Calpain-cleaved collapsin response mediator protein-3 induces neuronal death after glutamate toxicity and cerebral ischemia

Abstract

Collapsin response mediator proteins (CRMPs) mediate growth cone collapse during development, but their roles in adult brains are not clear. Here we report the findings that the full-length CRMP-3 (p63) is a direct target of calpain that cleaves CRMP-3 at the N terminus (+76 amino acid). Interestingly, activated calpain in response to excitotoxicity in vitro and cerebral ischemia in vivo also cleaved CRMP-3, and the cleavage product of CRMP-3 (p54) underwent nuclear translocation during neuronal death. The expression of p54 was colocalized with the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive nuclei in glutamate-treated cerebellar granule neurons (CGNs) and in ischemic neurons located in the infarct core after focal cerebral ischemia, suggesting that p54 might be involved in neuronal death. Overexpression studies showed that p54, but not p63, caused death of human embryonic kidney cells and CGNs, whereas knock-down CRMP-3 expression by selective small interfering RNA protected neurons against glutamate toxicity. Collectively, these results reveal a novel role of CRMP-3 in that calpain cleavage of CRMP-3 and the subsequent nuclear translocation of the truncated CRMP-3 evokes neuronal death in response to excitotoxicity and cerebral ischemia. Our findings also establish a novel route of how calpain signals neuron death.

Figure 1.

CRMP-3 is cleaved toward the N terminus. Protein extracts from glutamate-treated CGNs (A) and ischemic brains (B) were subjected to SDS-PAGE (10% gel) and Western blotting with an antibody against CRMP-3. The full-length CRMP-3 appeared as a 63 kDa band, whereas the breakdown product of p63 had a molecular weight of 54 kDa. The p54 band appeared in glutamate-treated CGNs (A) and ischemia in the brain (B). MK-801 at 10 μm protected CGNs against glutamate toxicity and prevented the breakdown of CRMP-3 (A). Purified recombinant semaphorin 3A was added to CGNs at 5 μg/ml for 18 h before cell extract was collected for Western blotting with CRMP-3 antibody (A, last lane). Bands corresponding to p63 and p54 were cut out on a clean surface and subjected to mass spectrometry analysis. A representative mass spectrum of CRMP-3 is shown in C. The p54 band was also subjected to N-terminal protein sequencing to confirm the identity of p54 and the cleavage site of CRMP-3 (arrow in D). The cleavage site sequence of CRMP-3 was compared with the calpain cleavage site of spectrin, which shared >50% homology (D).

Figure 2.

CRMP-3 is a direct target of calpain. A, Proteins extracted from glutamate-treated CGNs either with or without preincubation with calpain inhibitors, ALLN or calpeptin, were subjected to Western blotting to detect changes in CRMP-3 breakdown. Calpain inhibitors ALLN at 10 μm and calpeptin at 50 μm protected CGNs and prevented the breakdown of CRMP-3 (A). B, Normal mouse brain protein extracts were stimulated with 5 mm CaCl₂ to activate endogenous calpain, which led to the cleavage of the endogenous CRMP-3, as shown by Western blotting. The addition of calpain inhibitors ALLN (10 μm) and calpeptin (50 μm) before calcium stimulation prevented calpain activation and the breakdown of CRMP-3. Western blotting for GAPDH was used to show equal protein loading (A, B). C, Active calpain I at the concentrations indicated were added into normal brain protein extracts without calcium stimulation. At the concentrations of 0.01 and 0.005 U, CRMP-3 was cleaved after 20 min incubation with calpain I, whereas lower concentrations of calpain were not sufficient to cleave CRMP-3. Calpain inhibitors ALLN and calpeptin effectively prevented the breakdown of CRMP-3 (last two lanes). D, Active calpain I at the concentrations indicated was mixed with 10 μg of recombinant CRMP-3 expressed in E. coli. At the concentrations of 0.01 and 0.005 U of calpain I used, CRMP-3 was cleaved into a p54 fragment.

Figure 3.

Altered expression of CRMP-3 during neuronal death. A–C, CGNs were treated with glutamate (Glut) (50 μm) in either the absence (B) or presence (C) of calpain inhibitor ALLN (10 μm). Untreated CGNs were used as a negative control (Ctl) (A). ALLN protected CGNs against glutamate toxicity (C). After 4–6 h of treatment with 50 μm glutamate, cells were either fixed and subjected to immunostaining for CRMP-3 (A–C) or double stained with TUNEL (E, F). Note that glutamate-treated CGNs had shorter neurites and higher levels of CRMP-3 immunostaining in areas in and around the nucleus (B) compared with those in the untreated and ALLN-protected neurons. Blue in A–C indicates Hoechst 32558-positive nuclei. Neuroprotection by calpain inhibitors was also quantified using PI staining as described in Materials and Methods (D). G–I, Nuclear fractions from glutamate-treated CGNs (G) and ischemic brains (H) were prepared as described in Materials and Methods and were probed with antibodies to CRMP-3. Histone H1 and GAPDH were also probed as loading controls for nuclear and cytosolic fractions, respectively. Total protein extracts were also used as positive controls. A clear p54 band appeared in the nuclear fraction of glutamate-treated CGNs (G) and ischemic brains (H). The intensity of each band was quantified using densitometry and plotted in I. Data represented the average of at least three repeats, with error bars representing the SD. Statistical analysis in D and I was performed using Student’s t test, and significant groups were identified using Tukey’s post hoc test. *p < 0.05; **p < 0.01. J–U, Mice were subjected to 1 h MCAO, followed by reperfusion. Brains were fixed with 4% paraformaldehyde and embedded in paraffin. Sections at 10 μm thickness were cut and subjected to immunostaining to detect CRMP-3. N and R show MCAO-induced nuclear localization of CRMP-3 (red) in ischemic neurons located in the ischemic side of the brain but not in the contralateral side (J). TUNEL staining was also performed on the same section (K, O, S), and images for CRMP-3 and TUNEL were merged (yellow in L, P, T). All sections were counterstained with Hoechst 32558 to show the nuclei (blue in M, Q, L). Scale bars, 100 μm.

Figure 4.

Overexpression of p54 induces cell death. A, Plasmid constructs of EGFP-tagged full-length CRMP-3 (pEGFP-p63), p54 (pEGFP-p54), and EGFP vector control (pEGFP) were transfected into HEK293 cells. The overexpressed proteins were detected by Western blotting (B). EGFP fusion protein with p63 and p54 had a molecular weight of 98 and 89 kDa, respectively (B). Proteins from HEK293 and PC12 cells were subjected to Western blotting to confirm that they did not express endogenous CRMP-3 (C). Cultured HEK293 cells were transfected with 10 μg of plasmids. After 2 d, cells were fixed and viewed under a fluorescent microscope. EGFP vector construct expressed EGFP in both the nucleus and the cytoplasm. In contrast, EGFP-p63 only expressed in the cytoplasm (E), whereas EGFP-p54 expressed mostly in the nucleus (F). Overexpression of p54 (pEGFP-p54) caused nuclear condensation (F), which overlapped with TUNEL positivity (F′ and F″). The number of TUNEL-positive cells were counted and plotted in G. Scale bar, 50 μm. **p < 0.01 by one-way ANOVA and post hoc Tukey’s test. H–M are CGNs infected with lentiviral constructs. Lentiviral vector expressing EGFP, EGFP-p63, and EGFP-p54 were made as described in Materials and Methods and were used to infect 7-d-old mature postmitotic CGNs for 72 h. Cell were examined live under a fluorescent microscope, followed by fixation with fresh Formalin and TUNEL staining (I, K, M). The number of TUNEL-positive cells were counted and plotted in N. Statistical analysis was performed using one-way ANOVA, and significant groups were identified by Tukey’s post hoc analysis. **p < 0.01.

Figure 5.

Downregulation of CRMP-3 expression protects neurons against glutamate toxicity. CGNs were cultured in a 24-well plate for 7 d, followed by transfection for 2 d with 1 μg of CRMP-3-specific siRNA (1 or 2) or a control siRNA (Ctl-siRNA) using FuGene 6. CGNs were subsequently collected for Western blotting to show the selective downregulation of CRMP-3 (A, B) but not CRMP-1, CRMP-2, or CRMP-4 (C) with GAPDH as a loading control. Densitometry measurement of the CRMP-3 band intensity in A showed a clear downregulation of CRMP-3 by siRNA-1 (B; values represent the average of two independent repeats). Transfection of neurons by siRNA was confirmed by examining the fluorescence of Alexa Fluor 546 (D–F). Immunostaining of these CGNs showed that siRNA-1 (I), but not the control siRNA (Ht), reduced expression of CRMP-3 in neurons. The siRNA-1 was subsequently used to transfect 7-d-old CGNs for 2 d. CGNs were treated with 50 μm glutamate and followed by CFDA staining to detect live cells in culture using a plate reader. Data in J represent at least four independent repeats. Statistical analysis was performed using one-way ANOVA, and significant groups were determined by Tukey’s post hoc analysis. **p < 0.01.