title: A neuronal death model: overexpression of neuronal intermediate filament protein peripherin in PC12 cells.
abstract: BACKGROUND
  Abnormal accumulation of neuronal intermediate filament (IF) is a pathological indicator of some neurodegenerative disorders. However, the underlying neuropathological mechanisms of neuronal IF accumulation remain unclear. A stable clone established from PC12 cells overexpressing a GFP-Peripherin fusion protein (pEGFP-Peripherin) was constructed for determining the pathway involved in neurodegeneration by biochemical, cell biology, and electronic microscopy approaches. In addition, pharmacological approaches to preventing neuronal death were also examined.
  
  
  RESULTS
  Results of this study showed that TUNEL positive reaction could be detected in pEGFP-Peripherin cells. Swollen mitochondria and endoplasmic reticulum (ER) were seen by electron microscopy in pEGFP-Peripherin cells on day 8 of nerve growth factor (NGF) treatment. Peripherin overexpression not only led to the formation of neuronal IF aggregate but also causes aberrant neuronal IF phosphorylation and mislocation. Western blots showed that calpain, caspase-12, caspase-9, and caspase-3 activity was upregulated. Furthermore, treatment with calpain inhibitor significantly inhibited cell death.
  
  
  CONCLUSIONS
  These results suggested that the cytoplasmic neuronal IF aggregate caused by peripherin overexpression may induce aberrant neuronal IF phosphorylation and mislocation subsequently trapped and indirectly damaged mitochondria and ER. We suggested that the activation of calpain, caspase-12, caspase-9, and caspase-3 were correlated to the dysfunction of the ER and mitochondria in our pEGFP-Peripherin cell model. The present study suggested that pEGFP-Peripherin cell clones could be a neuronal death model for future studies in neuronal IFs aggregate associated neurodegeneration.
authors.affiliation: Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Jen-Ai Road, Taipei, 100, Taiwan.
authors.forename: Wen-Ching
authors.initials: WC
authors.lastname: Lee
authors.forename: Yun-Yu
authors.initials: YY
authors.lastname: Chen
authors.forename: Daphne
authors.initials: D
authors.lastname: Kan
authors.forename: Chung-Liang
authors.initials: CL
authors.lastname: Chien
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D007381:Intermediate Filament Proteins / Q000235:genetics* / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria; D009414:Nerve Growth Factors / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D009474:Neurons / Q000502:physiology* / Q000648:ultrastructure; D016716:PC12 Cells; D064531:Peripherins; D010766:Phosphorylation; D051381:Rats; D014162:Transfection
chemical_list: D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D064531:Peripherins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002154:Calpain; D020169:Caspases