Braun, J. E. A. | Search bioRxiv

bioRxiv2023

Cysteine String Protein alpha in Extracellular Vesicle Subtypes: a Proteomic Analysis

Cysteine string protein (CSP /DnaJC5) is a presynaptic J-domain protein (JDP) that prevents neurodegeneration. CSP/DnaJC5 is reported to facilitate export of distinct, highly oligomeric, disease-causing proteins in addition to wild-type TDP-43, tau and -synuclein. Yet, detailed mechanistic knowledge of the full CSP/DnaJC5 secreted proteome is lacking. Understanding the CSP/DnaJC5 export pathway has implications for a growing number of neurodegenerative diseases. In humans, Leu115Arg or Leu116deletion mutations cause adult-onset neuronal ceroid lipofusinosis (ANCL), a rare neurodegenerative disorder. In the present study, we examined extracelular vesicles (EVs) released from CSP/DnaJC5 expressing cells. Cells are known to secrete many types of EVs of different sizes and origins into the extracellular space. EV subpopulations were separated by their sedimentation speed and subjected to proteomic analysis. We find that CSP/DnaJC5 and the CSP/DnaJC5 mutants, Leu115Arg or Leu116del are enriched in multiple EV subpopulations. The exported protein profile is determined by proteomics. We report that several other J-domain proteins (JDPs), such as DnaJC7, DnaJA1 and DnaJA2 are exported and speculate that export of JDPs may facilitate the secretion of diverse client proteins. Our work provides a platform for further inquiry into the role of secreted CSP/DnaJC5 and other JDPs in proteostasis.

cell biology↗

bioRxiv2018

Cargo-Loading of Misfolded Proteins into Extracellular Vesicles: The Role of J Proteins

Extracellular vesicles (EVs) are secreted vesicles of diverse size and cargo that are implicated in the cell-to-cell transmission of disease-causing-proteins in several neurodegenerative diseases. Mutant huntingtin, the disease-causing entity in Huntingtons disease, has an expanded polyglutamine track at the N terminus that causes the protein to misfold and form toxic intracellular aggregates. In Huntingtons disease, mutant huntingtin aggregates are transferred between cells by an unknown route. We have previously identified a cellular pathway that is responsible for the export of mutant huntingtin via extracellular vesicles, given the heterogeneity of EVs, here we examine the specific EVs involved. In this work we expressed a form of polyglutamine expanded huntingtin (GFP-tagged 72Qhuntingtinexon1) in cells to assess the EVs involved in cellular export. We demonstrate that the molecular chaperone, cysteine string protein (CSP; DnaJC5), mediates export of disease-causing-polyglutamine-expanded huntingtin cargo via two distinct vesicle populations of 180-240nm and 15-30m. In doing so, our data links the molecular chaperone, CSP, and the packaging of pathogenic misfolded huntingtin into two separate extracellular vesicles pathways.

neuroscience↗