Proteomic identification of synaptic caspase substrates

Abstract

The dismantling and elimination of excess neurons and their connections (pruning) is essential for brain development and may be aberrantly reactivated in some neurodegenerative diseases. Growing evidence implicates caspase-mediated apoptotic and nonapoptotic cascades in the dysfunction and death of neurons in neurodegenerative disorders such as Alzheimer's, Parkinson, and Huntington's diseases. It is the cleaved caspase substrates that are the effectors of synapse elimination. However, their identities, specific cleavage sites, and functional consequences of cleavage are largely unknown. An important gap in our knowledge is a comprehensive catalog of synapsespecific or synapse-enriched caspase targets. Traditional biochemical approaches have revealed only a small number of neuronal caspase targets. Instead, we utilized a gel-based proteomics approach to enable the first global analysis of caspase-mediated cleavage events in mammalian brain synapses, employing both an in vitro system with recombinant activated caspases and an in vivo model of ethanol-induced neuronal apoptosis. Of the more than 70 putative cleavage substrates that were identified, 22 were previously known caspase substrates. Among the novel targets identified and validated by Western blot were the proton pump ATPase subunit ATP6V1B2 and the N-ethylmaleimide-sensitive fusion protein (NSF). Our work represents the first comprehensive, proteome-wide screen for proteolytic targets of caspases in neuronal synapses.Our discoveries will have significance for both furthering basic understanding of roles of caspases in synaptic plasticity and synaptic loss in neurodegeneration, and on a more immediately practical level, may provide candidate biomarkers for measuring synapse loss in human disease states. K E Y W O R D Sapoptosis, caspase, cleavage, mass spectrometry, presynaptic, proteolytic, synaptic | I N TR ODU C TI ONCaspases (cysteine-dependent aspartate-directed proteases) represent a family of intracellular proteases that have a conserved cysteine residue in their active site and display a preference for substrates with an aspartic residue in the P1 position. These proteolytic enzymes and their substrates have traditionally been viewed as playing an essential role in apoptosis, or programmed cell death, a process fundamental to all multicellular organisms and critical to development, homeostasis, and disease. Caspases cleave a large number of specific target proteins, not only for the explicit purpose of destruction, but also to induce new functional activities that orchestrate the orderly transformation of intact cells into fragments that may be removed from the body by phagocytic cells. With respect to brain function and development, apoptotic neuronal death has received considerable attention. For example, following an ischemic stroke event, accounting for 85% of all cases, caspase-based apoptosis persists for days to weeks, per-

GPRIN1 modulates neuronal signal transduction and affects mouse-learning behavior

“…Interestingly our interactome analysis showed that endogenous GPRIN1 specifically interacts with caspase-3 in primary neurons ( Fig. S3G ) as previously reported at synaptic level (Victor et al, 2018). In line with these findings we showed that GPRIN1 interacted with caspase3 in SHSY-5S neuroblastoma cell lines and AMPA stimulation likely increased this interaction ( Fig.…”

“…In particular, a specific function has been proposed for GPRIN1 as a scaffold protein capable of driving the interaction between α4 nAChRs and Gαi in T cells (Nordman et al, 2014) and, recently, of mediating the anti-inflammatory properties of α7 nAChRs/Gαq signaling in microglia cells (King et al, 2017). Finally a recent proteomic study identified GPRIN1 as a novel synaptic substrate for caspase 3-dependent proteolytic cleavage, the major early event in synapse loss and neurodegeneration (Victor et al, 2018). Considering our findings showed increased level of activated caspase 3 in neurons lacking GPRIN1, further investigation needs to be done to determine whether in healthy neurons GPRIN1 inhibit caspase 3 activity in neurons by sequestering it and whether GPRIN1 might be itself a proteolytic substrate of caspase 3.…”

Caspase-3 Cleaves Extracellular Vesicle Proteins During Auditory Brainstem Development

“…Official gene symbols of protein sets were submitted to the online tool Draw Venn Diagram (Bioinformatics and Evolutionary Genomics, Ghent University 1 ) to obtain the numbers of unique proteins found in each set and their overlaps. This site was used to generate Figure 3 comparing ABC3 substrates, the brainstem proteome, and previously identified caspase-3 substrates (Lüthi and Martin, 2007;Rawlings et al, 2010;Crawford et al, 2013;Victor et al, 2018). For all other Venn diagrams, the numbers in set overlap from Draw Venn Diagram were used to create Venn diagrams with proportionally accurate areas using the Euler package on R.…”

“…Auditory Brainstem Caspase-3 Activity Cleaves Both Novel and Known Substrates, Especially Known Substrates Observed in Non-apoptotic Contexts Next, we investigated whether our ABC3 substrates have been previously reported as caspase-3 substrates and whether they are associated with apoptotic or non-apoptotic caspase activity. We consulted four sources of known caspase-3 substrates: MEROPS (Rawlings et al, 2010), CASBAH (Lüthi and Martin, 2007), DegraBase (Crawford et al, 2013), and Victor et al, 2018. We used a hypergeometric test to calculate the probability of obtaining the observed number of proteins that were caspase-3 substrates both in the above databases and in our ABC3 dataset, assuming each is randomly selected from a brainstem proteome of 5,653 proteins containing 288 caspase-3 substrates. Nearly one third (91) of our 288 ABC3 substrates were present in at least one of the four datasets (p = 2.45 × 10 −10 ), suggesting that caspase-3 cleaves many of the same proteins in the developing auditory brainstem as in other conditions (Figure 3, Table 2, Supplementary Data 2).…”

“…Non-apoptotic caspase functions, in contrast, are often more transient and difficult to verify conclusively. Some studies have explored caspase activity in cellular compartments relevant to neural development, such as synaptosomes (Victor et al, 2018), but only a handful of substrates have been demonstrated to be important for non-apoptotic neurodevelopmental functions in living systems (Kellermeyer et al, 2018). An unbiased investigation of non-apoptotic neurodevelopmental caspase substrates that approximates the depth and breadth of screens for apoptotic substrates has never been accomplished.…”