Translational inhibition of α-synuclein by Posiphen normalizes distal colon motility in transgenic Parkinson mice

Abstract

Parkinson disease (PD) is a neurodegenerative disease with motor as well as non-motor symptoms, including gastrointestinal dysfunction. In humans, these precede the motor symptoms by decades. Previously developed and characterized transgenic mice expressing the mutant human α-synuclein gene (SNCA) (either A53T or A30P), but not the endogenous mouse Snca, serve as models for familial PD. These animals demonstrate both robust abnormalities in enteric nervous system (ENS) function as well as synuclein-immunoreactive aggregates in ENS ganglia by 3 months of age, recapitulating early gastrointestinal abnormalities seen before the gait impairment characteristics of human and murine PD. Posiphen is a translational inhibitor of α-synuclein that targets the 5' untranslated region (UTR) of SNCA mRNA and could be a potential drug for the treatment of PD. However, its efficacy in ameliorating symptoms of PD has not yet been evaluated. Here, we used these transgenic mouse models to investigate the efficacy of Posiphen in reversing the gastrointestinal dysfunction. We show that Posiphen normalizes the colonic motility of both transgenic mouse models, although it did not affect the Whole Gut Transit Time (WGTT). Pharmacokinetics studies revealed that Posiphen is more abundant in the brain than in blood, in agreement with its lipophilicity, and the main metabolite is N⁸-NorPosiphen, a molecule with similar properties as Posiphen. The brain Posiphen levels necessary to effect optimal function were calculated and compared with efficacious brain levels from previous studies, showing that a 2-3 mM concentration of Posiphen and metabolites is sufficient for functional efficacy. Finally, 10 mg/kg Posiphen reduced α-synuclein levels in the gut of hSNCA^A53T mice treated for twenty-one weeks, while 50 and 65 mg/kg Posiphen reduced α-synuclein levels in the brain of hSNCA^A53T mice treated for twenty-one days. In conclusion, this is the first study showing the preclinical efficacy of Posiphen in normalizing the colonic motility in mouse models of gastrointestinal dysfunction in early PD. This result is in agreement with the ability of Posiphen to reach the nervous system, and its mechanism of action, the translational inhibition of α-synuclein expression. These significant findings support further development of Posiphen as a drug for the treatment of PD.

Keywords: Parkinson’s disease; Posiphen; colonic motility; gastrointestinal dysfunction; α-synuclein.

Figure 1

Posiphen decreases latency time of colonic motility in two mouse models of PD. (A) hSNCA ^A53T, Snca ^+/+, and Snca ^-/-, and (B) hSNCA ^A30P, Snca ^+/+, and Snca ^-/- are compared. Daily IP injection of 3 or 10 mg/kg Posiphen starting at six weeks of age until four or seven months of age decreases the prolonged bead expulsion times in hSNCA ^A53T and hSNCA ^A30P mice, in comparison to their saline-treated counterparts (0 mg/kg); statistically significant differences are indicated on the graphs. Mean and SEM are shown. N = on average 9 mice per group. Unpaired, two-tailed Mann-Whitney tests were performed. SEM; standard error of the mean.

Figure 2

Colonic motility after Posiphen discontinuation. (A) hSNCA ^A53T, Snca ^+/+, and Snca ^-/-, and (B) hSNCA ^A30P, Snca ^+/+, and Snca ^-/- are compared. Mice were treated with daily IP injections of 0, 3 or 10 mg/kg Posiphen in saline from six weeks to seven months of age, at which point the treatment stopped. Nine and sixteen weeks after the discontinuation of treatment, the mice described in Figure 1 were tested again with the bead expulsion test to assess their colonic motility. The statistically significant differences found between saline-treated vs. Posiphen-treated hSNCA ^A53T and hSNCA ^A30P mice after treatment discontinuation are indicated on the graphs. Mean and SEM are shown. N = on average 10 mice per group. Unpaired, two-tailed Mann-Whitney tests were performed. SEM; standard error of the mean.

Figure 3

WGTT in hSNCA ^A53T male mice treated with daily IP injections of 0, 3 or 10 mg/kg Posiphen in saline from six weeks to seven months of age. Posiphen did not affect the WGTT of hSNCA ^A53T mice, as determined by unpaired, two-tailed t-tests. Mean and SEM are shown. N is indicated on the graph. SEM; standard error of the mean.

Figure 4

Motor function assessment by rotarod testing. Mice treated with daily IP injections of 0 or 10 mg/kg Posiphen starting at six weeks until seven months of age were assessed by rotarod testing at seven months of age. Repeated measures two-way ANOVA, with Tukey’s multiple comparisons test, was performed for all treatment groups. (A) Rotarod performance of the saline treated animals is shown. The hSNCA ^A53T genotype did not affect the time the mice stayed on the accelerating rotator in a statistically significant manner, as compared to the control mice, with the exception of a single difference between hSNCA ^A53T vs. Snca ^+/+ (day 1 - test 4). (B) Rotarod performance of the 0 and 10 mg/kg Posiphen-treated mice at the last rotarod trial (day 3 - test 4). Posiphen treatment has no effect on motor function of either Snca ^+/+, Snca ^-/- or hSNCA ^A53T mice. Mean and SEM are shown. N = on average 13 per group for (A), and 11 per group for (B). SEM; standard error of the mean.

Figure 5

Weights of mice at seven months of age, after daily IP injections of 0, 3, or 10 mg/kg Posiphen starting at six weeks old. Posiphen treatment did not affect the weight of treated mice, as determined by Kruskal-Wallis test with Dunn’s multiple comparisons test. Mean and SEM are shown. N = on average 12 per group. SEM; standard error of the mean.

Figure 6

Distribution of Posiphen and its metabolites in brain and blood of Snca ^+/+, Snca ^-/-, and hSNCA ^A53T mice. Mice were treated with daily IP injections of 10 mg/kg Posiphen starting at six weeks old for 21 days. Concentrations of Posiphen (A, D) and its main metabolites N¹-NorPosiphen (B, E) and N⁸-NorPosiphen (C, F) in brain cerebellum (A-C) and blood (D-F) were measured by LC-MS/MS. Mean and SEM are shown. N = 3 per group. SEM; standard error of the mean.

Figure 7

Concentration of Posiphen and metabolites in the brain of transgenic mouse models of PD (A) and AD (B), where Posiphen was effective in ameliorating impairments in colonic motility and memory, respectively. (A) hSNCA ^A53T mice were treated with 10 mg/kg Posiphen IP for 3 weeks, starting at 6 weeks of age. (B) APP/PS1 transgenic mice (carrying the human genes for APP and PS1 (Presenilin 1) with mutations associated with familial AD) were treated with 25 mg/kg PO for 2 weeks, starting at 3 months of age (adapted from [30]). Concentrations of Posiphen and metabolites were measured in the cerebellum by LC-MS/MS in both cases. Mean and SEM are shown. The numbers above the columns are the means, in ng/g. N = 3 per group. PO; per os. SEM; standard error of the mean.

Figure 8

α-synuclein gut levels following treatment of hSNCA ^A53T mice with 10 mg/kg Posiphen for 21 weeks or 21 days, starting at six weeks of age. A. α-synuclein Western blot of gut samples of Snca ^-/- mice, and hSNCA ^A53T mice treated with saline or 10 mg/kg Posiphen (ko; knock out Snca ^-/- mice, as negative control). B. Semi-quantization of the α-synuclein Western blot for hSNCA ^A53T mice. 21 weeks but not 21 days of Posiphen treatment significantly reduced α-synuclein levels, by 50.6%. *P ≤ 0.05, by unpaired, two-tailed t-test. Mean and SEM are shown. N = 4 per group. αSYN; α-synuclein. SEM; standard error of the mean.

Figure 9

Daily IP Posiphen treatment of hSNCA ^A53T transgenic mice for 21 days, starting at six weeks of age, reduces α-synuclein brain levels in a dose-dependent manner. (A) α-synuclein Western blot of mouse brain extracts, following treatment with 0, 5, 20, 35, 50 or 65 mg/kg Posiphen in saline. β-actin was used as loading control. In the first blot, where β-actin signals were variable, Ponceau S stain indicates equal loading. (B) Semi-quantization of the Western blots in (A) after normalization with β-actin. Since the three blots comparing 0 vs. 65 mg/kg, 5 vs. 50 mg/kg and 20 vs. 35 mg/kg were run separately, without a common sample, they were plotted separately. Unpaired, two-tailed t-tests were performed for each pair, and significance is indicated on the graphs. *P ≤ 0.05. The numbers in the plots indicate the percent reduction of the α-synuclein level by the highest Posiphen dose, as compared to the lowest, in each pair. Data without normalization with β-actin look similar (not shown), with 50.6%, 33.7%, and 12.2% reduction, respectively, and unpaired two-tailed t-tests as follows: 0 vs. 65 mg/kg; P = 0.0858 (trend that does not reach significance), and 5 vs. 50 mg/kg; **P ≤ 0.01. C) α-synuclein ELISA of brain extracts from the same treatment groups, showing that the two highest doses reduced α-synuclein levels by roughly 28%. Unpaired, two-tailed t-tests were performed between 0 mg/kg and the various Posiphen doses. *P ≤ 0.05. In case of 35 vs. 0 mg/kg, there is a trend that does not reach significance: P = 0.0683. Mean and SEM are shown. N = 5 mice per group. αSYN; α-synuclein. SEM; standard error of the mean.