Treatment with extracellular HSP70/HSC70 protein can reduce polyglutamine toxicity and aggregation.
The accumulation of insoluble protein aggregates is a feature of neurodegenerative disease. Overexpression of Heat Shock Protein 70 (HSP70) can protect cells with protein aggregates from apoptosis. Another trait of HSP70 is its ability to cross the plasma membrane. Therefore, we purified a preparation of HSP70/HSC70 from bovine muscle and used it in a model of Huntington's disease. Human neuroblastoma SK-N-SH cells were transfected with huntington exon 1 with short (25) or long (103) CAG trinucleotide repeats coupled to green flourescent protein (GFP). Cells transfected with the long polyCAG repeat had insoluble protein aggregates and died through apoptosis. Biotinylated HSP70/HSC70 incorporated into the culture medium appeared inside the cells within 3-6 h of incubation. This incorporation correlated with a reduction in apoptotic cells by 40-50%. Confocal microscopy revealed that labelled internalized HSP70/HSC70 co-localized with the polyglutamine inclusions. The measurement of the number and size of inclusions showed that HSP70/HSC70 was able to reduce both these parameters. A filter trap assay and immunoblotting demonstrated that the introduction of HSP70/HSC70 also decreased protein aggregation. Together with earlier data on the effects of exogenously administered HSP70/HSC70 on cultured cells and on animals, these data show that preparations based on HSP70 may have some potential as therapies for a variety of neurodegenerative pathologies.
Novoselova
Tatiana V
TV
Laboratory of Cell Protection Mechanisms, Institute of Cytology Russian Academy of Science, St Petersburg, Russia.
Margulis
Boris A
BA
Novoselov
Sergey S
SS
Sapozhnikov
Alexander M
AM
van der Spuy
Jacqueline
J
Cheetham
Michael E
ME
Guzhova
Irina V
IV
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western / Q000379:methods; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002449:Cell Aggregation / Q000187:drug effects / Q000502:physiology; D002452:Cell Count / Q000379:methods; D045744:Cell Line, Tumor; D048429:Cell Size / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles; D018613:Microscopy, Confocal / Q000379:methods; D009447:Neuroblastoma; D010455:Peptides / Q000037:antagonists & inhibitors / Q000633:toxicity*; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection / Q000379:methods; D019680:Trinucleotide Repeat Expansion / Q000502:physiology
D004791:Enzyme Inhibitors; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D007211:Indoles; D010455:Peptides; D049452:Green Fluorescent Proteins; C097188:polyglutamine; C007293:DAPI; D020169:Caspases; D019311:Staurosporine