Beyond the synapse: Exploring CSPα’s role in proteostasis, SNAP-25 stability, and cellular differentiation in PC12 cells

Abstract

The Cysteine String Protein alpha (CSPα) is a presynaptic co-chaperone that is essential for the maintenance of synapses and for presynaptic proteostasis. CSPα, via its interactions with the Heat shock cognate 70 (Hsc70) and the Small Glutamate Tetrapeptide repeat protein (SGT) acts to maintain the conformation of client proteins, including key regulators of exo- and endocytic reactions, SNAP-25 and Dynamin-1. CSPα is described as having “antineurodegenerative properties” and has been implicated in a number of neurodegenerative disorders, including Alzheimer’s, Parkinson’s, Huntington's, and Motor neuron disease. CSPα -/- mice have a reduced life span and associated neurodegeneration and progressive loss of synapses. The CSPα -/- neurons have increased levels of misfolded proteins and ubiquitination.
Increased levels of ubiquitination and ubiquitin-induced degradation of SNAP-25 have been detected in CSPα -/- mice. There must be an E3 ligase responsible for the ubiquitination and associated degradation; as such, we planned to identify E3 ligases that interact with SNAP25 and have the potential to lead to its increased degradation in the absence of CSPα. As such, the first aim was to identify E3 ligases by a bioinformatics-based approach and use two biochemical approaches: a BioID proximity-dependent labelling protocol and Myc pull-down to identify potential E3 ligase-SNAP-25 interacting proteins. This approach evidenced known interactions with SNARE proteins and also showed a new interaction with E3-ligase Parkin. This interaction with SNAP-25 was independent of Parkin’s E3 ligase activity, as an interaction between the Myc-BioID -SNAP-25 construct and both WT-HA-Parkin and the ligase-dead C431S-HA-Parkin was observed.


Secondly, CRISPR Cas was utilised to generate two independent CSPα-deficient PC12 cell lines, which were characterised with the aim of investigating SNAP-25-E3 ligase interactions in this context.
Before using these reagents, we observed that knockdown of CSPα led to hyperproliferation. This was associated with a loss of neurotrophin responsiveness and corresponding loss of differentiation ability and an alteration in the expression of Trk A and Trk B, the plasma membrane receptors for the neurotrophic factors NGF and BDNF. This unexpected consequence of CSPα was investigated using label-free whole-cell proteomics. This identified large-scale protein alterations between the parental and CSPα-deficient lines. The phenotypic and proteomic changes observed in the absence of CSPα provide insight into potential roles for CSPα beyond its neuronal chaperone function. They implicate an apparent critical role in cellular homeostasis that controls signalling and metabolic fluxes that regulate proliferation. My observation is consistent with an emerging nascent literature that describes clear potential for a role of this co-chaperone beyond the role of maintenance of proteostasis, supporting transmitter release in differentiated neurons.

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Identifiers

ORCID for Vincent O'Connor: orcid.org/0000-0003-3185-5709

ORCID for Katrin Deinhardt: orcid.org/0000-0002-6473-5298

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