University of South Florida Health neuroscientists discover a defect early in autophagy that may help develop SSH1 inhibitors to treat Alzheimer’s and other neurodegenerative diseases

When the physiological process of autophagy runs smoothly, cellular waste is routinely collected for disposal so it does not pile up like garbage at the curbside.

TAMPA, Fla (Oct. 12, 2020) — In a healthy brain, the multistep waste clearance process known as autophagy routinely removes and degrades damaged cell components – including malformed proteins like tau and toxic mitochondria. This cellular debris would otherwise pile up like uncollected trash to drive the death of brain cells (neurons), ultimately destroying cognitive abilities like thinking, remembering and reasoning in patients with Alzheimer’s and certain other neurodegenerative diseases.

The protein p62, a selective autophagy cargo receptor, plays a major role in clearing misfolded tau proteins and dysfunctional mitochondria, the energy powerhouse in all cells including neurons. Through autophagy (meaning “self-eating” in Greek) old or broken cellular material is ultimately digested and recycled in lysosomes, membrane-bound structures that work like mini-waste management plants.

Now, neuroscientists at the University of South Florida Health (USF Health) Byrd Alzheimer’s Center report for the first time that the protein phosphatase Slingshot-1, or SSH1 for short, disrupts p62’s ability to function as an efficient “garbage collector” and thereby impairs the disposal of both damaged tau and mitochondria leaking toxins. In a preclinical study, the researchers showed that SSH1’s influence in halting p62-mediated protective clearance of tau was separate from SSH1’s role in activating cofilin, an enzyme that plays an essential part in worsening tau pathology.

Their findings were published Oct. 12  in Autophagy.

David Kang, PhD, professor of molecular medicine at the USF Health Byrd Alzheimer’s Center, was senior author of the study published in Autophagy.

First author Cenxiao Fang, MD, PhD

“Slingshot-1 is an important player in regulating the levels of tau and neurotoxic mitochondria, so it’s important to understand exactly what’s going wrong when they accumulate in the brain,” said the paper’s senior author David Kang, PhD, professor of molecular medicine at the USF Health Morsani College of Medicine, who holds the Fleming Endowed Chair in Alzheimer’s Disease and serves as the director of basic research at the Byrd Alzheimer’s Center. “This study provides more insight into a defect stemming from the p62 pathway, which will help us develop SSH1 inhibitors (drugs) to stop or slow Alzheimer’s disease and related neurodegenerative disorders.”

First enzyme leading to p62 deactivation

At the start of their study, Dr. Kang’s team, including first author and doctoral student Cenxiao (Catherine) Fang, MD, already knew that, in the case of clearing bad mitochondria (known as mitophagy), the enzyme TBK1 transiently adds phosphate to p62. Phosphate is specifically added at the site of amino acid 403 (SER403), which activates p62. However, no scientist had yet discovered what enzyme removes phosphate from p62, known as dephosphorylation. Tightly controlled phosphorylation is needed to strike a balance in p62 activation, an early step key in priming the cargo receptor’s ability to recognize and collect chunks of cellular waste labelled as “garbage” by a ubiquitin tag. Put simply, when autophagy works well, ubiquitinated tau and ubiquitinated mitochondria are selectively targeted for collection and then delivered for destruction and recycling by autophagosomes (the garbage trucks in this dynamic process). But, garbage collector p62 doesn’t touch the cell’s healthy (untagged) proteins and organelles.

In a series of gene deactivation and overexpression experiments using human cell lines, primary neurons, and a mouse model of tauopathy, Dr. Kang’s team discovered SSH1, acting specifically on SER403, as the first enzyme to remove this key phosphate off p62, causing p62 deactivation.

.

“When something shifts out of balance, like overactivation of Slingshot-1 by Alzheimer’s-related protein Aβ for example, then SSH1 starts to remove the phosphate off the garbage collector p62, essentially relaying the message ‘stop, don’t do your job.’  That leads to bad consequences like accumulation of damaged tau proteins and toxic mitochondria,” Dr. Kang said. “If we can bring phosphorylation regulation back into balance through inhibitors that dampen overactive Slingshot-1, we can increase p62’s normal activity in removing the toxic garbage.”

Learning more from a surprising result

This latest study builds upon previous USF Health research showing that Aβ-activated cofilin, which occurs through SSH1, essentially kicks tau from the microtubules providing structural support to neurons, thereby boosting the build-up of tau tangles inside dying nerve cells. In the displacement process, cofilin gets transported to mitochondria and damage to the energy-producing mitochondria ensues.

Following up on that collateral cofilin-triggered damage, Dr. Kang’s team expected to find a widespread mitophagy upon SSH1 expression — a typical response to clear out the damaged mitochondria.

“However, we found the opposite of what we expected. That is, SSHI expression suppressed the mitophagy response, which meant that Slingshot-1 was suppressing mitophagy through another mechanism,” Dr. Kang said. “That mechanism turned out to be inactivation of p62, which occurs simultaneously with cofilin activation.”

The researchers showed that two major and entirely separate signaling pathways implicated in tau pathology – one for p62 and another for cofilin – are both regulated by the same enzyme, SSH1.

“In addition to the SSH1-cofilin activation pathway in promoting tau displacement from microtubules, this study highlights the divergent SSH1-p62 inhibitory pathway in impairing autophagic clearance of misfolded tau,” the study authors report.

Cellular autophagy illustration showing the fusion of a lysosome (upper left) with an autophagosome.

The USF Health study was supported by grants from the NIH’s National Institute on Aging and National Institute of Neurological Disorders and Stroke, the U.S. Department of Veterans Affairs, and the Florida Department of Health. This research represented a major part of the doctoral thesis of the first author Cenxiao Fang, MD, PhD, who recently received her PhD degree from USF and is now a postdoctoral scholar at the University of Minnesota.