Overexpression of HDJ-2 protects astrocytes from ischemia-like injury and reduces redistribution of ubiquitin staining in vitro.
HDJ-2, a member of the HSP40 family, functions as a cochaperone to promote protein folding both by binding to unfolded polypeptides and by regulating the activity of HSP70. This study tested whether HDJ-2 overexpression alone could provide significant protection from ischemia-like injury. Primary mouse astrocyte cultures were infected with an HDJ-2 encoding retroviral vector or control retrovirus lacking HDJ-2. Expression of HDJ-2 was confirmed by immunohistochemical staining and immunoblotting. Injury paradigms to mimic ischemia were glucose deprivation (GD) for 24 hours and oxygen-glucose deprivation (OGD) for 8 hours. Cell death was determined by trypan blue exclusion and cell counting. Overexpression of HDJ-2 alone significantly reduced astrocyte injury after both GD and OGD, independent of an elevation in HSP70. To further search for the mechanism of HDJ-2 protection, cultured astrocytes allowed to recover 16 hours after 8 hours GD were labeled with a monoclonal antiubiquitin antibody that recognizes both free ubiquitin and ubiquitinated proteins. The immunolabeling pattern changed from a relatively even distribution in both nuclei and cytoplasm in control cells to heterogeneous aggregates and marked reduction of nuclear staining in most of the cells after GD. When HDJ-2 was overexpressed, the number of cells with evidence of protein aggregation was significantly reduced. Thus, blocking protein aggregation may be an important mechanism by which HDJ-2 protects cells from damage.
Qiao
Yanli
Y
Department of Anesthesia, Stanford University School of Medicine, California 94305, USA.
Ouyang
Yi-Bing
YB
Giffard
Rona G
RG
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D002545:Brain Ischemia / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002478:Cells, Cultured; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000378:metabolism*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D051379:Mice; D015427:Reperfusion Injury / Q000378:metabolism*; D013194:Staining and Labeling; D025801:Ubiquitin / Q000378:metabolism*
D002352:Carrier Proteins; C107481:DNAJA1 protein, human; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D025801:Ubiquitin