A Drosophila Model of Autosomal Dominant Adult-Onset Neuronal Ceroid Lipofuscinosis (ANCL/CLN4) Links Toxicity to CSP Activity

Abstract

Autosomal dominant adult onset neuronal ceroid lipofuscinoses (ANCL/CLN4) is a rare neurodegenerative disorder caused by mutations in the human gene DNAJC5 which encodes cysteine string protein alpha (CSPα). ANCL is characterized by the appearance of aberrant lysosomal storage material in the post-mortem brains of patients, who usually die from widespread neuronal loss within 10 years from the onset of symptoms. CSPα is a neuroprotective co-chaperone specifically localized to synaptic vesicles (SVs) and is evolutionarily conserved in all animals. CSPα forms a chaperone complex with HSC70 to properly fold a limited number of synaptic proteins. Complete loss of CSP leads to neurodegeneration and reduced lifespans in flies and mice. However, the mechanism of degeneration induced by ANCL mutations is currently unknown and there are no available animal models to study the dysfunctional proteins in situ. In this thesis, I describe the generation and subsequent characterization of the first animal model of ANCL, using the fruit fly Drosophila melanogaster. First, I show that human CSPα (hCSPα) is conserved functionally from humans to flies. Wildtype hCSPα expressed in flies localizes properly to SVs and is able to rescue lifespan defects in CSP null mutant flies. Overexpression of hCSPα proteins with the ANCL causing L115R and L116Δ mutations recapitulates numerous phenotypes consistent with human disease pathology. This includes the appearance of high molecular weight (HMW) SDS-resistant aggregates on western blots, accumulation of aberrant osmophilic membrane structures observed via electron microscopy, and a dose-dependent reduction in adult viability. Mutant hCSPα is mislocalized from SVs to enlarged abnormal endosomes, which accumulate in neuronal axons and somata. These endosomes strongly co-localize with the endosomal sorting required for transport (ESCRT) complex protein HRS, contain large amounts of ubiquitinated proteins, and lack markers of lysosomal maturation. This suggests that the ANCL causing mutations may cause disruptions in endo-lysosomal trafficking via an ESCRT related mechanism. To probe the genetic nature of the mutant alleles I expressed the mutant hCSPα transgenes with various doses of endogenous Drosophila CSP (dCSP). I show that loss of dCSP suppresses toxicity, as well as the aberrant endosomal accumulations and HMW aggregates induced by overexpression of mutant hCSPα. Additionally, expression of a combination of the wildtype and mutant hCSPα showed a super-additive effect on viability and HMW aggregates. This suggests that the disease-causing mutations may act as hypermorphic gain of function alleles, contrary to existing models, which suggest a dominant-negative mechanism. I also performed an F1 candidate screen for genetic modifiers of toxicity, using a robust and easy-to-score adult eye morphology and pigmentation phenotype. Using this approach, I discovered several strong interactors, both enhancers and suppressors, including member of the ESCRT trafficking pathway and other known CSP-interacting proteins. Of particular interest was the CSP co-chaperone Hsc70, which had several loss of function alleles among the strongest observed suppressors. Loss of Hsc70 also greatly reduces toxicity and endosomal accumulations of overexpressed mutant hCSPα but interestingly does not have a significant effect on the levels of HMW CSPα aggregates. This further supports the model that ANCL mutations act as hypermorphs, with a toxic mechanism involving CSP’s endogenous interactions with HSC70. Finally, I discuss the implications of these findings in relation to previous studies of the ANCL causing mutations and endogenous CSPα/HSC70 function and propose a novel mechanistic disease model. This model postulates that mutant CSP is properly trafficked to synapses but, after a brief lifespan as a properly functioning HSC70 co-chaperone, is then ubiquitinated and localized onto endosomes. Ubiquitinated mutant CSP is then clustered by HRS but is unable to mature properly through an ESCRT dependent degradation pathway. These endosomes are retrogradely trafficked through the axon to the soma where they fuse, accumulate, and persist, eventually leading to cellular toxicity via an unknown mechanism. The hypermorphic nature of the mutants can be explained by the novel observation that normal endogenous CSP also traffics through a retrograde ESCRT dependent pathway, where it intersects and co-accumulates with mutant CSP, potentially contributing to toxicity.