L444P Gba1 mutation increases formation and spread of ?-synuclein deposits in mice injected with mouse ?-synuclein pre-formed fibrils.
ABSTRACT: Parkinson disease is the most common neurodegenerative movement disorder, estimated to affect one in twenty-five individuals over the age of 80. Mutations in glucocerebrosidase 1 (GBA1) represent the most common genetic risk factor for Parkinson disease. The link between GBA1 mutations and ?-synuclein accumulation, a hallmark of Parkinson disease, is not fully understood. Following our recent finding that Gba1 mutations lead to increased ?-synuclein accumulation in mice, we have studied the effects of a single injection of mouse ?-synuclein pre-formed fibrils into the striatum of Gba1 mice that carry a L444P knock-in mutation. We found significantly greater formation and spread of ?-synuclein inclusions in Gba1-transgenic mice compared to wild-type controls. This indicates that the Gba1 L444P mutation accelerates ?-synuclein pathology and spread.
Project description:Mutations in glucocerebrosidase 1 (GBA1) represent the most prevalent risk factor for Parkinson's disease. The molecular mechanisms underlying the link between GBA1 mutations and Parkinson's disease are incompletely understood. We analysed two aged (24-month-old) Gba1 mouse models, one carrying a knock-out mutation and the other a L444P knock-in mutation. A significant reduction of glucocerebrosidase activity was associated with increased total alpha-synuclein accumulation in both these models. Gba1 mutations alone did not alter the number of nigral dopaminergic neurons nor striatal dopamine levels. We then investigated the effect of overexpression of human alpha-synuclein in the substantia nigra of aged (18 to 21-month-old) L444P Gba1 mice. Following intraparenchymal injections of human alpha-synuclein carrying viral vectors, pathological accumulation of phosphorylated alpha-synuclein occurred within the transduced neurons. Stereological counts of nigral dopaminergic neurons revealed a significantly greater cell loss in Gba1-mutant than wild-type mice. These results indicate that Gba1 deficiency enhances neuronal vulnerability to neurodegenerative processes triggered by increased alpha-synuclein expression.
Project description:BACKGROUND:Mutations in glucocerebrosidase (GBA) cause Gaucher disease (GD) and increase the risk of developing Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB). Since both genetic and environmental factors contribute to the pathogenesis of sporadic PD, we investigated the susceptibility of nigrostriatal dopamine (DA) neurons in L444P GBA heterozygous knock-in (GBA +/L444P ) mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a selective dopaminergic mitochondrial neurotoxin. METHOD:We used GBA +/L444P mice, ?-synuclein knockout (SNCA -/- ) mice at 8 months of age, and adeno-associated virus (AAV)-human GBA overexpression to investigate the rescue effect of DA neuronal loss and susceptibility by MPTP. Mitochondrial morphology and functional assay were used to identify mitochondrial defects in GBA +/L444P mice. Motor behavioral test, immunohistochemistry, and HPLC were performed to measure dopaminergic degeneration by MPTP and investigate the relationship between GBA mutation and ?-synuclein. Mitochondrial immunostaining, qPCR, and Western blot were also used to study the effects of ?-synuclein knockout or GBA overexpression on MPTP-induced mitochondrial defects and susceptibility. RESULTS:L444P GBA heterozygous mutation reduced GBA protein levels, enzymatic activity and a concomitant accumulation of ?-synuclein in the midbrain of GBA +/L444P mice. Furthermore, the deficiency resulted in defects in mitochondria of cortical neurons cultured from GBA +/L444P mice. Notably, treatment with MPTP resulted in a significant loss of dopaminergic neurons and striatal dopaminergic fibers in GBA +/L444P mice compared to wild type (WT) mice. Levels of striatal DA and its metabolites were more depleted in the striatum of GBA +/L444P mice. Behavioral deficits, neuroinflammation, and mitochondrial defects were more exacerbated in GBA +/L444P mice after MPTP treatment. Importantly, MPTP induced PD-like symptoms were significantly improved by knockout of ?-synuclein or augmentation of GBA via AAV5-hGBA injection in both WT and GBA +/L444P mice. Intriguingly, the degree of reduction in MPTP induced PD-like symptoms in GBA +/L444P ?-synuclein (SNCA) -/- mice was nearly equal to that in SNCA -/- mice after MPTP treatment. CONCLUSION:Our results suggest that GBA deficiency due to L444P GBA heterozygous mutation and the accompanying accumulation of ?-synuclein render DA neurons more susceptible to MPTP intoxication. Thus, GBA and ?-synuclein play dual physiological roles in the survival of DA neurons in response to the mitochondrial dopaminergic neurotoxin, MPTP.
Project description:Accumulating evidence suggests that ?-synuclein (?-syn) occurs physiologically as a helically folded tetramer that resists aggregation. However, the mechanisms underlying the regulation of formation of ?-syn tetramers are still mostly unknown. Cellular membrane lipids are thought to play an important role in the regulation of ?-syn tetramer formation. Since glucocerebrosidase 1 (GBA1) deficiency contributes to the aggregation of ?-syn and leads to changes in neuronal glycosphingolipids (GSLs) including gangliosides, we hypothesized that GBA1 deficiency may affect the formation of ?-syn tetramers. Here, we show that accumulation of GSLs due to GBA1 deficiency decreases ?-syn tetramers and related multimers and increases ?-syn monomers in CRISPR-GBA1 knockout (KO) SH-SY5Y cells. Moreover, ?-syn tetramers and related multimers are decreased in N370S GBA1 Parkinson's disease (PD) induced pluripotent stem cell (iPSC)-derived human dopaminergic (hDA) neurons and murine neurons carrying the heterozygous L444P GBA1 mutation. Treatment with miglustat to reduce GSL accumulation and overexpression of GBA1 to augment GBA1 activity reverse the destabilization of ?-syn tetramers and protect against ?-syn preformed fibril-induced toxicity in hDA neurons. Taken together, these studies provide mechanistic insights into how GBA1 regulates the transition from monomeric ?-syn to ?-syn tetramers and multimers and suggest unique therapeutic opportunities for PD and dementia with Lewy bodies.
Project description: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 ?-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 ?-synuclein pathology, and that rescuing brain glucocerebrosidase activity might represent a therapeutic strategy for GBA1-associated synucleinopathies.
Project description:Mutations in GBA1 encountered in Gaucher disease are a leading risk factor for Parkinson disease and associated Lewy body disorders. Many GBA1 mutation carriers, especially those with severe or null GBA1 alleles, have earlier and more progressive parkinsonism. To model the effect of partial glucocerebrosidase deficiency on neurological progression in vivo, mice with a human A53T ?-synuclein (SNCAA53T) transgene were crossed with heterozygous null gba mice (gba+/-). Survival analysis of 84 mice showed that in gba+/-//SNCAA53T hemizygotes and homozygotes, the symptom onset was significantly earlier than in gba+/+//SNCAA53T mice (p-values 0.023-0.0030), with exacerbated disease progression (p-value <0.0001). Over-expression of SNCAA53T had no effect on glucocerebrosidase levels or activity. Immunoblotting demonstrated that gba haploinsufficiency did not lead to increased levels of either monomeric SNCA or insoluble high molecular weight SNCA in this model. Immunohistochemical analyses demonstrated that the abundance and distribution of SNCA pathology was also unaltered by gba haploinsufficiency. Thus, while the underlying mechanism is not clear, this model shows that gba deficiency impacts the age of onset and disease duration in aged SNCAA53T mice, providing a valuable resource to identify modifiers, pathways and possible moonlighting roles of glucocerebrosidase in Parkinson pathogenesis.
Project description:BACKGROUND: Mutations of the glucocerebrosidase (GBA) gene have reportedly been associated with Parkinson disease (PD) in various ethnic populations such as Singaporean, Japanese, Formosan, Canadian, American, Portuguese, Greek, Brazilian, British, Italian, Ashkenazi Jewish, southern and southwestern Chinese. The purpose of this study is to determine in central China whether or not the reported GBA mutations remain associated with PD. METHODS: In this project, we conducted a controlled study in a cohort of 208 central Chinese PD patients and 298 controls for three known GBA mutations (L444P, N370S and R120W). RESULTS: Our data reveals a significantly higher frequency of L444P mutation in GBA gene of PD cases (3.4%) compared with the controls (0.3%) (P = 0.007, OR = 10.34, 95% CI = 1.26 - 84.71). Specifically, the frequency of L444P mutation was higher in the late onset PD (LOPD) cases compared with that in control subjects. The N370S and R120W mutations were detected in neither the PD group nor the control subjects. CONCLUSIONS: Our observations demonstrated that the GBA L444P mutation confers genetic risk for PD, especially LOPD, among the population in the central China area.
Project description: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 ?-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 ?-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 ?-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 ?-synuclein mice reduced the levels of soluble ?-synuclein, suggesting that increasing the glycosidase activity can modulate ?-synuclein processing and may modulate the progression of ?-synucleinopathies. Hence, increasing glucocerebrosidase activity in the CNS represents a potential therapeutic strategy for GBA1-related and non-GBA1-associated synucleinopathies, including PD.
Project description:Mutations in the GBA1 gene are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). GBA1 encodes the lysosomal lipid hydrolase glucocerebrosidase (GCase), and its activity has been linked to accumulation of ?-synuclein. The current study systematically examines the relationship between GCase activity and both pathogenic and non-pathogenic forms of ?-synuclein in primary hippocampal, cortical, and midbrain neuron and astrocyte cultures, as well as in transgenic mice and a non-transgenic mouse model of PD. We find that reduced GCase activity does not result in aggregation of ?-synuclein. However, in the context of extant misfolded ?-synuclein, GCase activity modulates neuronal susceptibility to pathology. Furthermore, this modulation does not depend on neuron type but rather is driven by the level of pathological ?-synuclein seeds. This study has implications for understanding how GBA1 mutations influence PD pathogenesis and provides a platform for testing novel therapeutics.
Project description:Numerically the most important risk factor for the development of Parkinson's disease (PD) is the presence of mutations in the glucocerebrosidase GBA1 gene. In vitro and in vivo studies show that GBA1 mutations reduce glucocerebrosidase (GCase) activity and are associated with increased ?-synuclein levels, reflecting similar changes seen in idiopathic PD brain. We have developed a neural crest stem cell-derived dopaminergic neuronal model that recapitulates biochemical abnormalities in GBA1 mutation-associated PD. Cells showed reduced GCase protein and activity, impaired macroautophagy, and increased ?-synuclein levels. Advantages of this approach include easy access to stem cells, no requirement to reprogram, and retention of the intact host genome. Treatment with a GCase chaperone increased GCase protein levels and activity, rescued the autophagic defects, and decreased ?-synuclein levels. These results provide the basis for further investigation of GCase chaperones or similar drugs to slow the progression of PD.
Project description:Mutations in GBA1, the gene for glucocerebrosidase (GCase), are genetic risk factors for Parkinson disease (PD). ?-Synuclein (?-Syn), a protein implicated in PD, interacts with GCase and efficiently inhibits enzyme activity. GCase deficiency causes the lysosomal storage disorder Gaucher disease (GD). We show that saposin C (Sap C), a protein vital for GCase activity in vivo, protects GCase against ?-syn inhibition. Using nuclear magnetic resonance spectroscopy, site-specific fluorescence, and Förster energy transfer probes, Sap C was observed to displace ?-syn from GCase in solution and on lipid vesicles. Our results suggest that Sap C might play a crucial role in GD-related PD.