Mechanism and rescue of lysosomal defects in Adult-onset Neuronal Ceroid Lipofuscinosis

Adult-onset neuronal ceroid lipofuscinosis (ANCL) is a fatal lysosomal storage disorder known to be caused by autosomal dominant mutations (CSPαL115R, CSPαL116del) in the gene encoding cysteine string protein-alpha (CSPα). There are currently no treatments that can reverse or slow ANCL progression, which leads to progres- sive cognitive and motor impairment, epileptic seizures, dementia, and premature death. CSPα has no estab- lished connection to lysosomal function and is known to form a chaperone complex with SGT (small glutamine- rich tetratricopeptide repeat-containing protein) and Hsp70/Hsc70 (heat shock protein/cognate protein 70 kDa), which chaperones the synaptic SNARE protein SNAP-25. My preliminary data establishes SNAP-23, a homo- log of SNAP-25, as a previously unknown client of the CSPα/SGT/Hsc70 chaperone complex. This interaction was found via (i) immunoprecipitation of CSPα from wild type mouse brain followed by tandem mass spectrom- etry identification of SNAP-23, (ii) reduced SNAP-23 protein levels in CSPα knockout (CSPα-/-) mouse brains, and (iii) co-immunoprecipitation of SNAP-23 with each member of the CSPα/SGT/Hsc70 chaperone complex. Importantly, SNAP-23 forms a SNARE-complex with VAMP7 and syntaxin-4, the formation of which mediates Ca2+-dependent lysosomal exocytosis. Accordingly, I found diminished Ca2+-dependent lysosomal exocytosis in CSPα-/- primary neurons by measuring cell surface exposure of the LAMP-1 luminal domain. This preliminary data provides a novel, direct link between ANCL mutations in CSPα and the lysosomal pathology observed in ANCL, via impaired SNAP-23 function. The proposed study aims to (a) delineate the specific molecular mecha- nism(s) by which CSPα mutations impair SNAP-23 function, leading to the lysosomal pathology of ANCL, and (b) to evaluate the lysosomal SNARE-protein SNAP-23, a novel client of the CSPα/SGT/Hsc70 chaperone complex, as a potential therapeutic target for ANCL. I hypothesize that ANCL-causing mutations CSPαL115R and CSPαL116del reduce the ability of the CSPα/SGT/Hsc70 complex to effectively chaperone lysosomal SNARE protein SNAP-23, leading to defects in SNARE-complex assembly and lysosomal exocytosis which can be rescued by increasing SNAP-23 protein levels. In this proposal, aim 1 will examine how ANCL-causing mutations in CSPα impact the ability of the CSPα/SGT/Hsc70 complex to chaperone SNAP-23, and how loss of CSPα chaperone activity impacts SNAP-23 stability, lysosomal SNARE-complex assembly, and lysosomal exocytosis. Aim 2 will evaluate the potential of genetic and pharmacological strategies that in- crease SNAP-23 protein levels to rescue (a) SNARE-complex and lysosomal defects resulting in primary neu- rons from CSPα-/- mice and (b) premature lipofuscinosis and neurodegeneration in CSPα-/- mice. Successful completion of these studies will inform the development of rational therapeutic strategies for ANCL patients. Project Number: 1F31NS143430-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Samantha McDonough | Institution: WEILL MEDICAL COLL OF CORNELL UNIV, NEW YORK, NY | Award Amount: $49,538 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F03C-F (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11312883