Zonisamide attenuates α-synuclein neurotoxicity by an aggregation-independent mechanism in a rat model of familial Parkinson's disease

Abstract

The anti-epileptic agent zonisamide (ZNS) has been shown to exert protective effects in neurotoxin-based mouse models of Parkinson disease. However, it is unknown whether ZNS can attenuate toxicity of familial Parkinson's disease-causing gene products. In this study, we investigated the effects of ZNS on neurodegeneration induced by expression of A53T α-synuclein in the rat substantia nigra using a recombinant adeno-associated virus vector. Expression of A53T α-synuclein yielded severe loss of nigral dopamine neurons and striatal dopamine nerve terminals from 2 weeks to 4 weeks after viral injection. Oral administration of ZNS (40 mg/kg/day) significantly delayed the pace of degeneration at 4 weeks after viral injection as compared with the vehicle group. This effect lasted until 8 weeks after viral injection, the final point of observation. ZNS treatment had no impact on the survival of nigrostriatal dopamine neurons in rats expressing green fluorescent protein. Quantification of striatal Ser129-phosphorylated α-synuclein-positive aggregates showed that these aggregates rapidly formed from 2 weeks to 4 weeks after viral injection. This increase was closely correlated with loss of nigrostriatal dopamine neurons. However, ZNS treatment failed to alter the number of all striatal Ser129-phosphorylated α-synuclein-positive aggregates, including small dot-like and large round structures. The number of these aggregates was almost constant at 4 weeks and 8 weeks after viral injection, although ZNS persistently prevented loss of nigrostriatal dopamine neurons during this period. Also, ZNS treatment did not affect the number of striatal aggregates larger than 10 µm in diameter. These data show that ZNS attenuates α-synuclein-induced toxicity in a manner that is independent of the formation and maturation of α-synuclein aggregates in an in vivo model of familial Parkinson's disease, suggesting that ZNS may protect nigrostriatal dopamine neurons by modulating cellular damage or a cell death pathway commonly caused by neurotoxins and α-synuclein.

Figure 1. rAAV-mediated expression of A53T α-synuclein in the rat nigrostriatal system.

(A) rAAV-A53T α-synuclein was unilaterally injected in the substantia nigra pars compacta. Immunohistochemical images of the rat substantia nigra (SN) and striatum at 2 weeks after viral injection. Upper panels show sections immunostained with LB509 antibody that recognizes total human α-synuclein, including phosphorylated and nonphosphorylated forms. Middle and bottom panels show sections immunostained with antibodies for tyrosine hydroxylase (TH) and dopamine transporter (DAT), respectively. Left and right panels show sections of rats treated with vehicle and zonisamide (ZNS), respectively. Scale bars, 1 mm. (B) Left panels shows western blotting of the rat ventral midbrains at 2 weeks after viral injection. 1% Triton X-100-soluble extracts (5 µg/lane) were analyzed by western blotting with Syn-1 (total α-synuclein), LB509 (human total α-synuclein), and EP1536Y (phosphorylated α-synuclein) antibodies. For loading control, the same amounts of samples were blotted with β-actin antibody. Right graphs show the ratios of the total α-synuclein and phosphorylated α-synuclein expression levels in the ZNS group relative to those in the vehicle group. Band intensities were normalized to β-actin to correct loading variations between lanes. Data represent the means ± SD.

Figure 2. Stereological assessments of the number of nigral dopamine neurons in rats treated with vehicle or ZNS.

Rats were unilaterally injected with rAAV-A53T α-synuclein (A, C) or rAAV-GFP (B, D). The sections were immunostained with antibody for TH (A, B) or DAT (C, D). In rats injected with rAAV-A53T α-synuclein (A, C), photomicrographs show representative immunostainings of the injected and uninjected sides of the substantia nigra at 2 weeks after injection (left panels), and those of the injected sides at 4 weeks (middle panels) and 8 weeks (right panels) after injection. In rats injected with rAAV-GFP (B, D), photomicrographs show representative immunostainings of the injected and uninjected sides of the substantia nigra at 4 weeks (left panels) and 8 weeks (right panels) after injection. Scale bars, 0.5 mm. (A–D) Graphs show quantitative analysis of TH or DAT-positive nigral neurons. As compared with the vehicle group, ZNS treatment significantly delayed loss of TH-positive nigral neurons at 4 weeks (P = 0.004) and 8 weeks (P = 0.010) after rAAV-A53T α-synuclein injection. Loss of DAT-positive nigral neurons in the ZNS group was also delayed at 4 weeks (P = 0.008) and 8 weeks (P = 0.040) after viral injection. The open bars illustrate the vehicle group and the black bars illustrate the ZNS group. Data represent the mean ± SD and P-values were estimated by two-way ANOVA, followed by unpaired t test (*P<0.05).

Figure 3. Semiquantification of the optical densities of striatal dopamine nerve terminals in rats treated with vehicle or ZNS.

Rats were unilaterally injected with rAAV-A53T α-synuclein (A, C) or rAAV-GFP (B, D). The sections were immunostained with antibody for TH (A, B) or DAT (C, D). In rats injected with rAAV-A53T α-synuclein (A, C), photomicrographs show representative immunostainings of the injected and uninjected sides of the substantia nigra at 2 weeks after injection (left panels), and those of the injected sides at 4 weeks (middle panels) and 8 weeks (right panels) after injection. In rats injected with rAAV-GFP (B, D), photomicrographs show representative immunostainings of the injected and uninjected sides of the substantia nigra at 4 weeks (left panels) and 8 weeks (right panels) after injection. Scale bars, 1.0 mm. (A–D) Graphs show semiquantitative analysis of striatal TH or DAT-positive fibers. As compared with the vehicle group, ZNS significantly suppressed loss of striatal TH-positive fibers at 4 weeks (P<0.001) and 8 weeks (P = 0.004) after rAAV-A53T α-synuclein injection. Decreased densities of DAT-positive fibers were significantly suppressed in the ZNS group at 4 weeks (P<0.001) and 8 weeks (P<0.001) after viral injection. The open bars illustrate the vehicle group and the black bars illustrate the ZNS group. Data represent the mean ± SD and P-values were estimated by two-way ANOVA, followed by a Bonferroni’s post hoc test (*P<0.05).

Figure 4. Biochemical analysis of the expression levels of TH and DAT in rats unilaterally injected with rAAV-A53T α-synuclein.

The striatum tissues were collected at 4 weeks after viral injection, and divided into the injected and uninjected sides. (A) 1% Triton X-100-soluble extracts (10 µg/lane) were analyzed by western blotting with TH, DAT, and LB509 (human total α-synuclein) antibodies. Representative blot data of the vehicle and ZNS groups are shown. Human α-synuclein is observed only in the rAAV-injected sides. Although the DAT blot of vehicle-treated samples is separated from the blot of ZNS-treated ones, these blots are originally derived from the same blot. (B) Graphs show the ratios of TH and DAT expression levels in the ZNS group relative to those in the vehicle group. As compared with vehicle treatment, ZNS significantly retained the expression levels of TH (n = 4 for each group, P = 0.013) and DAT (n = 4 for each group, P<0.001). Data represent the means ± SD. P-values were estimated by unpaired t test (*P<0.05).

Figure 5. Effects of late treatment with ZNS on A53T α-synuclein-induced loss of nigrostriatal neurons.

ZNS administration was started 7 days after rAAV-A53T α-synuclein injection, and the treatment was performed until 4 weeks after viral injection. Sections were immunostained with antibody for TH. Graphs show the quantitative analysis of the number of TH-positive nigral neurons (A) and the semiquantitative analysis of the optical density of TH-positive striatal fibers (B). As compared with vehicle treatment, ZNS significantly suppressed loss of TH-positive nigral neurons (P = 0.012). Decreased densities of TH-positive nigral fibers were significantly suppressed in the ZNS group (P = 0.020). Data represent the mean ± SD. P-values were estimated by unpaired t test (*P<0.05).

Figure 6. Analysis of Ser129-phosphorylated α-synuclein-positive aggregates in the striatum.

(A–C) The sections were immunostained with antibody that specifically recognizes Ser129-phosphorylated α-synuclein. (A) Photomicrographs show representative immunostainings of the injected and uninjected sides of the substantia nigra at 2 weeks after rAAV-A53T α-synuclein injection. Left panels show images at low magnification, and right panels show enlarged images of the injected side of the substantia nigra. Scale bars indicate 1 mm and 200 µm in left and right panels, respectively. (B) High magnification photomicrographs show representative immunostainings of the injected sides of the striatum at 4 weeks after viral injection. Right photograph shows the enlarged image of the indicated area of left photograph. Scale bars, 50 µm (left panel) and 20 µm (right panel). (C) Photomicrographs show representative immunostainings of the injected sides of the striatum at 4 weeks (left panels) and 8 weeks (right panels) after viral injection in rats, which were treated with vehicle (upper panels) or ZNS (lower panels). Arrowheads indicate Ser129-phosphorylated α-synuclein-positive aggregates larger than 10 µm in diameter. Scale bars, 50 µm. (D) Graph shows quantitative analysis of all striatal Ser129-phosphorylated α-synuclein-positive aggregates, including small dot-like and large round structures, despite their size. Two-way ANOVA (group × time) demonstrated neither main effect of group [F(1,30) = 0.642, P = 0.429] nor significant group × time interaction [F(2,30) = 0.451, P = 0.641]. (E) Graph shows quantitative analysis of striatal Ser129-phosphorylated α-synuclein-positive aggregates larger than 10 µm in diameter. Two-way ANOVA (group × time) demonstrated neither main effect of group [F(1,20) = 1.195, P = 0.287] nor significant group × time interaction [F(1,20) = 3.202, P = 0.089]. The open bars illustrate the vehicle group and the black bars illustrate the ZNS group. Data represent the mean ± SD.