A calpain-2 selective inhibitor enhances learning & memory by prolonging ERK activation

Abstract

While calpain-1 activation is required for LTP induction by theta burst stimulation (TBS), calpain-2 activation limits its magnitude during the consolidation period. A selective calpain-2 inhibitor applied either before or shortly after TBS enhanced the degree of potentiation. In the present study, we tested whether the selective calpain-2 inhibitor, Z-Leu-Abu-CONH-CH2-C6H3 (3, 5-(OMe)2 (C2I), could enhance learning and memory in wild-type (WT) and calpain-1 knock-out (C1KO) mice. We first showed that C2I could reestablish TBS-LTP in hippocampal slices from C1KO mice, and this effect was blocked by PD98059, an inhibitor of ERK. TBS resulted in PTEN degradation in hippocampal slices from both WT and C1KO mice, and C2I treatment blocked this effect in both mouse genotypes. Systemic injection of C2I 30 min before training in the fear-conditioning paradigm resulted in a biphasic dose-response curve, with low doses enhancing and high doses inhibiting freezing behavior. The difference between the doses needed to enhance and inhibit learning matches the difference in concentrations producing inhibition of calpain-2 and calpain-1. A low dose of C2I also restored normal learning in a novel object recognition task in C1KO mice. Levels of SCOP, a ERK phosphatase known to be cleaved by calpain-1, were decreased in dorsal hippocampus early but not late following training in WT mice; C2I treatment did not affect the early decrease in SCOP levels but prevented its recovery at the later time-point and prolonged ERK activation. The results indicate that calpain-2 activation limits the extent of learning, an effect possibly due to temporal limitation of ERK activation, as a result of SCOP synthesis induced by calpain-2-mediated PTEN degradation.

Keywords: Calpain-1; Calpain-2; ERK; Hippocampus; Learning; SCOP.

Figure 1. A calpain-2 selective inhibitor rescued TBS-induced LTP impairment in hippocampal slices from C1KO mice, an effect blocked by PD9805, and increased HFS-induced LTP in WT mice

A. Vehicle (grey) or calpain-2 inhibitor (200 nM, blue) was applied for 10 min after TBS; PD98059 (50 μM) was applied alone (brown) for 20 min starting 10 min before TBS; PD98059 (50 μM) and calpain-2 inhibitor (200 nM) (green). B. Vehicle (grey) or Calpain-2 inhibitor (200 nM, blue) was applied for 10 min before HFS (*p<0.05, as compared to vehicle). Results are means ± S.E.M. of 4–6 slices from 3 animals.

Figure 2. Blockade of TBS-induced PTEN degradation by calpain-2 inhibitor treatment in hippocampal slices from WT and C1KO mice

A. PTEN immunoreactivity 10 min after TBS delivered in slices treated with vehicle or calpain-2 inhibitor (200 nM) for 10 min after TBS. Scale bar: 20 μm. B. Quantitative analysis of PTEN immunostaining in slices from WT mice; results are means ± S.E.M. of 4–6 slices from 3 animals (***p< 0.001, as compared to WT group; ###p< 0.001, as compared to WT-TBS group). C. Quantitative analysis of PTEN immunostaining in slices from C1KO mice; results are means ± S.E.M. of 4–6 slices from 3 animals (**p< 0.01, as compared to C1KO group; #p< 0.05, as compared to C1KO-TBS group).

Figure 3

Biphasic effects of a selective calpain-2 inhibitor on learning and memory in WT mice WT mice were treated with C2I (A&B 0.03–10 mg/kg;) 30 min before training. Results are expressed as percent time mice exhibited freezing behavior. A. Quantitative analysis of percent freezing for each group in context memory. B. Quantitative analysis of percent freezing for each group in tone memory. Results represent means ± S.E.M. of 6–18 mice. *p< 0.05, **p<0.01, as compared to vehicle controls.

Figure 4. A calpain-2 selective inhibitor enhanced learning and memory in WT mice and rescued learning and memory impairment in C1KO mice

WT and C1KO mice were treated with vehicle or calpain-2 inhibitor (0.3mg/kg) 30 min before training. Discrimination index for novel-object recognition was determined one hour (A) and 24 hour (B), after training. Results are means ± S.E.M. of 6 mice for each group (***p< 0.001 as compared to WT group; #p< 0.05, ##p< 0.01, ###p< 0.001, as compared to vehicle group).

Figure 5. A calpain-2 selective inhibitor prolonged ERK activation in dorsal hippocampus

A. Experimental design: Calpain-2 inhibitor (0.3 mg/kg) or Vehicle were injected (i.p.) in WT mice 30 min before training; dorsal and ventral hippocampi were collected 5 min and 50 min after training. B. Representative western blots for SCOP, Akt, p-ERK and ERK under various experimental conditions. C&D. Quantitative analysis of the levels of SCOP (normalized to Akt values) and p-ERK/ERK ratios. * p < 0.05 **p<0.01.