, partial, duplicate, or even triplicate copies of this gene, while others have only a small mutation. With so many potential causes, researchers have been in desperate need of a way to accurately and efficiently model genetic diseases like PMD in human cells.

By recapitulating multiple stages of the disease in their laboratory, the researchers established a broad platform for testing new therapeutics at the molecular and cellular level. They were also able to link defects in brain cell function to patient genetics.

“Stem cell technology allowed us to grow cells that make myelin in the laboratory directly from individual PMD patients. By studying a wide spectrum of patients, we found that there are distinct patient subgroups.

This suggests that individual PMD patients may require different clinical treatment approaches,” said Paul Tesar, PhD, study lead, Dr. Donald and Ruth Weber Goodman Professor of Innovative Therapeutics, and Associate Professor of Genetics and Genome Sciences at Case Western Reserve University School of Medicine.

The researchers watched in real-time as the patients’ stem cells matured in the laboratory. “We leveraged the ability to access patient-specific brain cells to understand why these cells are dysfunctional. We found that a subset of patients exhibited an overt dysfunction in certain cellular stress pathways,” said Zachary Nevin, first author of the study and MD/PhD student at Case Western Reserve University School of Medicine. “We used the cells to create a screening platform that can test medications for the ability to restore cell function and myelin. Encouragingly, we identified molecules that could reverse some of the deficits.” The promising finding provides proof-of-concept that medications that mend a patient’s cells in the laboratory could be advanced to clinical testing in the future.

The stem cell platform could also help other researchers study and classify genetic diseases with varied causes, particularly other neurologic disorders. Said Tesar, “Neurological conditions present a unique challenge, since the disease-causing cells are locked away in patients’ brains and inaccessible to study. With these new patient-derived stem cells, we can now model disease symptoms in the laboratory and begin to understand ways to reverse them.”

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Brain’s ‘GPS’ does a lot more than just navigate

The part of the brain that creates mental maps of one’s environment plays a much broader role in memory and learning than was previously thought, according to new research published this week in the journal Nature by researchers at Princeton University.

“Almost 40 years of research suggested that a certain region of the brain was devoted to spatial navigation,” said David Tank, Princeton’s Henry L. Hillman Professor in Molecular Biology and co-director of the Princeton Neuroscience Institute. “We found that this same region is also involved when navigating not only spatial environments but also cognitive ones.”

The study looked at a region of the brain called the hippocampus that has been known since the 1970s to become active when rats travel around their environments. That research, and related work showing that cells in the nearby entorhinal cortex fire when animals reach specific locations, led to the finding that the brain creates an internal representation of the outside world — a sort of mental positioning system — that tells an animal where it is in its environment. These findings earned three scientists the 2014 Nobel Prize in Physiology or Medicine.

Now researchers at Princeton have found that those same brain regions are active when the brain is exploring a very different kind of environment, one involving listening to sounds. The researchers monitored neural activity as the rats listened and responded to certain sounds, and found similar firing patterns to those seen when rats are exploring their environments.

The research addresses a longstanding mystery in neuroscience, how the hippocampus could be associated both with making maps of the external environment and with making new memories. People with damage to the hippocampus, such as the amnesia patient known by the initials H.M. who participated in five decades of studies until his death in 2008, lack the ability to form new memories.

In previous studies where scientists monitored the electrical activity of cells in the hippocampus, they found that the cells fired in sequences that represented where the animal was, which direction its head was facing, which direction it was traveling and where it was relative to a boundary, according to Dmitriy Aronov, first author on the paper who conducted the work while a postdoctoral researcher at the Princeton Neuroscience Institute and who is now an assistant professor of neuroscience at Columbia University. “The mystery was, what do these firing patterns have to do with memory?”

The researchers theorized that perhaps the hippocampus and the nearby entorhinal cortex, which work together to make these mental maps, were in fact not specific to mapping per se but were involved in more general cognitive tasks, and that mapping was just one aspect of larger cognitive tasks involving learning and memory. Perhaps the reason previous studies only turned up the location-finding tasks is because rats spend most of their time exploring their environments as they forage for food.

By giving the rats a different task, such as exploring sounds, the researchers might see evidence of cognitive activities in the hippocampal-entorhinal circuit. The researchers chose sound as an analogy to space because both can vary along a continuum: the rats can explore ever-increasing frequencies the way they would move forward along a lengthy corridor.

To test the theory, the researchers monitored the electrical activity of neurons in the hippocampal and entorhinal regions while the rats manipulated sounds and learned to associate certain sound frequencies with rewards. Tank and Aronov teamed with undergraduate Rhino Nevers, Class of 2018, to conduct the work. The researchers first taught the rats to depress a lever to increase the pitch, or frequency, of a tone being played over a speaker. The rats learned that if they released the lever when the tone reached a predetermined frequency range, they would receive a reward.

The team observed that the patterns of neuronal firing corresponded to the rats’ behaviors during the task. Sequences of neural activity were produced as the rats advanced through the progression of frequencies, analogous to the sequences produced during traversing a progression of places in space. There were even patterns that corresponded to particular sound frequencies. The neurons involved in these firing patterns were identical to those involved in mapping and navigation. These cells included hippocampal place cells, so named because they fire when the rat is in a particular place, and entorhinal grid cells, which fire when the rats pass through certain locations.

The findings suggest that there are common mechanisms in the hippocampal-entorhinal system that can represent diverse sorts of tasks, said Tank, who is also director of the Simons Collaboration on the Global Brain. “The implication from our work is that these brain areas don’t represent location specifically, but rather they can represent other relevant features of the animal’s experience. When those features vary in a continuous way, sequences of neural activation are produced,” Tank said.

The discovery fits with how we think about mapping our environment in the context of learning about new places and forming memories of experiences, said Aronov. “When you visit a new location, you don’t only make a mental map, but you also form memories of your location. We feel that this study solves the mystery of the hippocampus in representing both memory and location, in that these neurons are general purpose neurons capable of representing any relevant information.”

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