Abstract
BackgroundAbnormal 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.ResultsResults 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.ConclusionsThese 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.
Highlights
Abnormal accumulation of neuronal intermediate filament (IF) is a pathological indicator of some neurodegenerative disorders
Establishment of pEGFP-Peripherin stable cell lines To examine the effect of exogenous peripherin on neuronal IF structures and neuronal functions, the cDNA of rat peripherin tagged with enhanced green fluorescence protein (EGFP-Peripherin) was first transfected into PC12 cells by electroporation
We found that protein levels of nonphosphorylated and phosphorylated NF-H and NF-M were higher in pEGFP-Peripherin cells than that observed in PC12 cells
Summary
Abnormal accumulation of neuronal intermediate filament (IF) is a pathological indicator of some neurodegenerative disorders. In amyotrophic lateral sclerosis (ALS) patients, peripherin together with neuronal IFs were detected in the majority of abnormal IF inclusion bodies in mature or aging motor neurons [14,15,16]. A transgenic study showed that mice overexpressing peripherin developed a late-onset motor neuron death and IF inclusions resembling axonal spheroids found in ALS patients [18]. A recent study indicated that overexpressing peripherin can cause defective axonal transport of type IV neurofilament proteins in cultured dorsal root ganglion (DRG) neurons from peripherin transgenic embryos [20]. These studies suggest that peripherin may be an important etiological factor in neurodegenerative diseases
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