Knockdown of glutamate-cysteine ligase by small hairpin RNA reveals that both catalytic and modulatory subunits are essential for the survival of primary neurons

Glutathione deficiency is an early biochemical feature that occurs during apoptotic neuronal death associated with certain neurological disorders such as Parkinson disease. However, whether specific targeting of glutathione biosynthesis in neurons is sufficient to trigger neurodegeneration remains undetermined. To address this issue, we used a vector-based small hairpin RNA (shRNA) strategy to knock down each subunit of glutamate-cysteine ligase (GCL; gamma-glutamylcysteine synthetase), the heterodimeric enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Independent targeting of the catalytic and modulatory subunits by shRNA caused disruption of GCL as assessed by Northern and Western blotting, enzyme activity, and glutathione concentrations. Silencing each subunit in primary cortical neurons spontaneously elicited time-dependent apoptotic death, an effect that was synergistic with glutamate or nitric oxide treatment. Moreover, neuronal apoptosis by GCL knockdown was rescued by expressing the corresponding subunit full-length cDNA carrying silent mutations within the shRNA target cDNA sequence and by incubating neurons with gamma-glutamylcysteine or glutathione ethyl ester. In contrast, supplying glutathione precursors to neurons from co-cultured astrocytes did not prevent the apoptotic death triggered by GCL knockdown. Finally, overexpressing the catalytic (but not modulatory) GCL subunit full-length cDNA increased enzyme activity and glutathione concentrations, yielding neurons more resistant to glutamate- or nitric oxide-mediated apoptosis. Thus, specific and independent disruption of each subunit of GCL in neurons can be said to cause a primary decrease in glutathione that is sufficient to promote neurodegeneration.