Augmenting CNS glucocerebrosidase activity as a therapeutic strategy for parkinsonism and other Gaucher-related synucleinopathies

Abstract

Mutations of GBA1, the gene encoding glucocerebrosidase, represent a common genetic risk factor for developing the synucleinopathies Parkinson disease (PD) and dementia with Lewy bodies. PD patients with or without GBA1 mutations also exhibit lower enzymatic levels of glucocerebrosidase in the central nervous system (CNS), suggesting a possible link between the enzyme and the development of the disease. Previously, we have shown that early treatment with glucocerebrosidase can modulate α-synuclein aggregation in a presymptomatic mouse model of Gaucher-related synucleinopathy (Gba1(D409V/D409V)) and ameliorate the associated cognitive deficit. To probe this link further, we have now evaluated the efficacy of augmenting glucocerebrosidase activity in the CNS of symptomatic Gba1(D409V/D409V) mice and in a transgenic mouse model overexpressing A53T α-synuclein. Adeno-associated virus-mediated expression of glucocerebrosidase in the CNS of symptomatic Gba1(D409V/D409V) mice completely corrected the aberrant accumulation of the toxic lipid glucosylsphingosine and reduced the levels of ubiquitin, tau, and proteinase K-resistant α-synuclein aggregates. Importantly, hippocampal expression of glucocerebrosidase in Gba1(D409V/D409V) mice (starting at 4 or 12 mo of age) also reversed their cognitive impairment when examined using a novel object recognition test. Correspondingly, overexpression of glucocerebrosidase in the CNS of A53T α-synuclein mice reduced the levels of soluble α-synuclein, suggesting that increasing the glycosidase activity can modulate α-synuclein processing and may modulate the progression of α-synucleinopathies. Hence, increasing glucocerebrosidase activity in the CNS represents a potential therapeutic strategy for GBA1-related and non-GBA1-associated synucleinopathies, including PD.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Progressive accumulation of tau aggregates in the brains of Gba1D409V/D409V mice. (A) Images show immunostaining with an anti-tau serum (green) and nuclear staining (DAPI; blue) in the hippocampi of 2-, 6-, and 12-mo-old Gba1D409V/D409V and age-matched wild-type (WT) mice. (Scale bar, 500 μm.) (B) Quantification of Tau-5 immunoreactivity in WT and Gba1D409V/D409V hippocampi at 2, 6, and 12 mo shows progressive accumulation of aggregates with age (n ≥ 5 per group). (C) Shown are representative immunoblots of hippocampal lysates from 18-mo-old Gba1D409V/D409V mice and age-matched controls for AT8, AT180, AT270, Tau-5, and β-tubulin. Each lane represents an independent mouse brain. Clone AT8 antibody shows increased tau phosphorylation (S202/T205) in aged Gba1D409V/D409V mice. No differences between mutant and wild-type mice were observed in total tau levels (Tau-5) or other phosphorylated species (AT180 or AT270). The results are represented as the means ± SEM. Bars marked with different letters are significantly different from each other (P < 0.05).

Fig. 2.

CNS administration of AAV–GBA1 reduces GlcSph levels and reverses memory deficits. Four-mo-old and 12-mo-old Gba1D409V/D409V mice were given bilateral hippocampal injections of either AAV–EV or AAV–GBA1. Uninjected Gba1D409V/D409V littermates were euthanized at the time of surgeries as baselines for biochemical and histological endpoints (n = 8). Age-matched, uninjected wild-type (WT; n = 9) mice were used as a positive control. In both cohorts, tissues were collected for biochemical and pathological analysis at 6 mo postinjection. (A) Hippocampal expression of the recombinant enzyme 6 mo after stereotaxic injections. Image shows glucocerebrosidase immunoreactivity (red) and nuclear (DAPI; blue) stains in an AAV–GBA1-injected Gba1D409V/D409V mouse. (Scale bar, 400 μm.) Inset depicts glucocerebrosidase and nuclear staining in an AAV–EV-injected mouse. (B and C) Hippocampal administration of AAV–GBA1 into Gba1D409V/D409V mice increased glucocerebrosidase activity (B, red; n = 11, P < 0.05) and promoted clearance of GlcSph to WT levels (C, red; n = 11, P < 0.05), whereas AAV–EV-treated Gba1D409V/D409V mice showed no change in glucocerebrosidase activity (B, blue; n = 12, P > 0.05) and continued to accumulate GluSph compared with baseline levels (C, black; n = 8, P < 0.05). (D) Presurgical evaluation of 4-mo-old wild-type (WT) and Gba1D409V/D409V mice revealed no object preference when exposed to two identical objects. The results from trial 1 (training) are shown as white (WT) and purple (Gba1D409V/D409V mice) filled bars. After a 24-h retention period, mice were presented with a novel object. In trial 2 (testing, hatched bars), WT mice investigated the novel object significantly more frequently (n = 9, P < 0.05). In contrast, Gba1D409V/D409V mice (n = 11; purple hatched bar) showed no preference for the novel object, indicating a cognitive impairment. (E) At 2 mo postinjection, mice were subjected to the novel object recognition (NOR) test. AAV–GBA1-treated Gba1D409V/D409V mice (n = 10; blue hatched bar), but not AAV–EV-treated animals (n = 9; red hatched bar), exhibited a reversal of their memory deficit when presented with the novel object during the testing trial. (F) A separate cohort of 12-mo-old Gba1D409V/D409V mice were injected with AAV–EV (n = 12) or AAV–GBA1 (n = 12). Similar to the 4-mo-old cohort, reversal of the memory dysfunction was observed when these animals were tested at 2 mo postinjection (14 mo of age). The results are represented as means ± SEM. (D–F) The horizontal line demarcates 50% target investigations, which represents no preference for either object (*, significantly different from 50%, P < 0.05). (B and C) Bars with different letters are significantly different from each other (P < 0.05).

Fig. 3.

Expression of glucocerebrosidase in symptomatic Gba1D409V/D409V mouse hippocampi slows accumulation of aggregated α-synuclein and tau. Two cohorts of Gba1D409V/D409V mice were injected with either AAV–EV or –GBA1 bilaterally into the hippocampus at 4 or 12 mo of age. Age-matched, uninjected WT mice were left untreated as positive controls. Gba1D409V/D409V littermates were harvested at the time of the injections as a baseline group. Injected animals were killed 6 mo after surgery. Graphs represent hippocampal quantifications of ubiquitin (A), proteinase K-resistant α-synuclein (B), and tau immunoreactivity (C) for the cohorts injected at 4 (Left) or 12 (Center) mo of age. Glucocerebrosidase augmentation in the CNS of symptomatic Gba1D409V/D409V mice reduced the levels of aggregated proteins, although this treatment was less effective in older animals. Images (Right) show ubiquitin (A, green), proteinase K-resistant α-synuclein (B, red) and tau (C, green) immunoreactivity in the hippocampi of 18-mo-old Gba1D409V/D409V mice treated with AAV–EV or –GBA1. DAPI nuclear staining is shown in blue. (Scale bar, 100 μm.) The results are represented as means ± SEM, with n ≥ 8 per group. Bars with different letters are significantly different from each other (P < 0.05).

Fig. 4.

Glucocerebrosidase augmentation in A53T α-synuclein mouse brain decreases α-synuclein levels. A53T α-synuclein transgenic mice exhibit decreased brain glucocerebrosidase activity. (A) The activity of various lysosomal enzymes was determined in cortical homogenates from homozygous (n = 9) and heterozygous (n = 8) α-synuclein transgenics and wild-type littermates (n = 9). Glucocerebrosidase activity was inversely correlated with α-synuclein levels, with homozygous mice showing a greater reduction of hydrolase activity. The enzymatic activities of two other lysosomal hydrolases, hexosaminidase and β-galactosidase, remained unchanged by the expression of A53T α-synuclein. (B) Four-mo-old A53T α-synuclein mice were each injected with either AAV–GFP (n = 6) or AAV–GBA1 (n = 5) unilaterally into the right striatum. The left striatum was used as an uninjected control for each animal to reduce the variability in α-synuclein levels between subjects. Four mo later, mice were euthanized, and both striata were collected. Robust glucocerebrosidase activity was observed in the AAV–GBA1-injected striatum (sevenfold over the uninjected contralateral side). Expression of glucocerebrosidase promoted decreased α-synuclein levels in the cytosolic fraction (Tris-soluble, non-membrane–associated; P < 0.05). (C) Newborn (P0) A53T α-synuclein mice were injected with either AAV–GFP or –GBA1 into the lumbar spinal cord. As expected, robust glucocerebrosidase activity was noted in AAV–GBA1-injected mice (threefold over controls). As in the striatum, expression of glucocerebrosidase reduced α-synuclein levels in the cytosolic fraction (Tris-soluble, nonmembrane associated; n = 7 per group, P < 0.05). Data are represented as means ± SEM. *P < 0.05.