Neuroprotective adeno-associated virus Bcl-xL gene transfer in models of motor neuron disease.
Recent work implicates excitotoxicity-induced apoptosis as the mechanism triggering motor neuron death in amyotrophic lateral sclerosis (ALS). Our laboratory has previously utilized glutamate excitotoxicity in vitro to study this process. The present experiment tests whether overexpression of the gene for Bcl-xL can inhibit excitotoxicity in this model system. To track Bcl-xL expression, the gene for green fluorescent protein (GFP) was inserted in-frame, upstream of the Bcl-xL gene. The GFP-Bcl-xL gene was then cloned into an adeno-associated viral (AAV2) vector. GFP expression in both SH-SY5Y and embryonic day 15 (E15) motor neurons (MNs) peaked 48 hours after infection. Bcl-xL expression in SH-SY5Y cells significantly reduced terminal deoxy-UTP nick-end labeling (TUNEL)-positive cells and maintained cell density after glutamate exposure. Similarly, Bcl-xL expression inhibited the development of TUNEL staining in E15 MNs and supported cell density after glutamate exposure. These findings suggest that AAV-mediated expression of genes for antiapoptotic proteins may provide a means for ALS gene therapy.
Garrity-Moses
Mary E
ME
Department of Neurological Surgery, Lerner Research Institute, Cleveland Clinic Foundation, NB 2 120, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA.
Teng
Qingshan
Q
Liu
James
J
Tanase
Diana
D
Boulis
Nicholas M
NM
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western / Q000379:methods; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D002478:Cells, Cultured; D000229:Dependovirus / Q000502:physiology*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000502:physiology; D018698:Glutamic Acid / Q000009:adverse effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D020287:In Situ Nick-End Labeling / Q000379:methods; D016472:Motor Neuron Disease / Q000235:genetics / Q000517:prevention & control*; D009447:Neuroblastoma; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014162:Transfection / Q000379:methods; D051020:bcl-X Protein / Q000235:genetics* / Q000378:metabolism*
D051020:bcl-X Protein; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases