Targeted Overexpression of α-Synuclein by rAAV2/1 Vectors Induces Progressive Nigrostriatal Degeneration and Increases Vulnerability to MPTP in Mouse

<div><p>Mutations, duplication and triplication of <i>α-synuclein</i> genes are linked to familial Parkinson’s disease (PD), and aggregation of α-synuclein (α-syn) in Lewy bodies (LB) is involved in the pathogenesis of the disease. The targeted overexpression of α-syn in the substantia nigra (SN) mediated by viral vectors may provide a better alternative to recapitulate the neurodegenerative features of PD. Therefore, we overexpressed human wild-type α-syn using rAAV2/1 vectors in the bilateral SN of mouse and examined the effects for up to 12 weeks. Delivery of rAAV-2/1-α-syn caused significant nigrostriatal degeneration including appearance of dystrophic striatal neurites, loss of nigral dopaminergic (DA) neurons and dissolving nigral neuron bodies in a time-dependent manner. In addition, the α-syn overexpressed mice also developed significant deficits in motor function at 12 weeks when the loss of DA neurons exceeded a threshold of 50%. To investigate the sensitivity to neurotoxins in mice overexpressing α-syn, we performed an MPTP treatment with the subacute regimen 8 weeks after rAAV injection. The impact of the combined genetic and environmental insults on DA neuronal loss, striatal dopamine depletion, dopamine turnover and motor dysfunction was markedly greater than that of either alone. Moreover, we observed increased phosphorylation (S129), accumulation and nuclear distribution of α-syn after the combined insults. In summary, these results reveal that the overexpressed α-syn induces progressive nigrostriatal degeneration and increases the susceptibility of DA neurons to MPTP. Therefore, the targeted overexpression of α-syn and the combination with environmental toxins may provide valuable models for understanding PD pathogenesis and developing related therapies.</p></div

α-syn aggregation and nuclear distribution in nigral neurons after MPTP treatment.

<p>Double Immunofluorescence staining for NeuN (green) and <b>α</b>-syn (red) was performed to assess the condition of <b>α</b>-syn (antibody against <b>α</b>-syn, Santa Cruz, sc-7011-R). Small arrows denote dot-like accumulation of <b>α</b>-syn surround the nucleus after rAAV- <b>α</b>-syn transduction (E). More <b>α</b>-syn accumulation in cytoplasm (small arrows, K) and nucleus distribution (big arrow, K) in neurons of the <b>α</b>-syn and MPTP combined treatments. A-C: CON (rAAV-GFP+saline); D-F: SYN (rAAV- <b>α</b>-syn+saline); G-I: MPTP (rAAV-GFP+MPTP); J-L: SYN-MPTP (rAAV- <b>α</b>-syn+MPTP). Scale bar: 50um (A, B, D, E, G, H, J, K); 20μm (C, F, I, L).</p

Striatal dopamine depletion after MPTP treatment.

<p>*: <i>P</i><0.05,</p><p>***: <i>P</i><0.001 compared to CON;</p><p>##: <i>P</i><0.01,</p><p>###: <i>P</i><0.001 compared to SYN.</p><p>One-way ANOVA Newman-Keuls <i>post-hoc</i> test.</p><p>Striatal dopamine depletion after MPTP treatment.</p

Motor dysfunction assessment and numbers of TH positive neurons in the SNpc after MPTP treatment.

<p>Body weights of mice during MPTP injection (5 consecutive days: A). *, compared to CON (rAAV-GFP+saline); +, compared to SYN (rAAV-α-syn+saline); #, compared to MPTP (rAAV-GFP+MPTP); SYN-MPTP (rAAV-α-syn+MPTP). ANOVAMotor dysfunction assessment: pole test was performed 1 and 14 days after the last MPTP injection (B), and locomotor activities as well as rearing activities in open field test were recorded 2 and 14 days after the last MPTP injection (C-D), data are means±SEM of 9 mice, *, +, #<i>P</i><0.05, ++<i>P</i><0.01, ***<i>P</i><0.001 (one-way ANOVA Newman-Keuls <i>post-hoc</i> test). Representative images and quantification for TH positive neurons in the SNpc after MPTP treatment (E-I), scale bar: 400μm. Data are means±SEM of 6 mice, **<i>P</i><0.01; ***, +++, ### <i>P</i><0.001 (one-way ANOVA Newman-Keuls <i>post-hoc</i> test).</p

Accumulation of phosphorylated α-syn in TH positive neurons of SN and increased protein levels of phosphorylated α-syn in midbrain.

<p>Double Immunofluorescence staining for phosphorylated <b>α</b>-syn (P-S129) (green) and TH (red) was performed. Phosphorylated forms were observed in TH positive neurons of SYN (D-F) and MPTP (G-I) mice, furthermore, notable accumulation was also detected in DA neurons of the SYN-MPTP mice (J-L). Scale bar: 20μm. Phosphorylated <b>α</b>-syn (P-S129) was quantified by western blotting (M) and the accumulation in midbrain of SYN-MPTP mice was significant (N). Data are presented as mean±SEM of 3 mice; *<i>P</i><0.05, ***<i>P</i><0.001 compared to SYN-MPTP (one-way ANOVA Newman-Keuls <i>post-hoc</i> test).</p

Loss of striatal protein TH and increased dopamine turnover after MPTP treatment.

<p>Protein blots for striatal TH were analyzed TH/β-actin ratio and normalized by the results of CON (A, B). Dopamine turnover, which was analyzed by DOPAC/DA and HVA/DA ratio, exhibited significant increase after MPTP treatment (C). Data are presented as mean±SEM of 3 mice; *, compared to CON; #, compared to SYN; +, compared to MPTP. *, +, #: <i>P</i><0.05; **, ++, ##: <i>P</i><0.01; ***, ###, +++: <i>P</i><0.001 (one-way ANOVA Newman-Keuls <i>post-hoc</i> test).</p

Immunohistological analysis of DA neurons in the SNpc and TH positive neurites in the striatum.

<p>Representative micrographs of nigral sections immunostained for TH in mice at 4, 8 and 12 weeks after delivery of rAAV-GFP (CON: A, C and E) and rAAV-α-syn (SYN: B, D and F) vectors. Scale bar: 400μm. Illustration of striatal TH positive fiber density over time (H, J, L: rAAV-GFP; I, K, M: rAAV-α-syn). Scale bar: 50μm. Quantification of TH positive neurons in the SNpc and striatal TH immunoreactive fiber density was shown (G, N). Data are means ± SEM of 6 mice, **<i>P</i><0.01, ***<i>P</i><0.001 as compared to rAAV-GFP control (2-way ANOVA, Bonferroni <i>post hoc</i> test).</p

Overexpression of α-syn in the SNpc after rAAV-α-syn transduction.

<p>Western blotting showed a significant expression of human-<b>α</b>-syn in HEK293 cells 96 hours after transduction (A). The position of SNpc was indicated (B) and TH positive staining (Red) indicated a loss of DA neurons in the SNpc 8 weeks after rAAV-<b>α</b>-syn injection, however, the co-staining of human-<b>α</b>syn (Green) was also observed in the existing DA neurons (I-K). The overexpression of GFP was also detected in TH-positive neurons in the SNpc after rAAV2/1-GFP injection (F-H). And no α-syn staining was observed in the none-injected mouse (C-E). TH staining confirmed no loss of DA neurons in GFP and none injected group. Antibody against human α-syn was used (CST). Scale bar: 100μm.</p