Survival effects of pigment epithelium-derived factor expressed by a lentiviral vector in rat cerebellar granule cells.
We have previously shown that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cells (CGCs), by blocking apoptotic death, and can also protect these cells against glutamate-induced neurotoxicity. In preparation for gene therapy studies, pseudotyped HIV-1-based lentiviral vectors containing the PEDF gene, as well as either green fluorescent protein or beta-galactosidase, were prepared. These bicistronic vectors are unique in that they express two genes efficiently under one promoter. Primary cell cultures of CGCs from postnatal day 8 rats were infected with the vectors encoding PEDF. RT-PCR demonstrated expression of mRNA and Western blot analysis confirmed that infected CGCs secrete PEDF protein to the medium. Assays for cell survival demonstrated that PEDF-infected cells were significantly more protected compared with mock-infected controls for 6-8 days in culture, as well as against induced apoptosis. The PEDF vectors expressing tat (trans-acting transcription factor) provided more protection than the tat(-) vectors. These results demonstrate that while the lentiviral vectors expressing PEDF are as neuroprotective as the protein itself for CGCs, the vectors have the advantage of providing long-lasting expression of PEDF protein, which will be more effective in in vivo studies. The present results suggest that this system may be useful for gene therapy for neurodegenerative disorders.
Nomura
T
T
Neurotrophic Factors Section, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Md. 20892-4126, USA.
Yabe
T
T
Mochizuki
H
H
Reiser
J
J
Becerra
S P
SP
Schwartz
J P
JP
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D005136:Eye Proteins; D018507:Gene Expression Regulation, Developmental; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics; D005822:Genetic Vectors; D007150:Immunohistochemistry; D007763:Lac Operon; D016086:Lentivirus; D009414:Nerve Growth Factors; D011506:Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015843:Serpins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D015696:Gene Products, tat; D009414:Nerve Growth Factors; D011506:Proteins; D012333:RNA, Messenger; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor