Effects of Novel Calpain Inhibitors in Transgenic Animal Model of Parkinson's disease/dementia with Lewy bodies

Abstract

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders of the aging population characterized by the accumulation of α-synuclein (α-syn). The mechanisms triggering α-syn toxicity are not completely understood, however, c-terminus truncation of α-syn by proteases such as calpain may have a role. Therefore, inhibition of calpain may be of value. The main objective of this study was to evaluate the effects of systemically administered novel low molecular weight calpain inhibitors on α-syn pathology in a transgenic mouse model. For this purpose, non-tg and α-syn tg mice received the calpain inhibitors - Gabadur, Neurodur or a vehicle, twice a day for 30 days. Immunocytochemical analysis showed a 60% reduction in α-syn deposition using Gabadur and a 40% reduction using Neurodur with a concomitant reduction in c-terminus α-syn and improvements in neurodegeneration. Western blot analysis showed a 77% decrease in α-spectrin breakdown products (SBDPs) SBDPs with Gabadur and 63% reduction using Neurodur. There was a 65% reduction in the active calpain form with Gabadur and a 45% reduction with Neurodur. Moreover, treatment with calpain inhibitors improved activity performance of the α-syn tg mice. Taken together, this study suggests that calpain inhibition might be considered in the treatment of synucleinopathies.

Figure 1

Immunoblot analysis of the effects of calpain inhibitors on α-syn tg mice. α-Syn tg mice were injected ip twice a day for 30 days with either Gabadur (1 mg/mouse; N = 10), Neurodur (1 mg/mouse; N = 10), or PBS vehicle control (N = 10) and compared to vehicle-treated non-tg mice (N = 8). (a) Immunoblot and (b) quantification of calpain levels. Calpain levels were significantly increased in vehicle-treated α-syn tg mice compared to vehicle-treated non-tg mice. Treatment with either Gabadur or Neurodur significantly reduced calpain protein levels in α-syn tg mice compared to vehicle-treated α-syn tg mice. (c) Immunoblot and (d) quantification of α-spectrin breakdown products (α-SBDPs). α-SBDPs levels were significantly increased in vehicle-treated α-syn tg mice compared to vehicle-treated non-tg mice. Treatment with either Gabadur or Neurodur significantly reduced α-SBDPs protein levels in α-syn tg mice compared to vehicle-treated α-syn tg mice. Statistical analysis was conducted using one-way ANOVA post hoc Dunnett’s test for comparison with vehicle-treated non- tg mice (*p < 0.05) and Tukey–Kramer test for comparison with vehicle-treated α-syn tg mice (^#p < 0.05).

Figure 2

Effects of Gabadur and Neurodur on total α-syn levels in the neocortex and hippocampus of α-syn tg mice. α-Syn tg mice were injected ip twice a day for 30 days with either Gabadur (1 mg/mouse; N = 10), Neurodur (1 mg/mouse; N = 10), or PBS vehicle control (N = 10) and compared to vehicle-treated non-tg mice (N = 8). (a) Representative photomicrographs and quantitation of total α-syn immunoreactivity in the (b) neocortex and (c) hippocampus. Immunoreactivity of total α-syn was significantly increased in vehicle-treated α-syn tg mice compared to vehicle-treated non-tg mice in both the neocortex and hippocampus. Treatment with either Gabadur or Neurodur significantly reduced total α-syn immunoreactivity in α-syn tg mice compared to vehicle-treated α-syn tg mice. Scale bar = 35 µm. Statistical analysis was conducted using one-way ANOVA post hoc Dunnett’s test for comparison with vehicle-treated non- tg mice (*p < 0.05) and Tukey–Kramer test for comparison with vehicle-treated α-syn tg mice (^#p < 0.05).

Figure 3

Effects of Gabadur and Neurodur on c-terminus α-syn levels in the neocortex and hippocampus of α-syn tg mice. α-Syn tg mice were injected ip twice a day for 30 days with either Gabadur (1 mg/mouse; N = 10), Neurodur (1 mg/mouse; N = 10), or PBS vehicle control (N = 10) and compared to vehicle-treated non-tg mice (N = 8). (a) Representative photomicrographs and quantitation of c-terminus α-syn immunoreactivity in the (b) neocortex and (c) hippocampus. Immunoreactivity of c-terminus α-syn was significantly increased in vehicle-treated α-syn tg mice compared to vehicle-treated non-tg mice in both the neocortex and hippocampus. Treatment with either Gabadur or Neurodur significantly reduced c-terminus α-syn immunoreactivity in α-syn tg mice compared to vehicle-treated α-syn tg mice. Scale bar = 35 µm. Statistical analysis was conducted using one-way ANOVA post hoc Dunnett’s test for comparison with vehicle-treated non- tg mice (*p < 0.05) and Tukey–Kramer test for comparison with vehicle-treated α-syn tg mice (^#p < 0.05).

Figure 4

Immunoblot analysis of the effects of Gabadur and Neurodur on total and C-terminus α-syn protein levels in α-syn tg mice. α-Syn tg mice were injected ip twice a day for 30 days with either Gabadur (1 mg/mouse; N = 10), Neurodur (1 mg/mouse; N = 10), or PBS vehicle control (N = 10) and compared to vehicle-treated non-tg mice (N = 8). Hemibrains were homogenized and divided into cytosolic (soluble) and particulate (membrane) fractions and analyzed by immunoblot. (a) Representative immunoblots of the cytosolic fraction analyzed for total and c-terminus α-syn protein levels. (b) Quantitative analysis of total α-syn and c-terminus α-syn protein levels. Total α-syn protein levels were significantly increased in vehicle-treated α-syn tg mice compared to vehicle-treated non-tg mice. Treatment with either Gabadur or Neurodur increased total and reduced c-terminus α-syn protein levels in α-syn tg mice compared to vehicle-treated α-syn tg mice. (c) Representative immunoblots of the particulate fraction analyzed for total and c-terminus α-syn protein levels. (d) Quantitative analysis of total α-syn and c-terminus α-syn protein levels. Treatment with either Gabadur or Neurodur decreased total and c-terminus α-syn protein levels in α-syn tg mice compared to vehicle-treated α-syn tg mice. Statistical analysis was conducted using one-way ANOVA post hoc Dunnett’s test for comparison with vehicle-treated non- tg mice (*p < 0.05) and Tukey–Kramer test for comparison with vehicle-treated α-syn tg mice (^#p < 0.05).

Figure 5

Effects of Gabadur and Neurodur on neurodegeneration (NeuN) in the neocortex and hippocampus of α-syn tg mice. α-Syn tg mice were injected ip twice a day for 30 days with either Gabadur (1 mg/mouse; N = 10), Neurodur (1 mg/mouse; N = 10), or PBS vehicle control (N = 10) and compared to vehicle-treated non-tg mice (N = 8). (a) Representative photomicrographs and quantitation of NeuN-positive neurons in the (b) neocortex and (c) hippocampus. The number of NeuN-positive neurons was significantly reduced in vehicle-treated α-syn tg mice compared to vehicle-treated non-tg mice in both the neocortex and hippocampus. Treatment with either Gabadur or Neurodur significantly increased the number of NeuN-immunoreactive neurons in both the neocortex and hippocampus. Scale bar = 35 µm. Statistical analysis was conducted using one-way ANOVA post hoc Dunnett’s test for comparison with vehicle-treated non- tg mice (*p < 0.05) and Tukey–Kramer test for comparison with vehicle-treated α-syn tg mice (^#p < 0.05).

Figure 6

Effects of Gabadur and Neurodur on astrogliosis and microgliosis in α-syn tg mice. α-Syn tg mice were injected ip twice a day for 30 days with either Gabadur (1 mg/mouse; N = 10), Neurodur (1 mg/mouse; N = 10), or PBS vehicle control (N = 10) and compared to vehicle-treated non-tg mice (N = 8). (a) Representative photomicrographs and quantitation of GFAP-positive astrocytes in the (b) neocortex and (c) hippocampus. The number of GFAP-positive neurons was significantly increased in vehicle-treated α-syn tg mice compared to vehicle-treated non-tg mice in both the neocortex and hippocampus. Neither treatment with Gabadur nor Neurodur significantly affected the number of GFAP-immunoreactive astrocytes in either the neocortex or hippocampus compared to vehicle-treated α-syn tg mice. (d) Representative photomicrographs and quantitation of Iba-1-positive microglia in the (e) neocortex and (f) hippocampus. The number of Iba-1-positive microglia was significantly increased in vehicle-treated α-syn tg mice compared to vehicle-treated non-tg mice in both the neocortex and hippocampus. Treatment with Gabadur or Neurodur significantly reduced the number of Iba-1-immunoreactive microglia in the neocortex and hippocampus. Scale bar = 35 µm. Statistical analysis was conducted using one-way ANOVA post hoc Dunnett’s test for comparison with vehicle-treated non- tg mice (*p < 0.05) and Tukey–Kramer test for comparison with vehicle-treated α-syn tg mice (^#p < 0.05).

Figure 7

Effects of Gabadur and Neurodur on behavioral measures in α-syn tg mice. α-Syn tg mice were injected ip twice a day for 30 days with either Gabadur (1 mg/mouse; N = 10), Neurodur (1 mg/mouse; N = 10), or PBS vehicle control (N = 10) and compared to vehicle-treated non-tg mice (N = 8). Testing for (a) total spontaneous locomotor activity, (b) lateral activity, (c) rearing, and (d) thigmotaxis. Compared to vehicle-treated non-tg mice, vehicle-treated α-syn tg mice had a significant increase in total spontaneous activity. Gabadur and Neurodur significantly reduced total spontaneous locomotor compared to vehicle-treated α-syn tg mice. There were no significant differences between treatment groups in any of the other behavioral tests. Statistical analysis was conducted using one-way ANOVA post hoc Dunnett’s test for comparison with vehicle-treated non- tg mice (*p < 0.05) and Tukey–Kramer test for comparison with vehicle-treated α-syn tg mice (^#p < 0.05).