Investigating the role of CSPalpha in presynaptic autophagy

SUMMARY
We have characterized Cysteine String Protein α (CSPα), a resident synaptic vesicle (SV) co-chaperone whose
activity is essential for presynaptic proteostasis. CSPα chaperones key presynaptic proteins and deletion of
CSPα triggers the loss of SVs, synapse loss, neurodegeneration, and early lethality. Significantly, mutations in
CSPα causes cause a neurodegenerative disease with presenile dementia (CLN4) and implicated in ADRDs.
We discovered that CSPα knockout (KO) brains exhibit increased number of presynaptic autophagosomes that
contain SVs as cargo, suggesting that CSPα regulates SV turnover through autophagy. Our single-nucleus RNA-
seq on wildtype (WT) and CSPα KO cortex also show elevated autophagy gene expression in neurons.
Therefore, we aim to investigate the links between SV degradation, autophagy, and neurodegeneration. Based
on these compelling data, we will test the hypothesis that CSPα is important for maintaining the composition of
SVs and deletion or CLN4 mutations of CSPα result in SV turnover through autophagy. The objective of this
application is to elucidate the mechanisms through which CSPα regulates synaptic autophagy and
neurodegeneration. We will use electron microscopy (EM), quantitative mass spectrometry, live imaging Keima
assays, western blotting, and immunocytochemistry to test our hypothesis using WT, CSPα KO, CLN4 models.
In Aim 1, we will characterize SVs, SVs targeted for autophagy, and synaptic autophagosomes purified from WT
and CSPα KO brains by mass spectrometry and EM. In Aim 2, we will investigate the relationship between the
SV cycle and autophagosome biogenesis by tagging SV proteins with the Keima autophagy reporter in WT,
CSPα KO, and CLN4 cultures. We will also perform EM to monitor distribution of autophagosomes in CLN4
cultures. We expect that realization of this proposal will have major impacts on our knowledge of presynaptic
autophagy, SV turnover, and CLN4. Achievement of our aims is likely to advance therapeutic interventions for
CLN4 and ADRDs, given the genetic and functional links of CSPα to these neurodegenerative diseases.

Grant Number: 1R21AG093151-01
NIH Institute/Center: NIH
Principal Investigator: Sreeganga Chandra