Neurotoxicity Research

, Volume 19, Issue 3, pp 374–388

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

  • Richard A. Harbison
  • Kristen R. Ryan
  • Heather M. Wilkins
  • Emily K. Schroeder
  • F. Alexandra Loucks
  • Ron J. Bouchard
  • Daniel A. Linseman


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.


Cerebellar granule neurons MPP(+) Calpain Oxidative stress Neurotoxicity Bcl-2 


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Richard A. Harbison
    • 1
  • Kristen R. Ryan
    • 2
  • Heather M. Wilkins
    • 1
  • Emily K. Schroeder
    • 3
  • F. Alexandra Loucks
    • 4
  • Ron J. Bouchard
    • 3
  • Daniel A. Linseman
    • 1
    • 3
    • 5
  1. 1.Department of Biological Sciences and Eleanor Roosevelt InstituteUniversity of DenverDenverUSA
  2. 2.Toxicology Program, Department of Pharmaceutical SciencesUniversity of Colorado DenverAuroraUSA
  3. 3.Research ServiceVeterans Affairs Medical CenterDenverUSA
  4. 4.Neuroscience ProgramUniversity of California San FranciscoSan FranciscoUSA
  5. 5.Division of Clinical Pharmacology and Toxicology, Department of MedicineUniversity of Colorado DenverAuroraUSA

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