Calpain 1 Knockdown Increases Smn Protein Level in Cultured Spinal Cord MNs
Calpains are involved in several muscle and neurodegenerative disorders, including SMA, and Smn is a direct target of calpain cleavage in muscle tissue [24, 25]. To analyze the effect of calpain reduction on Smn in spinal cord MNs, we used a lentiviral RNA interference method to downregulate the calpain protein level in these cells. Embryonic (E12.5) spinal cord MNs were isolated and plated in culture wells. Three hours later, we transduced them with lentivirus containing EV or short hairpin RNA sequences targeting specific sites of mouse calpain 1 (shCalp1)  and maintained in the presence of the neurotrophic factors cocktail (NTFs) (1 ng/ml brain-derived neurotrophic factor; 10 ng/ml glial cell line-derived neurotrophic factor, 10 ng/ml ciliary neurotrophic factor, 10 ng/ml cardiotrophin-1 and 10 ng/ml hepatocyte growth factor). At 3, 6, and 9 days after transduction, cell lysates were collected and submitted to western blot using an anti-SMN antibody. We observed a significantly increased Smn protein level in shCalp1 cells after 3 (1.30 ± 0.09, p < 0.05), 6 (1.69 ± 0.19, p < 0.005), and 9 (1.75 ± 0.46, p < 0.05) days of transduction, compared with the EV (Fig. 1a). Control blot analysis using an anti-calpain antibody demonstrated that calpain protein was reduced in shCalp1 condition compared to EV. To analyze whether this Smn reduction is preferentially localized in cell soma or neurites, we measured Smn levels with immunofluorescence using confocal microscopy. MNs were plated on glass coverslips and transduced using the EV or shCalp1 constructs. Six days after transduction, cells were fixed and Smn immunostaining with anti-SMN antibody was performed. Using the NIH ImageJ software, quantification of the average of fluorescence units (AFU) showed that Smn increases in both MN soma (AFU 115.80 ± 7.27, p < 0.001) and neurites (AFU 12.29 ± 2.21, p < 0.0005) in shCalp1 cells, compared to EV (AFU 85.43 ± 5.34 and 6.18 ± 0.56, respectively) (Fig. 1b).
To elucidate the effect of calpain reduction on MNs viability, survival experiments were performed. Cells were plated at low density to avoid cell contact-mediated effects (~ 8000 cells/cm2) and were cultured under several conditions: non-transduced cells in the absence (NBM) or the presence of NTFs, and EV- or shCalp1-transduced cells in the presence of NTFs. At 3, 6, and 9 days after plating, cells were counted in three wells of each condition. Considering 100% the initial number of cells present in a well on day 0, survival was estimated as the percentage of remaining cells in the same well. As shown in Fig. 1c, using the phase-contrast microscope, we observed that after 9 days without NTFs, MNs were largely degenerated and had a significantly lower percentage of surviving cells (day 3, NBM 3.38 ± 5.86% vs. NTFs 64.38 ± 3.90%, p < 0.001). However, the presence of EV or shCalp1 did not significantly modify MN survival, compared to the NTFs condition (day 9, 40.76 ± 2.63%, 44.11 ± 5.12%, and 42.35 ± 2.51%, respectively). Therefore, endogenous calpain reduction did not reduce MNs viability.
Calpain 1 Knockdown Prevents Smn Reduction Caused by Membrane Depolarization
Addition of high K+ to the culture medium induces chronic depolarization of the neuronal plasma membrane, which in turn activates voltage-gated calcium channels (VGCCs), resulting in Ca2+ influx and elevation of intracellular Ca2+ concentration [26, 28]. Reaching the appropriate intracellular Ca2+ level induces the activation of Ca2+-dependent proteases such as calpains. To elucidate the effect of intracellular Ca2+ increase on Smn level and the mediating role of calpain, we isolated E12.5 mice spinal cord MNs and established a primary culture in the presence of NTFs. Cells were transduced with EV or shCalp1 constructs. Six days later, cells were treated with 30 mM of KCl (30K) for 3 h and protein extracts were obtained and submitted to western blot using anti-SMN antibody. Smn protein was significantly reduced in 30K EV-treated cultures (0.5 ± 0.15-fold, p < 0.0001) compared to non-treated EV control (Fig. 2). As expected, shCalp1 (1.5 ± 0.15-fold) treatment induces Smn increase when compared with EV control (p < 0.005). However, no significant differences of Smn level were observed in 30K shCalp1, compared to shCalp1 condition (1.5 ± 0.4- and 1.5 ± 0.15-fold, respectively), suggesting that high K+ treatment induced Smn protein reduction through calpain activity in cultured spinal cord MNs.
Calpeptin Treatment Increases Smn Protein Level in Cultured Spinal Cord MNs
To further assess the role of calpain activity on Smn protein level in MNs, we used the cell-permeable inhibitor calpeptin . We first analyzed the effect of calpeptin in basal culture conditions. E12.5 MNs were isolated and cultured in the presence of NTFs. Six days later, plating culture medium was changed to fresh medium containing NTFs or NTFs plus 25 μM calpeptin. Total cell lysates were obtained at 3, 9, 16, 20, and 24 h after treatment and submitted to western blot protein analysis using an anti-Smn antibody. Smn protein level was significantly increased after 3, 9, 16, and 20 h of calpeptin treatment, compared to non-treated controls (3.34 ± 0.56, p < 0.005; 4.40 ± 0.58, p < 0.005; 2.89 ± 0.61, p < 0.05; and 2.06 ± 0.50, p < 0.05, respectively; Fig. 3a). No changes of Smn protein were observed in cells treated 24 h, indicating that calpain inhibition using calpeptin increases Smn up to 24 h of treatment in cultured MNs. No changes in Smn protein were observed in cells treated 24 h with calpeptin, indicating that inhibition of calpain by calpeptin increased Smn up to 24 h of treatment in cultured MNs. This temporary effect may have been due to the degradation of calpeptin; the providers recommend that the aqueous solution not be stored for more than 1 day. Future research should consider the effect of calpeptin degradation by refreshing the media and/or varying other experimental conditions.
To study whether calpeptin treatment reverts Smn reduction caused by chronic membrane depolarization, primary CD1 MN cultures were established. Six days after plating, cells were treated with NTFs (control) or NTFs plus 30K or 25 μM calpeptin or 25 μM calpeptin+30K. Three hours after treatment, cell lysates were obtained and submitted to western blot using anti-α-fodrin or anti-SMN antibodies. Degradation of α-fodrin to the 150/145 kDa-specific fodrin breakdown products indicates calpain activation . The levels of 150/145 kDa product in cells treated with NTFs plus 30K (30K) were significantly increased compared to NTF control condition (1.33 ± 0.13, p < 0.0001). However, the addition of calpeptin to NTF control or 30K conditions significantly reduced fodrin cleavage products (0.66 ± 0.05, p < 0.001, and 0.63 ± 0.07, p < 0.001, respectively) (Fig. 3b). This result suggests that calpain is activated after membrane depolarization in mouse MNs. Smn protein level in calpeptin+30K-treated cells (1.67 ± 0.26, p < 0.05) was significantly increased compared to 30K and control conditions (Fig. 3b). These results together indicate that Smn is reduced in 30K-treated cells, and this effect can be prevented by calpeptin treatment.
High Potassium Treatment Induces Smn Cleavage in Cultured MNs
In muscle tissue and in U2-OS cell lines, SMN is processed by calpain leading to the production of N- and C- terminal cleavage products [24, 25]. To identify some of these cleavage products in cultured MNs, we isolated E12.3 MNs from CD1 mice and cultured for 6 days in the presence of NTFs. Cells were treated for 3 h with 30 mM KCl (30K) or with 25μM calpeptin or 30K plus 25 μM calpeptin, and total lysates were analyzed by western blot using two monoclonal antibodies, anti-SMN (Clone 8) (BD Bioscience) and anti-SMN1 antibody (9F2) (Cell Signaling), to detect the cleavage products. Anti-SMN antibody (Clone 8) recognizes full-length and N-terminal fragments, and anti-SMN1 (9F2) antibody recognizes C-terminal fragments. Quantification of full-length Smn (~ 37 kDa) showed that 30K treatment significantly reduced Smn protein level (0.59 ± 0.07, p < 0.0001), whereas calpeptin treatment increased Smn (2.27 ± 0.50, p < 0.05), compared to non-treated control. When the same membranes were overexposed, we detected an increase of N-terminal Smn cleavage product (~ 20 kDa) in 30K condition (1.98 ± 0.50, p < 0.05), compared to the untreated control, which was prevented by addition of 25 μM calpeptin to the medium (0.53 ± 0.12, p < 0.05) (data not shown).
Using the anti-SMN1 antibody, C-terminal fragments (~ 17 kDa) were only evident when the membrane was overexposed. As shown in Fig. 4, Smn C-terminal fragments were significantly increased in 30K (1.49 ± 0.29; p < 0.05), compared to control. Conversely, calpeptin treatment significantly reduced C-terminal fragments in 30K and control conditions (0.22 ± 0.04 p < 0.05, and 0.27 ± 0.04, p < 0.0001, respectively). These results together suggest that high K+ treatment reduces full-length Smn and increases C-terminal fragments.
Calpeptin Treatment Increases Smn Protein Level and Prevents Neurite Degeneration in SMA Mutant MNs
To determine whether calpeptin treatment regulates Smn in MNs from E12.5 embryos of the severe SMA transgenic mouse model (FVB·Cg-Tg (SMN2)89AhmbSmn1tm1Msd/J), mice were genotyped and the spinal cords of wild-type (WT) and mutant (mutSMA) were dissected. WT and mutSMA isolated MNs were cultured in the presence of NTFs, carefully following the same protocol used for CD1 cells: six days after plating, WT and mutSMA cells were treated with 25 μM calpeptin during 3 h. Protein extracts were collected and submitted to western blot analysis using anti-SMN antibody. Results indicate that Smn is increased in calpeptin-treated WT condition (2.13 ± 0.50-fold induction, p < 0.05) compared to WT control (Fig. 5a, left graph). Furthermore, in mutSMA cultures, calpeptin treatment increased Smn protein, compared to mutSMA non-treated cells (5.18 ± 1.48-fold induction, p < 0.05) (Fig. 5a, right graph).
Focal axonal swellings have been previously described in Smn-reduced mouse tissues and MNs [27, 35]. To determine the presence of neurite degeneration in MNs obtained from the severe SMA mouse model (Smn−/−; SMN2+/+), cells were isolated from the mutSMA and WT mice at E12.5 and cultured at a low density (~ 5000 cells/cm2). We assessed neurite degeneration by quantifying neurite segments in a determinate area of the plate using phase-contrast microscopy at days 6, 9, and 12. Neurites were considered degenerated if they showed evidence of swellings and/or blebbing (see “Materials and Methods”) . Nine and 12 days after plating in the presence of NTFs, mutSMA MNs showed a higher percentage of degeneration compared to WT MNs (9 days, WT 15.68 ± 6.67% of degenerated neurites and mutSMA 30.13 ± 13.10%, p < 0.05; 12 days, WT 24.10 ± 7.37% and mutSMA 43.08 ± 4.71%, p < 0.01) (data not shown). In order to analyze the effect of calpain reduction on neurite degeneration, mutSMA and WT cells were transduced with the lentivirus carrying shCalp1 or EV constructs. After 6 days of transduction, no significant differences were observed between groups. However, 9 days after transduction, we detected a significant difference in neurite morphology when the shCalp1 mutSMA (16.64 ± 5.67%) was compared with the EV mutSMA (26.17 ± 8.22%, p < 0.01) cultures. After 12 days, the signs of degeneration increased to 36.55 ± 2.28% in EV mutSMA cultures, whereas shCalp1 mutSMA cultures showed significantly reduced degeneration (29.24 ± 4.89%, p < 0.0005), compared to the EV mutSMA control (Fig. 5b). This percentage did not differ from those observed in EV WT (26.29 ± 4.69%) and shCalp1 WT (25.62 ± 3.61%). All these results together demonstrate that calpain regulates Smn protein level and neurite degeneration in Smn-reduced MNs.
Calpeptin Administration Extends Survival of Severe SMA and SMNdelta7 SMA Mice
The evidences suggested that calpain regulates Smn protein level positively in MNs. Therefore, we decided to examine whether calpeptin administration had a positive effect on SMA mice. To further assess this hypothesis, we started a treatment protocol using two different SMA mouse models: the FVB·Cg-Tg (SMN2)89AhmbSmn1tm1Msd/J (mutSMA) and the FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg (SMN2*delta7)4299Ahmb/J (SMNdelta7). To determine if calpeptin affected the lifespan and the body weight of these mice, animals were tattooed and genotyped at postnatal day 0 (P0). WT and mutants littermates were grouped by sham or treatment and the subcutaneous injections were started at P1, with a daily dose of 6 μg calpeptin per gram of weight. Results showed that calpeptin administration significantly improved lifespan of the mutSMA-calpeptin group (average days 9 ± 2.97, n = 11), compared with the mutSMA-sham (average days 4.08 ± 2.54, n = 12, p < 0.0001) (Fig. 6a). Likewise, lifespan of SMNdelta7-calpeptin group (average days 14.41 ± 2.90, n = 15) was significantly increased compared to SMNdelta7-sham (average days 9.8 ± 2.89, n = 10, p < 0.0001) mice (Fig. 6b).
When the weight of the animal groups was measured, we did not find significant differences in calpeptin-treated mutant compared to non-treated mice. In contrast, weight was slightly increased in calpeptin-treated WT groups compared with sham-treated WT groups from day 13 of treatment to the end of the experiment (Fig. 6c, d).
Effect of Calpeptin Treatment on Motor Function in SMA Mice
Because mutSMA and SMNdelta7 mouse models are characterized by severe motor function impairment, we analyzed whether the enhancement in lifespan observed in both models after calpeptin treatment was correlated with motor functional improvement. Two behavior tests were performed: the righting reflex test and the tube test [32, 36]. The righting reflex response is based on the ability of neonatal mice to return to their four paws after having been placed in supine position. It can be measured in pups as early as P1-P2 and evaluated until P9-P10. The test was performed on mice in the sham- or calpeptin-treated WT and sham- or calpeptin-treated mutant (mutSMA and SMNdelta7) groups. The test was designed with a maximum time of 30 s and measurements were taken daily before the calpeptin injection from P1 to P10, done in triplicate for each animal with 5 min resting time between tests. Results obtained in WT groups showed that the righting reflex time was consistently faster (~ 10 s) than in mutant groups (~ 30 s). In calpeptin-treated mutSMA, there were no significant differences from the beginning of the treatment (P1) to P7 compared to sham-treated mutSMA. However, from P7 to the end of the experiment the righting reflex time progressively decreased in calpeptin-treated mutSMA and SMNdelta7 (~ 20 s) (Fig. 7a, b).
The tube test is a non-invasive motor function test specifically designed for neonatal rodents. It evaluates the proximal hind-limb muscle strength, weakness, and fatigue. The animal age range for this test is from P2 to P8. Two parameters were evaluated in the present study: latency to fall from the edge of the tube (in seconds, latency to fall graphs) and the hind-limb score (HLS graphs) which assess the positioning of the legs and tail. Mice with motor weakness show reduced time to fall and low score of HLS. The test was performed in the same sham and treated groups of mice described. The test was designed with a maximum time of 30 s, and measures were performed in triplicate daily before the calpeptin injection from P1 to P8. When the latency time was analyzed in the mutSMA model, we observed no differences in time-to-fall from P1 to P5. Nevertheless, on days 6 (P6) and 7 (P7) after treatment, calpeptin-treated mutSMA mice (P6, 15.62 ± 10.66 s, n = 7) showed significantly increased time-to-fall compared to sham-treated mutSMA group (P6, 3.84 ± 1.92, n = 3, p < 0.05) and no significant differences compared to WT groups (P6 WT-SHAM, 19.4 ± 9.5) (Fig. 7c). In mutant SMNdelta7 mice, calpeptin treatment significantly increased latency-to-fall compared to sham-treated controls from P3 to the end of the experiment (p < 0.05) (Fig. 7d). In both SMA models, HLS measures revealed that calpeptin treatment of mutant mice significantly increased the score compared to sham mutants (mutSMA P2 to P8, p < 0.001; SMNdelta7 P6 to P8, p < 0.05) (Fig. 7e, f). Altogether, the results obtained from the behavior tests suggested that calpeptin treatment ameliorates motor function of mutant mice in both SMA models analyzed.