Roles of the miR-137-3p/CAPN-2 gene pair in ischemia-reperfusion-induced neuronal apoptosis through modulation of p35 cleavage and subsequent caspase-8 overactivation

Background: Neuron survival after ischemia-reperfusion (IR) injury is the primary determinant of motor function prognosis. MicroRNA (miR)-based gene therapy has gained attention. Our previous work explored the mechanisms by which miR-137-3p modulates neuronal apoptosis in both in vivo and in vitro IR models. Methods: IR-induced motor dysfunction and spinal calpain (CAPN) subtype expression and subcellular distribution were detected within 12 h post IR. Dysregulated miRs, including miR-137-3p, were identified by miR microarray analysis and confirmed by PCR. Luciferase assay confirmed that CAPN-2 is a corresponding target of miR-137-3p, and their modulation of motor function was evaluated by intrathecal infection with synthetic miRs. CAPN-2 activity was measured by the intracellular Ca 2+ concentration and mean fluorescence intensity in vitro . Neuronal apoptosis was detected by flow cytometry and lactate dehydrogenase (LDH) release. The activities of p35, p25, Cdk5 and caspase-8 were evaluated by ELISA and Western blotting after transfection with specific inhibitors and miRs. Results: The IR-induced motor dysfunction time course was closely associated with CAPN-2 protein upregulation, which was mainly distributed in neurons. The miR-137-3p/CAPN-2 gene pair was confirmed by luciferase assay. miR-137-3p mimic significantly improved IR-induced motor dysfunction and decreased CAPN-2 expression, even in combination with recombinant rat calpain-2 (rr-CALP2) injection, whereas miR-137-3p inhibitor reversed these effects. Similar changes were observed in the intracellular Ca 2+ concentration and CAPN-2 expression and activity when cells were exposed to OGD/R and transfected with synthetic miRs in vitro

apoptosis was effective in preserving hindlimb motor function in rodent models [5,9]. Recently, some studies have shown that disturbed ionic homeostasis, such as ischemic or mechanical injury-induced excessive intracellular calcium ion concentration ([Ca 2+ ]) in neurons, could eventually trigger neuronal apoptosis by influencing vital biological functions and metabolism [10][11][12]. Thus, preserving intracellular calcium homeostasis may represent a promising treatment for attenuating neuronal apoptosis after IR insult.
Increased intracellular Ca 2+ can activate a verity of proteases [13]. Belonging to a family of calcium-dependent neutral proteases, calcium-activated neutral proteinases (CANPs, also called calpains) are the most well-known effector that reacts to intracellular Ca 2+ dysregulations through calcium-binding subunits [14,15]. Eleven types of calpain isoforms have been identified in humans so far, of which calpain-1 (µ-calpain, CAPN-1) and calpain-2 (m-calpain, CAPN-2) are the most widely ubiquitous isoforms in the central nervous system (CNS) [13]. Being distributed in the same subcellular localization (cytoplasm) and sharing a common small subunit (known as CAPN-4) upon activation, CAPN-1 and CAPN-2 seem to have similar biochemical properties [13,16]. CAPN-1 and CAPN-2 have previously been demonstrated to be overactivated in various models of neurodegenerative diseases and injury, although they require micromolar and millimolar calcium levels for activation, respectively [13,[14][15][16]. However, in contrast to traditional views, some studies have recently suggested that CAPN-1 activation plays prosurvival roles whereas CAPN-2 plays neurodegenerative roles, based on their opposite functions in promoting neuronal plasticity following CNS injury [17][18][19]. The most notable characteristic of calpains is their ability to perform partial truncation, which is a proteolytic cleavage of protein substrates, such as cytoskeleton proteins, membrane-bound proteins and protein kinases, at specific sites [13]. Commonly, the downstream products of CAPN-mediated truncation are bioactive, which can further amplify neurotoxic insults or oxidative stress by activating a subsequent signaling pathway [13,20]. For example, the membrane-bound protein p35, known as a specific neuronal activator for cyclin-dependent kinase-5 (Cdk5), has been demonstrated to be a major substrate exclusively regulated by CAPNs in the pathogenesis of neurodegenerative disease [13,19,20]. Further exploration of downstream targets in rodent in vivo and in vitro experiments revealed that Cdk5 overactivation induced by cleaved p35 occurred exclusively in the presence of CAPN-2 [19][20][21]. In those studies, overexpressed CAPN-2 precisely cleaved the normally membrane-bound p35 into the more stable p25 form, which finally led to inappropriate increases in p25/Cdk5 activation and protein levels of caspase-3, a final executioner of neuronal apoptosis [19,22,23]. Acting as an upstream activator of caspase-3, caspase-8 is implicated in various models of CNS diseases and is critical for neuronal apoptosis [24,25]. Thus, preventing caspase-8 proteolysis is especially important for controlling a series of broad caspase activations [24,25].
Consistently, the p35 protein from baculovirus effectively blocked the apoptosis cascade by forming a p35-caspase-8 complex via a thioester bond [24,26]. The structural experiments further identified the N terminus of p35 as the major element necessary to preserve the intact covalent bond within the p35-caspase-8 crystal structure [26]. Thus, an increase in CAPN-2-mediated p35 cleavage may be reasonably inferred to also lead to conformational changes in p35 to further initiate caspase-8 and downstream caspase activation. Collectively, strong evidence suggests that the destructive functions of CAPN-2 during CNS injury are greatly attributed to its catalyzed substrates and downstream signaling pathways [17,19,20,27]. However, no study has explored the abovementioned hypothesis in spinal cord IR injury using methods specifically targeting CAPN-2.
MicroRNAs (miRs) are a group of small, endogenous, noncoding RNAs [28]. miRs are widely expressed in the CNS and able to negatively regulate target genes by either degradation or posttranscriptional repression [5,6,28]. In our previous studies, we identified hundreds of aberrant miRs in injured spinal cords by microarray analysis [5,6,29]. Intrathecal pretreatment with synthetic miR mimic resulted in significant improvement in neurological deficits by recovering the altered miR expression [5,6,29]. These findings suggest promising miR-based gene therapy targeting CAPN-2. In this context, we first searched bioinformatical databases, identify potential miRs that may have binding sites with CAPN-2 among all dysregulated miRs detected in microarray analysis. Our present study suggested that miR-137-3p and miR-124-3p had target interactions with CAPN-2, which is supported by another study that explored the roles of miR-137-3p in rescuing motoneuron degeneration after brachial plexus root avulsion injury [30]. Then, we studied the functions and mechanisms by which the miR-137-3p/CAPN-2 gene pair regulates neuronal apoptosis by pretreatment of in vivo and in vitro models with synthetic miRs, a selective CAPN-2 inhibitor, recombinant rat calpain-2 (rr-CALP2) or a specific caspase-8 inhibitor.

Experimental animals
Sprague-Dawley rats weighting 200-to 250g were obtained from the Animal Center of China Medical University (Shenyang, China). All rats were preacclimatized 7 days before surgery. They were housed in standard cages under a 12-h light/dark cycle with the temperature at 23-24°C, humidity at 40-50%. The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (United States National Institutes of Health publication number 85-23, National Academy Press, Washington DC, revised 1996).

Rat IR model establishment and experimental groups
The rat IR model was preformed by occluding the aortic arch for 14 minutes [4,29]. Briefly, after being anesthetized, the rats were catheterized at the left carotid artery and the tail artery to measure proximal and distal blood pressure (BP), respectively. Following exposing the aortic arch, the clamp was placed between the left common carotid artery and the left subclavian artery for 14 min to induce ischemia. The ischemia was confirmed as a 90% decrease in distal BP. Then the clamp was removed to induce the reperfusion for 12h. The sham-operated rats were preformed the same procedures without inducing ischemia.

MiR microarray analysis
As we previously reported, the rat miRNA microarray analysis was performed with the miRNeasy mini kit (Qiagen, West Sussex, United Kingdom) [29,31]. The L4-6 segments of the spinal cord were collected at 4h after reperfusion. According to the manufacturers' instructions, 2.5μg total RNA samples were firstly labeled with the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) and hybridized on a miRCURY™ LNA Array (version 18.0, Exiqon, Vedbaek, Denmark).
After removing the nonspecific bindings, the fluorescent images of microarray slides were scanned by an Axon GenePix 4000B microarray scanner (Axon Instruments, CA, USA). The fluorescent intensity of the scanned images were loaded into the GenePix Pro 6.0 program (Axon Instruments) for feature extraction. The average of the replicated miRs with intensities of 50 or more were used to calculate a normalization factor. After normalized by the median normalization method, the significantly different miRs were identified by Volcano Plot filtering. Finally, the hierarchical clustering was performed to determine the differences of the miR expression by MEV software (version 4.6, TIGR ).

Intrathecal injection and drug delivery
All treatments in vivo including the synthetic miRs (Dharmacon,Chicago, IL, USA) and recombinant rat calpain-2 (rr-CALP2,B71107, 150U/L, Calbiochem, China ) were diluted into 20 µl in total volume and intrathecally injected, as we previously described [5,6]. Briefly, the needle of a 25μ microsyringe was inserted into the L 5-6 segment of spinal cords by the sign of a tail flick. Then, the concentration of 100 μmol/L of miR-137-3p mimic, 125 μmol/L miR-137-3p inhibitor or 100 μmol/L negative control (NC) was co-administered with Lipofectamine 3000 (Invitrogen, USA) at a 24h-interval for five consecutive days before surgery. Likewise, the rr-CALP was dissolved into a final concentration of 75 U/L immediately before injection.
The number of days and the dosage used in this stud were evaluated by the overall effects in vivo by PCR and Western blotting in preliminary experiments. Only the rats displayed normal motor function were included for further study.

Motor function assessment
All being fully preacclimatized to the testing environment, the hind-limb motor functions were scored with Tarlov scores by two observers by the double-blind method [5].

Oxygen-glucose deprivation and reperfusion (OGD/R) model
As we previously performed, the OGD/R model was established in 70-80% confluent VSC4.1 neurons to mimic the IR insult in vivo [5]. After twice washes and replacement the medium with glucose-free Hank's Balanced Salt Solution (HBSS), the neurons were kept in an anaerobic chamber (95% N2 and 5% CO2) at 37 °C for 6h. Then the medium was changed into initial medium and air condition for another 18h to induce reoxygenation. The control neurons were cultured in normal and atmosphere for 24h without depriving oxygen and glucose.
For in vitro experiment, the neurons were pretreated with the synthetic miR and specific inhibitors 24h before underwent OGD/R insults [5]. As we previously, after seeded at a concentration of 4×10 5 per well, the miR-137-3p mimic (50 nmol/L) or NC (50 nmol/L) was cotransfected with 5 μL Lipofectamine 3000, whereas for inhibitor experiments, the Roscovitine (10 µM, Cdk5 inhibitor, Sigma-Aldrich Co., China) or Z-IETD-FMK (50 µM, caspase-8 inhibitor, R&D Systems,United States) was added into culture medium alone. The concentration of each treatment and the in vitro effects were determined by PCR in preliminary experiments.

Detection of CAPN-2 activity
The tensin homolog (PTEN) is a selective substrate for CAPN-2. PTEN is degraded as a result of CAPN-2 activation and is widely used for quantitative analysis of neuronal CAPN-2 activity in vivo and in vitro [19,32]. As previously described,

Lactate dehydrogenase (LDH) assay
The LDH released from VSC4.1 neurons was detected by a commercial LDH Assay Kit (Abcam, CA, USA). According to the manufacturer's instructions, 50 µL medium was collected at 24h after each treatment and measured at the absorbance of 450 nm.

Detection of Caspase-8 activity
The caspase-8 activity was detected by the caspase-8 assay kit (Abcam, CA, USA), which is based on the spectrophotometric detection of p-nitroaniline (pNA) moiety after it is cleaved from the labeled substrate Ac-IETD by caspase-8. The sample were measured in triplicate at the absorbance at 405 nm.
According to the manufactures instructions, the activities in supernatants were measured at 450 nm after each treatment. Each sample were performed in triplicate and the average was presented as ng/L.

Detection of neuronal apoptosis by flow cytometry
The apoptotic neurons were by detected by BD FACSCalibur flow cytometry (BD Bioscience, MA, USA) with excitation at 488 nm and emission at 530 nm [5]. Briefly, the 1×10 5 neurons were first stained with 10 µl Annexin V-fluorescein isothiocyanate (FITC) at 37 ℃ for 15 min and then counterstained with 5 µl propidium iodide (PI) for 30 min in the dark. The fluorescence was excitation at 488 nm and emission at 530 nm. Each sample was prepared in triplicate.

Statistical analysis
The data were expressed as the mean±standard deviation (SD) and analyzed using SPSS 19.0 software (SPSS, Chicago,USA). Statistical comparisons between two groups were assessed by t tests or Mann-Whitney tests, whereas comparisons among three or more groups were performed one-or two-way ANOVA followed by the Tukey-Kramer test. A P value <0.05 was considered statistically significant.

Temporal changes in motor dysfunction and spinal CAPN subtype expression post IR
All rats exhibited normal motor function before undergoing IR surgery. As shown in Figure 1A, compared with sham-operated rats, the rats in the IR groups displayed

IR-induced aberrant spinal miR-137-3p expression and negative regulation of CAPN-2 expression in vivo
Microarray analysis showed that several aberrant miRs were greatly dysregulated in injured spinal cords at 4 h post IR (Figure 2A). Among these miRs, miR-137-3p has been indicated to be closely associated with neurodevelopment and CNS disease and to be highly expressed in the brain [30,33]. Thus, we hypothesized that miR-137-3p was also widely expressed in spinal cord tissues and confirmed that it This negative target interaction was further confirmed by a luciferase reporter assay, in which the miR-137-3p mimic significantly decreased the luciferase activity in cells containing the wild-type (WT) 3¢-UTR but not the mutated (MT) 3¢-UTR ( Figure 2C, P < 0.05). As we previously reported, we assessed the potential in vivo interactions by intrathecal pretreatment with synthetic miRs [5,6]. Consistently, compared with the IR group, the group given intrathecal injection of miR-137-3p mimic had significantly lower CAPN-2 protein and mRNA expression, whereas the group pretreated with miR-137-3p inhibitor injection had significantly higher CAPN-2 expression ( Figure 2D-E, P<0.05). As expected, the synergistic upregulation in CAPN-2 expression post IR that occurred with injection of rr-CALP2, a recombinant CAPN-2 that specifically upregulates CAPN-2 expression, was partly reversed by miR-137-3p mimic injection (P<0.05). No significant changes were detected when injected with miR-137-3p NC injection had no significant effects on CAPN-2 expression (P>0.05).

Effects of the miR-137-3p/CAPN-2 pair on IR-induced hindlimb motor dysfunction
To further clarify the regulatory roles of the miR-137-3p/CAPN-2 pair in vivo, the hindlimb motor function was assessed ( Figure 2F). As expected, compared with baseline and sham-operated rats, all IR-injured rats showed obvious hindlimb motor dysfunction during the reperfusion period (P<0.05). Compared with the timematched injured rats in the IR group, the rats injected with the miR-137-3p mimic exhibited higher average Tarlov scores, whereas the rats injected with the miR-137-3p inhibitor showed lower Tarlov scores (P<0.05). Likewise, in conjunction with the mRNA and protein levels of CAPN-2, rr-CALP2 injection reversed the improvement in motor function, indicated by Tarlov scores comparable to those for the IR group (P>0.05). There were no detectable differences between the IR-injured rats with or without miR-137-3p NC treatment at any observed time point (P>0.05). PTEN is a selective substrate for CAPN-2 [32]. Therefore, the OGD/R-induced changes in CAPN-2 expression and activity were further confirmed by assessment of PTEN at the same observed time points. As shown by representative images of double fluorescent staining, both PTEN and p35 fluorescent labels were predominantly distributed in the cytoplasm and nucleus of VSC 4.1 neurons ( Figure   3B). Consistent with previous studies [19,32]
Additionally, as the final executor of the apoptotic network, caspase-3 protein expression was measured in each treatment group at the same time point. The Western blotting results showed that the protein expression of caspase-3 changed in accordance with the change in the protein expression of caspase-8 ( Figure 5C, D). In addition, changes in LDH release from injured neurons exhibited the same pattern as changes in flow cytometry among the treatment groups, suggesting an important role of the miR-137-3p/CAPN-2 pair in regulating subsequent Cdk5 and caspase-8 overactivation and neuronal apoptosis ( Figure 6C, P<0.05). No significant differences were observed between the injured neurons with or without miR-137 NC treatment (P > 0.05).

Discussion
Our previous studies revealed that IR-induced dysregulated miRs in spinal cords played important roles in driving pathogenesis during the reperfusion period and finally caused severe motor and sensory dysfunction [5,6,29]. Recently, an increasing number of studies have suggested that miR-based gene therapy is a promising treatment for neurological recovery by effectively preventing neuronal apoptosis. In the present study, we investigated the function and mechanisms of miR-137-3p and its target CAPN-2 in both in vivo and in vitro IR models to better understand the pathophysiological mechanisms and find better treatments in the clinic.
Previous studies have explored the crucial roles of CAPNs in determining neuronal survival during CNS injury [16, 17-19, 27, 30]. Some studies have observed prosurvival roles for CAPN-1 activation but destructive roles for CAPN-2 activation in retinal ganglion cell degeneration [17,19]. However, in a recent rat contusive spinal cord injury model, CAPN-1 was activated and contributed to impaired locomotor function [16]. These differences might be explained by the preferential participation of CAPN isoforms to different cellular functions even in different substructures of the same cells [14,35]. Thus, the contradictory roles of the activation of these two isoforms provide great challenges for our detailed understanding of the pathophysiological mechanisms of IR injury. In this context, we examined CAPN-1 and CAPN-2 protein expression and assessed motor function using Tarlov scores at several time points post IR. Our results showed that only the temporal expression patterns of CAPN-2 were negatively correlated with IR-induced motor dysfunction, with initial significant differences detected at 0.5 h post IR and reaching the maximum at 4 h post IR (Fig. 1). This finding was consistent with a previous study on spinal cord injury, in which the progressively increased calpain content in the lesion was first detected as early at 30 min after trauma and reached a 91% increase at 4 h after trauma [36]. We further explored the cellular distribution of CAPN-2 in major spinal cord cell types by double immunofluorescence at 4 h post IR when CAPN-2 expression reached its peak. Representative images and quantification showed that CAPN-2 was primarily expressed in spinal neurons, indicating that neuronal CAPN-2 might be the major effector during the reperfusion period.
MiRs are small RNA molecules that negatively regulate gene expression by binding with the 3′-UTR of targets via complementary base pairs [28]. MiRs are widely expressed in the CNS and have been implicated in multiple pathological processes, including IR [29]. We have suggested that some miRs, including miR-187-3p, miR-27 and miR-125b, that are highly expressed in spinal cords may provide new insights for research and clinical treatment [5,6,29]. Likewise, in this study, using miR microarray analysis and luciferase assay, we found that miR-137-3p expression was greatly changed at 4 h post IR and had a target interaction with CAPN-2 (Fig. 2).
Continuous intrathecal injection of synthetic miRs before IR was previously reported to effectively regulate miR expression and corresponding target gene expression in vivo [ 5,6,29]. Given the complicated cellular crosstalk in vivo, we defined the overall effects of the miR-137-3p/CAPN-2 gene pair by assessing motor function in a rat model. As expected, in contrast to the decrease in CAPN-2 protein expression, intrathecal injection of miR-137-3p mimic greatly increased Tarlov scores, indicating decreased motor dysfunction, whereas treatment with miR-137-3p inhibitor or NC did not have these effects. Moreover, to further confirm the above interaction, a direct delivery of exogenous CAPN-2 (rr-CALP2) was intrathecally performed.
Consistent with the ability of exogenous CAPN-2 to activate the intrinsic CAPN-2 in uninjured nerves [35,37], the synergistic increase in CAPN-2 expression caused by exogenous rr-CALP2 injection was significantly prevented by miR-137-3p mimic, as comparable CAPN-2 protein levels and similar behavioral assessments were observed throughout the reperfusion period in injured rats with or without combined injection with rr-CALP2 (Fig. 2). These findings suggested that miR-137-3p acted as a functional regulator of CAPN-2 in the spinal cord.
As a trigger, CAPN-2 requires millimolar (0.250-0.750 mM) calcium concentrations for its activation [13]. Thus, parallel in vitro experiments were performed to better  [14,38]. Consistently, our current data showed that the synthetic miR-137 mimic also prevented the increase in the intracellular [Ca 2+ ] and decreased CAPN-2 expression and activity in stressed neurons. In addition, PTEN is a selective substrate for CAPN-2 [32] and is thus widely used to quantitatively measure neuronal CAPN-2 activity in vivo and in vitro [19,32]. As expected, PTEN and CAPN-2 were identically distributed in the cytoplasm of neurons, but their protein levels and immunoreactivities changed in opposite directions in response to transfection with the different treatments.
Decreased PTEN expression indicates an increase in CAPN-2 activation; therefore, these results suggest that OGD/R-induced CAPN-2 overactivation was regulated by synthetic miR.
Previous in vitro and in vivo studies have shown that CAPN-2 upregulation triggers neuronal apoptosis [20,39]. These studies found that activated CAPN-2 directly and precisely cleaves its substrate, membrane-bound protein p35 into p25, which consequently results in Cdk5 activation in cultured primary neurons and retinal ganglion cells [13,20,39]. Similarly, our in vitro immunofluorescent staining ( Fig. 4) revealed that the cytoplasmic and nuclear labels for p35 and p25 fluorescence completely overlapped with the CAPN-2 labels in VSC4.1 neurons. In a previous study, during polybrominated diphenyl ether-153-induced neuronal apoptosis, p35 was found to accumulate in the perinuclear region and plasma membrane, and p25 was localized in both the cytoplasm and nucleus [20]. Given that the p35/Cdk5 complex mainly exerts its function in the nucleus, these mislocalizations of p35 and p25 might be a sign of the formation of the p25/Cdk5 complex [20,40,41]. Additionally, our Western blotting results showed that the expression pattern of Cdk5 protein in each group changed in agreement with the levels of the p25 and CAPN-2 protein and opposite to the levels of the p35 protein ( Fig. 4). On the other hand, the changes in p35 and p25 activities in accordance with the protein level of CAPN-2 in neurons transfected with miR-137-3p mimic or NC also demonstrated that the conversion of p35 into p25 requires the presence of CAPN-2. Additionally, selective inhibition of CAPN-2 expression has been shown to preserve both the structure and function of vulnerable neurons [14,17,19]. In this study, pretreatment with miR-137-3p and Cdk5 inhibitor comparably inhibited the number of neurons located in the A4 and A2 quadrants of the flow cytometry dotplot graphs and LDH leakage in culture medium after OGD/R insult (Fig. 6). These findings all support the hypothesis that the dynamic localization of p35 and p25 are considered a signal for p25/Cdk5 activation-induced apoptosis.
Caspase-cascade activation has been suggested to be central for neuronal apoptosis during IR injury [5,9]. Acting as the apical member of the caspase family, caspase-8 overactivation has been shown to hold special importance in controlling a series of broad caspase-cascade networks by initiating downstream caspase activation, such as activation of caspase-3 [24,42]. Meanwhile, as a cysteine protease, caspase-8 requires proteolytic cleavage before activation. Under normal conditions, caspase-8 forms the p35-caspase-8 complex through a covalent bond with the N terminus of p35 [26]. The N terminus of p35 is known to be a major element necessary for preserving the p35-caspase-8 complex, and the baculovirus p35 protein has been demonstrated to effectively block the apoptosis cascade by preventing caspase-8 proteolysis and activation [24,26]. In contrast, CAPN-2 overexpression-mediated p35 cleavage may cause conformational changes in p35 and consequently initiate aberrant caspase-8 activation. In agreement with the above hypothesis, a previous study showed that CAPN-2 was required to initiate endoplasmic reticulum stressinduced apoptosis mediated by a caspase-8-dependent pathway [25]. Similar in our in vitro immunofluorescence and Western blotting results, the fluorescent labels for p35 in the cytoplasm were identically distributed with the caspase-8 labels in neurons, and the protein expression of p35 and caspase-8 were changed in opposing directions when CAPN-2 expression was downregulated by pretreatment with miR-137-3p mimic (Fig. 5). In addition, our hypothesis that CAPN-2 initiates caspase-8-mediated apoptosis was additionally supported by the similar results observed in neurons transfected with miR-137-3p mimic and those transfected with the caspase-8-specific inhibitor Z-IETD-FMK. Both treatments exhibited comparable inhibitory effects on the protein level and activity of caspase-8, the number of injured neurons in the A4 and A2 quadrants of the dot-plot graphs and the amount of LDH leakage (Figs. 5 and 6). Acting as the final executor of the caspase family, caspase-3 expression was equally decreased in neurons transfected with miR-137-3p mimic and Z-IETD-FMK, supporting the assumption that CAPN-2-induced caspase-8 activation may simultaneously lead to caspase-3 activation. Indeed, similar observations were made in a study of hydrogen peroxide-induced apoptotic pathways [25].
Of note, transfection with the Cdk5 inhibitor, roscovitine, had no significant effects on the expression of CAPN-2 or p35, possibly due to the eventual modulation of the CAPN-2/p35-p25/Cdk5 pathway [20]. Roscovitine is known to be highly specific for Cdk5 and is therefore unlikely to influence the activity of the apical members of the signaling pathway [20]. Undoubtedly, whether caspase-8 directly activates caspase-3 by proteolytic cleavage or by activating caspase-3 activators needs to be elucidated in further study.

Conclusion
This study highlights the roles of CAPN-2 in triggering motor dysfunction after spinal cord IR injury and investigates the target interactions with miR-137-3p in both in vivo and in vitro models. The effects of the miR-137-3p/CAPN-2 gene pair on neuronal apoptosis may be attributed to CAPN-2 inhibition resulting in an inhibition of the cleavage of the substrate p35, consequently preventing the overactivation of p25/Cdk5 and the initiation of the caspase-8-mediated caspase cascade.

Ethics approval and consent to participate
The animal experiments in this study were approved by the Ethics Committee of  Modulation of VSC4.1 neuronal apoptosis by the miR-137-3p/CAPN-2 gene pair after OGD/R. A