Elevated cholesterol in ATAD3 mutants is a compensatory mechanism that leads to membrane cholesterol aggregation.

Aberrant cholesterol metabolism causes neurological disease and neurodegeneration, and mitochondria have been linked to perturbed cholesterol homeostasis via the study of pathological mutations in the ATAD3 gene cluster. However, whether the cholesterol changes were compensatory or contributory to the disorder was unclear, and the effects on cell membranes and the wider cell were also unknown. Using patient-derived cells, we show that cholesterol perturbation is a conserved feature of pathological ATAD3 variants that is accompanied by an expanded lysosome population containing membrane whorls characteristic of lysosomal storage diseases. Lysosomes are also more numerous in Drosophila neural progenitor cells expressing mutant Atad3, which exhibit abundant membrane-bound cholesterol aggregates, many of which co-localize with lysosomes. By subjecting the Drosophila Atad3 mutant to nutrient restriction and cholesterol supplementation, we show that the mutant displays heightened cholesterol dependence. Collectively, these findings suggest that elevated cholesterol enhances tolerance to pathological ATAD3 variants; however, this comes at the cost of inducing cholesterol aggregation in membranes, which lysosomal clearance only partly mitigates.

α-Synuclein expression in response to bacterial ligands and metabolites in gut enteroendocrine cells: an in vitro proof of concept study.

Caudo-rostral migration of pathological forms of α-synuclein from the gut to the brain is proposed as an early feature in Parkinson's disease pathogenesis, but the underlying mechanisms remain unknown. Intestinal epithelial enteroendocrine cells sense and respond to numerous luminal signals, including bacterial factors, and transmit this information to the brain via the enteric nervous system and vagus nerve. There is evidence that gut bacteria composition and their metabolites change in Parkinson's disease patients, and these alterations can trigger α-synuclein pathology in animal models of the disorder. Here, we investigated the effect of toll-like receptor and free fatty acid receptor agonists on the intracellular level of α-synuclein and its release using mouse secretin tumour cell line 1 enteroendocrine cells. Secretin tumour cell line 1 enteroendocrine cells were treated for 24 or 48 h with toll-like receptor agonists (toll-like receptor 4 selective lipopolysaccharide; toll-like receptor 2 selective Pam3CysSerLys4) and the free fatty acid receptor 2/3 agonists butyrate, propionate and acetate. The effect of selective receptor antagonists on the agonists' effects after 24 hours was also investigated. The level of α-synuclein protein was measured in cell lysates and cell culture media by western blot and enzyme-linked immunosorbent assay. The level of α-synuclein and tumour necrosis factor messenger RNA was measured by quantitative reverse transcription real-time polymerase chain reaction. Stimulation of secretin tumour cell line 1 enteroendocrine cells for 24 and 48 hours with toll-like receptor and free fatty acid receptor agonists significantly increased the amount of intracellular α-synuclein and the release of α-synuclein from the cells into the culture medium. Both effects were significantly reduced by antagonists selective for each receptor. Toll-like receptor and free fatty acid receptor agonists also significantly increased tumour necrosis factor transcription, and this was effectively inhibited by corresponding antagonists. Elevated intracellular α-synuclein increases the likelihood of aggregation and conversion to toxic forms. Factors derived from bacteria induce α-synuclein accumulation in secretin tumour cell line 1 enteroendocrine cells. Here, we provide support for a mechanism by which exposure of enteroendocrine cells to specific bacterial factors found in Parkinson's disease gut dysbiosis might facilitate accumulation of α-synuclein pathology in the gut.

Unexpected phenotypic and molecular changes of combined glucocerebrosidase and acid sphingomyelinase deficiency.

Heterozygous variants in GBA1, encoding glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson's disease (PD). Moreover, sporadic PD patients also have a substantial reduction of GCase activity. Genetic variants of SMPD1 are also overrepresented in PD cohorts, whereas a reduction of its encoded enzyme (acid sphingomyelinase or ASM) activity is linked to an earlier age of PD onset. Despite both converging on the ceramide pathway, how the combined deficiencies of both enzymes might interact to modulate PD has yet to be explored. Therefore, we created a double-knockout (DKO) zebrafish line for both gba1 (or gba) and smpd1 to test for an interaction in vivo, hypothesising an exacerbation of phenotypes in the DKO line compared to those for single mutants. Unexpectedly, DKO zebrafish maintained conventional swimming behaviour and had normalised neuronal gene expression signatures compared to those of single mutants. We further identified rescue of mitochondrial Complexes I and IV in DKO zebrafish. Despite having an unexpected rescue effect, our results confirm ASM as a modifier of GBA1 deficiency in vivo. Our study highlights the need for validating how genetic variants and enzymatic deficiencies may interact in vivo.

The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines.

Sequence variants or mutations in the GBA gene are numerically the most important risk factor for Parkinson disease (PD). The GBA gene encodes for the lysosomal hydrolase enzyme, glucocerebrosidase (GCase). GBA mutations often reduce GCase activity and lead to the impairment of the autophagy-lysosomal pathway, which is important in the turnover of alpha-synuclein, accumulation of which is a key pathological hallmark of PD. Although the E326K variant is one of the most common GBA variants associated with PD, there is limited understanding of its biochemical effects. We have characterized homozygous and heterozygous E326K variants in human fibroblasts. We found that E326K variants did not cause a significant loss of GCase protein or activity, endoplasmic reticulum (ER) retention or ER stress, in contrast to the L444P GBA mutation. This was confirmed in human dopaminergic SH-SY5Y neuroblastoma cell lines overexpressing GCase with either E326K or L444P protein. Despite no loss of the GCase activity, a significant increase in insoluble alpha-synuclein aggregates in E326K and L444P mutants was observed. Notably, SH-SY5Y overexpressing E326K demonstrated a significant increase in the lipid droplet number under basal conditions, which was exacerbated following treatment with the fatty acid oleic acid. Similarly, a significant increase in lipid droplet formation following lipid loading was observed in heterozygous and homozygous E326K fibroblasts. In conclusion, the work presented here demonstrates that the E326K mutation behaves differently to the common loss of function GBA mutations; however, lipid dyshomeostasis and alpha-synuclein pathology are still evident.

Glucocerebrosidase-associated Parkinson disease: Pathogenic mechanisms and potential drug treatments.

Dysfunction of the endolysosomal system is implicated in the pathogenesis of both sporadic and familial Parkinson disease (PD). Variants in genes encoding lysosomal proteins have been estimated to be associated with more than half of PD cases. The most common genetic risk factor for PD are variants in the GBA gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), which is involved in sphingolipid metabolism. In this review we will describe the clinical symptoms and pathology of GBA-PD, and how this might be affected by the type of GBA variant. The putative mechanisms by which GCase deficiency in neurons and glia might contribute to PD pathogenesis will then be discussed, with particular emphasis on the accumulation of α-synuclein aggregates and the spread of pathogenic α-synuclein species between the cell types. The dysregulation of not only sphingolipids, but also phospholipids and cholesterol in the misfolding of α-synuclein is reviewed, as are neuroinflammation and the interaction of GCase with LRRK2 protein, another important contributor to PD pathogenesis. Study of both non-manifesting GBA carriers and GBA-PD cohorts provides an opportunity to identify robust biomarkers for PD progression as well as clinical trials for potential treatments. The final part of this review will describe preclinical studies and clinical trials for increasing GCase activity or reducing toxic substrate accumulation.

Glucocerebrosidase deficiency promotes release of α-synuclein fibrils from cultured neurons.

Mutations in the GBA gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the most important genetic risk factor for Parkinson disease (PD). GCase activity is also decreased in sporadic PD brains and with normal ageing. Loss of GCase activity impairs the autophagy lysosomal pathway resulting in increased α-synuclein (α-syn) levels. Furthermore, elevated α-syn results in decreased GCase activity. Although the role of α-syn in PD remains unclear, evidence indicates that aggregated α-syn fibrils are a pathogenic species in PD, passing between neurons and inducing endogenous native α-syn to aggregate; spreading pathology through the brain. We have investigated if preformed α-syn fibrils (PFFs) impair GCase activity in mouse cortical neurons and differentiated dopaminergic cells, and whether GCase deficiency in these models increased the transfer of α-syn pathology to naïve cells. Neurons treated with PFFs induced endogenous α-syn to become insoluble and phosphorylated at Ser129 to a greater extent than monomeric α-syn-treatment. PFFs, but not monomeric α-syn, inhibited lysosomal GCase activity in these cells and induced the unfolded protein response. Neurons in which GCase was inhibited by conduritol ß-epoxide did not increase the amount of insoluble monomeric α-syn or its phosphorylation status. Instead the release of α-syn fibrils from GCase deficient cells was significantly increased. Co-culture studies showed that the transfer of α-syn pathology to naïve cells was greater from GCase deficient cells. This study suggests that GCase deficiency increases the spread of α-syn pathology and likely contributes to the earlier age of onset and increased cognitive decline associated with GBA-PD.

The role of glucocerebrosidase in Parkinson disease pathogenesis.

GBA encodes the lysosomal enzyme glucocerebrosidase (GCase), an enzyme involved in sphingolipid metabolism. Mutations in the GBA gene are numerically the most important risk factor for developing Parkinson disease (PD) accounting for at least 5% of all PD cases. Furthermore, loss of GCase activity is found in sporadic PD brains. Lysosomal dysfunction is thought to play a principal role in PD pathogenesis and in particular its effect on the metabolism of α-synuclein. A hallmark of PD is the presence of intraneuronal protein inclusions called Lewy bodies, which are composed mainly of α-synuclein. Cellular and animal models of GCase deficiency result in lysosomal dysfunction, and in particular the autophagy lysosome pathway, resulting in the accumulation of α-synuclein. Some forms of mutant GCase unfold in the endoplasmic reticulum activating the unfolded protein response, which might also contribute to PD pathogenesis. It has also been suggested that accumulation of GCase substrates glucosylceramide/glucosylsphingosine may contribute to GBA-PD pathogenesis. Mitochondrial dysfunction and neuroinflammation are associated with GCase deficiency and have also been implicated in the aetiology of PD. This review discusses these points and highlights potential treatments that might be effective in treating GCase deficiency in PD.

DJ-1 is a redox sensitive adapter protein for high molecular weight complexes involved in regulation of catecholamine homeostasis.

DJ-1 is an oxidation sensitive protein encoded by the PARK7 gene. Mutations in PARK7 are a rare cause of familial recessive Parkinson's disease (PD), but growing evidence suggests involvement of DJ-1 in idiopathic PD. The key clinical features of PD, rigidity and bradykinesia, result from neurotransmitter imbalance, particularly the catecholamines dopamine (DA) and noradrenaline. We report in human brain and human SH-SY5Y neuroblastoma cell lines that DJ-1 predominantly forms high molecular weight (HMW) complexes that included RNA metabolism proteins hnRNPA1 and PABP1 and the glycolysis enzyme GAPDH. In cell culture models the oxidation status of DJ-1 determined the specific complex composition. RNA sequencing indicated that oxidative changes to DJ-1 were concomitant with changes in mRNA transcripts mainly involved in catecholamine metabolism. Importantly, loss of DJ-1 function upon knock down (KD) or expression of the PD associated form L166P resulted in the absence of HMW DJ-1 complexes. In the KD model, the absence of DJ-1 complexes was accompanied by impairment in catecholamine homeostasis, with significant increases in intracellular DA and noraderenaline levels. These changes in catecholamines could be rescued by re-expression of DJ-1. This catecholamine imbalance may contribute to the particular vulnerability of dopaminergic and noradrenergic neurons to neurodegeneration in PARK7-related PD. Notably, oxidised DJ-1 was significantly decreased in idiopathic PD brain, suggesting altered complex function may also play a role in the more common sporadic form of the disease.

Parkinson disease-linked GBA mutation effects reversed by molecular chaperones in human cell and fly models.

GBA gene mutations are the greatest cause of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase) but the mechanisms by which loss of GCase contributes to PD remain unclear. Inhibition of autophagy and the generation of endoplasmic reticulum (ER) stress are both implicated. Mutant GCase can unfold in the ER and be degraded via the unfolded protein response, activating ER stress and reducing lysosomal GCase. Small molecule chaperones that cross the blood brain barrier help mutant GCase refold and traffic correctly to lysosomes are putative treatments for PD. We treated fibroblast cells from PD patients with heterozygous GBA mutations and Drosophila expressing human wild-type, N370S and L444P GBA with the molecular chaperones ambroxol and isofagomine. Both chaperones increased GCase levels and activity, but also GBA mRNA, in control and mutant GBA fibroblasts. Expression of mutated GBA in Drosophila resulted in dopaminergic neuronal loss, a progressive locomotor defect, abnormal aggregates in the ER and increased levels of the ER stress reporter Xbp1-EGFP. Treatment with both chaperones lowered ER stress and prevented the loss of motor function, providing proof of principle that small molecule chaperones can reverse mutant GBA-mediated ER stress in vivo and might prove effective for treating PD.

Autophagic lysosome reformation dysfunction in glucocerebrosidase deficient cells: relevance to Parkinson disease.

Glucocerebrosidase (GBA1) gene mutations increase the risk of Parkinson disease (PD). While the cellular mechanisms associating GBA1 mutations and PD are unknown, loss of the glucocerebrosidase enzyme (GCase) activity, inhibition of autophagy and increased α-synuclein levels have been implicated. Here we show that autophagy lysosomal reformation (ALR) is compromised in cells lacking functional GCase. ALR is a cellular process controlled by mTOR which regenerates functional lysosomes from autolysosomes formed during macroautophagy. A decrease in phopho-S6K levels, a marker of mTOR activity, was observed in models of GCase deficiency, including primary mouse neurons and the PD patient derived fibroblasts with GBA1 mutations, suggesting that ALR is compromised. Importantly Rab7, a GTPase crucial for endosome-lysosome trafficking and ALR, accumulated in GCase deficient cells, supporting the notion that lysosomal recycling is impaired. Recombinant GCase treatment reversed ALR inhibition and lysosomal dysfunction. Moreover, ALR dysfunction was accompanied by impairment of macroautophagy and chaperone-mediated autophagy, increased levels of total and phosphorylated (S129) monomeric α-synuclein, evidence of amyloid oligomers and increased α-synuclein release. Concurrently, we found increased cholesterol and altered glucosylceramide homeostasis which could compromise ALR. We propose that GCase deficiency in PD inhibits lysosomal recycling. Consequently neurons are unable to maintain the pool of mature and functional lysosomes required for the autophagic clearance of α-synuclein, leading to the accumulation and spread of pathogenic α-synuclein species in the brain. Since GCase deficiency and lysosomal dysfunction occur with ageing and sporadic PD pathology, the decrease in lysosomal reformation may be a common feature in PD.

Mitochondrial dysfunction associated with glucocerebrosidase deficiency.

The lysosomal hydrolase glucocerebrosidase (GCase) is encoded for by the GBA gene. Homozygous GBA mutations cause Gaucher disease (GD), a lysosomal storage disorder. Furthermore, homozygous and heterozygous GBA mutations are numerically the greatest genetic risk factor for developing Parkinson's disease (PD), the second most common neurodegenerative disorder. The loss of GCase activity results in impairment of the autophagy-lysosome pathway (ALP), which is required for the degradation of macromolecules and damaged organelles. Aberrant protein handling of α-synuclein by the ALP occurs in both GD and PD. α-synuclein is the principle component of Lewy bodies, a defining hallmark of PD. Mitochondrial dysfunction is also observed in both GD and PD. In this review we will describe how mitochondria are affected following loss of GCase activity. The pathogenic mechanisms leading to mitochondria dysfunction will also be discussed, focusing on the likely inhibition of the degradation of mitochondria by the ALP, also termed mitophagy. Other pathogenic cellular processes associated with GBA mutations that might contribute, such as the unfolding of GCase in the endoplasmic reticulum, calcium dysregulation and neuroinflammation will also be described. Impairment of the ALP and mitochondria dysfunction are common pathogenic themes between GD and PD and probably explain why GBA mutations increase the risk of developing PD that is very similar to sporadic forms of the disease.

Mitochondrial and lysosomal biogenesis are activated following PINK1/parkin-mediated mitophagy.

Impairment of the autophagy-lysosome pathway is implicated with the changes in α-synuclein and mitochondrial dysfunction observed in Parkinson's disease (PD). Damaged mitochondria accumulate PINK1, which then recruits parkin, resulting in ubiquitination of mitochondrial proteins. These can then be bound by the autophagic proteins p62/SQSTM1 and LC3, resulting in degradation of mitochondria by mitophagy. Mutations in PINK1 and parkin genes are a cause of familial PD. We found a significant increase in the expression of p62/SQSTM1 mRNA and protein following mitophagy induction in human neuroblastoma SH-SY5Y cells. p62 protein not only accumulated on mitochondria, but was also greatly increased in the cytosol. Increased p62/SQSMT1 expression was prevented in PINK1 knock-down cells, suggesting increased p62 expression was a consequence of mitophagy induction. The transcription factors Nrf2 and TFEB, which play roles in mitochondrial and lysosomal biogenesis, respectively, can regulate p62/SQSMT1. We report that both Nrf2 and TFEB translocate to the nucleus following mitophagy induction and that the increase in p62 mRNA levels was significantly impaired in cells with Nrf2 or TFEB knockdown. TFEB translocation also increased expression of itself and lysosomal proteins such as glucocerebrosidase and cathepsin D following mitophagy induction. We also report that cells with increased TFEB protein have significantly higher PGC-1α mRNA levels, a regulator of mitochondrial biogenesis, resulting in increased mitochondrial content. Our data suggests that TFEB is activated following mitophagy to maintain autophagy-lysosome pathway and mitochondrial biogenesis. Therefore, strategies to increase TFEB may improve both the clearance of α-synuclein and mitochondrial dysfunction in PD. Damaged mitochondria are degraded by the autophagy-lysosome pathway and is termed mitophagy. Following mitophagy induction, the transcription factors Nrf2 and TFEB translocate to the nucleus, inducing the transcription of genes encoding for autophagic proteins such as p62, as well as lysosomal and mitochondrial proteins. We propose that these events maintain autophagic flux, replenish lysosomes and replace mitochondria.

Endoplasmic reticulum and lysosomal Ca²âº stores are remodelled in GBA1-linked Parkinson disease patient fibroblasts.

Mutations in ß-glucocerebrosidase (encoded by GBA1) cause Gaucher disease (GD), a lysosomal storage disorder, and increase the risk of developing Parkinson disease (PD). The pathogenetic relationship between the two disorders is unclear. Here, we characterised Ca(2+) release in fibroblasts from type I GD and PD patients together with age-matched, asymptomatic carriers, all with the common N370S mutation in ß-glucocerebrosidase. We show that endoplasmic reticulum (ER) Ca(2+) release was potentiated in GD and PD patient fibroblasts but not in cells from asymptomatic carriers. ER Ca(2+) signalling was also potentiated in fibroblasts from aged healthy subjects relative to younger individuals but not further increased in aged PD patient cells. Chemical or molecular inhibition of ß-glucocerebrosidase in fibroblasts and a neuronal cell line did not affect ER Ca(2+) signalling suggesting defects are independent of enzymatic activity loss. Conversely, lysosomal Ca(2+) store content was reduced in PD fibroblasts and associated with age-dependent alterations in lysosomal morphology. Accelerated remodelling of Ca(2+) stores by pathogenic GBA1 mutations may therefore feature in PD.

No evidence for substrate accumulation in Parkinson brains with GBA mutations.

BACKGROUND: To establish whether Parkinson's disease (PD) brains previously described to have decreased glucocerebrosidase activity exhibit accumulation of the lysosomal enzyme's substrate, glucosylceramide, or other changes in lipid composition. METHODS: Lipidomic analyses and cholesterol measurements were performed on the putamen (n = 5-7) and cerebellum (n = 7-14) of controls, Parkinson's disease brains with heterozygote GBA1 mutations (PD+GBA), or sporadic PD. RESULTS: Total glucosylceramide levels were unchanged in both PD+GBA and sporadic PD brains when compared with controls. No changes in glucosylsphingosine (deacetylated glucosylceramide), sphingomyelin, gangliosides (GM2, GM3), or total cholesterol were observed in either putamen or cerebellum. CONCLUSIONS: This study did not demonstrate glucocerebrosidase substrate accumulation in PD brains with heterozygote GBA1 mutations in areas of the brain with low α-synuclein pathology.

Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression.

Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinson's disease (PD). This gene encodes a mitochondrial serine/threonine kinase, which is partly localized to mitochondria, and has been shown to play a role in protecting neuronal cells from oxidative stress and cell death, perhaps related to its role in mitochondrial dynamics and mitophagy. In this study, we report that increased mitochondrial PINK1 levels observed in human neuroblastoma SH-SY5Y cells after carbonyl cyanide m-chlorophelyhydrazone (CCCP) treatment were due to de novo protein synthesis, and not just increased stabilization of full length PINK1 (FL-PINK1). PINK1 mRNA levels were significantly increased by 4-fold after 24h. FL-PINK1 protein levels at this time point were significantly higher than vehicle-treated, or cells treated with CCCP for 3h, despite mitochondrial content being decreased by 29%. We have also shown that CCCP dissipated the mitochondrial membrane potential (Δψm) and induced entry of extracellular calcium through L/N-type calcium channels. The calcium chelating agent BAPTA-AM impaired the CCCP-induced PINK1 mRNA and protein expression. Furthermore, CCCP treatment activated the transcription factor c-Fos in a calcium-dependent manner. These data indicate that PINK1 expression is significantly increased upon CCCP-induced mitophagy in a calcium-dependent manner. This increase in expression continues after peak Parkin mitochondrial translocation, suggesting a role for PINK1 in mitophagy that is downstream of ubiquitination of mitochondrial substrates. This sensitivity to intracellular calcium levels supports the hypothesis that PINK1 may also play a role in cellular calcium homeostasis and neuroprotection.