Inhibition of rho kinase enhances survival of dopaminergic neurons and attenuates axonal loss in a mouse model of Parkinson's disease

Abstract

Axonal degeneration is one of the earliest features of Parkinson's disease pathology, which is followed by neuronal death in the substantia nigra and other parts of the brain. Inhibition of axonal degeneration combined with cellular neuroprotection therefore seem key to targeting an early stage in Parkinson's disease progression. Based on our previous studies in traumatic and neurodegenerative disease models, we have identified rho kinase as a molecular target that can be manipulated to disinhibit axonal regeneration and improve survival of lesioned central nervous system neurons. In this study, we examined the neuroprotective potential of pharmacological rho kinase inhibition mediated by fasudil in the in vitro 1-methyl-4-phenylpyridinium cell culture model and in the subchronic in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. Application of fasudil resulted in a significant attenuation of dopaminergic cell loss in both paradigms. Furthermore, dopaminergic terminals were preserved as demonstrated by analysis of neurite network in vitro, striatal fibre density and by neurochemical analysis of the levels of dopamine and its metabolites in the striatum. Behavioural tests demonstrated a clear improvement in motor performance after fasudil treatment. The Akt survival pathway was identified as an important molecular mediator for neuroprotective effects of rho kinase inhibition in our paradigm. We conclude that inhibition of rho kinase using the clinically approved small molecule inhibitor fasudil may be a promising new therapeutic strategy for Parkinson's disease.

Figure 1

Survival of tyrosine hydroxylase-immunopositive neurons after 20 µM MPP⁺ or PBS control treatment for 48 h. (A) MPP⁺-treated cultures supplemented with fasudil 20 µM (Fas20) showed higher numbers of surviving tyrosine hydroxylase-immunopositive neurons compared with vehicle-treated cells (Ctrl). Bars represent means ± SEM. *P < 0.05, ***P < 0.001. (B) Representative micrographs of midbrain dopaminergic neurons cultures labelled against tyrosine hydroxylase (Cy3, red). Scale bar = 200 µm. n.s = not significant; TH = tyrosine hydroxylase.

Figure 2

Quantification of tyrosine hydroxylase-immunopositive neurites 48 h after 20 µM MPP⁺ or PBS control treatment. (A and B) Supplementation with fasudil 20 µM (Fas20) results in an increased neurite length per cell (A) and in an increased cumulative neurite length (B) both with and without MPP⁺ stress. Bars represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ^§P = 0.08. (C) Representative micrographs of midbrain dopaminergic neuron cultures labelled against tyrosine hydroxylase (Cy3, red). Scale bar = 50 µm. Ctrl = control.

Figure 3

Neurite outgrowth of tyrosine hydroxylase-immunopositive cells from the lesion site 48 h after mechanical scratch with and without concomitant MPP⁺ treatment. (A) MPP⁺ (20 µM) toxicity leads to an overall reduction in neurite outgrowth. Supplementation with fasudil 20 µM (Fas20) results in an increased outgrowth both with PBS and with MPP⁺ stress, whereas fasudil 100 µM (Fas100) does enhance neurite outgrowth only under PBS treatment. Bars represent means ± SEM. *P < 0.05, n.s = not significant. (B) Representative micrographs of midbrain dopaminergic neuron cultures labelled for tyrosine hydroxylase (white). Arrow indicates site of mechanical scratch lesion. Scale bar = 100 µm. Ctrl = control.

Figure 4

Nigral neuronal protection by 2 weeks rho kinase inhibition in MPTP-treated mice. (A and B) Quantification of nigral cells immunopositive for tyrosine hydroxylase (TH, A) or NeuN (B) in mice treated with vehicle (Vehicle Ctrl), MPTP and vehicle (MPTP Ctrl) or fasudil (100 mg/kg) and MPTP (Fas MPTP). (C) Representative micrographs of substantia nigra mouse brain sections labelled against tyrosine hydroxylase (black). Bars represent means ± SD. ***P < 0.001. Scale bar = 1 mm. Fas = fasudil; SN = substantia nigra.

Figure 5

Nigral neuronal protection by 6 weeks rho kinase inhibition in MPTP-treated mice. (A and B) Quantification of nigral cells immunopositive for tyrosine hydroxylase (TH, A) or of Nissl-positive neurons (B) in mice treated with vehicle (Vehicle Ctrl), MPTP and vehicle (MPTP Ctrl), fasudil 30 mg/kg and MPTP (Fas30 MPTP) or fasudil 100 mg/kg and MPTP (Fas100 MPTP). (C) Representative micrographs of substantia nigra (SN) mouse brain sections labelled against tyrosine hydroxylase (black). Bars represent means ± SEM. *P < 0.05, **P < 0.01. n.s = not significant. Scale bar = 1 mm.

Figure 6

Striatal protection by 2 weeks rho kinase inhibition in MPTP-treated mice. (A) Quantification of striatal tyrosine hydroxylase-immunopositive fibre innervation in mice treated with vehicle (Vehicle Ctrl), MPTP and vehicle (MPTP Ctrl) or fasudil (100 mg/kg) and MPTP (Fas MPTP). (B) Representative micrographs of striatal fibres immunopositive for tyrosine hydroxylase (black). (C–F) HPLC analysis of striatal dopamine and its metabolites. (G) Striatal MPP⁺levels 90 min after MPTP injection. Bars represent means ± SEM. **P < 0.01, ***P < 0.001. DOPAC = dihydroxyphenylacetic acid; HVA = homovanillic acid; met.ratio = metabolite ratio; n.s = not significant. Scale bar = 1 mm.

Figure 7

Striatal protection by 6 weeks rho kinase inhibition in MPTP-treated mice. (A) Quantification of striatal tyrosine hydroxylase-immunopositive fibre innervation in mice treated with vehicle (Vehicle Ctrl), MPTP and vehicle (MPTP Ctrl), fasudil 30 mg/kg and MPTP (Fas30 MPTP) or fasudil 100 mg/kg and MPTP (Fas100 MPTP). (B) Representative micrographs of striatal fibres immunopositive for tyrosine hydroxylase (TH, black). (C–F) HPLC analysis of striatal dopamine and its metabolites. Bars represent means ± SEM. *P < 0.05, ***P < 0.001, ^§P = 0.07. DOPAC = Dihydroxyphenylacetic acid; HVA = homovanillic acid; n.s = not significant. Scale bar = 1 mm.

Figure 8

Behavioural analysis of mice treated with the rho kinase inhibitor fasudil for 2 weeks after the initial MPTP application. (A) Quantification of the percentage of rears against the wall with both paws, only right paw, only left paw or free rears. (B) Quantification of the average running time out of three runs on an accelerating rotarod. Bars represent means ± SEM. *P < 0.05, ***P < 0.001, ^§P = 0.06. n.s = not significant.

Figure 9

Behavioural analysis of mice treated with the rho kinase inhibitor fasudil for 6 weeks after the initial MPTP application. (A) Quantification of the percentage of rears against the wall with both paws, only right paw, only left paw or free rears. (B) Quantification of the average running time out of three runs on an accelerating rotarod. Bars represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. n.s = not significant.

Figure 10

Activation of survival signalling pathways after rho kinase inhibition by fasudil 20 µM (Fas) or Y-27632 10 µM in midbrain dopaminergic neurons treated with or without MPP⁺. (A) Representative immunoblots showing the regulation of Akt, pAkt, MAPK, pMAPK, STAT3, pSTAT3, Bcl2 and GAP43. (B–I) Quantification of immunoblot bands from three independent experiments for (total) Akt and phosphorylated Akt/(total) Akt (B and C), (total) MAPK and phosphorylated MAPK/(total) MAPK (D and E), (total) STAT3 and phosphorylated STAT3/(total) STAT3 (F and G), Bcl2 (H) and GAP43 (I). Bars represent means ± SEM. **P < 0.01.