Team Solves Decades-Old Mystery of How Cells Keep from Bursting

A team led by scientists at The Scripps Research Institute (TSRI) has identified a long-sought protein that facilitates one of the most basic functions of cells: regulating their volume to keep from swelling excessively.

The identification of the protein, dubbed SWELL1, solves a decades-long mystery of cell biology and points to further discoveries about its roles in health and disease—including a serious immune deficiency that appears to result from its improper function.

“Knowing the identity of this protein and its gene opens up a broad new avenue of research,” said the study’s principal investigator Ardem Patapoutian, a Howard Hughes Medical Institute (HHMI) Investigator and professor at TSRI’s Dorris Neuroscience Center and Department of Molecular and Cellular Neuroscience.

The report appears as the cover story in the April 10, 2014 issue of the journal Cell.

Unraveling the Mystery

Water passes through the membrane of most cells with relative ease and tends to flow in a direction that evens out the concentration of dissolved molecules or “solutes.” “Water in effect follows the solutes,” explained Zhaozhu Qiu, a member of the Patapoutian laboratory who was first author of the study. “Any decrease in the solute concentration outside a cell or an increase within the cell will make the cell swell with water.”

For decades, experiments have demonstrated the existence of a key relief valve for this swelling: an unidentified ion channel in the cell membrane, dubbed VRAC (volume-regulated anion channel). VRAC opens in response to cell swelling and permits an outflow of chloride ions and some other negatively charged molecules—which water molecules follow, thus reducing the swelling.

“For the past 30 years, scientists have known that there is this VRAC channel, and yet they haven’t known its molecular identity,” said Patapoutian.

Finding the proteins that make VRAC and their genes was a goal that had eluded prior attempts because of the technical hurdles involved. However, in the new study, Qiu and his colleagues were able to set up a rapid, “high-throughput” screening test based on fluorescence. They engineered human cells to produce a fluorescent protein whose glow would be quenched when the cells became swollen and VRAC channels opened.

With the help of automated screening specialists at the La Jolla-based Genomics Institute of the Novartis Research Foundation (GNF), which recently began a broad new collaboration agreement with TSRI, the team cultured large arrays of the cells and, using a technique known as RNA interference, blocked the activity of a different gene for each clump of cells.

The idea was to watch for the groups of cells that continued to glow—indicating that the gene inactivation had disrupted VRAC.

In this way, with several rounds of tests, the team sifted through the human genome and ultimately found one gene whose disruption reliably terminated VRAC activity. It was a gene that had been discovered in 2003 and catalogued as “LRRC8.” Although it appeared to code for a cell-membrane-spanning protein—as one would expect for an ion channel—almost nothing else was known about it.

The team renamed it SWELL1.

Potential Roles in Disease

Investigating further, the researchers showed that SWELL1 does indeed localize to the cell membrane as an ion channel protein would. Experiments by Adrienne Dubin, a staff scientist at TSRI, showed that certain mutations of SWELL1 alter the VRAC channel’s ion-passing properties—indicating that SWELL1 is a central feature of the ion channel itself.

“It is at least a major part of the VRAC channel for which cell biologists have been searching all this time,” said Patapoutian.

Patapoutian, Qiu and their colleagues now will study SWELL1 further, including an examination of what happens to lab mice that lack the protein in various cell types.

Curiously, the gene for SWELL1 was first noted by scientists because a mutant, dysfunctional form of it causes a very rare type of agammaglobulinemia—a lack of antibody-producing B cells, which leaves a person unusually vulnerable to infections. That suggests that SWELL1 is somehow required for normal B-cell development.

“There also have been suggestions from prior studies that this volume-sensitive ion channel is involved in stroke because of the brain-tissue swelling associated with stroke and that it may be involved as well in the secretion of insulin by pancreatic cells,” said Patapoutian. “So there are lots of hints out there about its relevance to disease—we just have to go and figure it all out now.”

Researchers discover underlying genetics, marker for stroke, cardiovascular disease

Scientists studying the genomes of nearly 5,000 people have pinpointed a genetic variant tied to an increased risk for stroke, and have also uncovered new details about an important metabolic pathway that plays a major role in several common diseases. Together, their findings may provide new clues to underlying genetic and biochemical influences in the development of stroke and cardiovascular disease, and may also help lead to new treatment strategies.

"Our findings have the potential to identify new targets in the prevention and treatment of stroke, cardiovascular disease and many other common diseases," said Stephen R. Williams, Ph.D., a postdoctoral fellow at the University of Virginia Cardiovascular Research Center and the University of Virginia Center for Public Health Genomics, Charlottesville.

Dr. Williams, Michele Sale, Ph.D., associate professor of medicine, Brad Worrall, M.D., professor of neurology and public health sciences, all at the University of Virginia, and their team reported their findings March 20, 2014 in PLoS Genetics. The investigators were supported by the National Human Genome Research Institute (NHGRI) Genomics and Randomized Trials Network (GARNET) program (www.genome.gov/27541119).

Stroke is the fourth leading cause of death and a major cause of adult disability in this country, yet its underlying genetics have been difficult to understand. Numerous genetic and environmental factors can contribute to a person having a stroke. “Our goals were to break down the risk factors for stroke,” Dr. Williams said.

The researchers focused on one particular biochemical pathway called the folate one-carbon metabolism (FOCM) pathway. They knew that abnormally high blood levels of the amino acid homocysteine are associated with an increased risk of common diseases such as stroke, cardiovascular disease and dementia. Homocysteine is a breakdown product of methionine, which is part of the FOCM pathway. The same pathway can affect many important cellular processes, including the methylation of proteins, DNA and RNA. DNA methylation is a mechanism that cells use to control which genes are turned on and off, and when.

But clinical trials of homocysteine-lowering therapies have not prevented disease, and the genetics underlying high homocysteine levels - and methionine metabolism gone awry - are not well defined.

Dr. Williams and his colleagues conducted genome-wide association studies of participants from two large long-term projects: the Vitamin Intervention for Stroke Prevention (VISP), a trial looking at ways to prevent a second ischemic stroke, and the Framingham Heart Study (FHS), which has followed the cardiovascular health and disease in a general population for decades. They also measured methionine metabolism - the ability to convert methionine to homocysteine - in both groups. In all, they studied 2,100 VISP participants and 2,710 FHS subjects.

In a genome-wide association study, researchers scan the genome to identify specific genomic variants associated with a disease. In this case, the scientists were trying to identify variants associated with a trait - the ability to metabolize methionine into homocysteine.

Investigators identified variants in five genes in the FOCM pathway that were associated with differences in a person’s ability to convert methionine to homocysteine. They found that among the five genes, one - the ALDH1L1 gene - was also strongly associated with stroke in the Framingham study. When the gene is not working properly, it has been associated with a breakdown in a normal cellular process called programmed cell death, and cancer cell survival.

They also made important discoveries about the methionine-homocysteine process. “GNMT produces a protein that converts methionine to homocysteine. Of the five genes that we identified, it was the one most significantly associated with this process,” Dr. Williams said. “The analyses suggest that differences in GNMT are the major drivers behind the differences in methionine metabolism in humans.”

"It’s striking that the genes are in the same pathway, so we know that the genomic variants affecting that pathway contribute to the variability in disease and risk that we’re seeing," he said. "We may have found how genetic information controls the regulation of GNMT."

The group determined that the five genes accounted for 6 percent of the difference in individuals’ ability to process methionine into homocysteine among those in the VISP trial. The genes also accounted for 13 percent of the difference in those participants in the FHS, a remarkable result given the complex nature of methionine metabolism and its impact on cerebrovascular risk. In many complex diseases, genomic variants often account for less than 5 percent of such differences.

"This is a great example of the kinds of successful research efforts coming out of the GARNET program," said program director Ebony Madden, Ph.D. "GARNET scientists aim to identify variants that affect treatment response by doing association studies in randomized trials. These results show that variants in genes are associated with the differences in homocysteine levels in individuals."

The association of the ALDH1L1 gene variant with stroke is just one example of how the findings may potentially lead to new prevention efforts, and help develop new targets for treating stroke and heart disease, Dr. Williams said.

"As genome sequencing becomes more widespread, clinicians may be able to determine if a person’s risk for abnormally high levels of homocysteine is elevated," he said. "Changes could be made to an individual’s diet because of a greater risk for stroke and cardiovascular disease."

The investigators plan to study the other four genes in the pathway to try to better understand their potential roles in stroke and cardiovascular disease risk.

Substance in Humans is Effective Fighting Stroke Damage

A molecular substance that occurs naturally in humans and rats was found to “substantially reduce” brain damage after an acute stroke and contribute to a better recovery, according to a newly released animal study by researchers at Henry Ford Hospital.

The study, published online before print in Stroke, the journal of the American Heart Association, was the first ever to show that the peptide AcSDKP provides neurological protection when administered one to four hours after the onset of an ischemic stroke.

This type of stroke occurs when an artery to the brain is blocked by a blood clot, cutting off oxygen and killing brain tissue with crippling or fatal results.

“Stroke is a leading cause of death and disability worldwide,” said Li Zhang, M.D., a researcher at Henry Ford and lead author of the study. “Our data showed that treatment of acute stroke with AcSDKP alone or in combination with tPA substantially reduced neurovascular damage and improved neurological outcome.”

Commonly called a “clot-buster,” tPA, or tissue plasminogen activator, is the only FDA-approved treatment for acute stroke.

However, tPA must be given shortly after the onset of stroke to provide the best results. It also has the potential to cause a brain hemorrhage.

The Henry Ford study found that this narrow “therapeutic window” is extended for up to four hours after stroke and the therapeutic benefit of tPA is amplified when tPA is combined with AcSDKP. Further, the researchers discovered that AcSDKP alone is an effective treatment if given up to one hour after the brain attack.

The researchers tested the actions of both substances on laboratory rats in which acute stroke had been induced. It was already known that the peptide AcSDKP provides anti-inflammatory effects and helps protect the heart when used to treat a variety of cardiovascular diseases. The Henry Ford scientists reasoned that the peptide may have similar neurological benefits.

Significantly, they found that AcSDKP can readily cross the so-called “blood brain barrier” that blocks other neuroprotective substances.

A battery of behavioral tests was given to the lab rats both before and after stroke was induced to measure the effects of AcSDKP administered alone one hour after onset and combined with tPA four hours after stroke.

Besides finding that both methods “robustly” decreased neurological damage associated with stroke, they did so without increasing the incidence of brain hemorrhage or the formation of additional blood clots.

“With the increased use of clot-busting therapy in patients with acute stroke, both the safety and effectiveness of the combined treatment shown in our study should encourage the development of clinical trials of AcSDKP with tPA,” Dr. Zhang says.

Researchers find brain’s ‘sweet spot’ for love in neurological patient

A region deep inside the brain controls how quickly people make decisions about love, according to new research at the University of Chicago.

The finding, made in an examination of a 48-year-old man who suffered a stroke, provides the first causal clinical evidence that an area of the brain called the anterior insula “plays an instrumental role in love,” said UChicago neuroscientist Stephanie Cacioppo, lead author of the study.

In an earlier paper that analyzed research on the topic, Cacioppo and colleagues defined love as “an intentional state for intense [and long-term] longing for union with another” while lust, or sexual desire, is characterized by an intentional state for a short-term, pleasurable goal.

In this study, the patient made decisions normally about lust but showed slower reaction times when making decisions about love, in contrast to neurologically typical participants matched on age, gender and ethnicity. The findings are presented in a paper, “Selective Decision-Making Deficit in Love Following Damage to the Anterior Insula,” published in the journal Current Trends in Neurology.

“This distinction has been interpreted to mean that desire is a relatively concrete representation of sensory experiences, while love is a more abstract representation of those experiences,” said Cacioppo, a research associate and assistant professor in psychology. The new data suggest that the posterior insula, which affects sensation and motor control, is implicated in feelings of lust or desire, while the anterior insula has a role in the more abstract representations involved in love.

In the earlier paper, “The Common Neural Bases Between Sexual Desire and Love: A Multilevel Kernel Density fMRI Analysis,” Cacioppo and colleagues examined a number of studies of brain scans that looked at differences between love and lust.

The studies showed consistently that the anterior insula was associated with love, and the posterior insula was associated with lust. However, as in all fMRI studies, the findings were correlational.

“We reasoned that if the anterior insula was the origin of the love response, we would find evidence for that in brain scans of someone whose anterior insula was damaged,” she said. 

In the study, researchers examined a 48-year-old heterosexual male in Argentina, who had suffered a stroke that damaged the function of his anterior insula. He was matched with a control group of seven Argentinian heterosexual men of the same age who had healthy anterior insula.

The patient and the control group were shown 40 photographs at random of attractive, young women dressed in appealing, short and long dresses and asked whether these women were objects of sexual desire or love. The patient with the damaged anterior insula showed a much slower response when asked if the women in the photos could be objects of love.

“The current work makes it possible to disentangle love from other biological drives,” the authors wrote. Such studies also could help researchers examine feelings of love by studying neurological activity rather than subjective questionnaires.

MIT robot may accelerate trials for stroke medications

The development of drugs to treat acute stroke or aid in stroke recovery is a multibillion-dollar endeavor that only rarely pays off in the form of government-approved pharmaceuticals. Drug companies spend years testing safety and dosage in the clinic, only to find in Phase III clinical efficacy trials that target compounds have little to no benefit. The lengthy process is inefficient, costly, and discouraging, says Hermano Igo Krebs, a principal research scientist in MIT’s Department of Mechanical Engineering.

“Most drug studies failed and some companies are getting discouraged,” Krebs says. “Many have recently abandoned the neuro area [because] they have spent so much money on developing drugs that don’t work. They end up focusing somewhere else.”

Now a robot developed by Krebs and his colleagues may help speed up drug development, letting pharmaceutical companies know much earlier in the process whether a drug will ultimately work in stroke patients.

To receive approval from the Food and Drug Administration, a company typically has to enroll 800 patients to demonstrate that a drug is effective during a Phase III clinical trial; this sample size is determined, in part, by the accuracy of standard outcome measurements, which quantify a patient’s ability over time to, say, lift her arm past a certain point. A clinical trial can take several years to enroll appropriate patients, run tests, and perform analyses.

The study’s authors found that by using a robot’s measurements to gauge patient performance, companies might only have to test 240 patients to determine whether a drug works — a reduction of 70 percent that Krebs says would translate to a similar reduction in time and cost.

While pharmaceutical companies would still have to adhere to the FDA’s established guidelines and outcome measurements to receive final drug approval, Krebs says they could use the robot measurements to guide early decisions on whether to further pursue or abandon a certain drug. If, after 240 patients, a drug has no measurable effect, the company can pursue other therapeutic avenues. If, however, a drug improves performance in 240 robot-measured patients, the pharmaceutical company can continue investing in the trial with confidence that the drug will ultimately pass muster.

The researchers have published their results in the journal Stroke.

Creating a translator for stroke recovery

In their study, Krebs and his colleagues explored the robot MIT-Manus as a tool for evaluating patient improvement over time. The robot, developed by the team at MIT’s Newman Laboratory for Biomechanics and Human Rehabilitation, has mainly been used as a rehabilitation tool: Patients play a video game by maneuvering the robot’s arm, with the robot assisting as needed.

While the robot has mainly been used as a form of physical therapy, Krebs says it can also be employed as a measurement tool. As a patient moves the robot’s arm, the robot collects motion data, including the patient’s arm speed, movement smoothness, and aim. For the current study, the researchers collected such data from 208 patients who worked with the robot seven days after suffering a stroke, and continued to do so for three months.

The researchers created an artificial neural network map that relates a patient’s motion data to a score that correlates with a standard clinical outcome measurement.

The authors then selected a separate group of nearly 3,000 stroke patients who did not use the robot, but who went through standard clinical tests. In particular, the researchers calculated the “effect size” — the difference in patient performance from the beginning to the end of a trial, divided by the standard deviation, or variability, of improvement among these patients. To determine whether a drug works, the FDA will often look to a study’s effect size.

Using the robot-derived neural network map, the group calculated the effect size at twice the rate usually achieved with standard clinical outcome measurements, indicating that the robot scale demonstrated greater sensitivity in measuring patient recovery.

The study’s authors went one step further and performed a power analysis that determines the optimal sample size for a given technique, finding that the robot scale would require only 240 patients to determine a drug’s effectiveness — a reduction in sample size that would save a company up to 70 percent in time and cost.

“Such a savings would be fantastic,” says David Reinkensmeyer, a professor of physical medicine and rehabilitation at the University of California at Irvine. “Robotic measurements will help us identify promising treatments with smaller numbers of patients and provide better insight into the mechanisms of the treatments, so that we can target those mechanisms and improve the treatments.”

Currently, only a few stroke drugs are in the late stages of development. However, once a company reaches a Phase III clinical trial, Krebs says it may use the MIT-Manus robot as a more efficient way to evaluate the drug’s impact by employing the measurement techniques on a smaller group of patients.