Non-lysosomal β-glucocerebrosidase inhibition with ambroxol hydrochloride in an animal model of amyotrophic lateral sclerosis

A. Henriques¹, A. Bouscary², M. Spedding¹, J. Loeffler². ¹Spedding Research Solutions, Le Vesinet, France, ²INSERM U1118, University of Strasbourg, Strasbourg, France.

Title: Neuroprotection and axonal plasticity after inhibition of non-lysosomal glucocerebrosidase with ambroxol hydrochloride in an animal model of amyotrophic lateral sclerosis

Introduction: Metabolomic and transcriptomic reports connect glucosylceramide and other glycosphingolipides to neurodegeneration and muscle denervation in amyotrophic lateral sclerosis (ALS) (1). Glucosylceramide is degraded by GBA1 and GBA2 β-glucocerebrosidases and their inhibition with conduritol B epoxide (CBE) is beneficial in an animal model of ALS (2). Ambroxol hydrochloride is a safe drug known to inhibit GBA2, a non-lysosomal form of β-glucocerebrosidase. The aim of the study was to investigate whether ambroxol hydrochloride could be a drug candidate for amyotrophic lateral sclerosis.

Method: In vitro β-glucocerebrosidase activities were determined using an assay based on 4-Methylumbelliferyl-β-D-glucopyranoside (n=5-6/group). Adult FVB female SOD1^G86R mice and non-transgenic mice were used. In vivo experiments followed current European Union regulations and were approved by the local ethic committee (Aafis#4555). Ambroxol hydrochloride was given through drinking water (160mg/kg/day, vehicle: drinking water). Muscle innervation was assessed by histology (n=4 WT; n=7/group SOD1^G86Rmice). Disease progression was monitored daily (n=14/group). In vitro axonal plasticity was studied with a co-culture of spinal cord explants and myoblast cells (n=3-7 spinal explant/group). Data was expressed as the mean±SEM. Difference among groups was assessed with ANOVA followed by two-tailed Fisher’s LSD or Log-rank test. p-values<0.05 were considered significant.

Results: Compared to controls, spinal cord of SOD1^G86R mice had higher GBA2 activity (100%±16 versus 301.9%±78.5, n=6). Ambroxol hydrochloride (100μM) did not affect GBA1 activity but inhibited 50% of GBA2 activity (Fig. 1A, B). In vivo, ambroxol hydrochloride improved muscle innervation (Fig. 1C) and extended survival (Fig. 1D) in symptomatic SOD1^G86R mice. In vitro, ambroxol hydrochloride (100μM) enlarged functional neurite network (Fig. 1E).

Figure 1. Ambroxol hydrochloride supports axonal plasticity and improves survival in SOD1^G86R mice. A-B. CBE and ambroxol hydrochloride’s effect on in vitro GBA1 (A) or GBA2 (B) activities. C. AMB’s effect on muscle innervation. D; Kaplan-Meier showing survival of SOD1^G86R mice. E. Neurite network of spinal explants. NMJs, neuromuscular junctions. AMB, ambroxol hydrochloride. *, p<0.05.

Conclusions: Glycosphingolipids play an important role in muscle innervation, which degenerates in ALS from a very early disease stage. Ambroxol hydrochloride is a safe drug which may prove to be effective in this devastating disease.

References:

[1] Henriques A, et al. (2015). Hum.Mol.Genet. 25:7390-7405

[2] Henriques A et al. (2017) Scientific Reports. 7:5235