Rho Kinase Inhibition with Fasudil in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis-Symptomatic Treatment Potential after Disease Onset

Abstract

Despite an improved understanding of the genetic background and the pathomechanisms of amyotrophic lateral sclerosis (ALS) no novel disease-modifying therapies have been successfully implemented in clinical routine. Riluzole still remains the only clinically approved substance in human ALS treatment with limited efficacy. We have previously identified pharmacological rho kinase (ROCK) inhibitors as orally applicable substances in SOD1.G93A transgenic ALS mice (SOD1^G93A), which are able to extend survival time and improve motor function after presymptomatic treatment. Here, we have evaluated the therapeutic effect of the orally administered ROCK inhibitor Fasudil starting at a symptomatic disease stage, more realistically reflecting the clinical situation. Oral Fasudil treatment was initiated at a symptomatic stage at 80 days of life (d80) with 30 or 100 mg/kg body weight in both female and male mice. While baseline neurological scoring and survival were not influenced, Fasudil significantly improved motor behavior in male mice. Spinal cord pathology of motoneurons (MN) and infiltrating microglial cells (MG) at disease end-stage were not significantly modified. Although treatment after symptom onset was less potent than treatment in asymptomatic animals, our study shows the therapeutic benefits of this well-tolerated substance, which is already in clinical use for other indications.

Keywords: Fasudil; ROCK; SOD1; mouse model of ALS; neuroprotection.

Figure 1

Electrophysiological analysis including NCV, CMAP and DML of the sciatic nerve. Depicted are histograms for d70 (A–D) and for d100 old (E–H) SOD1^WT and SOD1 ^G93A mice (SOD1^WT d70 n = 3, SOD1 ^G93A d70 n = 3, SOD1^WT d100 n = 6, SOD1 ^G93A d100 n = 6). Bar represents means ± SEM; ^*P < 0.05.; ^**P < 0.01.

Figure 2

Weight dynamics in SOD1^G93A transgenic mice orally treated with 30 mg/kg Fasudil (Fas30), 100 mg/kg Fasudil (Fas100) or with vehicle (Veh). Depicted are female (Fas30 n = 14, Fas100 n = 15, Veh n = 14) (A,B) and male (Fas30 n = 11, Fas100 n = 12, Veh n = 12) (C,D) cohorts.

Figure 3

Progression of neurological symptoms and disease duration in SOD1^G93A mice treated with 30 mg/kg Fasudil (Fas30), 100 mg/kg Fasudil (Fas100), or with vehicle (Veh) as control. Displayed are neurological scores and disease duration for female (Fas30 n = 14, Fas100 n = 15, Veh n = 14) (A,B) and male (Fas30 n = 11, Fas100 n = 12, Veh n = 12) (C,D) cohorts.

Figure 4

Survival in SOD1^G93A mice treated with 30 mg/kg Fasudil (Fas30), 100 mg/kg Fasudil (Fas100), or with vehicle (Veh). Depicted is the cumulative probability of survival of female (Fas30 n = 14, Fas100 n = 15, Veh n = 14) (A) and male (Fas30 n = 11, Fas100 n = 12, Veh n = 12) (B) mice.

Figure 5

ROCK inhibition with Fasudil stabilizes motor performance in SOD1^G93A male mice. Displayed are mean durations on the rotarod of female (Fas30 n = 14, Fas100 n = 15, Veh n = 14) (A) and male (Fas30 n = 11, Fas100 n = 12, Veh n = 12). (B) SOD1 ^G93A mice treated with 30 mg/kg Fasudil (Fas30), 100 mg/kg Fasudil (Fas100), or with vehicle (Veh). Data represent means ± SEM. ^* P < 0.05; ^**P < 0.01, ^***P < 0.001.

Figure 6

Motoneuronal survival in the spinal cord in end-stage stage SOD1^G93A transgenic mice. (A,B) Numbers of ChAT-immunopositive motoneurons (MN) per section in the spinal cord anterior horn of female (Fas30 n = 3, Fas100 n = 3, Veh n = 3) and male (Fas30 n = 3, Fas100 n = 3, Veh) mice treated with 30 mg/kg Fasudil (Fas30), 100 mg/kg Fasudil (Fas100), or with vehicle (Veh). (C,D) Representative micrographs of ChAT-immunopositive MNs in the spinal cord anterior horn of female (C) and male (D) mice. [ChAT/Cy3 (red), DAPI (blue)]. Bars represent means ± SEM. Scale bar: 100 μm.

Figure 7

Infiltration of microglial cells in the lumbar spinal cord anterior horn in end-stage stage SOD1^G93A mice. (A,B) Numbers of Iba1-immunopositive microglia (MG) per section in the spinal cord anterior horn of female (Fas30 n = 3, Fas100 n = 3, Veh) (A) and male (Fas30 n = 3, Fas100 n = 3, Veh) (B) mice treated with 30 mg/kg Fasudil (Fas30), 100 mg/kg Fasudil (Fas100), or with vehicle (Veh). (C,D) Representative micrographs of Iba1-immunopositive MG in spinal cord anterior horn of female (C) and male (D) mice. [Iba1/Cy3 (red), DAPI (blue)]. Bars represent means ± SEM. Scale bar: 100 μm.