Synapses must be preserved throughout an organism's lifespan to allow for normal brain function and behavior. Synapse maintenance is challenging given the long distances between the termini and the cell body, reliance on axonal transport for delivery of newly synthesized presynaptic proteins, and high rates of synaptic vesicle exo- and endocytosis. Hence, synapses rely on efficient proteostasis mechanisms to preserve their structure and function. To this end, the synaptic compartment has specific chaperones to support its functions. Without proper synaptic chaperone activity, local proteostasis imbalances lead to neurotransmission deficits, dismantling of synapses, and neurodegeneration. In this review, we address the roles of four synaptic chaperones in the maintenance of the nerve terminal, as well as their genetic links to neurodegenerative disease. Three of these are Hsp40 co-chaperones (DNAJs): Cysteine String Protein alpha (CSPα; DNAJC5), auxilin (DNAJC6), and Receptor-Mediated Endocytosis 8 (RME-8; DNAJC13). These co-chaperones contain a conserved J domain through which they form a complex with heat shock cognate 70 (Hsc70), enhancing the chaperone's ATPase activity. CSPα is a synaptic vesicle protein known to chaperone the t-SNARE SNAP-25 and the endocytic GTPase dynamin-1, thereby regulating synaptic vesicle exocytosis and endocytosis. Auxilin binds assembled clathrin cages, and through its interactions with Hsc70 leads to the uncoating of clathrin-coated vesicles, a process necessary for the regeneration of synaptic vesicles. RME-8 is a co-chaperone on endosomes and may have a role in clathrin-coated vesicle endocytosis on this organelle. These three co-chaperones maintain client function by preserving folding and assembly to prevent client aggregation, but they do not break down aggregates that have already formed. The fourth synaptic chaperone we will discuss is Heat shock protein 110 (Hsp110), which interacts with Hsc70, DNAJAs, and DNAJBs to constitute a disaggregase. Hsp110-related disaggregase activity is present at the synapse and is known to protect against aggregation of proteins such as α-synuclein. Congruent with their importance in the nervous system, mutations of these co-chaperones lead to familial neurodegenerative disease. CSPα mutations cause adult neuronal ceroid lipofuscinosis, while auxilin mutations result in early-onset Parkinson's disease, demonstrating their significance in preservation of the nervous system.

Parkinson’s disease is characterized by the aggregation of the presynaptic protein α-synuclein and its deposition into pathologic Lewy bodies. While extensive research has been carried out on mediators of α-synuclein aggregation, molecular facilitators of α-synuclein disaggregation are still generally unknown. We investigated the role of molecular chaperones in both preventing and disaggregating α-synuclein oligomers and fibrils, with a focus on the mammalian disaggregase complex. Here, we show that overexpression of the chaperone Hsp110 is sufficient to reduce α-synuclein aggregation in a mammalian cell culture model. Additionally, we demonstrate that Hsp110 effectively mitigates α-synuclein pathology in vivo through the characterization of transgenic Hsp110 and double-transgenic α-synuclein/Hsp110 mouse models. Unbiased analysis of the synaptic proteome of these mice revealed that overexpression of Hsp110 can override the protein changes driven by the α-synuclein transgene. Furthermore, overexpression of Hsp110 is sufficient to prevent endogenous α-synuclein templating and spread following injection of aggregated α-synuclein seeds into brain, supporting a role for Hsp110 in the prevention and/or disaggregation of α-synuclein pathology.

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein–associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

Author contributions: ELG, HRZ, VC, and GSW conducted experiments. ELG and SSC designed experiments. ELG and JB performed data analysis. TTL conducted mass spectrometry. ELG and SSC wrote paper. All authors read and edited text. Highlights• 10% of palmitoylated proteins are palmitoyl protein thioesterase 1 (PPT1) substrates • Unbiased proteomic approaches identify 9 broad classes of substrates, including synaptic adhesion molecules and endocytic proteins • PPT1 depalmitoylates several transmembrane proteins in their extracellular domains • Depalmitoylation allows for disulfide bond formation in some PPT1 substrates • Protein degradation does not require depalmitoylation by PPT1 • Other palmitoylated Neuronal Ceroid Lipofuscinosis proteins are impacted by deficiency of PPT1, indicating a common disease pathway SUMMARYWe report here the identification of substrates of the depalmitoylating enzyme PPT1 by quantitative mass spectrometry. We first used a stringent Acyl Resin-Assisted Capture (Acyl RAC) screen in which PPT1 knockout (KO) mouse brain proteins showing increased in vivo palmitoylation are identified as putative PPT1 substrates. We then validated hits by directly depalmitoylating with PPT1 to confirm bona fide substrates. This novel twostep screen identified >100 substrates not previously known to be depalmitoylated by PPT1, including a wide range of channels/transporters, G-proteins, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Interestingly, many sites of depalmitoylation on transmembrane proteins were located in extracellular domains facing the synaptic cleft. For this group of substrates, depalmitoylation appears to facilitate disulfide bond formation. Collectively, these diverse substrates may explain the many facets of CLN1 disease caused by the loss of PPT1 function.

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