005-22 – Investigating how ANCL-causing mutations lead to neurodegeneration

Cysteine string protein (CSP) encoded by DNAJC5 is a member of the DnaJ/Hsp40 family of co-chaperones, highly enriched in neurons. Three disease-causing mutations have been identified in CSP, all of which give rise to a rare neurodegenerative disease: adult-onset neuronal ceroid lipofuscinosis (ANCL). An accepted role for CSP is to maintain the correct conformation of neuronal presynaptic proteins involved in synaptic exo/endocytosis, notably SNAP-25. However, a second role for CSP has been proposed, whereby misfolded intracellular proteins such as α-synuclein are processed by CSP for secretion from cells, thus CSP may have a more general relevance for neurodegeneration. In this study, Caenorhabditis elegans were utilised to generate the first ANCL animal disease model to express ANCL-causing mutations in CSP as a single endogenous chromosomal copy, utilising CRISPR-cas9. Additionally, we investigated the protein interactome of CSP, utilising the proximity labelling tool, miniTurbo. We demonstrate that ANCL-causing mutations cause a significantly reduced lifespan, impaired chemotaxis, and age-dependent defects in locomotion. Interestingly, we demonstrate that these deficits are more severe in CSP null C. elegans, compared to ANCL mutant C. elegans, suggesting ANCL-causing mutations do not cause a complete loss of function of CSP. Additionally, miniTurbo proximity labelling revealed insights into wild-type CSP interactions, bypassing issues of CSP’s tendency to self-aggregate into stable oligomers in vitro. BioID revealed known interacting proteins such as Hsc70 and SNAP-25, but also revealed novel interactions. As some, but not all, of these interactions were absent in ANCL mutant CSP, this may help to explain how ANCL-causing mutations lead to neurodegeneration. These findings have provided insights into the impact of ANCL-causing mutations on CSP interactions, which will ultimately facilitate the identification of potential therapeutics for ANCL.