Calpastatin overexpression attenuates amyloid-beta-peptide toxicity in differentiated PC12 cells.
Amyloid beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). Abeta is toxic to neurons, possibly through causing initial synaptic dysfunction and neuronal membrane dystrophy, promoted by increased cellular Ca(2+). Calpain (Ca(2+)-dependent protease) and caspase have been implicated in AD. Previously, we used calpain and caspase pharmacological inhibitors to study effects of Abeta25-35 (sAbeta) on neuronal-like differentiated PC12 cells. We reported that sAbeta-treated cells exhibited calpain activation and protein degradation (due to both calpain and caspase-8). We have now found that overexpression of the calpain specific inhibitor calpastatin in differentiated PC12 cells significantly inhibited the sAbeta-induced calpain activation and decreased the protease activity. Calpastatin overexpression inhibited the sAbeta-promoted degradation of fodrin, protein kinase Cepsilon, beta-catenin (membrane structural proteins and proteins involved in signal transduction pathways), and prevented the sAbeta-induced alteration of neurite structure (manifested by varicosities). Overexpression of calpastatin also inhibited Ca(2+)-promoted calpain activation and protein degradation; this is consistent with the notion that the Abeta-induced increase in calpain activity results from a rise in cellular Ca(2+), provided the calpastatin level is not so high as to strongly inhibit calpain. Carrying out transfection without selection allowed the comparison in the same culture of calpastatin-overexpressing with non-overexpressing cells. In cultures transfected with green fluorescent protein (GFP)-calpastatin plasmid, calpastatin overexpression (indicated by GFP-labeling) led to inhibition in sAbeta-induced membrane propidium iodide (PI) permeability, whereas non-transfected, GFP-unlabeled cells exhibited PI permeability. Overall, the results demonstrate that the effects of Abeta-toxicity studied here were attenuated to a large extent by calpastatin overexpression, indicating that the protease calpain is involved in Abeta-toxicity (obviating a primary, direct role for caspases). Increased expression of calpastatin and/or decrease in calpain may serve as one of the means for ameliorating some of the early symptoms of AD.
Vaisid
T
T
Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel.
Barnoy
S
S
Kosower
N S
NS
D016229:Amyloid beta-Peptides / Q000633:toxicity*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D008840:Microfilament Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism*; D016716:PC12 Cells / Q000187:drug effects; D010446:Peptide Fragments / Q000633:toxicity*; D011419:Propidium; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D051381:Rats; D014162:Transfection / Q000379:methods; D051176:beta Catenin / Q000378:metabolism; D013049:Spectrin
D016229:Amyloid beta-Peptides; D002118:Calcium; D002135:Calcium-Binding Proteins; D002154:Calpain; D002352:Carrier Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011419:Propidium; D051744:Protein Kinase C-epsilon; D051176:beta Catenin; D013049:Spectrin; C074636:amyloid beta-protein (25-35); C033668:calpastatin