Fig. 7. Lithium reduces the vulnerability of CbCln3^{Δex7/8/Δex7/8} cerebellar cells to starvation-induced cell death.

(A) Effect of lithium on the sensitivity of CbCln3^{Δex7/8/Δex7/8} cerebellar cells to starvation-induced cell death. CbCln3^+/+ and CbCln3^{Δex7/8/Δex7/8} cerebellar cells were incubated in amino acid-free medium for 12 h in the absence or presence of LiCl, after which the incidences of cell death were determined using a Live/Dead cell assay. Bars indicate means ± S.D. (n > 3). (B) Suppression of CLN3 mutation-induced cell death by lithium. SH-SY5Y cells were transiently transfected with pcDNA, pCLN3Δ1, pCLN3Δ2, pCLN3Δ3, pCLN3 AS or pCLN3, after which cells were treated for 24 h with 10 mM NaCl or 10 mM LiCl. Cell death assays were performed as in (A). (C and D) Suppression of amino aciddeprivation- induced cell death by L390,330 IMPase inhibitor. CbCln3^+/+ (C) and CbCln3^{Δex7/8/Δex7/8} (D) cerebellar cells were incubated for the indicated times in amino acid-free medium in the absence or presence of 10 mM NaCl, 10 µM SB216763, 10 µM L690,330 or 10 mM LiCl. Cell death assays were performed as in (A) and values indicate means ± S.D. (n = 3). (E) Reducing IMPase expression suppresses amino acid deprivation-induced cell death. CbCln3^+/+ and CbCln3^{Δex7/8/Δex7/8} cells were transiently transfected for 48 h with IMPase1 shRNA, IMPase2 shRNA or both, after which they were incubated for 9 h with complete medium or amino acid-free medium (HBSS). Cell death assays were performed as in (A) and values indicate means ± S.D. (n = 3) (left). Expression levels of IMPase1 and IMPase2 were determined by Western blotting using IMPase1 and IMPase2 antibodies (right). Asterisks indicate significant difference from control (p < 0.05).