Knockdown of m‐calpain increases survival of primary hippocampal neurons following NMDA excitotoxicity

Abstract

The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms to the various forms of injury has not been determined as available calpain inhibitors are not isoform-specific. In this study, we evaluated the relative role of m-calpain and l-calpain in a primary hippocampal neuron model of NMDA-mediated excitotoxicity. Baseline mRNA expression for the catalytic subunit of m-calpain (capn2 ) was found to be 50-fold higher than for the l-calpain catalytic subunit (capn1) based on quantitative real-time PCR. Adeno-associated viral vectors designed to deliver short hairpin RNAs targeting capn1 or capn2 resulted in 60% and 90% knockdown of message respectively.Knockdown of capn2 but not capn1 increased neuronal survival after NMDA exposure at 21 days in vitro. Nuclear translocation of calpain substrates apoptosis inducing factor, p35/p25 and collapsin response mediator protein (CRMP) 2-4 was not detected after NMDA exposure in this model. However, nuclear translocation of CRMP-1 was observed and was prevented by capn2 knockdown. These findings provide insight into potential mechanisms of calpain-mediated neurodegeneration and have important implications for the development of isoform-specific calpain inhibitor therapy.

Gel-based Protease Proteomics for Identifying the Novel Calpain Substrates in Dopaminergic Neuronal Cell

“…Potentially Protective Effect for ASNA1, Optineurin, and Peripherin in MPP ϩ -induced Cell Death-Previous reports have indicated that calpain activation following neurotoxin challenge is related to various cell death signaling pathways and that many calpain-cleaved substrates play a critical role in maintaining cell survival under physiological conditions (23,28,29). We have also demonstrated that MPP ϩ -mediated cell death is blocked in the presence of calpeptin and overexpressed calbindin-D28K (15,24).…”

Calpains and neuronal damage in the ischemic brain: The swiss knife in synaptic injury

“…The excitotoxic response resulting from the excessive influx of Ca 2+ through NMDAR channels, with a consequent [Ca 2+ ]i overload, is partly mediated by activation of calpains (Bano et al, 2005;Bevers et al, 2009;Curcio et al, 2015;Roberts-Lewis et al, 1994;Seubert et al, 1989;Wei et al, 2012;Zhou and Baudry, 2006). Calpain activity was indeed shown to contribute to the demise process upon exposure of different types of neurons to toxic concentrations of NMDA (D'Orsi et al, 2012;Jang et al, 2009;Wang and Huang, 2012;Xu et al, 2007;Zhou and Baudry, 2006).…”

Calpains Are Downstream Effectors ofbax-Dependent Excitotoxic Apoptosis

“…Moreover, calpain inhibition was neuroprotective in in vivo models of excitotoxic injury (Wu et al, 2004), ischemic stroke, and neurodegeneration (Lee et al, 1991;Rami and Krieglstein, 1993;Bartus et al, 1994;Hong et al, 1994). It has also been reported that knockdown of calpain II (mcalpain) increased survival of primary hippocampal neurons after NMDA excitotoxicity (Bevers et al, 2009). Importantly, previous studies did not fully address the question under which cell-death conditions calpain inhibition is neuroprotective and when in the cell death cascade calpains became activated.…”