Glucosylceramide synthase inhibition alleviates aberrations in synucleinopathy models

Abstract

Mutations in the glucocerebrosidase gene (GBA) confer a heightened risk of developing Parkinson's disease (PD) and other synucleinopathies, resulting in a lower age of onset and exacerbating disease progression. However, the precise mechanisms by which mutations in GBA increase PD risk and accelerate its progression remain unclear. Here, we investigated the merits of glucosylceramide synthase (GCS) inhibition as a potential treatment for synucleinopathies. Two murine models of synucleinopathy (a Gaucher-related synucleinopathy model, Gba^D409V/D409V and a A53T-α-synuclein overexpressing model harboring wild-type alleles of GBA, A53T-SNCA mouse model) were exposed to a brain-penetrant GCS inhibitor, GZ667161. Treatment of Gba^D409V/D409V mice with the GCS inhibitor reduced levels of glucosylceramide and glucosylsphingosine in the central nervous system (CNS), demonstrating target engagement. Remarkably, treatment with GZ667161 slowed the accumulation of hippocampal aggregates of α-synuclein, ubiquitin, and tau, and improved the associated memory deficits. Similarly, prolonged treatment of A53T-SNCA mice with GZ667161 reduced membrane-associated α-synuclein in the CNS and ameliorated cognitive deficits. The data support the contention that prolonged antagonism of GCS in the CNS can affect α-synuclein processing and improve behavioral outcomes. Hence, inhibition of GCS represents a disease-modifying therapeutic strategy for GBA-related synucleinopathies and conceivably for certain forms of sporadic disease.

Keywords: GBA mutations; Gaucher disease; Lewy body dementia; Parkinson’s disease; glucosylceramide synthase.

Fig. 1.

The glucosylceramide synthase (GCS) inhibitor, GZ667161 reduces CNS glycosphingolipids in the mouse model of Gaucher-related synucleinopathy. (A) Structure of GZ667161 (quinuclidin-3-yl-N-[1-[3-(4-fluorophenyl)phenyl]-1-methyl-ethyl]carbamate). (B) Schematic of glucosylceramide synthase inhibition by GZ667161. Gba^D409V/D409V mice were fed GZ667161 as described in SI Materials and Methods, littermates were fed a control diet, and age-matched, wild-type untreated mice were positive controls. (C and D) GZ667161 treatment reduced brain GlcCer in Gba^D409V/D409V mice (cohorts 1 and 2, Materials and Methods). (E and F) Gba^D409V/D409V mice accumulate the glucocerebrosidase substrate, GlcSph, but GZ667161 significantly reduced their GlcSph. The results are represented as means ± SEM, with n ≥ 10 per group (*P < 0.05; **P < 0.01).

Fig. S1.

Timeline for treatment and endpoints. (A) Two Gba^D409V/D409V cohorts were studied. In cohort 1 (presymptomatic), drug administration was initiated after pups were weaned at 4 wk of age and continued until killing at 10 mo of age. Gba^D409V/D409V cohort 2 (symptomatic) was administered GZ667161 starting at 6 mo of age until killing at 13 mo of age. (B) A53T–SNCA mice were randomized to GZ667161 or control diet at 6 wk of age. Treatment continued until the end of the study at 8 mo of age. Abbreviations: CM, contextual memory; NOR, novel object recognition; GSL, glycosphingolipid quantification; IHC, immunohistochemistry (Proteinase K-resistant α-synuclein, ubiquitin, and tau).

Fig. 2.

GCS inhibition ameliorates memory deficits in a mouse model of Gaucher-related synucleinopathy. Two independent randomized cohorts of Gba^D409V/D409V mice received GZ667161 or control diet. Age-matched WT mice served as positive controls. (A) Cohort 1 mice were subjected to the novel object recognition test 2 mo after treatment initiation. None of the groups showed an object preference after exposure to two identical objects during training (clear white, blue, and red bars). After 24 h, the mice were presented with a novel object. In the testing trial (hatched bars), WT mice investigated the novel object significantly more (P < 0.01), Gba^D409V/D409V mice (blue hatched bar) showed no preference for the novel object (cognitive impairment), and GZ667161-treated Gba^D409V/D409V mice showed a modest but significant increase in investigation of the novel target (red hatched bars, P < 0.05). (B) The memory impairment of cohort 1 Gba^D409V/D409V mice was corroborated by decreased freezing responses in the contextual fear conditioning test at 9 mo of age (blue bar, P < 0.05), whereas littermate mice treated with GZ667161 showed a better contextual memory response (red bar). (C and D) Cognitively impaired Gba^D409V/D409V mice were randomized to receive GZ667161 (n = 15) or control diet (n = 14) from 6 to 13 mo of age (cohort 2, symptomatic). GZ667161 treatment attenuated the memory impairment in the novel object recognition and contextual fear tasks. The horizontal line demarcates 50% target investigations, which represents no preference for either object. The results are represented as means ± SEM, with n ≥ 10 per group (*P < 0.05; **P < 0.01).

Fig. S2.

The glucosylceramide synthase (GCS) inhibitor, GZ667161, reduces CNS glucosylceramide and does not affect memory in wild-type animals. C57BL/6 mice were fed GZ667161 as described in SI Materials and Methods, littermates were fed a standard control diet. (A) Treatment with GZ667161 reduced brain GlcCer. (B) Treatment of wild-type mice with GZ667161 did not affect the contextual memory response. Data are presented as mean ± SEM (t test, **P < 0.01, significantly different from one another). NS, not significant.

Fig. 3.

GCS inhibition reduces pathological aberrations in a mouse model of Gaucher-related synucleinopathy. Brain sections from WT and Gba^D409V/D409V mice were stained for proteinase K-resistant α-synuclein (A), ubiquitin (B), and tau (C) aggregates. GZ667161 reduced aggregated protein levels of Gba^D409V/D409V mice. The representative images (Right) show proteinase K-resistant α-synuclein immunoreactivity (A, red), ubiquitin (B, green), and tau (C, green) in the hippocampi of GZ667161-treated and control Gba^D409V/D409V mice of cohort 1. DAPI-stained cell nuclei fluoresce blue. All data represent the mean ± SEM, with n ≥ 8 per group (*P < 0.05; **P < 0.01).

Fig. 4.

GCS inhibition reduces GlcCer and affects cognition in the A53T–SNCA mouse model of synucleinopathy. A53T–SNCA mice were fed GZ667161 from 6 wk of age to 8 mo. Equivalent littermates were fed a control diet to monitor disease progression, and age-matched untreated WT mice were positive controls. (A) GZ667161 treatment reduced brain GlcCer in A53T–SNCA mice. (B) All mice subjected to the novel object recognition test at 4.5 mo of age showed no object preference after exposure to two identical objects during training (clear white, blue, and red bars). WT mice investigated the novel object significantly more frequently than A53T–SNCA mice (white hatched bars, P < 0.01), but A53T–SNCA mice (blue hatched bar) showed no such preference, indicating cognitive impairment. GZ667161-treated A53T–SNCA mice showed a nonsignificant trend toward cognitive improvement (red hatched bars, P = 0.11). (C) The memory impairment of A53T–SNCA mice was corroborated by a decreased freezing response in the contextual fear-conditioning test at 8 mo of age (blue bar, P < 0.05). Treatment of A53T–SNCA mice with GZ667161 significantly attenuated the contextual memory responses at this later time point (red bars, P < 0.05). Bars with different letters are significantly different from one another (P < 0.05). All data represent the mean ± SEM, with n ≥ 10 per group (*P < 0.05; **P < 0.01).

Fig. 5.

GCS inhibition affects α-synuclein membrane distribution and reduces pathological aberrations in the A53T–SNCA mouse model of synucleinopathy. A53T–SNCA mice were randomized to receive GZ667161 or control diet (n = 12 in each group) from 6 wk of age to 8 mo. Homogenized cerebral cortices provided cytosolic and membrane-associated fractions. (A) GCS inhibition decreased human α-synuclein in the membrane-associated fractions (red bars; P < 0.05). (B and C) GZ667161 reduced accumulation of hippocampal aggregated ubiquitin (B) and tau (C) in these mutant mice, with littermates killed at 6 wk of age to provide a histological baseline (n = 6). The images show ubiquitin (B, green) and tau (C, green) immunoreactivity in hippocampi of control or GZ667161-treated 8-mo-old A53T–SNCA mice. DAPI stains cell nuclei blue. All data represent the mean ± SEM, with n ≥ 8 per group (*P < 0.05; **P < 0.01).