In this study we report on the effective use of a novel ‘top-down’ approach for identifying individual proteins and functional pathways responsible for regulating neurodegeneration in synaptic and axonal compartments of neurons. By undertaking a series of comparative quantitative proteomic screens on degenerating synapse-enriched fractions isolated from the mouse brain we identified 47 proteins with robustly modified expression levels during the early stages of neurodegeneration. We showed that molecular responses to degeneration occurring in synapse-enriched fractions following injury were recapitulated in synapse-enriched fractions undergoing pathological changes as a result of disease-causing genetic mutations. We also used our proteomic data to design molecular genetic screens in Drosophila that revealed roles for 6 proteins in regulating synaptic and axonal degeneration in vivo. These findings further our understanding of mechanisms regulating the active degeneration of synapses and axons, providing a basis from which to develop novel neuroprotective strategies for a range of neurodegenerative conditions.
An initial comparison of the 6 individual proteins found to directly mediate synaptic and axonal stability and degeneration in our Drosophila
screen reveals a diverse range of biological functions. For example, both DNAJC5/CSP and DNAJC6 belong to the evolutionarily conserved DNAJ/HSP40 family of proteins that regulate molecular chaperone activity by stimulating ATPase activity 
, whereas CALB2/calretinin is an intracellular calcium-binding protein 
and ROCK2 is a Rho kinase belonging to a family of serine/threonine kinases involved in structural remodeling of the cytoskeleton 
. Despite this apparent heterogeneity, it should be noted that the in silico
analysis of data generated by our proteomics experiments highlighted significant clustering of proteins within functional networks that regulate synaptic transmission. This finding is further reinforced by comparisons of the biological roles of the 6 proteins found to independently regulate degeneration in our Drosophila
screen, 5 of which have been implicated in the control of synaptic function: both CALB2/Calretinin and ALDHA1 modulate synaptic long-term potentiation (LTP) 
, DNAJC6 has been implicated in clatherin-mediated synaptic vesicle recycling 
, DNAJC5/CSP plays a role in SNARE-complex assembly 
, and ROCK2 levels influence synaptic transmission and plasticity 
. Taken together with previous reports linking perturbations in synaptic transmission with synaptic degeneration 
(also see below), our findings suggest that endogenous neuronal proteins and pathways regulating synaptic function play an important role in modulating neurodegenerative pathways.
It is worth noting, however, that at least one of the other proteins found to influence degeneration in our Drosophila
screen (HIBCH; 3-hydroxyisobutyryl-CoA hydrolase) is unlikely to impact directly on synaptic transmission pathways. HIBCH plays an important role in valine catabolism, disruption of which is sufficient to induce progressive infantile neurodegeneration in humans 
. Thus, multiple cellular and molecular pathways are likely to converge on mechanisms regulating synaptic and axonal degeneration. This finding is supported by our in silico
analysis revealing that several of the proteins identified in our screen also contribute to pathways regulating neurite development. This supports previous observations from Drosophila
models linking ubiquitin-mediated developmental processes with neurodegenerative processes occurring in axonal compartments of neurons 
. Thus, although proteins and pathways involved in synaptic transmission are likely to contribute significantly to neurodegeneration, other distinct molecular pathways also appear to be capable of influencing synaptic and axonal degeneration in vivo
Only one of proteins we identified as a direct mediator of degeneration, DNAJC5/CSP, belongs to the small group of endogenous genes and proteins previously reported to directly affect synaptic stability and degeneration in vivo
. DNAJC5/CSP has been implicated in synaptic degeneration contributing to the pathogenesis of neurodegenerative diseases 
. However, our findings are partially inconsistent with previously published studies examining the role of DNAJC5/CSP in animal models. For example, Fernández-Chacón and colleagues reported that loss of CSP expression in mice caused synaptic degeneration in the CNS, leading them to conclude that increased levels of the protein may be neuroprotective 
. By contrast, we found that DNAJC5/CSP levels are robustly and consistently increased
in degenerating synapse-enriched fractions following injury and in synapse-enriched fractions from mouse models of neurodegenerative disease. Moreover, a thorough genetic analysis in Drosophila
using well-defined mutants in DNAJC5/CSP revealed that loss of CSP is neuroprotective, delaying degeneration in axonal and synaptic compartments. Thus, whilst it is clear that DNAJC5/CSP needs to be regarded as a critical regulator of-neuronal stability and degeneration in vivo
, precise details correlating expression levels with its role in stabilizing distal axons and synapses during disease-induced degeneration remain to be determined.
Given that only partial coverage of the entire synaptic proteome is possible through the coupling of subcellular fractionation with current proteomics technologies, alongside the stringent 20% cut off threshold employed, the refinement methodologies applied in the current study and the limited number of viable fly lines that we screened, it is highly likely that additional genes and proteins capable of regulating neurodegeneration remain to be discovered. Our uncovering of molecular responses underlying neurodegeneration in distal compartments of neurons, alongside the identification of 5 novel mediators of degeneration and new experimental insights into the role of DNAJC5/CSP, suggests that combining proteomic screens on synapse-enriched fractions with axonal/synaptic degeneration assays in Drosophila provides a powerful approach for elucidating mechanisms of neurodegeneration in vivo.