Here, we tested our hypothesis that the neuroprotective effects of 3 MA may have been achieved through cas pase 3 suppression. To assess caspase 3 activation, we the examined the proteolysis of an endogenous caspase 3 substrate and caspase 3 enzymatic activity assay. The aII spectrin breakdown profile using total anti aII spectrin antibody showed an increase of the caspase 3 generated spectrin breakdown product of 120 kDa at 24 h following treatment of cere bellar neurons with NMDA in culture. Increases in the calpain generated SBDP150 and SBDP145 were also observed at 24 hours with NMDA treated cultures, sug gesting the involvement of calpains as well. Staurosporine treated cultures were used as posi tive controls for caspase 3 activation and SBDP120 gen eration.
To further confirm that the 120 kDa band was caspase 3 generated, immunoblots were analyzed using anti SBDP120 specific antibody developed in house. The blots confirmed the appearance of Inhibitors,Modulators,Libraries the SBDP120 at 24 hours in the NMDA treated cultures but not in the controls. 3 MA co treatment suppressed the increased SBDP120 levels to near normal levels. Densito metric analysis of the immunoblots showed a significant Inhibitors,Modulators,Libraries reduction in the caspase 3 mediated SBDP120 levels in NMDA 3 MA co treated cultures as compared to NMDA treated cells. Interestingly, calpain mediated SBDP150 and SBDP145 were not attenuated by 3 MA. To assay the caspase 3 protease activity N acetyl Asp Glu Val Asp AMC was incubated with protease inhibitor free cell lysates under various conditions.
Cas pase 3 activity was significantly increased in NMDA treated cultures at 12 and 24 hours when compared to control cultures. On the other hand, this caspase 3 activity was significantly reduced by 3 MA co treatment when compared to NMDA treatment alone. ATG7 disruption results in neuroprotection following Inhibitors,Modulators,Libraries NMDA exposure Since 3 MA might have non autophagy related effects, ATG7 siRNA was generated and transfected into the neurons in culture to further investigate the effects of autophagy inhibition on NMDA neurotoxicity. First, we observed that atg 7 siRNA partially but significantly reduced Atg 7 protein levels as well as LC3 II levels at 72 h after siRNA treatment. Scrambled ATG7 siRNA had no effect. Inhibitors,Modulators,Libraries We then further analyzed the effects of ATG7 siRNA and scrambled ATG7 siRNA on NMDA exposure induced Inhibitors,Modulators,Libraries cell death, as measured by LDH release assay.
Silencing the Atg7 protein expression in neurons resulted in a significant reduction of LDH release in the medium compared to neurons transfected with NMDA with scrambled ATG7 siRNA or NMDA alone. ATG7 siRNA and scrambled siRNA alone did not significantly increase LDH above control cells. Since NMDA toxicity has been demonstrated to induce apoptotic cell death, we incu Y-27632 2HCL bated neurons with pan caspase inhibitor IDN6556 to study if we achieved neuroprotection.