Authors :
Presenting Author: Dylan Ukasik, BS – Wayne State University School of Medicine
Eishi Asano, MD, PhD – Pediatrics – Wayne State University School of Medicine, Children’s Hospital of Michigan; Daniela Cukovic, M.Sc – Pediatrics – Wayne State University School of Medicine; Alan Dombkowski, PhD – Translational Neuroscience Program, Pediatrics – Wayne State University School of Medicine; Nicole Doyon Reale, BS – Pediatrics – Wayne State University School of Medicine; Aimee Luat, MD – Pediatrics – Wayne State University School of Medicine, Central Michigan University College of Medicine, Children's Hospital of Michigan; Paul Stemmer, PhD – Institute of Environmental Health Sciences – Wayne State University; Krish Upadhyay, HS Student – Cranbrook Kingswood High School
Rationale:
Tuberous Sclerosis Complex (TSC) is a genetic disorder caused by mutations in the TSC1 and/or TSC2 genes that leads to the formation of benign tumors in the brain known as cortical tubers. A subset of tubers are capable of inducing seizures; however, the molecular mechanism responsible for this process has yet to be fully elucidated. Recent evidence suggests exosomes may play a role. Exosomes are small extracellular vesicles (EV) that transport molecular cargo between cells. Recent studies have demonstrated the importance of exosomes in intercellular communication and disease pathology. Exosome cargo includes RNA, DNA, protein, and lipids, which can be selectively packaged into the vesicles by the source cell and provoke a functional response in recipient cells. The protein cargo in exosomes of human epileptogenic brain tissue has not been characterized. We used quantitative proteomics to compare EV cargo proteins in epileptogenic and non-epileptogenic TSC tubers.
Methods:
We isolated exosomes from archived, frozen cortical tissue that was surgically resected during the treatment of drug-resistant epilepsy in TSC patients. Specimens included four epileptogenic tubers (ET), three non-epileptogenic tubers (NT), and three non-tuber controls (NC). Protein cargo was extracted from vesicles and analyzed using quantitative LC-MS/MS proteomics. Statistical analysis with control of the false discovery rate was used to identify proteins differentially abundant among the groups. Differently expressed proteins were analyzed to identify those potentially involved in altered electrophysiology and seizures. This research was approved by the Wayne State University Human Subject Institutional Review Board.
Results:
Isolated vesicles were comprised primarily of exosomes (< 150 nm), and 1866 proteins were quantified. Principal component analysis demonstrates that exosomal protein cargo is distinct in ET compared to NT and NC controls. Statistical analysis identified 216 proteins differentially abundant in epileptogenic exosomes. Pathway analysis revealed enrichment for proteins involved in vesicle-mediated transport, including syntenin-1 and YKT6. Syntenin-1 was previously reported as increased in TSC neurons and caused a reduction in spine synapse density. We also identified elevated proteins in exosomes secreted from ET that were associated with alterations in electrophysiological properties and seizures.
Conclusions:
Epileptogenic tubers secrete exosomes with altered protein cargo. Differentially expressed proteins suggest increased vesicle-mediated transport. Elevated proteins are also associated with altered electrophysiology, synapse morphology, and seizures. Our results warrant further investigation of the role of the altered exosomal proteins in intercellular signaling and epilepsy.
Funding:
This research was supported by the TSC Alliance (A19-0151-001), National Institutes of Health (R56 NS079429), and the Department of Defense (W81XWH-22-1-0798).