Abstract Details

Title A Gene Linked to Transverse Myelitis Is Associated with Increased Entry of AQP4 into Exosomes

Topic Autoimmune Neurology

Presentation(s) P7 - Poster Session 7 (5:00 PM-6:00 PM)

Poster/Presentation
Number 8-006

Objective We investigated changes in exosome export, morphology and cargo associated with alterations in VPS37A expression to assess if this protein may lead to changes in exosome function.

Background

Transverse myelitis (TM) is a demyelinating disorder frequently of unknown etiology. Previous work in the Levy lab discovered a unique mutation in VPS37A in a pair of siblings with TM. VPS37A is a protein component of the ESCRT pathway, important for exosome formation.

Exosomes are a subcategory of extracellular vesicles, and have been shown as carriers of antigens in the periphery, and notably have AQP4 has been detected in exosomes isolated from the blood of patients. Therefore, exosomes and their biogenesis have been increasingly investigated given their implications as biomarkers and involvement in immune responses.

Design/Methods HEK293T cells or AQP4 (aquaporin-4)-GFP HEK 293T cells were transfected with VPS37A shRNA, scramble shRNA (vehicle), VPS37A plasmid, VPS37AL234I (associated with TM) plasmid, or VPS37AK382N (associated with HSP70). Cell lysates and supernatants were collected at d5. Filtered supernants were imaged with NanoSight for exosome quantification. Protein was isolated from exosomes and cell lysates with lysis buffer and run on western blots with appropriate staining for exosome markers.

Results

Overexpression of VPS37AL234I (associated with TM) plasmid and VPS37AK382N (associated with HSP70) in HEK293T cells was associated with up to 78.7% and 93.5% increased exosome production respectively as compared to vehicle transfected cells. We additionally show evidence of increases in AQP4 protein in exosomes isolated from a AQP4-GFP HEK 293T cells transfected with either VPS37A mutation.

Conclusions VPS37A mutations and knockdown alter the number of exosomes exported in culture. Importantly, VPS37AL234I (associated with TM), was associated with increased AQP4 cargo in exported exosomes, suggesting that this mutation may increase the number of exosomes exported and may be associated with an increased antigenic load. This may have downstream consequences to immune activation.

Authors/Disclosures
Monique Anderson, MD, PhD (Mass General Hospital) PRESENTER	Dr. Anderson has nothing to disclose.
Takahisa Mikami, MD (Massachusetts General Hospital)	Dr. Mikami has nothing to disclose.
Natalia Drosu	Ms. Drosu has nothing to disclose.
Philippe-Antoine Bilodeau, MD (Massachusetts General Hospital)	Dr. Bilodeau has nothing to disclose.
Natasha Bobrowski-Khoury, PhD (Mass General Hospital/Harvard Medical School)	Dr. Bobrowski-Khoury has nothing to disclose.
Shuhei Nishiyama, MD, PhD (Tohoku University Graduate school of Medicine)	Dr. Nishiyama has nothing to disclose.
Michael Levy, MD, PhD, FAAN (Massachusetts General Hospital/Harvard Medical School)	Dr. Levy has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Mitsubishi Pharma. Dr. Levy has received personal compensation in the range of $500-$4,999 for serving as a Consultant for UCB Pharma. Dr. Levy has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Sanofi. Dr. Levy has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Alexion. Dr. Levy has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Horizon. Dr. Levy has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Genentech. Dr. Levy has received personal compensation in the range of $500-$4,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Elsevier. Dr. Levy has received personal compensation in the range of $10,000-$49,999 for serving as an Expert Witness for Various law firms. The institution of Dr. Levy has received research support from National Institutes Health.

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