Combining Comparative Proteomics and Molecular Genetics Uncovers Regulators of Synaptic and Axonal Stability and Degeneration In Vivo

Abstract

Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel “top-down” approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.

Molecular neuropathology of the synapse in sheep with CLN5 Batten disease

“…Wishart et al 2012; 2014). We adapted the subcellular fractionation protocol previously used for fresh mouse brain for frozen brain samples from the sheep.…”

“…2012). In these experiments, we compared levels of eight individual proteins previously shown to be present in mouse synapses, and whose expression levels were robustly modified in synapses undergoing degeneration: α ‐synuclein, calretinin (Calb2), 2′,3′‐cyclic nucleotide 3′ phosphodiesterase (CNP), cysteine‐string protein alpha (CSP‐ α ; DNAJC5), neurofascin, rho‐associated, coiled‐coil containing protein kinase 2 (ROCK2), sirtuin 2 (SIRT2), and ubiquitin protein ligase E3 component n‐recognin 4 (UBR4) (Wishart et al.…”

“…In these experiments, we compared levels of eight individual proteins previously shown to be present in mouse synapses, and whose expression levels were robustly modified in synapses undergoing degeneration: α ‐synuclein, calretinin (Calb2), 2′,3′‐cyclic nucleotide 3′ phosphodiesterase (CNP), cysteine‐string protein alpha (CSP‐ α ; DNAJC5), neurofascin, rho‐associated, coiled‐coil containing protein kinase 2 (ROCK2), sirtuin 2 (SIRT2), and ubiquitin protein ligase E3 component n‐recognin 4 (UBR4) (Wishart et al. 2012). Direct comparison of these proteins in synaptosome preparations from mice and sheep, using fluorescent Western blotting, confirmed that all eight proteins were conserved between the two species and were detectable using the same primary antibodies (Fig.…”

“…Moreover, the finding that molecular mechanisms underlying synaptic pathophysiology are conserved across several different neurodegenerative conditions (Wishart et al. 2012) suggests that studies examining mechanisms of synaptic pathology in one condition (such as the NCLs) may also have applicability to other related neurodegenerative conditions.…”

“…Similar links have been established for many of the other proteins we examined in our study, where previous data from a combined mouse/ Drosophila study identified them as potential regulators of synaptic and axonal degeneration (Wishart et al. 2012). Thus, many of the molecular regulators of synaptic pathophysiology identified in studies using lower organisms appear to be conserved in the sheep model of CLN5 Batten disease, establishing a clear path from small animal to large animal to human patients with neurodegenerative disease.…”