, 2010). These results strongly suggest that with repeated firing and multiple rounds of the synaptic vesicle cycle, CSPα KO synapses likely accrue incorrect conformations of CSPα clients, eventually leading to synaptic dysfunction and loss. Recent biochemical analyses of CSPα KO mice showed that the t-SNARE SNAP-25 is a protein

substrate or client of the Hsc70-CSPα chaperone complex and that deletion of CSPα leads to a 50% decrease in SNAP-25 levels (Chandra et al., 2005 and Sharma et al., 2011). Nonetheless, SNAP-25 heterozygous mice, which also have a similar decrease in SNAP-25 levels and function, are phenotypically normal (Washbourne et al., 2002), suggesting that other unknown client proteins contribute to the CSPα KO phenotypes. Identification of these clients is critical to understanding CSPα-dependent mechanisms Dolutegravir of synapse maintenance. The decrease of SNAP-25 levels in CSPα KO brains suggests that misfolded clients are degraded

and that additional clients can be screened for on the basis of lowered synaptic protein amounts in CSPα KO brains. It should be noted that several proteins that Pifithrin-�� bind CSPα have been identified in different model systems, including the SNARE syntaxin, Gαs, rab3b, and synaptotagmin 9 (Boal et al., 2011, Magga et al., 2000, Natochin et al., 2005, Nie et al., 1999 and Sakisaka et al., 2002), but none of these proteins have been unambiguously demonstrated to be clients of the Hsc70-CSPα chaperone complex. In this study,

we use unbiased, systematic proteomics to identify CSPα client proteins and show that SNAP-25 and the endocytic GTPase dynamin 1 are key clients of the Hsc70-CSPα chaperone complex. We additionally demonstrate that CSPα promotes the self-assembly of dynamin 1, thereby regulating synaptic vesicle endocytosis. Finally, we show that the levels of CSPα chaperone complex are decreased in AD brains. Our results reveal that CSPα participates in an essential presynaptic quality control mechanism that allows for the activity-dependent maintenance Fossariinae of synapses. Chaperones are critical for protein homeostasis; they help refold nonnative proteins and allow for conformational switches of folded proteins (Fujimoto and Nakai, 2010 and Voisine et al., 2010). In their absence, misfolded proteins are either targeted for degradation or form aggregates, leading to a decrease in native protein amounts. We therefore hypothesized that the levels of CSPα clients should be reduced in CSPα KO brains. To identify the repertoire of CSPα clients in the presynaptic terminal, we performed an unbiased quantitative comparison of the synaptic proteomes of wild-type and CSPα KO brains. We employed two proteomic methods: DIGE (2D fluorescence Difference Gel Electrophoresis) and iTRAQ (Isobaric Tag for Relative and Absolute Quantitation).