, 1995, 2000) and are implicated in synaptic plasticity and memory (Bingol and Sheng,

compound screening assay 2011; Li et al., 2010; Lynch et al., 2007). Calpain inhibitors rescued PAIP2A degradation, while proteasome and caspase-3 inhibitors failed to do so (Figures 4B and 4C). Consistent with these results, strong tetanic stimulation (using TBS) in acute hippocampal slices led to a decrease (55.25% ± 5.99% of control levels) in PAIP2A levels in the CA1 region, which was blocked by the calpain inhibitor (Figure 4D). Weak stimulation (using 1HFS) had a smaller effect on PAIP2A levels (76.58% ± 4.73% of control levels). Thus, calpain-mediated PAIP2A proteolysis is induced by synaptic activity in situ. To determine whether PAIP2A levels also decrease in vivo in response to activity, mice were trained in the contextual fear-conditioning task and lysates from dorsal hippocampi were subjected to western blot analysis. Control mice received the equivalent foot shock without pairing to the context. PAIP2A was reduced to 84.64% ± 2.2% of basal level at ∼1.5 min after

the end of training (2.5 min after foot shock) and was significantly lower than in untrained mice (untrained: 98.15% ± 3.7%, p < 0.05; Figure 4E). PAIP2A returned to prestimulation selleck levels 20 min after training. To examine whether downregulation of PAIP2A in vivo is mediated by calpains, we infused a mixture of calpain inhibitors into the dorsal hippocampus of bilaterally cannulated rats before subjecting them to the contextual fear conditioning training. PAIP2A levels decreased in vehicle-treated rats to 80.3% ± ADAMTS5 5% relative to untrained animals but did not change when calpain inhibitors were added (Figure 4F), demonstrating that

PAIP2A degradation is mediated by calpain. In accordance with previous studies documenting a critical role for calpain in synaptic plasticity and memory formation (Lynch and Baudry, 1984; Shimizu et al., 2007), we also found that inhibiting calpain activity impaired long-term contextual fear memory without affecting short-term memory (Figure S5). To investigate calpain-mediated degradation in vitro, we treated immunoprecipitated PAIP2A from mouse brain with purified calpains I and II. PAIP2A levels decreased after 1 hr, showing that both calpain isoforms proteolyze PAIP2A (Figure 4G). Degradation of PAIP2A in brain lysates was calcium dependent (Figure 4H) and was prevented by calpain inhibitor but not by proteasome and caspase-3 inhibitors. To examine the role of calpain-mediated PAIP2A degradation in L-LTP, we tested the effect of calpain inhibition on L-LTP in WT and Paip2a−/− hippocampal slices. In accordance with previous reports ( del Cerro et al., 1990; Vanderklish et al.