Involvement of the miR-137-3pCAPN-2 Interaction in Ischemia-Reperfusion-Induced Neuronal Apoptosis through Modulation of p35 Cleavage and Subsequent Caspase-8 Overactivation

Abstract EN

Background.

Neuron survival after ischemia-reperfusion (IR) injury is the primary determinant of motor function prognosis.

MicroRNA- (miR-) based gene therapy has gained attention recently.

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 localization were detected within 12 h post IR.

Dysregulated miRs, including miR-137-3p, were identified by miR microarray analysis and confirmed by PCR.

A luciferase assay confirmed CAPN-2 as a corresponding target of miR-137-3p, and their modulation of motor function was evaluated by intrathecal injection with synthetic miRs.

CAPN-2 activity was measured by the intracellular Ca2+ concentration and mean fluorescence intensity in vitro.

Neuronal apoptosis was detected by flow cytometry and TUNEL assay.

The activities of p35, p25, Cdk5, and caspase-8 were evaluated by ELISA and Western blot after transfection with specific inhibitors and miRs.

Results.

The IR-induced motor dysfunction time course was closely associated with upregulated expression of the CAPN-2 protein, which was mainly localized in neurons.

The miR-137-3p/CAPN-2 interaction was confirmed by luciferase assay.

The 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 the miR-137-3p inhibitor reversed these effects.

Similar changes in the intracellular Ca2+ concentration, CAPN-2 expression, and CAPN-2 activity were observed when cells were exposed to oxygen-glucose deprivation and reperfusion (OGD/R) and transfected with synthetic miRs in vitro.

Moreover, double fluorescence revealed identical neuronal localization of CAPN-2, p35, p25, and caspase-8.

The decrease in CAPN-2 expression and activity was accompanied by the opposite changes in p35 activity and protein expression in cells transfected with the miR-137-3p mimic, roscovitine (a Cdk5 inhibitor), or Z-IETD-FMK (a caspase-8 inhibitor).

Correspondingly, the abovementioned treatments resulted in a higher neuron survival rate than that of untreated neurons, as indicated by decreases in the apoptotic cell percentage and p25, Cdk5, caspase-8, and caspase-3 protein expression.

Conclusions.

The miR-137-3p/CAPN-2 interaction modulates neuronal apoptosis during IR injury, possibly by inhibiting CAPN-2, which leads to p35 cleavage and inhibition of subsequent p25/Cdk5 and caspase-8 overactivation.