It’s a simple statement, but it’s true: “Perseverance is the key to scientific success,” says Pinchas “Hassy” Cohen, dean of the USC Leonard Davis School of Gerontology and USC Distinguished Professor of Gerontology, Medicine and Biological Sciences.
He would know. Nearly 25 years ago, Cohen — a professor at UCLA at the time — led a study on a growth hormone called insulin-like growth factor (IGF) that unintentionally revealed the existence of a small peptide called humanin. But this tiny protein wasn’t coded for in the DNA within the cell’s nucleus; it arose from the separate, smaller genome within the mitochondria, organelles known primarily for their function as energy-producing “powerhouses” of cells.
Humanin’s source in the mitochondrial genome, 16S rRNA, wasn’t initially thought to be a region that could code for proteins at all. The notion that it could code for humanin and similar “microproteins” was initially met with a great deal of skepticism from others in the scientific community, recalls Cohen, who joined the USC Leonard Davis School in 2012. It took many years of subsequent research from Cohen and colleagues for the wider field to accept that 16S, as well as other similar regions of nuclear and mitochondrial DNA, could produce tiny proteins with huge roles in metabolism, aging and age-related diseases.
“People now recognize that there are these things called small open reading frames, and that they can express small proteins, or microproteins. This opens up the human genome, originally thought to contain only 20,000 genes, and increases it by at least two orders of magnitude,” Cohen explains. “And in the mitochondrial DNA, where we primarily work, there used to be thought to exist only 13 protein-coding genes that are all involved in the mitochondrial biology. Now, we think there are approximately 700 of them.”
What started as an inadvertent finding has, after weathering years of skepticism and sparking the interest of more and more researchers, started a new era in biology and drug discovery, he says.
“The discovery of microproteins that we made 25 years ago and have built upon now was a real, unique body of work that represents a new chapter in biology,” Cohen says. “It really changes how we look at genetics and transcriptomics and proteomics. It completely reshuffles the deck, if you will.”
Mitochondria’s Multifaceted Role
Mitochondria aren’t simply energy factories for our cells; they also have important roles in metabolism, cell death, communication between cells and more. Their small, circular genome and their complex abilities reflect their evolutionary origin — before they were mitochondria, they were bacteria that were engulfed by larger cells to form a symbiotic relationship, which happened 1.5 billion years ago. This significantly upgraded ancient organisms’ ability to extract energy from food sources, allowing a more complex system to exist.
“Because mitochondria used to be bacteria themselves, they appear to retain some ability to sense their environment and communicate information to other mitochondria,” Cohen says.
Mitochondria’s communication skills are of particular interest to Changhan David Lee, associate professor of gerontology at the USC Leonard Davis School. Originally trained as a microbiologist and bacterial geneticist, he became more interested in the mitochondrial genome as he completed his PhD in genetic, molecular and cell biology at USC. In 2012, he joined Cohen’s group and began searching for new, small genes in the mitochondrial DNA that expanded the observations made in the Cohen lab. Lee was particularly excited about this concept as he was investigating new genes in bacterial genomes during his undergraduate training.
“At the time, mitochondria were still largely thought about as ‘just making energy,’ but it didn’t really make sense that that was the only thing that mitochondria would have evolved to have become,” Lee says. “I knew there was something more to it. And then I found Hassy’s research, and it just clicked. So, I joined his lab, and we set off to find some new genes.”
One of the genes they discovered coded for a protein called MOTS-c. Found during a screening for peptide activity in response to metabolic changes, the microprotein was first described in 2015 for its role as an “exercise mimetic,” restoring insulin sensitivity and counteracting diet-induced and age-dependent insulin resistance. Subsequent studies of MOTS-c led by Cohen, Lee and colleagues have greatly expanded the microprotein’s job description, uncovering its role as both sender and receiver in intracellular communication during cellular stress and its protective effect against the muscle loss that often accompanies obesity and aging, as well as highlighting how the hormone is expressed in the brain to help regulate metabolism.
In 2021, Research Assistant Professor of Gerontology Hiroshi Kumagai discovered a naturally occurring mutation found in 8% of Japanese individuals that predisposes them to Type 2 diabetes. Kumagai also recently discovered a key mechanism for the action of MOTS-c by unraveling the molecule CK2, with which MOTS-c interacts in muscle.
USC Leonard Davis School research has also shed light on how MOTS-c plays a role in immune system regulation. In mice that had been genetically engineered to develop autoimmune diabetes, a model of Type 1 diabetes in humans, treatment with injections of MOTS-c protected pancreatic cells from being attacked by immune cells and prevented the onset of the disease, per a 2021 study. And new research from Lee’s lab posits that MOTS-c is the first mitochondria-encoded peptide found to be a host-defense peptide, a protein that directly combats bacteria and regulates immune function.
“This shines a bit more of an evolutionary light on what the mitochondria, from its humble bacterial origin, may have had to do during evolution to work out a symbiotic relationship with a bigger cell and protect itself,” Lee says.
Mitochondrial Genetics and Aging
Research Associate Professor of Gerontology Kelvin Yen has long been interested in aging, conducting research on caloric restriction and longevity in mice as an undergraduate at the University of California, Berkeley, and studying the role of insulin and IGF signaling in extending the lifespan of worms as a PhD student at Mount Sinai School of Medicine. In 2010, as a postdoctoral researcher at the University of Massachusetts, Yen attended a talk given by Cohen on novel mitochondrial peptides and their role in aging and was immediately intrigued.
“It was super cool and very exciting,” Yen recalls. “I’d never heard of these microproteins before in my life, much less a mitochondria-specific microprotein.” Yen reached out to Cohen following the talk to ask about postdoctoral opportunities; he relocated to Los Angeles and joined the Cohen lab in 2011.
Since then, interest in mitochondrial microproteins has expanded rapidly, not only as USC Leonard Davis researchers identified more proteins but also as mass spectrometry technology improved, enabling more thorough confirmation of the new peptides, Yen explains. And as the body of research has grown, it has further highlighted the connections among mitochondrial biology, aging and age-related diseases.
In a 2016 study, the Cohen group uncovered the genes for six new mitochondrial microproteins, which were dubbed small humanin-like peptides (SHLP, pronounced “schlep”) 1 through 6, and described their possible protective roles versus age-related diseases, including cancer. Of the six, SHLP 2 has been particularly interesting, with the initial study suggesting that it has insulin-sensitizing, anti-diabetic effects.
Subsequent research published in 2024 showed how a variant of the SHLP 2 gene with a single-nucleotide polymorphism (SNP, or “snip”) — a difference in just one “letter” of the protein’s genetic code — increased both its expression and stability and cut the risk of Parkinson’s disease by 50%. The paper, first-authored by Adjunct Research Professor of Gerontology Su-Jeong Kim, noted that the variant was found in 1% of people of European descent.
Similarly, the mitochondrial microprotein MENTSH, recently characterized by Yen, appears to have a SNP variant associated with an increased risk of diabetes. The variant is more commonly found in people of Native American descent, Yen says.
Mitochondrial genetics offers unique insight into aging across various populations due to its location outside the nucleus of the cell, Cohen says. “With every other gene in the body, there are two copies; every person is an admixture of their two parents,” he says. “But mitochondrial DNA only comes from our mother; we only get one copy, and there is no admixture. This allows us to use mitochondrial DNA to trace our maternal ancestry.”
In addition, while mitochondrial DNA is passed directly from mother to child, it also undergoes evolution at 10 times the rate of nuclear DNA. As a result, “there’s a lot of diversity within the mitochondrial DNA that’s directly linked to maternal ethnicity,” Cohen explains.
Cross-Disciplinary Strength
As USC Leonard Davis researchers have characterized more microproteins, they have also been able to leverage the school’s leadership in biodemography, the incorporation of biological data into large population studies. With the genetic information that’s included in studies such as the Health and Retirement Study in the U.S., scientists can get a clearer picture of how mitochondrial DNA relates to health in humans, says USC University Professor and AARP Chair in Gerontology Eileen Crimmins, a pioneer in biodemography.
“Because we have built these large data sets with representative samples of individuals from different backgrounds, we can look up various genetic markers and we can see how they relate to health outcomes in real populations, with all the other competing things that affect their health,” Crimmins says. She and colleague Em Arpawong, research associate professor of gerontology and director of the Gerontology Bioinformatics Core, have invoked multiple data sources and data types, from genetic code to gene expression levels, to collaborate in unraveling how the mitochondrial genome works together with the nuclear genome to affect aging-related processes. They are co-authors on many USC Leonard Davis mitochondrial microprotein studies, designing analyses that take results from the lab bench and put them into real-life context. Brendan Miller, a 2022 USC PhD in neuroscience graduate, says the interdisciplinary nature of the Leonard Davis School was the “perfect environment” for his work. His fascination with mitochondria having their own genome and his interest in Alzheimer’s led him to the school and Cohen’s lab, and having Crimmins as a co-mentor helped him obtain his skills in statistical methods, population genomics and big-data analyses.
“As a result, we’ve been able to go into these large population databases and find different mitochondrial genes and variants of these genes and immediately bring them down into an experiment that we can test,” Miller says. “That was the biggest standout of USC: having multiple experienced investigators from different backgrounds working on the same question.”
Miller, now a postdoctoral scientist at the Salk Institute, was first author of a 2022 Cohen lab study that identified the mitochondrial peptide SHMOOSE. He used the techniques he learned at USC to identify a mutated version of the protein that increased Alzheimer’s disease risk and brain atrophy. Nearly a quarter of persons of European descent appear to have the mutation, which is associated with a 30% increase in Alzheimer’s risk.
“Ultimately, the goal for SHMOOSE would be to find ways to increase its sensitivity or stability, and pinpoint the exact mechanism that it is involved in,” Miller says, explaining that the peptide’s significant association with Alzheimer’s risk could make it an important drug candidate.
Microproteins in general are exciting potential treatments for age-related disease by nature of their size, he adds.
“Peptides offer a significant advantage in drug development because they’re specific as protein binders,” Miller explains. “There’s often many off-target effects from using larger drug templates. But for peptides, they are smaller and tend to be more specific.”
The tiny peptides produced from the mitochondrial genome appear to have big impacts on obesity, diabetes, frailty, Alzheimer’s and more, according to research from the Cohen lab. Additional recently discovered peptides also show promise against cancer, heart disease and eye disease.
Looking to the Future
Since Cohen arrived at the USC Leonard Davis School in 2012, he and his team have discovered and published research on a dozen mitochondrial microproteins, with many more in the pipeline, he says. The tools and techniques he and colleagues have developed continue to advance the field and propel discoveries toward translation.
“We know that these peptides have important roles in Alzheimer’s disease, Parkinson’s disease, cancer, obesity, diabetes, heart disease and probably multiple other issues related to health and aging. And we’ve created a pathway for discovery, characterization, IP protection, preclinical development and potential commercialization,” Cohen says.
He notes that an analog of MOTS-c has reached human clinical trials in a company he co-founded, with a Phase 1 study suggesting that MOTS-c treatment indeed has beneficial effects similar to what was seen in animal data. “It is overall a very exciting field with a lot of potential,” he says. “Naturally, it needs substantial investment to move to the next step, but the scientific foundation and rationale are only getting stronger and more compelling.”
Another part of growing the school’s strength in mitochondrial research has been recruiting and educating researchers who are excited about mitochondria and their roles in aging. Ana Silverstein and Melanie Flores, 2024 PhD in molecular biology graduates and postdoctoral researchers in the Cohen lab, both say they didn’t know much about mitochondria when starting their PhD program but broadly knew they wanted to study the immune system and cancer, respectively. Both note that learning about Cohen’s research during a presentation for molecular biology PhD students immediately sparked their interest.
“I saw immense overlap in my research interests in immunity and inflammation, and exploring questions related to mitochondrial function and the field of aging seemed like this exciting and nebulous expedition that I became eager to be a part of,” Silverstein says. “I jumped into longevity research and never looked back.”
Both researchers are already contributing to the rapidly growing body of mitochondrial microprotein research. Silverstein is working to characterize a peptide that’s a potential regulator of obesity and inflammation, while Flores is investigating a peptide that appears to be involved in regulating tumor growth and survival and could have potential implications for cancer therapeutics.
After more than two decades, the initial skepticism surrounding the discovery of mitochondrial microproteins has morphed into infectious excitement about this uncharted territory in biology — and the USC Leonard Davis School is blazing the trail.
“We’ve established multiple investigators within the school who are part of this process working on various different microproteins, all of which are important and relevant in aging, and have created the infrastructure to continue to use these tools to identify additional high-value mitochondrial microproteins that can be translated into potential interventions in diseases of aging,” Cohen says. “A lot of the people that I’ve trained have chosen this to be their field of study. And we’ve created a real force here, with many collaborations within USC and other collaborators in Los Angeles, around the country and around the world.”
