Calpain Plays a Central Role in 1-Methyl-4-phenylpyridinium (MPP(+))-Induced Neurotoxicity in Cerebellar Granule Neurons

Abstract

1-Methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity has previously been attributed to either caspase-dependent apoptosis or caspase-independent cell death. In the current study, we found that MPP(+) induces a unique, non-apoptotic nuclear morphology coupled with a caspase-independent but calpain-dependent mechanism of cell death in primary cultures of rat cerebellar granule neurons (CGNs). Using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay in CGNs exposed to MPP(+), we observed that these neurons are essentially devoid of caspase-dependent DNA fragments indicative of apoptosis. Moreover, proteolysis of a well recognized caspase-3 substrate, poly (ADP ribose) polymerase (PARP), was not observed in CGNs exposed to MPP(+). In contrast, calpain-dependent proteolysis of fodrin and pro-caspases-9 and -3 occurred in this model coupled with inhibition of caspase-3/-7 activities. Notably, several key members of the Bcl-2 protein family appear to be prominent calpain targets in MPP(+)-treated CGNs. Bid and Bax were proteolyzed to truncated forms thought to have greater pro-death activity at mitochondria. Moreover, the pro-survival Bcl-2 protein was degraded to a form predicted to be inactive at mitochondria. Cyclin E was also cleaved by calpain to an active low MW fragment capable of facilitating cell cycle re-entry. Finally, MPP(+)-induced neurotoxicity in CGNs was significantly attenuated by a cocktail of calpain and caspase inhibitors in combination with the antioxidant glutathione. Collectively, these results demonstrate that caspases do not play a central role in CGN toxicity induced by exposure to MPP(+), whereas calpain cleavage of key protein targets, coupled with oxidative stress, plays a critical role in MPP(+)-induced neurotoxicity. Our findings underscore the complexity of MPP(+)-induced neurotoxicity and suggest that calpain may play a fundamental role in causing neuronal death downstream of mitochondrial oxidative stress and dysfunction.

Mechanisms to prevent caspase activation in rotenone-induced dopaminergic neurodegeneration: role of ATP depletion and procaspase-9 degradation

“…Along with the line of recent report indicating a central role of calpain in MPP ? -induced neurotoxicity in cerebellar granule neurons [40], our data seem to extend previous observations of cross-talk among activated proteases as typified by the result that protease-mediated degradation and inactivation of procaspase-9 blocked formation of an adequate apoptosome complex and directly contributed to caspase-independent necrotic phenomena after the mitochondrial release of cytochrome c in rotenone-or MPP ? -induced neurodegeneration.…”

C-terminal binding proteins are essential pro-survival factors that undergo caspase-dependent downregulation during neuronal apoptosis

“…In response to apoptosis induced by toxin B or the related Clostridium sordellii lethal toxin which also inhibits Rac GTPase (Just et al, 1996), the observed downregulation of CtBP1 and CtBP2 was significantly blocked by caspase inhibition with BOC or zVAD but was unaffected by proteasome inhibition with MG132 (Figures 5B and 5C). In contrast to 5K and the Clostridial toxins which are each known to activate caspases in CGNs, we have previously shown that CGN death induced by MPP+ occurs through a caspase-independent mechanism (Harbison et al, 2011). In agreement with this, the downregulation of CtBP1 and CtBP2 induced by MPP+ in CGNs was unaltered by BOC but was significantly inhibited by MG132 (Figure 5D).…”

Calpain inhibition protected spinal cord motoneurons against 1-methyl-4-phenylpyridinium ion and rotenone

“…Relatively low doses of MPP + (1-150 μM) were capable of inducing damage to cells in culture, including rat cerebellar granule neurons (Du et al, 1997; Gonzalez-Polo et al, 2001; Harbison et al, 2011), MN9D (Choi et al, 1999; Chu et al, 2005; Luo et al, 2009), and rat ventral mesencephalic neurons (Dodel et al, 1998; Hartmann et al, 2000; Han et al, 2003, Jourdi et al, 2009), while almost 1000-fold higher doses of MPP + (mM range) were found deleterious for SH-SY5Y, PC12, N-27, and NT2 cells (Anantharam et al, 2007; Domingues et al, 2008; Wales et al, 2008; Cheng et al, 2009; Kim et al, 2009). These findings suggested diverse intracellular signaling pathways in MPP + -neurotoxicity.…”