OUR RESEARCH

Using the most advanced imaging and microscopy techniques, Iliff, then a postdoc at the University of Rochester Medical Center, observed waves of cerebrospinal fluid flowing around and through the brain, collecting waste from cells and flushing them out to the circulatory system.

And there was the kicker: the team found that this waste pick-up service for neurons goes into action during sleep. It turns off during the day when the brain is busy with other tasks.

The discovery by Iliff and his mentor at the time, Maiken Nedergaard, that this brain-wide network, called the “glymphatic system,” cleans the brain during sleep was one of Science magazine’s “Top 10 Breakthroughs” of 2013.

The research not only shined a light on the newly described glymphatic system, but also on the connection between sleep and brain health. Does the system work similarly in humans as it does in mice? Does the sanitation system in the brain clog or break down when sleep is impaired? If so, does waste that doesn’t get cleaned out of the brain trigger disease?

The discovery by Iliff and his mentor at the time, Maiken Nedergaard, that this brain-wide network, called the “glymphatic system,” cleans the brain during sleep was one of Science magazine’s “Top 10 Breakthroughs” of 2013.

The research not only shined a light on the newly described glymphatic system, but also on the connection between sleep and brain health. Does the system work similarly in humans as it does in mice? Does the sanitation system in the brain clog or break down when sleep is impaired? If so, does waste that doesn’t get cleaned out of the brain trigger disease?

Piecing the puzzle

In the past several years, Iliff – now an SIBCR-supported scientist, the University of Washington School of Medicine’s Arthur J. and Marcella McCaffery Professor in Alzheimer’s Disease, and Associate Director for Research at the VISN20 NW Mental Illness Research, Education, and Clinical Center (MIRECC) at the VA Puget Sound – has helped to piece together much of the puzzle.

In their studies in mice, Iliff and his research team have demonstrated that the glymphatic system, when working properly, indeed clears proteins implicated in neurodegenerative diseases such as Parkinson’s and Alzheimer’s. “In healthy people, these proteins are naturally produced and removed at near-equal rates,” he said. “But when this cleaning system is impaired, these proteins can pile up, spread and cause trouble.”

Iliff’s research group also found that in older mice and those with brain injuries, the cleaning system slowed, leading to the build-up of some of those same neurotoxic proteins.

And it’s no coincidence that a hallmark symptom of aging, traumatic brain injury and some neurodegenerative diseases is poor sleep.

The Alzheimer’s link

Notably, Iliff has found that among the wastes that accumulate in the brain when the glymphatic system is faulty, is amyloid beta, which has been implicated in Alzheimer’s disease. Moreover, other researchers have found that losing just a single night of sleep can increase amyloid in the brain.

So, should sleep and the glymphatic system be targets to treat or prevent neurodegenerative diseases?  Iliff believes so, particularly for Alzheimer’s. Currently-approved therapies, including the amyloid-focused drug aducanumab have been iffy at best and quite expensive. In the meantime, there are proven therapies, with or without drugs, to improve sleep.

Move to human trials

While Iliff’s lab will continue to study the glymphatic system in mice, more and more of his research has begun to focus on people. That’s just how successful science progresses.

Last fall, the US Department of Defense awarded $4.3 million for a three-year clinical trial of a device to speed up the natural system of brain cleansing that occurs with sleep. On the study, Iliff is working with University of Washington School of Medicine Department of Radiology researcher Dr. Swati Rane, along with collaborators at the University of North Carolina, Brain Electrophysiology Lab, the Oregon Health and Science University, and Montana State University.

“Our lab’s research over the past eight years on brain-waste clearance in animals has helped define glymphatic biology,” said Iliff. “Now we hope to see if we can use what we’ve learned to help people overcome poor or interrupted sleep and the brain dysfunction that follows.”

The technology in this study involves an easy-to-use, wireless, combined electrical brain wave monitoring (EEG)/stimulation headband that can be applied before sleep to monitor and improve deep sleep and glymphatic waste clearance.

Veterans’ research participation

Veterans will play an important role in Iliff’s future collaborative clinical studies. In the NW MIRECC at the VA Puget Sound, he works closely with Elaine Peskind, MD, and Murray Raskind, MD, both SIBCR scientists.

Dr. Peskind has been investigating mild traumatic brain injury in Iraq and Afghanistan war Veterans, while Dr. Raskind has pioneered the use of the drug prazosin, a medication approved for treatment of high blood pressure, for use in post-traumatic stress disorder (PTSD). While Dr. Iliff’s work in mice has shown that traumatic brain injury impairs brain waste clearance, and that the drug prazosin can improve sleep and glymphatic function, this team of SIBCR scientists now plans to extend these studies into Veteran populations to test whether prazosin can improve outcomes after traumatic brain injury by supporting brain waste clearance.

In parallel, Drs. Iliff, Rane and Peskind are conducting studies to develop better ways to measure these processes in the human brain, and to figure out if changes in waste clearance are reflected in spinal fluid biomarkers for brain toxic proteins implicated in neurodegenerative diseases. An initial study using these approaches to test the effects of poor sleep in young healthy volunteers is planned for the coming spring.

While it may be years before these studies yield new therapies for preventing and treating neurodegenerative disease, Iliff’s research supports some age-old advice: Get a good night’s sleep.

Many have witnessed death and destruction, while others suffered multiple concussions (mild traumatic brain injury) caused by the many explosions to which they were exposed.

While physical scars may not be obvious, behavioral disorders and problems caused by repetitive mild traumatic brain injury (mTBI) are all too apparent in many of these Veterans – the nightmares and sleep disturbance of post-traumatic stress disorder (PTSD), depression, anxiety, memory loss, irritability, impulsivity, persistent headaches and more.

As a laboratory scientist, Peskind can peer into the brain of living Veterans by using sophisticated neuroimaging and, in their group’s mouse blast mTBI model and brain tissue from deceased Veterans, at the microscopic level to see structural damage, neuron activity disruption and other dysfunction that may be the cause of these Veterans’ debilitating symptoms.

For the multi-faceted clinician-scientist, it’s been her way for nearly 40 years to: understand the anguish of her patients; get into the laboratory to pinpoint possible underlying causes; and offer whatever puzzle pieces that she finds so that research teams can develop new targets for prevention and treatment of these disorders.

Peskind is also driven by compassion and commitment to her Veteran patients, who become partners in research, too.  “I have so much respect for them,” said Peskind, an SIBCR-supported scientist. “Even after all that they’ve gone through, they remain selfless and devoted to serving the public good.”

Peskind is the Friends of Alzheimer’s Research Professor of Psychiatry in the Department of Psychiatry and Behavioral Sciences at the University of Washington School of Medicine; Co-Director of the VA VISN 20 Northwest Mental Illness Research, Education, and Clinical Center (MIRECC); and Director of both the MIRECC fellowship and a National Institute on Aging T32 institutional training grant.

While her academic title reflects her expertise in Alzheimer’s disease, the other disorders she studies – mild TBI and PTSD, both have been linked to increased risk of Alzheimer’s and other related neurodegenerative diseases. A prolific investigator, Peskind has published more than 350 studies.

Collecting crucial cerebrospinal fluid

One her pioneering studies in 2005 exploded the myth that lumbar punctures, or spinal, taps to draw cerebrospinal fluid (CSF) from research participants are risky, painful and uncomfortable.

Collecting CSF is important for studying fluid biomarkers, which are essential in research into neurodegenerative disease such as Alzheimer’s and Parkinson’s.

Peskind, a hands-on scientist who had performed more than a thousand lumbar punctures in the MIRECC, demonstrated and documented a new and safe method now used by research groups around the world.

Blocking agitation in people with dementia

It was in CSF, that Peskind and co-researchers discovered in 1995 that the brain’s system for storing and releasing the neurotransmitter norepinephrine is enhanced during normal aging and boosts even more in patients with Alzheimer’s disease. This adrenaline hyperactivity, which peaks at 4 p.m., explains the phenomenon of the late afternoon agitation and sometimes aggressive behavior (called “sundowning”), often seen in dementia patients, Peskind surmised.

There are no good treatments for this disruptive agitation for these patients, said Peskind. Atypical antipsychotics are often prescribed, but the benefits are questionable for this group of patients, and they have prominent negative side effects.

Now, with a target to aim – the brain’s adrenaline system – Peskind and longtime colleague Murray Raskind, also a SIBCR-supported investigator and Director of the VA Northwest MIRECC, developed clinical trials to test prazosin, an FDA-approved drug for hypertension, to treat Alzheimer’s disease patients with disruptive agitation that is common in the middle and late stages of the disease. Prazosin works by blocking norepinephrine in the brain – at a receptor called the alpha-1 adrenoreceptor.

Raskind and Peskind had also conducted clinical trials of prazosin to treat trauma nightmares and sleep disturbance in Veterans with PTSD. And both are teaming with Jeff Iliff, an SIBCR-supported neuroscientist, to test if prazosin can improve sleep and the brain’s own waste clearing (glymphatic) system in Veterans with traumatic brain injury.

Trauma from blasts

Today, Peskind turns most of her research attention to mild traumatic brain injury (mTBI), the “signature injury” of the Iraq and Afghanistan wars. It’s estimated that up to 500,000 service members in those wars came home with persistent post-concussive symptoms.

Those Veterans were frequently exposed to blasts, including from improvised explosive devices (IEDs), which send out highly intense shock waves that didn’t necessarily knock them over, but they were apparently harsh enough to rock the brain and cause concussion or mTBI.

In a study published in 2016, Peskind and co-researchers worked with 33 Veterans and found by functional imaging that the more blasts that they were exposed to, the more they showed chronic changes in neuronal activity in the cerebellum. This group of Veterans averaged 20 blast exposures that were severe enough to cause symptoms consistent with mTBI.

To examine these changes more closely, the research team studied the brains of mice that were exposed to mild blasts, and they also found neuron loss in the cerebellum. “It’s the same kind of effect that you see in boxers and other athletes who experience repeated blows to the head and are risk for chronic traumatic encephalopathy (CTE),” said Peskind.

The key in this study was pinpointing the cerebellum, long acknowledged as the center for sensory input and motor output, including movement coordination. Researcher now believe, however, that the cerebellum can influence person’s emotions and cognition.

“Problems with mood, irritability, anger dyscontrol, and impulsivity are very common in Veterans with repetitive mTBI,” said Peskind. “We need to pay more attention to how mTBI affects the cerebellum if we want to fully understand the emotional difficulties that our Veterans with mTBI are experiencing.”

She also noted that 75 percent of the mTBI Veterans she treats also have combat trauma PTSD.

“Something is going on in a very specific part of the brain that we don’t fully understand yet,” said Peskind.

True to form, Peskind’s research points the way for other scientists who may eventually design treatments for suffering Veterans.

The condition, called sarcopenia, is present in an estimated 25 percent of older individuals. While most people begin losing muscle in their 30s and 40s, the rate of decline dramatically increases between the ages of 65 and 80. Loss of stamina, trouble walking and climbing stairs, and poor balance are among the symptoms. These lead to increased falls and related-injuries, hospital stays and healthcare costs.

“Sadly, many people who suffer from muscle wasting disorders lose their independence and ability to perform simple daily tasks,” said Jose Garcia, MD, PhD, an SIBCR-supported researcher, and University of Washington Professor of Medicine in the Division of Gerontology and Geriatric Medicine.

A similar muscle wasting syndrome strikes cancer patients. Up to 75 percent of cancer patients suffer from the condition, called cachexia. In addition to loss of muscle mass and strength, hallmark symptoms include severe weight loss and fatigue. Moreover, these patients often are unable to tolerate treatments, such as chemotherapy. More than 20 percent of patients die from cachexia.

“Certainly, both these conditions are over-represented in the Veteran population,” said Garcia. “Although there are some clinical trials underway, we do not yet have proven treatments for these diseases that are all too prevalent in the general population as well.”

Garcia – Clinician Investigator and Acting Director, Geriatric Research, Education and Clinical Center and Director, Clinical Research Unit, VA Puget Sound Health Care System – has devoted much of his career to understanding the molecular mechanisms of these muscle wasting conditions and finding novel targets for treatment.

Hormone a culprit and treatment target

There is a constellation of biological factors to how muscles dwindle as we age or when we fall ill. And Garcia for nearly 20 years has focused on an anabolic hormone, ghrelin, and its receptor (GHSR), which play a role in food intake, fat deposition, muscle mass and function regulation and growth hormone release during aging and in cancer.

Produced largely in the stomach, ghrelin – dubbed the “hunger hormone” because it tells your brain that it’s time to eat – also is made in the brain, fat tissue and muscle.

Garcia has conducted numerous studies in laboratory mice of how ghrelin affects body weight, body composition and muscle mass and performance. In one key study, which he published in 2017 with former colleagues at the Baylor College of Medicine, the researchers compared aging mice without ghrelin (“knocked-out”) with “wild-type” mice that have ghrelin.

In line with what happens in humans, the old wild-type mice showed an increase in body weight and fat mass, and lower muscle strength and endurance. In the “knockout mice,” however, Garcia’s team showed for the first time that deleting ghrelin significantly prevented body weight and fat mass gain in older mice while maintaining muscle function. The team also identified a novel mechanism for ghrelin action in muscle during aging, which could be an important target for therapies to stem or prevent muscle wasting. He has also performed other animal studies showing that mice implanted with tumors develop cachexia and that this can be prevented by administering ghrelin.

“Short-term treatment of old mice with pharmacologic doses of ghrelin increased food intake, body weight, and muscle strength in mice; however, in the long run preventing ghrelin’s action may be more beneficial in certain settings like in aging-related sarcopenia.”

Studies like Garcia’s guide clinical researchers who are testing interventions for conditions like cancer cachexia, where an increase in fat mass is likely to be beneficial and to select outcomes to be measured, such as muscle fiber type and strength, appetite, and energy expenditure.

Making clinical trials more meaningful

While Garcia is a highly accomplished laboratory researcher, he is just as good a clinician and a clinical researcher.

Garcia has also led important clinical trials increasing our understanding and developing new treatments for cachexia in cancer patients.

“Even though scientists well understand that animal studies don’t always translate to humans, some are puzzled and disappointed by results of ghrelin-rooted treatments in humans”, said Garcia.

Some experimental therapies appear to increase body and muscle mass, and even some strength, in cancer papers. But they have failed to induce clinically significant improvements in physical function based on some of the measurement tools that researchers have come to rely upon.

“You can’t ask mice how they’re feeling,” said Garcia, who proposes tools that better measure not just muscle mass, but also those that measure what matter most to cancer patients – strength, function, and their quality of life.

In two separate publications in 2020, Garcia suggested that measurements of stair climb power, upper body strength, walking time, and other functions related to quality of life should be the focus instead of currently used measurements and biomarkers, such scans and blood tests.

“Tools to measure how patients feel and function that have been tested in clinical trials should be the main outcomes in future studies,” said Garcia. “Measuring muscle mass, strength and function altogether will offer a clearer and more comprehensive look at whether experimental treatments are working or not.”

The nematode – C. elegans – is about the size of a piece of lint, but its genes are remarkably similar to humans. They age from egg to adult in about three days, so researchers can observe entire lifetimes of thousands of worms rapidly.  C. elegans also are transparent, so scientists can peer through powerful microscopes to see their 300 neurons at work.

Brian Kraemer, an SIBCR-supported researcher and University of Washington Professor in the Division of Gerontology and Geriatric Medicine, began some 20 years ago to develop C. elegans models to understand the molecular mechanisms of neurodegenerative diseases associated with aging. Now, his research offers a novel approach to identify and develop drugs to treat Alzheimer’s disease

Targeting tau

The believed culprits and cause of Alzheimer’s are two proteins: amyloid, which triggers plaque, and tau, which forms tangles within brain cells.

“It’s thought that these two pathologies synergize to kill brain cells in Alzheimer’s disease,” said Kraemer, who also is Associate Director for Research, Geriatric Research, Education and Clinical Center at the VA Puget Sound Health Care System “What we’ve done is look in model organisms for genes that are important for the toxic cascade in Alzheimer’s disease.”

“We’ve specifically focused on the tau protein, which we think is more directly involved in the killing of brain cells,” said Kraemer.

After seeing some of his own family members suffer from Alzheimer’s disease, Kraemer has devoted his laboratory studies to “finding out what drives this terrible neurodegenerative disease.”

He started with worms

An advantage of a C. elegans model is that the worm can be easily engineered. Kraemer’s team in 2003 began taking the human tau gene that had a dementia-causing mutation and putting it into worms so that they “expressed” the human tau in their neurons. They then could see what happened if the worms had the same abnormal tau in their nerve cells as do humans with Alzheimer’s.

Sure enough, neurons in the worms stopped functioning normally – their movement slowed down and they become uncoordinated. When they looked biochemically at the worms, they also saw other changes that were similar to what happens in the brain cells of humans with dementia.

With this worm model, Kraemer and his team experimented with ways to make them well again by deleting genes – or “knocking them out” – and then looking for worms that appeared to be resistant to the ill effects of the mutated tau. They painstakingly watched under microscopes thousands of worms to see if any started moving normally again.

The team eventually identified a gene – MSUT-2 (mammalian suppressor of tauopathy 2) – that may prevent the breaking down of abnormal proteins and thus allow damage to build in the nervous system. Knocking out that gene cured worms, which maintained normal and robust movement.

From worms to mice models

With this knowledge from worms, Kraemer and his team took the studies up a notch by applying the experiments to mice, whose evolutionary distance to humans is  much closer than that between worms and humans.

In 2019, the team reported in the journal Science Translational Medicine that knocking out the MSUT2 gene in mice prevented the formation of the tau tangles that kill brain cells. Importantly, this lessened learning and memory problems in the mice as well.

The researchers also examined autopsy brain samples from Alzheimer’s patients and found that that cases with more severe disease lacked both MSUT2 protein and its partner protein – an RNA binding protein called PABPN1. They suggested that neurons that lose the MSUT2 -PABPN1 protein partnership may simply die during a patient’s life.

Moreover, mice lacking MSUT2 but possessing a normal complement of PABPN1 were strongly protected against abnormal tau and the resulting brain degeneration. Thus, the key to helping people with abnormal tau buildup is blocking MSUT2 while preserving PABPN1 activity, they found.

“The field has really been focused on amyloid for a long time, and there have been quite a few failed clinical trials,” said Kraemer. “At this point, maybe we need to intervene both on amyloid and tau simultaneously.”

Current treatments for Alzheimer’s mostly aim to clear amyloid plaques in patients’ brains. But this approach doesn’t seem to substantially improve cognition, and there still is limited evidence that it clinically slows disease progression. Amyloid plaques may be a symptom, rather than a cause of Alzheimer’s, said Kraemer.

“Given that tau is correlated with cognitive dysfunction, and amyloid is not, it seems logical to pay more attention to tau,” he said. “We feel that targeting tau may allow us to start to make changes to the way the disease progresses and actually impact people’s lives in the long term.”

Kraemer already is progressing on early-stage drug discovery towards MSUT2-based therapies. In April of 2022, the Washington Research Foundation (WRF), a leading technology transfer and grant-making organization, awarded Kraemer a $250,000 grant to advance his research and test small molecules that he has identified.

His studies will concentrate on the molecules’ ability to safely inhibit MSUT2 and cross blood-brain barrier. If successful, it could set the stage for the first human studies to develop new Alzheimer’s disease drugs using the novel technology.

But her basic science studies of pancreatic beta-cell dysfunction and death – metabolic hallmarks of high blood glucose – gained steam.

And some 19 years later, she’s still here – as a Research Associate Professor of Medicine at the University of Washington and investigator at the Diabetes Research Center, located at the VA Puget Sound Health Care System – and on a research path that may lead to novel therapeutic targets for type 2 diabetes.

Worldwide, nearly 537 million adults – 10.5 percent of the population — suffer from type 2 diabetes (T2D), and that number could soar to 783 million by 2045, according to the International Diabetes Federation.

Moreover, TD2 affects nearly 25 percent of the VA population, and it is the leading cause of blindness, end-stage renal disease and amputations for VA patients.

Hidden metabolic irregularities and numerous disease-causing pathways underlie this complex disease. Although a broad range of medications for TD2 have been developed in past decades, many have significant limitations and side effects. Scientists, thus, are on a constant search for precise drug targets or combinations of drugs to treat T2D.

For Zraika, it’s not just a scientific quest. It’s personal.

“Type 2 diabetes is often thought of as a disease caused by poor lifestyle habits like physical inactivity and overeating, said Zraika. “In reality, the risk of developing type 2 diabetes is increased by several different factors, some of which are not fully understood.

“There is, however, one factor that I understand all too well, and that is having a family history of diabetes,” she said. “Of my 10 maternal aunts and uncles, almost all have type 2 diabetes. As a child, I watched my maternal grandmother suffer from complications of type 2 diabetes at a time when there were few therapeutic options to treat the chronic disease.

“This experience had a profound impact on me – I developed a sense of responsibility to do something that might one day prevent members of my family, or even me, from developing type 2 diabetes.”

Early in her career, under the mentorship of current Diabetes Research Center director Steven Kahn, Zraika pursued important avenues of investigation, including mechanisms that lead to pancreatic beta-cell failure and loss in T2D and the role of specific proteins in modulating insulin secretion and blood sugar regulation.

Like most basic science research, Zraika’s has been arduous, but her work has been fruitful, too.

Her studies demonstrated that the protein neprilysin – a component of the renin-angiotensin system (RAS), which regulates blood pressure and blood volume – plays a role in beta cell function. In mice fed a high-fat diet, neprilysin was upregulated and this was associated with reduced glucose-stimulated insulin secretion.

In a series of studies published within the last few years, Zraika suggested that neprilysin inhibition – already an approved drug strategy for heart failure – could be repurposed to treat T2D. While Zraika highlighted the mechanisms underlying the beneficial glycemic effects of neprilysin deletion/inhibition, she also addressed effects that could limit the efficacy and safety of neprilysin inhibitors in the clinic.

“Their combination with angiotensin II receptor blockers is needed to circumvent deleterious consequences of neprilysin inhibition alone,” she concluded.

Successful research, breeds more research

Now, Zraika is conducting an NIH-funded project to investigate the beneficial effects of understudied angiotensin peptides on beta-cell function and survival. Revealing their previously unrecognized intracellular action on glycemic control could open a new target for T2D drugs.

Another of Zraika’s NIH-funded projects focuses on statins. While statins are the most commonly used cholesterol-lowering medication, they also increase the risk of T2D.  “With statin use up by 30 per cent in the U.S. in just 10 years, there is an urgent need to better understand how statins lead to demise of the β cells,” she said. “This knowledge will inform strategies aimed at reducing progression to T2D in patients on statin therapy.”

“Research is a roller coaster,” said Zraika. “Occasionally you generate exciting data and other times you generate data you don’t understand. It’s the latter that is most fascinating, as it leads to more questions and ideas that cause you to shift your focus and transition to the next thing.”

Her research creativity and progression are fueled by her collaborations with clinical scientists at the VA Puget Sound Health Care System and University of Washington.

“Basic scientists like me focus on minute details, and justifiably so when studying the molecular mechanisms of disease,” said Zraika. “However, it’s essential to understand why the minute details are important. Being surrounded by clinicians puts my basic research in perspective and gives it greater meaning.

“Collaborations with clinicians have also helped me better understand clinical challenges that are faced in diabetes diagnosis, treatment and management, and have driven my research in directions that address real-world problems,” she said.

And that world includes the VA Puget Sound Health Care System, where she has developed an appreciation for Veterans.

“Given that diabetes is over-represented in the Veteran population, I feel my work at the VA has more meaning and impact for Veterans.” said Zraika.  “Our Veterans have made countless sacrifices during times of war and peace. Their commitment is unparalleled and so they deserve the very best care and treatment for their ongoing battle with diabetes. My commitment to basic research may lead to better treatment options for Veterans.”

Veterans — and the general population – will be glad that Sakeneh Zraika has overstayed her trip to Seattle some 19 years ago.