Resistance to MPTP-Neurotoxicity in α-Synuclein Knockout Mice Is Complemented by Human α-Synuclein and Associated with Increased β-Synuclein and Akt Activation

Genetic and biochemical abnormalities of α-synuclein are associated with the pathogenesis of Parkinson's disease. In the present study we investigated the in vivo interaction of mouse and human α-synuclein with the potent parkinsonian neurotoxin, MPTP. We find that while lack of mouse α-synuclein in mice is associated with reduced vulnerability to MPTP, increased levels of human α-synuclein expression is not associated with obvious changes in the vulnerability of dopaminergic neurons to MPTP. However, expressing human α-synuclein variants (human wild type or A53T) in the α-synuclein null mice completely restores the vulnerability of nigral dopaminergic neurons to MPTP. These results indicate that human α-synuclein can functionally replace mouse α-synuclein in regard to vulnerability of dopaminergic neurons to MPTP-toxicity. Significantly, α-synuclein null mice and wild type mice were equally sensitive to neurodegeneration induced by 2′NH2-MPTP, a MPTP analog that is selective for serotoninergic and noradrenergic neurons. These results suggest that effects of α-synuclein on MPTP like compounds are selective for nigral dopaminergic neurons. Immunoblot analysis of β-synuclein and Akt levels in the mice reveals selective increases in β-synuclein and phosphorylated Akt levels in ventral midbrain, but not in other brain regions, of α-synuclein null mice, implicating the α-synuclein-level dependent regulation of β-synuclein expression in modulation of MPTP-toxicity by α-synuclein. Together these findings provide new mechanistic insights on the role α-synuclein in modulating neurodegenerative phenotypes by regulation of Akt-mediated cell survival signaling in vivo

Levels of α-synuclein affects β-synuclein expression and basal <i>Akt</i> phosphorylation <i>in vivo</i>.

<p><b><i>A.</i></b> Total tissue extracts from cortex and ventral midbrain of wild type (WT), moSyn-null mice (KO), and HuSyn transgenic (line I2-2) on moSyn-KO background (KO+) were immunobloted for endogenous β-synuclein, <i>Akt</i> (pSer473 and total) and GAPDH. <b><i>B and C.</i></b> Semi-quantitative analysis of β-synuclein <b>(B)</b> and pSer473-<i>Akt</i> levels <b><i>(C)</i></b>. The values are mean ± SEM from 4 animals (<i>*p>0.05, **p>0.01</i>, ANOVA with Newman-Keuls post-test).</p

Nigrostriatal system in transgenic wild type and mutant human α-synuclein overexpressing mice do not show increased sensitivity to acute MPTP intoxication.

<p>Transgenic mice expressing moderate levels (∼3-fold over endogenous levels) of wild type (WT, line I2-2), A30P mutant (line T3), and A53T mutant (line N2-5) human α-synuclein were subjected to acute paradigm of MPTP (18 mg MPTP/kg free base X4, every 2h). <i>A.</i> Striatal levels of dopamine and major metabolite DOPAC in transgenic and non-transgenic littermate cohorts were analyzed 7 days after the last MPTP injection. No differences were seen among transgenic and non-transgenic mice receiving the same treatments (saline or MPTP). <i>B.</i> Stereologic neuronal counts of TH-immunopositive and total neurons in transgenic and non-transgenic littermate cohorts analyzed two weeks following the last MPTP injection. While MPTP treatments caused a significant reduction in TH-immunopositive and total neurons, no significant differences in neuronal counts are observed between transgenic and non transgenic mice receiving the same treatment (saline or MPTP). Data represent mean ± SEM. *<i>p<0.05</i>, statistical significance versus saline controls using two way ANOVA, n = 5-6 per group, n.s not significant.</p

Effects of sub acute MPTP regimen, aging, and transgene expression level on the MPTP sensitivity of nigral dopaminergic neurons in human A53T α-synuclein transgenic mice.

<p><i>A.</i> Stereologic cell counts of total and TH-immunopositive neurons of SNpc in non transgenic and human A53T transgenic mice (line N2-5, ∼3-fold) analyzed two weeks after a sub-acute paradigm of MPTP (30 mg MPTP/kg free base once a day for 5 days). Although sub acute MPTP caused significant reductions in total and TH-positive neurons no significant differences were seen among transgenic and non transgenic mice receiving the same treatments (saline or MPTP). <i>B</i>. TH-positive and total neuronal counts in SNpc of 12-14 month old non transgenic and human A53T transgenic mice (line N2-5) after acute MPTP (15 mg MPTP/kg free base X4, every 2 h) at 7 days. Acute MPTP in older mice caused significant reduction in total and TH-positive neurons no significant differences were seen among transgenic and non transgenic mice receiving the same treatments (saline or MPTP). <i>C</i>. Cell counts of TH-positive and total neurons in high expressing lines of human A53T transgenic mice (G2-3 line, ∼6-fold) 7 days after acute MPTP intoxication (18 mg MPTP/kg free base X4, every 2 h). MPTP-intoxication resulted in a significant reduction of total and TH-positive neuronal counts in non transgenic and high expressing lines of A53T transgenic mice compared to saline treatments. A moderate increase in the vulnerability was observed in A53T transgenic mice. Data represent mean ± SEM. *<i>p<0.05</i>, statistical significance versus saline controls using two way ANOVA, n = 5-6 per group, n.s., not significant. #, significant using Neuman-Keuls post-test (<i>p<0.05</i>) but not with the Tukey-Kramer post test.</p

Levels of striatal dopamine and its metabolites in wild type and α-synuclein knock out mice following 2′NH₂MPTP treatment.

<p>Striatal levels of dopamine and its metabolites measured after 2 weeks of acute 2′NH₂MPTP (15 mg/kg, i.p.) administered four times every two hours in wild type and α-synuclein knock out mice. Data are expressed as mean (n = 5) ± SEM. Statistical analysis was performed by ANOVA, revealing no significant differences among group means. DA, dopamine; DOPAC, 3,4-dihydroxy-phenylacetic acid; HVA, homovanillic acid, WT, wild type; KO, knock out.</p

Human α-synuclein complements MPTP resistant phenotype of mouse α-synuclein null mice.

<p><b><i>A.</i></b> Expression of mouse and human α-synuclein in wild type (WT), mouse α-synuclein nulls (KO) and mice expressing wild type human α-synuclein (line I2-2) on a mouse α-synuclein null background (hWT/KO), total proteins isolated from 9 month old striata were subjected to immunoblot analysis for total α-synuclein (Syn-1), human α-synuclein (HuSyn), and GAPDH. <b><i>B and C.</i></b> TH-immunostaining staining of striatum (B) and SNpc (C) 7 days following saline or acute MPTP (20 mg/kg free base four times every 2 hour) treatment in wild type (WT), mouse α-Syn nulls (KO) and human α-Syn transgenic [wild type (hWT, line I2-2) and A53T (line N2-5)] on the mouse α-Syn null background (hWT/KO; A53T/KO). Only the KO is protected from MPTP toxicity. <b><i>D and E</i></b>. MPTP intoxication results in significant reductions in striatal DA-levels <b><i>(D)</i></b> and TH-positive neuronal cells <b><i>(E)</i></b> in mice with either mouse or human α-Syn expression. The α-Syn KO mice were completely protected against MPTP. The reduced basal number of DAergic neurons in α-Syn KO mice was not complemented by the human α-Syn expression <b><i>(E)</i></b>. Data represent mean ± SEM. ^@,** <i>p<0.05</i>, versus saline and ^#<i>p<0.05</i>, versus wild type MPTP, Two way ANOVA, n = 5-6, n.s not significant, scale bar: 200 µm.</p