Fig. 4.

An FTase inhibitor reduces α-synuclein accumulation and toxicity by a mechanism requiring the expression and activity of UCH-L1. (A) Cell viability was measured by MTT assay in SH-SY5Y cells infected with control virus (Con), virus expressing LacZ, or virus expressing α-synuclein (Syn) and treated with either DMSO or 100 nM FTI-277 (n = 3; *, P < 0.05). Values represent mean ± SE throughout. (B) α-Synuclein levels were quantified by Western blotting and normalization to actin in SH-SY5Y cells infected with α-synuclein-expressing virus and treated with either DMSO or 100 nM FTI-277 (n = 3; *, P < 0.05). (C) SH-SY5Y cells were transfected with either nonspecific (NS) or UCH-L1-specific siRNA oligos, followed by infection with α-synuclein-expressing virus and treatment with either DMSO or 100 nM FTI-277. Cell viability was assessed by flow cytometry and normalization against control cells that had undergone the same treatment, except that they were infected with vector instead of α-synuclein. UCH-L1 levels were analyzed by Western blot in a portion of cells before infection (n = 4; *, P < 0.01). (D) SH-SY5Y cells infected with α-synuclein-expressing virus were treated with 100 nM FTI-277, LDN-57414 (LDN), or their combination. Viable cells were counted by trypan blue staining (n = 3; *, P < 0.05) (E) Primary midbrain cultures were infected with either vector (Vec) or α-synuclein-expressing virus (Syn) and treated with DMSO or 100 nM FTI-277. Following treatment, tyrosine hydroxylase-positive (TH+) neurons were counted by using immunofluorescence microscopy. Four fields were counted and averaged in each of 3 independent experiments (n = 3; *, P < 0.01). (F) Endogenous α-synuclein levels were quantified as in B in cultured primary cortical neurons treated with either DMSO or 100 nM FTI-277 (n = 4; *, P < 0.05). Tubulin was used for normalization.