CNS expression of glucocerebrosidase corrects alpha-synuclein pathology and memory in a mouse model of Gaucher-related synucleinopathy

Abstract

Emerging genetic and clinical evidence suggests a link between Gaucher disease and the synucleinopathies Parkinson disease and dementia with Lewy bodies. Here, we provide evidence that a mouse model of Gaucher disease (Gba1(D409V/D409V)) exhibits characteristics of synucleinopathies, including progressive accumulation of proteinase K-resistant α-synuclein/ubiquitin aggregates in hippocampal neurons and a coincident memory deficit. Analysis of homozygous (Gba1(D409V/D409V)) and heterozygous (Gba1(D409V/+) and Gba1(+/-)) Gaucher mice indicated that these pathologies are a result of the combination of a loss of glucocerebrosidase activity and a toxic gain-of-function resulting from expression of the mutant enzyme. Importantly, adeno-associated virus-mediated expression of exogenous glucocerebrosidase injected into the hippocampus of Gba1(D409V/D409V) mice ameliorated both the histopathological and memory aberrations. The data support the contention that mutations in GBA1 can cause Parkinson disease-like α-synuclein pathology, and that rescuing brain glucocerebrosidase activity might represent a therapeutic strategy for GBA1-associated synucleinopathies.

Fig. 1.

Progressive accumulation of α-syn/ubiquitin aggregates in Gba1^D409V/D409V Gaucher mouse brains. (A) Frozen sections were pretreated without (Upper) or with (Lower) proteinase K (PK) to uncover endogenous α-syn aggregates (arrowheads). Images show α-syn (red) and ubiquitin (green) immunostaining and nuclear staining (blue) in the hippocampus of 12-mo-old Gba1^D409V/D409V mice. (Scale bar, 200 μm.) (B) Quantification of α-syn immunoreactivity in wild-type (WT) and Gba1^D409V/D409V hippocampi at 2, 6, and 12 mo following proteinase K treatment showed progressive accumulation of aggregates with age (n ≥ 5 per group). (C) Quantification of ubiquitin immunoreactivity in Gba1^D409V/D409V hippocampus at 2, 6, and 12 mo (n ≥ 5 per group) showed progressive accumulation with age, similar to α-syn. (D) Increased ubiquitination of high molecular weight proteins was detected in the hippocampus of Gba1^D409V/D409V mice. Shown is an immunoblot of hippocampal lysates from 12-mo-old Gba1^D409V/D409V mice and age-matched controls: ubiquitin (green) and β-tubulin (red). Each lane represents an independent mouse brain. (E) Quantitative analysis of ubiquitin/β-tubulin immunoblots in 12-mo-old Gba1^D409V/D409V mice and age-matched controls. All data represent mean ± SEM. Bars with * and # symbols are significantly different from each other (P < 0.05), as noted also in Table 1.

Fig. 2.

Ubiquitin and α-syn aggregates colocalize with neuronal markers. Sections of hippocampus in 12-mo-old Gba1^D409V/D409V brains, either pretreated with proteinase K (PK) (B) or not (A and C) before immunostaining. Immunostaining for NFH (A) exposed the presence of normal (arrowheads) and dystrophic (arrows) hippocampal neurites. (A) Colabeling of NFH and ubiquitin showed that ubiquitin aggregates were located in neurites. (B) Pretreating sections with proteinase K showed MAP2 colocalized with α-syn in Gba1^D409V/D409V hippocampus. (C) Immunostaining for GFAP and ubiquitin showed no colocalization of the astrocyte marker with the ubiquitin aggregates. DAPI nuclear staining is shown in blue. (Scale bar, 50 μm.)

Fig. 3.

Analysis of glucocerebrosidase substrates in Gba1^D409V/D409V mouse hippocampus shows progressive accumulation of glucosylsphingosine. The levels of GlcCer and GlcSph substrates in hippocampal lysates of 2-, 6-, and 12-mo-old wild-type and Gba1^D409V/D409V mice were quantified by mass spectrometry (n ≥ 6 per group). (A) No differences in the levels of GlcCer were noted between normal and Gaucher mice. (B) However, Gba1^D409V/D409V mice exhibited a marked and progressive accumulation of GlcSph. Data shown represent mean ± SEM. Bars with different symbols are significantly different from each other and from wild-type controls (P < 0.05).

Fig. 4.

Hippocampal memory deficits in Gba1^D409V/D409V mice. (A) Six-month-old wild-type and Gba1^D409V/D409V mice showed no object preference when exposed to two identical objects. Results from Trial 1 (training) are shown as white (wild-type mice) and blue (Gba1^D409V/D409V mice) solid bars. After a 24-h retention period, mice were presented with a novel object. In trial 2 (testing), wild-type mice (white hatched bar) investigated the novel object significantly more times (P < 0.05). In contrast, Gba1^D409V/D409V mice (blue hatched bar) showed no preference for the novel object, indicating a cognitive impairment (P = 0.92). The horizontal line demarcates 50% target investigations, which represents no preference for either object. (B and C) Open-field analysis showed no differences in ambulation or anxiety between Gba1^D409V/D409V and age-matched wild-type controls. (D and E) The memory impairment of Gba1^D409V/D409V mice was confirmed by decreased freezing responses in contextual and cued fear conditioning testing. All data represent mean ± SEM (*P < 0.05; **P < 0.01).

Fig. 5.

CNS administration of scAAV1-GBA1 ameliorates accumulation of GlcSph and ubiquitin/α-syn aggregates and corrects memory deficits in Gba1^D409V/D409V mice. Two-month-old Gba1^D409V/D409V mice were injected with either scAAV1-GFP (n = 8) or scAAV1-GBA1 (n = 12) bilaterally into the hippocampus. Age-matched wild-type (WT) uninjected mice were used as a positive control (n = 10). (A) Animals were subjected to the novel object-recognition test at 2 mo postinjection, as outlined in the legend to Fig. 4. scAAV1-GBA1–treated Gba1^D409V/D409V mice (purple hatched bar) but not scAAV1-GFP–treated animals (green hatched bar) showed improvement in the memory test. Four months postinjection, tissues were collected for biochemical and pathological analysis. Hippocampal administration of scAAV1-GBA1 to Gba1^D409V/D409V mice increased glucocerebrosidase activity (B), promoted clearance of GlcSph (C), and reduced the accumulation of hippocampal α-syn (D) and ubiquitin (E) aggregates. Results represent mean ± SEM. (A) Horizontal line demarcates 50% target investigations, which represents no preference for either object (*, significantly different from 50%, P < 0.05); (B–E) Bars with different symbols are significantly different from each other (P < 0.05).