The Novel Caspase-3 Substrate Gap43 is Involved in AMPA Receptor Endocytosis and Long-Term Depression

The cysteine protease caspase-3, best known as an executioner of cell death in apoptosis, also plays a non-apoptotic role in N-methyl-d-aspartate receptor-dependent long-term depression of synaptic transmission (NMDAR-LTD) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor endocytosis in neurons. The mechanism by which caspase-3 regulates LTD and AMPA receptor endocytosis, however, remains unclear. Here, we addressed this question by using an enzymatic N-terminal peptide enrichment method and mass spectrometry to identify caspase-3 substrates in neurons. Of the many candidates revealed by this proteomic study, we have confirmed BASP1, Dbn1, and Gap43 as true caspase-3 substrates. Moreover, in hippocampal neurons, Gap43 mutants deficient in caspase-3 cleavage inhibit AMPA receptor endocytosis and LTD. We further demonstrated that Gap43, a protein well-known for its functions in axons, is also localized at postsynaptic sites. Our study has identified Gap43 as a key caspase-3 substrate involved in LTD and AMPA receptor endocytosis, uncovered a novel postsynaptic function for Gap43 and provided new insights into how long-term synaptic depression is induced.Synaptic plasticity (the ability of synapses to change in strength) plays an important role in brain development and cognitive function, including learning and memory. N-methyl-d-aspartate receptor (NMDAR)¹-dependent long-term depression of synaptic transmission (LTD) is a major form of synaptic plasticity that leads to long-lasting decreases in synaptic strength. In NMDAR-LTD, synaptic depression is mainly mediated by removal of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors from the postsynaptic membrane through endocytosis (1–3).Caspases are cysteine-dependent proteases that cleave after an aspartate residue (4–6). Primary specificity for aspartate at the cleavage site is so rare in mammalian proteases that only granzyme B, a serine protease derived from lymphocytes, is known to also have such a property (7, 8). Caspases are best known for their pro-apoptotic function in programmed cell death, or apoptosis (9). Caspases activated at the end of a caspase cascade are “effector” caspases, and among them caspase-3 is the dominant one (10). In addition to apoptosis, caspases also play non-apoptotic roles such as in cell differentiation, dendritic development, and memory consolidation (11–14). For instance, our earlier studies show that NMDAR-LTD requires moderate and transient caspase-3 activation, which does not induce cell death (15, 16).In LTD, caspase-3 is activated by the mitochondrial pathway (15, 16). With the opening of NMDA receptors, calcineurin and protein phosphatase 1 are activated to dephosphorylate the Bcl-2 family protein BAD. Dephosphorylated BAD then translocates to the mitochondria, activating BAX, which is also a member of the Bcl-2 family protein. The subsequent release of cytochrome-c from mitochondria leads to the sequential activation of caspase-9 and caspase-3. Active caspase-3 induces AMPA receptor endocytosis, and therefore depression of synaptic strength. The mechanism by which caspase-3 promotes AMPA receptor endocytosis, however, remains to be determined.Caspases'' cellular functions are primarily mediated by the proteolysis of caspase substrates, resulting in change of their functions. Identification of specific proteins cleaved by caspases is therefore key to understanding the mechanisms mediating their biological functions. Several proteomic approaches, developed specifically for this purpose, have led to the identification of more than 2000 caspase cleavage sites (17–22). In particular, the recently developed subtiligase-based method for isolating proteolytic products has led to the identification of >1,000 putative caspase substrates in human samples (21, 23, 24). Subtiligase is an engineered peptide ligase that ligates esterified peptides onto the N termini of proteins or peptides through free α-amines (25). Because the majority of eukaryotic proteins are N-terminally acetylated—and therefore blocked from subtiligase labeling (26)—subtiligase can couple synthetic tagged peptides selectively to the free N-terminal α-amines of proteins derived from proteolysis. These peptide-conjugated proteolytic products can then be affinity-purified, digested with trypsin and sequenced by mass spectrometry. Identification of peptides ligated to the tagged peptide by subtiligase allows researchers to determine cleavage sites within the substrates.In this study, the subtiligase-based proteomic method was used to find capase-3 substrates in rat neurons, resulting in the identification of 81 putative aspartate cleavage sites in 56 proteins. Of these, 37 proteins (human and a single rat orthologs) were not previously reported in the CASBAH database (20), and 13 (human orthologs) are not included in the DegraBase data set compiled using the subtiligase methodology in non-neuronal tissue (21). Using complimentary methods, we further confirmed that, both in vivo and in vitro, caspase-3 cleaves three of these candidate substrates: growth associated protein 43 (Gap43), drebrin (Dbn1), and brain acid soluble protein 1 (BASP1). Surprisingly, we also found that AMPA receptor endocytosis and LTD induction both require caspase-3 to cleave Gap43, a protein well known for its presynaptic functions, at the sites identified by our study.