Neuroscience

A study in The Journal of General Physiology provides new evidence that the ubiquitous sodium pump is more complex—and more versatile—than we thought.

(Image caption: Structure of the sodium pump, which researchers reveal to be more versatile than previously thought)

The sodium pump is present in the surface membrane of all animal cells, using energy derived from ATP to transport sodium and potassium ions in opposite directions across the cell boundary. By setting up transmembrane gradients of these two ions, the pump plays a vital role in many important processes, including nerve impulses, heartbeats, and muscular contraction.

Now, Rockefeller University researchers Natascia Vedovato and David Gadsby demonstrate that, in addition to its role as a sodium and potassium ion transporter, the pump can simultaneously import protons into the cell. Their study not only provides evidence of “hybrid” function by the pump, it also raises important questions about whether the inflow of protons through sodium pumps might play a role in certain pathologies.

The sodium pump exports three sodium ions out of the cell and imports two potassium ions into the cell during each transport cycle. Vedovato and Gadsby show that, during this normal cycle, the pump develops a passageway that enables protons to cross the membrane. When the pump releases the first of the three sodium ions to the cell exterior, a newly emptied binding site becomes available for use by an external proton, allowing it to then make its way into the cytoplasm. The protons travel a distinct route, and proton inflow is not required for successful transport of sodium and potassium.

Import of protons is high when their extracellular concentration is high (pH is low) and membrane potential is negative. The authors therefore speculate that proton inflow might have important implications under conditions in which extracellular pH is lowered, such as in muscle during heavy exercise, in the heart during a heart attack, or in the brain during a stroke.

Researchers at the Centre for Addiction and Mental Health have discovered two new genes linked to intellectual disability, according to two research studies published concurrently in early March in the journals Human Genetics and Human Molecular Genetics.

“Both studies give clues to the different pathways involved in normal neurodevelopment,” says CAMH Senior Scientist Dr. John Vincent, who heads the MiND (Molecular Neuropsychiatry and Development) Laboratory in the Campbell Family Mental Health Research Institute at CAMH. “We are building up a body of knowledge that is informing us which kinds of genes are important to, and involved in, intellectual disabilities.”

In the first study, Dr. Vincent and his team used microarray genotyping to map the genes of a large consanguineous (intermarriage within the extended family) Pakistani family, in which five members of the youngest generation were affected with mild to moderate intellectual disability. Dr. Vincent identified a truncation in the FBXO31 gene, which plays a role in the way that proteins are processed during neuronal development, particularly in the cerebellar cortex.

In the second study, using the same techniques, Dr. Vincent and his team analyzed the genes of two consanguineous families, one Austrian and one Pakistani, and identified a disruption in the METTL23 gene linked to mild recessive intellectual disability. The METTL23 gene is involved in methylation—a process important to brain development and function.

About one per cent of children worldwide are affected by non-syndromic (i.e., the absence of any other clinical features) intellectual disability, a condition characterized by an impaired capacity to learn and process new or complex information, leading to decreased cognitive functioning and social adjustment. Although trauma, infection and external damage to the unborn fetus can lead to an intellectual disability, genetic defects are a principal cause.

These studies were part of an ongoing study of affected families in Pakistan, where the cultural tradition of large families and consanguineous marriages among first cousins increases the likelihood of inherited intellectual disability in offspring.

“Although it is easier to find and track genes in consanguineous families, these genes are certainly not limited to them,” Dr. Vincent points out. A recent study estimated that 13–24 per cent of intellectual disability cases among individuals of European descent have autosomal recessive causes, meaning that results of this study are very relevant to populations such as Canada.

Autosomal recessive gene mutations have traditionally been more difficult to trace, resulting in a paucity of research in this area. Parents of affected children show no symptoms, and the child must inherit one defective copy of the gene from each parent, so that only one in four offspring are likely to be affected. Smaller families, therefore, show a decreased incidence and are less amenable to this kind of study.

Dr. Vincent is currently engaged in a study that will screen Canadian populations with autism and intellectual disability for autosomal recessive gene mutations. Results will be available later this year.

A total of 42 genes linked to non-syndromic autosomal recessive forms of intellectual disability have now been identified; estimates suggest that up to 2,500 autosomal genes might be linked with intellectual disability, the majority being recessive.

Common psychiatric disorders, such as anxiety and addiction, likely result from changes in brain circuitry. Understanding structural and functional brain connections – and how they change in psychiatric disorders – could lead to novel preventive and therapeutic strategies.

The bed nucleus of the stria terminalis (BNST) has been linked to both anxiety and addiction, but its circuitry in humans has not been described. Jennifer Blackford, Ph.D., assistant professor of Psychiatry, and colleagues used two neuroimaging methods – diffusion tensor imaging and functional MRI – to identify patterns of connectivity between the BNST and other brain regions in healthy individuals. The BNST showed connections to multiple subcortical brain regions, including limbic, thalamic and basal ganglia structures, which matched reported connections in rodents. The researchers also identified two novel BNST connections: to the temporal pole and to the paracingulate gyrus.

The findings, reported in NeuroImage, provide a map of BNST neurocircuitry and lay the foundation for future studies of the circuits that mediate anxiety and addiction.

Memories are difficult to produce, often fragile, and dependent on any number of factors—including changes to various types of nerves. In the common fruit fly—a scientific doppelganger used to study human memory formation—these changes take place in multiple parts of the insect brain.

Scientists from the Florida campus of The Scripps Research Institute (TSRI) have been able to pinpoint a handful of neurons where certain types of memory formation occur, a mapping feat that one day could help scientists predict disease-damaged neurons in humans with the same specificity.

“What we found is that while a lot of the neurons will respond to sensory stimuli, only a certain subclass of neurons actually encodes the memory,” said Seth Tomchik, a TSRI biologist who led the study, which was published March 27, 2014, online ahead of print by the journal Current Biology.

The researchers examined a type of neuron called dopaminergic neurons—which respond to dopamine, a well-known neurotransmitter—and are involved in shaping diverse behaviors, including learning, motivation, addiction and obesity.

In the study, the scientists followed the stimulation of a large number of these neurons when an odor was paired with an aversive event such as a mild electric shock. The scientists then used imaging technology to follow changes in the brains of live flies, mapping the activation patterns of signaling molecules within the neurons and observing learning-related plasticity—in which neurons change and develop memory traces.

The scientists found that the neurons that did encode memories responded to a cellular signaling messenger known as cAMP (cyclic adenosine monophosphate) that is vital for many biological processes. cAMP is involved in a number of psychological disorders such as bipolar disorder and schizophrenia, and its dysregulation may underlie some cognitive symptoms of Alzheimer’s disease and Neurofibramatosis I.

In fact, the study pointed to a specific location in the brain—a particular lobe with a region known as the mushroom body—where the neurons appear to be particularly sensitive to elevated amounts of cAMP.

According to Tomchik, that’s an important finding in terms of human memory because olfactory memory formation in the fruit fly is very similar to human memory formation. 

“We have a good model in these two classes of neurons, one that encodes and one that doesn’t,” he said. “Now we know exactly where the memory formation should be and where to look to see how disease may disrupt it.”

Tamara Boto, the first author of the study and a member of Tomchik’s laboratory, added, “We know where, but we don’t yet know the mechanism of why only these subsets are affected. That’s our next job—to figure that out.”

Amyotrophic lateral sclerosis, also known as Lou Gehrig’s disease, is marked by a cascade of cellular and inflammatory events that weakens and kills vital motor neurons in the brain and spinal cord. The process is complex, involving cells that ordinarily protect the neurons from harm. Now, a new study by scientists in The Research Institute at Nationwide Children’s Hospital points to a potential culprit in this good-cell-gone-bad scenario, a key step toward the ultimate goal of developing a treatment.

Motor neurons, or nerve cells, in the brain and spinal cord control the function of muscles throughout the body. In amyotrophic lateral sclerosis (ALS), motor neurons die and muscles weaken. Patients gradually lose the ability to move and as the disease progresses, are unable to breathe on their own. Most people with ALS die from respiratory failure within 3 to 5 years from the onset of symptoms.

For the study, published recently online in Neuron, researchers examined a protein involved in transcriptional regulation, called nuclear factor-kappa B (NF-κB), known to play a role in the neuroinflammatory response common in ALS. NF-κB has also been linked to cancer and a number of other inflammatory and autoimmune diseases.

Using animal models, the researchers studied disease progression in mice in which NF-κB had been inhibited in two different cell types — astrocytes, the most abundant cell type in the human brain and supporters of neuronal function; and microglia, macrophages in the brain and spinal cord that act as the first and main form of defense against invading pathogens in the central nervous system. Inhibiting NF-κB in microglia in mice slowed disease progression by 47 percent, says Brian Kaspar, MD, a principal investigator in the Center for Gene Therapy at Nationwide Children’s and senior author of the new study.

“The field has identified different cell types in addition to motor neurons involved in this disease, so one of our approaches was to find out what weapons these cells might be using to kill motor neurons,” Dr. Kaspar says. “And our findings suggest that the microglia utilize an NF-κB-mediated inflammatory response as one of its weapons.”

Inhibiting the protein in astrocytes had no impact on disease progression, so the search for the weapons that cell type uses against motor neurons continues. These preliminary findings also don’t tell scientists how or why NF-κB turns the ordinarily protective microglia into neuron-killing molecules. But despite the mysteries that remain, the study moves scientists closer to finding a treatment for ALS.

The search for an ALS therapy has been focused in two directions: identifying the trigger that leads to disease onset and understanding the process that leads to disease progression. Changes in motor neurons are involved in disease onset, but disease progression seems to be dictated by changes to astrocytes, microglia and oligodendrocytes. Some cases of ALS are hereditary but the vast majority of patients have no family ties to the disease. The complexity of the disease and the lack of a clear familiar tie make screening before disease onset nearly impossible, highlighting the importance of slowing the disease, Dr. Kaspar says.

“Focusing on stopping the changes that occur in astrocytes and microglia has clinical relevance because most people don’t know they’re getting ALS, says Dr. Kaspar, who also is an associate professor of pediatrics and neurosciences at The Ohio State University College of Medicine. “We have identified a pathway in microglia that may be targeted to ultimately slow disease progression in ALS and are exploring potential therapeutic strategies and may have broader implications for diseases such as Alzheimer’s and Parkinson’s Disease amongst others.”

Long-term brain damage caused by stroke could be reduced by saving cells called pericytes that control blood flow in capillaries, suggest researchers from Oxford University, UCL and the University of Copenhagen.

Until now, many scientists believed that blood flow within the brain was solely controlled by changes in the diameter of arterioles, blood vessels that branch out from arteries into smaller capillaries.

In this new study, the UK and Danish researchers reveal that the brain’s blood supply is in fact chiefly controlled by the narrowing or widening of capillaries as pericytes tighten or loosen around them.

Their study, published this week in the journal Nature, shows not only that pericytes are the main regulator of blood flow to the brain, but also that they tighten and die around capillaries after stroke. This significantly impairs blood flow in the long term, causing lasting damage to brain cells.

The scientists showed that certain chemicals can halve pericyte death from simulated stroke in the lab, and they hope to develop these into drugs to treat stroke victims.

'This discovery offers radically new treatment approaches for stroke,' says study co-author Professor Alastair Buchan, Dean of Medicine and Head of the Medical Sciences Division at Oxford University. 'Importantly, we should now be able to identify drugs that target these cells. If we are able to prevent pericytes from dying, it should help restore blood flow in the brain to normal and prevent the ongoing slow damage we see after a stroke which causes so much neurological disability in our patients.'

Professor David Attwell of UCL, who led the study, explains: ‘At present, clinicians can remove clots blocking blood flow to the brain if stroke patients reach hospital early enough. However, the capillary constriction produced by pericytes may, by restricting the blood supply for a long time, cause further damage to nerve cells even after the clot is removed. Our latest research suggests that devising drugs to prevent capillary constriction may offer new therapies for reducing the disability caused by stroke.’

The new research also gives insight into the mechanisms underlying the use of functional magnetic resonance imaging to detect blood flow changes in the brain.

'Functional imaging allows us to see the activity of nerve cells within the human brain but until now we didn't quite know what we were looking at,' says Professor Martin Lauritzen of the University of Copenhagen. 'We have shown that pericytes initiate the increase in blood flow seen when nerve cells become active. So we now know that functional imaging signals are caused by a pericyte-mediated increase of capillary diameter. Knowing exactly what functional imaging shows will help us to better understand and interpret what we see.'

Working with genetically engineered mice, Johns Hopkins neuroscientists report they have identified what they believe is the cause of the vast disintegration of a part of the brain called the corpus striatum in rodents and people with Huntington’s disease: loss of the ability to make the amino acid cysteine. They also found that disease progression slowed in mice that were fed a diet rich in cysteine, which is found in foods such as wheat germ and whey protein.

Their results suggest further investigation into cysteine supplementation as a candidate therapeutic in people with the disease.

Up to 90 percent of the human corpus striatum, a brain structure that moderates mood, movement and cognition, degenerates in people with Huntington’s disease, a condition marked by widespread motor and intellectual disability. And while the genetic mutation underlying Huntington’s disease has long been known, the precise cause of that degeneration has remained a mystery.

In a report on their discovery in the advanced online publication of Nature on March 26, the Johns Hopkins researchers, led by Solomon Snyder, M.D., tracked the degenerative process to the absence of an enzyme, cystathionine gamma lyase, or CSE.

"Usually it’s very hard, if not impossible, to develop straightforward mechanisms that explain what’s going on in a disease. What’s even harder is even if you can find a mechanism that causes a tissue to rot, usually there’s nothing you can do about it,” says Snyder, a professor of neuroscience at the Johns Hopkins University School of Medicine. “In this case, there is."

Huntington’s disease, an inherited disorder, does its damage because of abnormal DNA coding for the amino acid glutamine. Healthy individuals have some 15 to 20 DNA “repeats” in that part of their genetic code, while Huntington’s disease gene carriers have more than 36 — and often upward of 100. Children born to a parent carrier have a 50/50 chance of inheriting the disorder, and the greater the number of repeats, the earlier the age of onset of the incurable disorder.

Bindu Diana Paul, Ph.D., a molecular neuroscientist and faculty instructor in Snyder’s laboratory, was studying mice lacking CSE, which helps make the amino acid cysteine and hydrogen sulfide that moderate blood pressure and heart function. Paul, who had previous research experience with Huntington’s disease, says she was startled to observe that her mutant mice also behaved a lot like those with the disease.

When a normal mouse is dangled upside down from its tail, it will twist and turn and try to bite the offending hand, she explains. But her CSE-knockout mice stayed relatively still and clasped their paws together — the same behavior she’d observed in mice with the rodent equivalent of Huntington’s disease. “It looked like there was a neurological deficit,” Paul says. “But nobody had looked at CSE in the brain.”

Paul and Snyder began monitoring CSE in mouse and human brain tissues and found considerably less CSE in all diseased tissues. All people carry some normal huntingtin protein made by the Huntington’s disease gene, although the protein’s function remains elusive. But people with Huntington’s disease also carry mutant huntingtin proteins. Snyder and his team saw that the mutant proteins were attaching themselves to a crucial protein responsible for turning the CSE gene on or off, which ultimately led the diseased rodent and human brain tissues to be deprived of cysteine.

To see if loss of cysteine was directly responsible for the symptoms associated with Huntington’s disease, the Johns Hopkins team turned to readily available sources of the substance in everyday foods and fed mice a cysteine-rich diet.

The results, Paul says, were striking. When those mice were dangled from their tails, they resumed struggling, although with a bit less vigor than their healthy peers. They were able to grip an object with greater strength, and they took longer to fall off a balancing apparatus than CSE-knockout mice. Their life expectancies increased one to two weeks.

Snyder and Paul say they are cautiously optimistic about the results, noting that although they suggest a possible treatment for Huntington’s disease, it’s clear that a high cysteine diet merely slows rather than halts the progression of the disease. Moreover, the results in live mice may not occur in humans.

The problems people with autism have with memory formation, higher-level thinking and social interactions may be partially attributable to the activity of receptors inside brain cells, researchers at Washington University School of Medicine in St. Louis have learned.

(Image caption: Learning, social interactions and higher-level thinking in people with autism may be adversely affected by receptors inside brain cells, scientists at Washington University School of Medicine have learned. The type of receptor they studied glows green on the surface of this cell. Inside the cell, the receptor covers a membrane stained red. Credit: Yuh-Jiin I. Jong)

Scientists were already aware that the type of receptor in question was a potential contributor to these problems – when located on the surfaces of brain cells. Until now, though, the role of the same type of receptor located inside the cell had gone unrecognized. Such receptors inside cells significantly outnumber the same type of receptors on the surface of cells.

The receptor under study, known as the mGlu5 receptor, becomes activated when it binds to the neurotransmitter glutamate, which is associated with learning and memory. This leads to chain reactions that convert the glutamate’s signal into messages traveling inside the cell.

In the new study, scientists working with cells in a dish linked mGlu5 receptors inside cells to processes that turn down the volume at which brain cells talk to each other. These volume changes, essential for learning and memory, may become exaggerated in people with autism.

Pharmaceutical companies have developed therapeutic compounds to decrease signaling associated with the mGlu5 receptor, moderating its effects on brain cells’ volume knobs. But the compounds were designed to target mGlu5 surface receptors. In light of the new findings, the scientists question if those drugs will reach the receptors inside cells.

“Our results suggest that to have the greatest therapeutic benefit, we may need to make sure we’re blocking all of this type of receptor, both inside and on the surface of the cell,” said senior investigator Karen O’Malley, PhD, professor of neurobiology.

The findings, published March 25 in The Journal of Neuroscience, also add a significant new dimension to basic brain cell function. Scientists have long assumed that brain cell receptors are only active on the surface of cells. The new study shows that receptors can be active inside cells, and their effects can be considerably different from the same receptors located on the cell surface.

“The traditional wisdom was that receptors inside the cell were either waiting to go to work on the surface or had just finished working there,” said O’Malley. “But when we compared how much of the mGlu5 receptor was on the surface of cells to how much was inside it, we were seeing so much more receptor inside the cell – at least 50 percent, and in some cases as much as 90 percent – that we wondered if the interior receptors had separate functions.”

In earlier studies, O’Malley and her collaborators showed that mGlu5 receptors on the cell surface sent completely different messages than the same receptors inside the cell.

The scientists knew that most autism studies were conducted with compounds that blocked mGlu5 receptors but could not get into the cell. To determine whether blocking inside receptors would have different effects, O’Malley collaborated with Yukitoshi Izumi, MD, PhD, research professor of psychiatry, and Charles F. Zorumski, MD, the Samuel B. Guze Professor and head of the Department of Psychiatry, who study cell-based models of learning and memory.

When the scientists examined these model systems using compounds that allowed them to activate only mGlu5 receptors within cells, they found that these receptors played a bigger role in turning down the volume of brain cell communications than did the cell surface receptors.

In the last few years, scientists have found that 20 or more types of brain cell receptors located on cell surfaces also are present at high levels inside cells, O’Malley noted.

“This should be a factor we consider when we design drugs to target brain cell receptors,” she said. “Do we want to reach cell surface receptors, receptors inside the cell or both?”

Following ischemic stroke, the integrity of the blood-brain barrier (BBB), which prevents harmful substances such as inflammatory molecules from entering the brain, can be impaired in cerebral areas distant from initial ischemic insult. This disruptive condition, known as diaschisis, can lead to chronic post-stroke deficits, University of South Florida researchers report.

(Image credit: Mosby’s Medical Dictionary, 8th edition. © 2009, Elsevier)

In experiments using laboratory rats modeling ischemic stroke, USF investigators studied the consequences of the compromised BBB at the chronic post-stroke stage. Their findings appear in a recent issue of the Journal of Comparative Neurology.

“Following ischemic stroke, the pathological changes in remote areas of the brain likely contribute to chronic deficits,” said neuroscientist and study lead author Svitlana Garbuzova-Davis, PhD, associate professor in the USF Health Department of Neurosurgery and Brain Repair. “These changes are often related to the loss of integrity of the BBB, a condition that should be considered in the development of strategies for treating stroke and its long-term effects.”

Edward Haller of the USF Department of Integrative Biology, the coauthor who performed electron microscopy and contributed to image analysis, emphasized that “major BBB damage was found in endothelial and pericyte cells, leading to capillary leakage in both brain hemispheres.” These findings were essential in demonstrating persistence of microvascular alterations in chronic ischemic stroke.

While acute stroke is life-threatening, the authors point out that survivors often suffer insufficient blood flow to many parts of the brain that can contribute to persistent damage and disability. Their previous investigation of subacute ischemic stroke showed far-reaching microvascular damage even in areas of the brain opposite from the initial stroke injury. While most studies of stroke and the BBB explore the acute phase of stroke and its effect on the blood-brain barrier, the present study revealed the longer-term effects in various parts of the brain.

The pathologic processes of stroke-induced vascular injury tend to occur in a “time-dependent manner,” and can be separated into acute (minutes to hours), subacute (hours to days), and chronic (days to months). BBB incompetence during post-stroke changes is well-documented, with some studies showing the BBB opening can last up to four to five days after stroke. This suggests that harmful substances entering the brain during this prolonged BBB leakage might increase post-ischemic brain injury.

In this study, the researchers used laboratory rats modeling ischemic stroke and observed injury not only in the primary area of the stroke, but also in remote areas, where persistent BBB damage could cause chronic loss of competence.

“Our results showed that the compromised BBB integrity detected in post-ischemic rat cerebral hemisphere capillaries — both ipsilateral and contralateral to initial stroke insult — might indicate chronic diaschisis,” Garbuzova-Davis said. “Widespread microvascular damage caused by endothelial cell impairment could aggravate neuronal deterioration. For this reason, chronic diaschisis poses as a therapeutic target for stroke.”

The primary focus for therapy development could be restoring endothelial and/or astrocytic integrity towards BBB repair, which may be “beneficial for many chronic stroke patients,” senior authors Cesar V. Borlongan and Paul R. Sanberg suggest. The researchers also recommend that cell therapy might be used to replace damaged endothelial cells.

“A combination of cell therapy and the inhibition of inflammatory factors crossing the blood-brain barrier may be a beneficial treatment for stroke,” Garbuzova-Davis said.