His team searches beyond the hallmark Alzheimer’s disease proteins for alternative treatments

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In his laboratory at the USF Health Byrd Alzheimer’s Center, neuroscientist David Kang, PhD, focuses on how different types of proteins damage the brain when they accumulate there. In the case of Alzheimer’s disease, decades of good science has zeroed in on amyloid and tau, as the two types of hallmark proteins driving the disease process that ultimately kills brain cells.

Dr. Kang and his team investigate molecular pathways leading to the formation large, sticky amyloid plaques between brain cells, and to the tau neurofibrillary tangles inside brain cells –including the interplay between the two proteins. But, he is quick to point out that amyloid and tau are “not the full story” in the quest to understand how normally aging brains go bad.

“Our goal is to understand as much of the entire Alzheimer’s disease process as possible and then target specific molecules that are either overactive or underactive, which is part of the drug discovery program we’re working on,” said Dr. Kang, professor of molecular medicine and director of basic research for the Byrd Alzheimer’s Center, which anchors the USF Health Neuroscience Institute.

Neuroscientist David Kang, PhD, (third from left)  stands with his team in his laboratory at the Byrd Alzheimer’s Center, which anchors the USF Health Neuroscience Institute.

Attacking dementia from different angles 

Dr. Kang’s group takes a multifaceted approach to studying the biological brain changes that impair thinking and memory in people with Alzheimer’s, the most common type of dementia, as well as Lewy body, vascular and frontotemporal dementias.

That includes examining how damaged mitochondria, the energy-producing power plants of the cell, contribute to pathology in all neurodegenerative diseases. “Sick mitochondria leak a lot of toxins that do widespread damage to neurons and other cells,” Dr. Kang said.

Dr. Kang’s team was the first to identify how mutations of a gene, called CHCHD10, which contributes to both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), cause both mitochondrial dysfunction and protein pathology called TDP-43. Their findings on the newly identified mitochondrial link to both neurodegenerative diseases were published in Nature Communications in 2017.

The role of selective degradation in ridding cells of abnormal proteins, old or damaged organelles (including mitochondria) and other debris is another key line of research pursued by Dr. Kang and colleagues.

A single stained nerve cell | Microscopic image courtesy of Kang lab

“We believe something more fundamental is going wrong in the brain during the aging process to tip the balance toward Alzheimer’s disease – beyond what we call proteinopathy” or deposits of malformed proteins like toxic amyloid and tau, said Dr. Kang, whose work is bolstered by nearly $8 million in grant funding from the National Institutes of Health (NIH), the Veterans Administration (VA merit awards) and the Florida Department of Health.

“I think one of the fundamental things happening is that the (cellular) plumbing system isn’t working to clear out all the accumulating junk,” he said. “That’s why we’re looking at the protective clearance mechanisms (autophagy and mitophagy) that would normally quickly remove misfolded proteins and dysfunctional mitochondria.”

Unfortunately, pharmaceutical trials to date have yielded no effective treatments for Alzheimer’s disease, the sixth leading cause of death in the U.S.  Most clinical studies have centered on developing medications to block or destroy the amyloid protein plaque formation, and a few have targeted the tau-containing neurofibrillary tangles. The five Alzheimer’s drugs currently available may provide temporary relief of symptoms, such as memory loss and confusion. But, they do not prevent or delay the mind-robbing disease as toxic proteins continue to build up and dismantle the brain’s communication network.

Lesson learned: The critical importance of intervening earlier

Some scientists argue that the “amyloid hypothesis” approach is not working. Dr. Kang is among those who maintain that amyloid plays a key role in initiating the disease process that leads to brain atrophy in Alzheimer’s – but that amyloid accumulation happens very early, as much as 10 to 20 years before people experience memory problems or other signs of dementia.

Early detection and treatment are key, Dr. Kang says, because as protein plaques and other lesions continue to accumulate in the brain, reversing the damage may not be possible.

“One reason we’ve been disappointed in the clinical trials is because so far they have primarily targeted patients who are already symptomatic,” Dr. Kang said. “Over the last decade we’ve learned that by the time someone is diagnosed with early Alzheimer’s disease, or even mild cognitive impairment, the brain has degenerated a lot. And once those nerve cells are gone they do not, for the most part, regenerate… The amyloid cascade has run its course.”

As protein plaques and other lesions continue to accumulate, becoming apparent with MRI imaging, reversing the damage may not be possible.  So, for anti-amyloid therapies – or even those targeting downstream tau – to work, patients at risk of Alzheimer’s need to be identified and treated very early, Dr. Kang said.

USF Health is recruiting healthy older adults with no signs of memory problems for a few prevention trials. A pair of Generation Program studies will test the effectiveness of investigational anti-Alzheimer’s drugs on those at high genetic risk for the disease before symptoms start. And, the NIH-sponsored Preventing Alzheimer’s with Cognitive Training (PACT) study is examining whether a specific type of computerized brain training can reduce the risk of mild cognitive impairment and dementias like Alzheimer’s disease in those age 65 and older.

To accelerate early intervention initiatives, more definitive tests are needed to pinpoint biomarkers that will predict Alzheimer’s disease development in genetically susceptible people. Dr. Kang is hopeful about the prospects.  His own team investigates how exosomes, in particular the lipid vesicles that shuttle proteins and other molecules from the brain into the circulating bloodstream, might be isolated and used to detect people at risk of proteinopathy.

“I think within the next five years, some type of diagnostic blood test will be available that can accurately identify people with early Alzheimer’s brain pathology, but not yet experiencing symptoms,” he said.

Graduate research assistant Yan Yan, a member of Dr. Kang’s research team, works at a cell culture hood.

Searching for alternative treatment targets

Meanwhile, Dr. Kang’s laboratory continues searching for other treatment targets in addition to amyloid and tau — including the enzyme SSH1, which regulates the internal infrastructure of nerve cells, called the actin cytoskeleton. SSHI, also known as slingshot, is needed for amyloid activation of cofilin, a protein identified by the USF Health neuroscientists in a recent study published in Communications Biology as a possible early culprit in the tauopathy process.

“Cofilin is overactive in the brains of Alzheimer’s patients so if we can inhibit cofilin by targeting slingshot, it may lead to a promising treatment,” Dr. Kang said.

Ultimately, as with other complex chronic diseases, Alzheimer’s may not be eliminated by a single silver-bullet cure.  Rather, Dr. Kang said, a combination of approaches will likely be needed to successfully combat the neurodegenerative disorder, which afflicts 5.8 million Americans.

“I think prevention through healthy living is definitely key, because brain aging is modifiable based on things like your diet as well as physical activity and brain exercises,” he said.  “Also, we need to focus on earlier diagnosis, before people become symptomatic, and develop next-generation drugs that can attack the disease on multiple fronts.”

Xingyu Zhao, PhD, a research associate in the Department of Molecular Medicine, is among the scientists in Dr. Kang’s laboratory studying the basic biology of the aging brain.

Fascinated by how the brain works — and malfunctions

Dr. Kang came to USF Health in 2012 after nearly 20 years as a brain researcher at the University of California San Diego, where he earned M.S. and PhD degrees in neurosciences and completed NIH National Research Service Award fellowships in the neuroplasticity of aging.

As an undergraduate Dr. Kang switched from studying engineering to a dual major in science/psychology. He began focusing on neurosciences in graduate school, he said, because tackling how the brain works and malfunctions was fascinating and always challenged him.

“With every small step forward, we learn something else about the basic biology of the aging brain,” said Dr. Kang, “It’s not just helpful in discovering what therapeutic approaches may work best against Alzheimer’s disease – we’re also learning more about other neurodegenerative conditions affecting the brain.”

In addition to leading day-to-day research operations at the Byrd Center and helping to recruit new Alzheimer’s investigators, Dr. Kang holds the Mary and Louis Fleming Endowed Chair in Alzheimer’s Research and serves as a research neurobiologist at the James A. Haley Veterans Haley Veterans’ Hospital.

He has authored more than 50 peer-reviewed journal articles on brain aging and Alzheimer’s disease research. A member of the NIH Clinical Neuroscience and Neurodegeneration Study Section since 2016, he has served on multiple national and international editorial boards, scientific panels and advisory boards.

Dr. Kang sits next to a computer monitor depicting stained microscopic images — a single neuron (far left) and the two hallmark pathological proteins for Alzheimer’s disease, tau tangles (center) and amyloid plaques (right).

Some things you may not know about Dr. Kang

• His parents were Presbyterian missionaries in Africa, so he spent nine years of his early life (third through 10^th grade) in Nigeria.
• Dr. Kang practices intermittent fasting, often forgoing breakfast and eating only within an 8-hour window. Animal studies indicate the practice may contribute to lifespan and brain health by improving cellular repair through the process of autophagy, he said. “Autophagy really kicks your cells’ plumbing system into gear to clear out all the waste.”

-Video and photos by Allison Long, USF Health Communications and Marketing

Understanding the sensory nerves involved in protective behaviors may lead to new therapies for respiratory, cardiovascular diseases

Think about the last time you stubbed a toe.

The sensory nerves activated when your toe slammed against a hard object initiated a defensive reflex leading you to withdraw your toe from the source of intense pain. Tom Taylor-Clark, PhD, associate professor in the Department of Molecular Pharmacology and Physiology, likens the pain-induced response to an early warning system that, if working properly, helps us avoid things that can cause damage.

“If you stub your toe once, sure it hurts so much,” he said, “but if you do it repeatedly, eventually you will break your toe.”

In his laboratory at the USF Health Morsani College of Medicine, Dr. Taylor-Clark studies the role of defensive, or nociceptive, sensory nerves in health and disease. Using a combination of electrophysiology, imaging and molecular biology techniques, he investigates how these peripheral nerves, which stimulate organs and penetrate nearly all the body’s tissues, sense their environment. That includes sensory nerve response to external stimuli, like extreme heat or cold, inhaled pollutants or a source of injury, and internal stimuli, such as inflammation, infection or organ damage.

Thomas Taylor-Clark, PhD, an associate professor in the Department of Molecular Pharmacology and Physiology, studies the role of defensive, sensory nerves in health and disease.

“We are interested in understanding the sensory nerves involved in protective behaviors, or defense, because they are the ones that go wrong in disease and injury,” Dr. Taylor-Clark said.

The protective role of airway sensory nerves in cough

His laboratory focuses primarily on the electrical excitability of sensory nerves of the airways. The researchers study the behavior of sensory nerves connecting the lungs with the brainstem, the primitive part of the brain that controls basic body functions such as breathing, swallowing and heart rate. In particular, Dr. Taylor-Clark works with colleagues to better understand the nerves involved in initiating the chronic cough associated with the asthma, a disease characterized by persistent airway inflammation.

Knowing more about how these airway sensory nerves work, including the interface between the conscious and unconscious in the brainstem networks that control cough, is important in understanding how they are disrupted by inflammatory disease. The information could help guide the design of new treatments for unresolved cough and associated symptoms, a major reason people visit primary care providers, Dr. Taylor-Clark said. In addition, better ways to treat cough are important, because for those with a variety of neuromuscular diseases impaired cough can cause an increase in pulmonary infections from aspiration.

Recently, Dr. Taylor-Clark’s team expanded their research to look into how pre-existing cardiovascular disease alters nerve-generated reflexes from the lungs to affect cardiovascular function.

 Dr. Taylor-Clark comments on one aspect of his laboratory’s sensory nerve research.

Stephen Hadley, a senior biological scientist in Dr. Taylor-Clark’s laboratory.

Three research awards totaling more than $2.85 million support their work. The studies are done using cell cultures as well as with the help of transgenic mice that selectively express red fluorescent protein in defensive neurons.

With a grant from the National Heart, Blood and Lung Institute, Dr. Taylor-Clark has investigated the connection between two well-known research findings to determine the downstream effects of mitochondria, the energy producers of the cell, on airway sensory nerve activation. The first finding, he said, was that airway sensory nerves respond to a type of inflammatory signaling that induces potentially damaging oxidative stress. The second was that mitochondria are located right next to signaling receptors in the sensory nerve cells.

“So, we hypothesized that perhaps mitochondria are not there just to produce energy, but to generate signaling,” Dr. Taylor-Clark said. “And we found that mitochondrial signaling activates the sensory nerves specifically by activating chili and wasabi receptors in airways.”

Hot on the trail of wasabi and chili receptors

These receptors for chili peppers (or capsaicin) and wasabi (allylisothiocyanate), officially known as TRPV1 and TRPA1 respectively, are expressed by every single defensive sensory nerve in your body, including those in your tongue, your skin – and your airways (nasal passages, bronchi, larynx). Together the TRPV1 and TRPA1 compounds contribute to involuntary cough reflex.

The USF work linking mitochondrial signaling and airway sensory nerve receptors, triggered by these TRPV1 and TRPA1 molecules that can generate pain as well as heat sensation, resulted in two major papers in the journal Molecular Pharmacology, one in 2013 and another in 2014. A supplementary biophysiological study defining how the wasabi (TRPA1) receptor works was published earlier this year in the Journal of General Physiology.

Above and below: Microscopic images from a transgenic mouse expressing the red fluorescent protein tdTomato  in defensive sensory nerve only.  This crosssection of the lung showing defensive nerve terminals (red)  innervating regions surrounding the small branches of bronchiles, or air tubes (green), within the lungs.

A slice of the brainstem showing central projections of defensive nerves (red) into the medulla, where the nerves transmit signals to brainstem networks to control various involuntary functions like breathing, cough, swallowing, heart rate and blood pressure.

Pollution-induced exacerbation of underlying cardiovascular disease

Another direction of scientific endeavor for Dr. Taylor-Clark is investigating how pre-existing cardiovascular disease may alter normal reflexes from the lungs to affect autonomic regulatory control of the heart. Seed funding from an earlier Morsani College of Medicine Research Office intramural BOOST grant helped his research group obtain a two-year American Heart Association award for this more recent area of research under the auspices of the USF Health Heart Institute.

In preliminary research presented last year at the Experimental Biology Conference, Dr. Taylor-Clark and colleagues reported that hypertensive rats exposed to wasabi, an irritant mimicking the effects of a pollutant like ozone when inhaled into the lungs, experience a much different cardiac response than healthy rats. The heart rate of healthy rats exposed to wasabi slows significantly as a protective mechanism to help slow the distribution of pollutants throughout the body. But given the same exposure, rats with chronic high blood pressure have periods of rapid heartbeats interspersed with a slow heart rate – which can evoke a potentially dangerous abnormal heart rhythm known as premature ventricular contractions.

“So you have a situation where you’ve gone from a healthy (cardiovascular) reflex to an aberrant reflex that may exacerbate pre-existing cardiovascular disease,” he said.

Working with researchers at the University of Florida, Dr. Taylor-Clark is a co-investigator for a recently awarded a three-year, $1.28M grant from the National Institutes of Health Common Fund’s Stimulating Peripheral Activity to Relieve Conditions (SPARC) funding program. The comprehensive project aims to improve maps of the peripheral nervous system —the electrical wiring that connects the brain and spinal cord with the rest of the body – so that more selective and minimally invasive “electroceutical” treatments might be developed for conditions such as heart disease, asthma and gastrointestinal disorders.

 USF’s involvement in NIH project charting defensive airway nerves.

Dr. Taylor-Clark and Stephen Hadley. Recently, Dr. Taylor-Clark’s laboratory expanded its research to look into how pre-existing cardiovascular disease alters nerve-generated reflexes from the lungs to affect cardiovascular function.

Mapping for the future of neuromodulation therapies

The UF-USF multidisciplinary team is focusing on functional mapping of peripheral and central neural circuits for airway protection and breathing.

Using cutting-edge genetic and neurophysiological approaches, they are characterizing the types of defensive airway nerves that control breathing, coughing and heart rate differently and finding where they connect into the brainstem network.

“We are trying to bridge the gap between what has been done (with nerve trafficking) in the lungs and what has been done in the brainstem, and then link them together,” Dr. Taylor-Clark said. “We have transgenic mice that make red fluorescent protein only in their defensive nerves, so now we can chart where targeted nerves are going with superior image quality.”

The team’s overall goal is to advance understanding of the neural pathways underlying respiratory control, laying the groundwork for future neuromodulation therapies to normalize lung function in people at risk.

“If we want to (preferentially) target these therapies for optimal effectiveness, we need to know where all these nerves go and what they do,” Dr. Taylor-Clark said.

Dr. Taylor Clark-received his PhD degree from University College London in 2004. He completed a postdoctoral fellowship at Johns Hopkins University Division of Allergy and Clinical Immunology and served as a medical faculty member at Hopkins for a year before joining USF’s medical school in 2009 as an assistant professor.

Dr. Taylor-Clark is associate chair for research in the Department of Molecular Pharmacology and Physiology. In 2015, he received the Award for Excellence in Teaching from USF’s Graduate PhD Program in Integrated Biological Sciences.

 How mapping neural circuits for airway protection and breathing may lead to novel therapies.

A combination of electrophysiology, imaging and molecular biology techniques are used to study behavior of sensory nerves connecting the lungs with the brainstem.

Some things you might not know about Dr. Taylor-Clark:

• In the mid-1990s, for two years before entering University College London as an undergraduate, he played bass guitar in a band that recorded and performed “very loud rock and roll” as part of the London music scene. These days, with wife Luciana as the audience, Dr. Taylor-Clark jams at home in his living room with daughter Ella, 9, who plays drums.

• Taylor-Clark’s PhD thesis involved a study of how the human nose congests. He measured the internal dimensions of people’s nasal passages with a sonar device at the end of a stick, recruiting family and friends, among others, as study volunteers. He induced sneezing and other symptoms of hay fever by spraying histamine into their nostrils. The shape of the nose and the interaction between nerves and blood vessels in the nose affected air flow and severity of symptoms, he discovered. “While writing the thesis, I began to realize how little was understood about nerves in the airways.”

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 Photos by Eric Younghans, USF Health Communications