alpha-synuclein promotes mitochondrial deficit and oxidative stress

Abstract

Abnormal accumulation of the presynaptic protein alpha-synuclein has recently been implicated in the pathogenesis of Alzheimer's and Parkinson's diseases. Because neurodegeneration in these conditions might be associated with mitochondrial dysfunction and oxidative stress, the effects of alpha-synuclein were investigated in a hypothalamic neuronal cell line (GT1-7). alpha-Synuclein overexpression in these cells resulted in formation of alpha-synuclein-immunopositive inclusion-like structures and mitochondrial alterations accompanied by increased levels of free radicals and decreased secretion of gonadotropin-releasing hormone. These alterations were ameliorated by pretreatment with anti-oxidants such as vitamin E. Taken together these results suggest that abnormal accumulation of alpha-synuclein could lead to mitochondrial alterations that may result in oxidative stress and, eventually, cell death.

Figure 1.

Characterization of patterns of murine α-synuclein expression in GT1-7 cells. A: Representative autoradiogram of the RPA showing murine α-synuclein mRNA levels in C (control nontransfected), AST (antisense transfected), VEC (vector-transfected), and ST (sense-transfected) cells. Protected fragment for murine α-synuclein was detected at 385 bp, whereas actin was detected at 85 bp. The left-most lane shows signal of undigested (u/d) radiolabeled riboprobes, whereas the other lanes contain riboprobes of RNase-digested samples. B: PhosphorImager analysis of murine α-synuclein mRNA signals (expressed as percent over control) showing that ST cells expressed ninefold over control, whereas AST cells expressed 50% of control levels. C: Representative autoradiogram of the Western blot showing that the α-synuclein antibody recognized a band at ∼18 kd. The left-most lane shows signal of the recombinant α-synuclein protein. C, control nontransfected; AST, antisense transfected; VEC, vector-transfected; and ST, sense-transfected cells. D: PhosphorImager analysis of α-synuclein signals from semiquantitative Western blots showing, in the cytosolic fractions, that ST cells expressed twofold over control, whereas AST cells expressed 50% of control levels.

Figure 2.

Confocal analysis of α-synuclein expression in transfected GT1-7 cells. Cells were double-immunolabeled with antibodies against murine α-synuclein (red) and the neuronal marker microtubule-associated protein 2 (green) and imaged with the laser-scanning confocal microscope. A: Control nontransfected cells showed mild punctuate α-synuclein immunostaining associated with the cytoplasm and some neuritic processes. B: ST cells showed intense immunoreactivity associated with granular cytoplasmic structures. C: Detail at higher magnification of the rectangular area presented in (B). D: Negative control with the primary antibodies inactivated. E, F, and G: ST cells showed intense immunoreactivity associated with granular cytoplasmic structures (*) and dense intracellular inclusions (arrows). H: AST cells showed microtubule-associated protein 2 immunoreactivity but minimal or none α-synuclein immunostaining.

Figure 3.

Ultrastructural analysis of mitochondrial alterations in transfected GT1-7 cells. Low power (original magnification, ×5,000; scale bar, 5 μm) (A) and high magnification (original magnification, ×20,000; scale bar, 1 μm) (B) views of vector-transfected cells showing mitochondria of normal characteristics (arrowheads). Low power (original magnification, ×5,000) (C) and high magnification (original magnification, ×20,000) (D) views of ST cells demonstrating abnormally enlarged mitochondria (arrowheads). ST cells also had mitochondria displaying vacuolization of the cristae (original magnification, ×10,000) (E) and distorted morphology (original magnification, ×15,000) (F).

Figure 4.

Ultrastructural analysis of other morphological alterations in transfected GT1-7 cells. Low power (original magnification, ×5,000; scale bar, 5 μm) (A) and high magnification (original magnification, ×20,000; scale bar, 1 μm) (B) views of VEC-transfected cells demonstrating normal organelles. Low power (original magnification, ×5,000) (C) and high magnification (original magnification, ×20,000) (D) views of ST cells demonstrating electrodense intracytoplasmic inclusions (arrowheads). E and F: Furthermore ST cells displayed large numbers of secondary lysosomes and laminated bodies in their cytoplasm and complete lack of neuritic processes (original magnification, ×15,000).

Figure 5.

MTT assay in transfected GT1-7 cells. α-Synuclein sense-transfected (ST) cells showed a significant 30% decrease in mitochondrial activity compared to nontransfected control (C). Treatment with the anti-oxidant vitamin E restored normal mitochondrial function in ST cells.

Figure 6.

DCF staining in GT1-7-transfected cells. All images were obtained by LSCM. A: Control cells showed mild fluorescence. B: Positive-control experiment where GT1-7 cells were exposed to 1 μmol/L hydrogen peroxide for 15 minutes showed a strong fluorescent signal (scale bar, 10 μm). C: α-Synuclein sense-transfected (ST) cells showed a strong fluorescent signal associated with the vesicular structures, indicative of oxidative stress. D: Treatment with the anti-oxidant vitamin E restored normal mitochondrial function in ST cells.

Figure 7.

Glutathione and GnRH levels in transfected GT1-7 cells. A: α-Synuclein sense-transfected (ST) cells showed a significant 85% increase in glutathione levels compared to nontransfected controls (C), vector-transfected cells (VEC), and antisense-transfected (AST) cells. B: α-Synuclein sense-transfected (ST) cells showed a significant 60% decrease in GnRH levels compared to nontransfected control (C). Treatment with the anti-oxidant vitamin E restored normal levels of GnRH secretion in ST cells.