Disease causing mutations in cysteine string protein-alpha disrupt SNARE-dependent lysosomal exocytosis

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PROJECT SUMMARY/ABSTRACT Adult onset neuronal ceroid lipofuscinosis (ANCL) is a fatal lysosomal storage disease caused by two known dominant mutations in the gene encoding cysteine string protein-α (CSPα): CSPαL115R and CSPαL116Δ. CSPα forms a chaperone complex with SGT (small glutamine-rich tetratricopeptide repeat-containing protein) and Hsp70/Hsc70 (heat shock protein/cognate 70 kDa) to chaperone the synaptic SNARE protein SNAP-25. It is surprising that mutations in CSPα lead to lysosomal pathology because its role has only been clarified in the context of synaptic function. I have recently found that SNAP-23, a homolog of SNAP-25, is also a 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 spectrometry identification of SNAP-23, (ii) reduced protein levels of SNAP-23 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 mediates Ca2+-dependent fusion of lysosomes with the plasma membrane by forming a SNARE-complex with VAMP-7 and syntaxin-4. In support of this function, I have identified diminished Ca2+-dependent lysosomal exocytosis in CSPα-/- primary neurons by measuring cell surface exposure of the LAMP-1 luminal domain following intracellular Ca2+ induction with ionomycin. Altogether, these preliminary data draw a new and direct connection between CSPα dysfunction and lysosomal pathology in ANCL by means of impaired SNAP-23 function. Key gaps remain in our understanding of how mutations in CSPα cause the pathological cascade of ANCL: a) how ANCL mutations in CSPα affect chaperoning of SNAP-23, and b) how SNAP-23 dysfunction leads to lysosomal pathology with lipofuscin accumulation. My hypothesis is that ANCL mutations in CSPα prevent the CSPα/SGT/Hsc70 complex from chaperoning the lysosomal SNARE protein SNAP-23, disrupting lysosomal exocytosis and leading to lipofuscin accumulation. This hypothesis will be addressed using a multi-disciplinary approach including primary cortical neurons from CSPα-/- mice, biochemical assays and lentiviral rescue experiments. Experiments will be carried out by means of two proposed specific aims: Aim 1 will clarify how ANCL mutations affect CSPα’s chaperoning of SNAP-23: Aim 2 will elucidate how ANCL mutations in CSPα affect lysosomal exocytosis, leading to lipofuscinosis. Completion of these aims will lead to a detailed understanding of the pathological cascade of ANCL, opening future avenues for testing therapeutics strategies.