Absence of α-synuclein affects dopamine metabolism and synaptic markers in the striatum of aging mice

Dopamine and its metabolite levels in striatum of 2-year-old wild type and synuclein null mutant mice. Striatal concentrations (ng/mg protein) of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in mutant animals were normalized to corresponding mean values for wild type animals (100%) in each experiment. Means±S.E.M. for 16 wild type (WT), 11 α-synuclein null mutant (alpha−/−) and 10 γ-synuclein null mutant (gamma−/−) animals from two separate experimental cohorts are shown. Statistical analysis revealed significant decrease of striatal DA in α-synuclein null mutant mice (*, p<0.05, one-way ANOVA with post hoc Fisher’s protected t-test) but no differences for other neurochemicals and genotypes (p>0.05).

The number of dopaminergic neurons in the substantia nigra and ventral tegmental area of 2-year-old wild type and synuclein null mutant mice and their performance in accelerated rotarod test. Bar chart shows means±S.E.M. of total number of TH-positive neurons in SNpc (A) and VTA (B). Neurons were counted separately in left and right SNpc and VTA of 9 wild type (WT), 9 α-synuclein null mutant (alpha−/−) and 10 γ-synuclein null mutant (gamma−/−) animals. Statistic analysis revealed significantly reduced number of neurons in SNpc for both types of mutant mice when compared to wild type mice (*, p<0.01, one-way ANOVA with post hoc Fisher’s protected t-test) and no difference in number of VTA neurons between all three groups (p>0.05). Bar chat in panel C shows results of an accelerated rotarod test that demonstrated no significant difference (p>0.05) in latency to fall between three groups of 2-year-old mice.

Immunohistochemical detection of tyrosine hydroxylase in the striatum of 2-year-old wild type and synuclein null mutant mice. Representative microphotographs of coronal sections of wild type, α-synuclein and γ-synuclein null mutant mouse brains at the Bregma 1.18mm level stained with antibody against TH (top panels) and high magnification images that reveal TH-positive fibers in the dorsal striatum (bottom panes, scale bar=10μm) are shown (A). Bar chart shows means±S.E.M. of TH staining densities (B). One-way ANOVA with post hoc Fisher’s protected t-test demonstrated that the density is significantly lower in the striatum of α-synuclein (alpha−/−) null mutant mice when compared to wild type (WT) mice (**p<0.01) or γ-synuclein (gamma−/−) null mutant mice (*p<0.05).

Synaptic marker proteins in the striatum of 2-year-old mice. Representative Western blots of striatal proteins extracted from wild type, α-synuclein and γ-synuclein null mutant mice are shown. Samples extracted from each dissected striatum individually were first normalized using anti-β-actin antibody as described in Section 2. Equal amount of total protein was loaded on each lane and probed with antibody against proteins shown left to each horizontal panel.

β-synuclein in the striatum of 2-year-old mice. (A) Bar chart shows means±S.E.M. of β-synuclein/GAPDH band density ratios normalized to the mean ratio for wild type animal samples as 100%. Individual striatum samples from 7 to 9 animals per genotype were analysed by Western blotting. No significant differences were found between groups (p>0.05). (B) A representative Western blot shows analysis of mixtures of the equal amounts of total protein samples from three striata for each genotype. The filters were probed simultaneously with anti-β-synuclein and anti-GAPDH antibody.