InteractiveFly: GeneBrief

Glucocerebrosidase 1b: Biological Overview | References

Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson's disease (PD). The spread of brain protein aggregates is associated with clinical disease progression, but how this occurs remains unclear. Mutations in glucosidase, beta acid 1 (GBA), which encodes glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies and associate with faster disease progression. To explore how GBA mutations influence pathogenesis, Drosophila model of GBA deficiency (Gba1b) was created that manifests neurodegeneration and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and altered protein composition of EVs was found in Gba1b mutants. Accordingly, it was hypothesized that GBA may influence pathogenic protein aggregate spread via EVs. It was found that accumulation of ubiquitinated proteins and Ref(2)P, Drosophila homologue of mammalian p62, were reduced in muscle and brain tissue of Gba1b flies by ectopic expression of wildtype GCase in muscle. Neuronal GCase expression also rescued protein aggregation both cell-autonomously in brain and non-cell-autonomously in muscle. Muscle-specific GBA expression reduced the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Perturbing EV biogenesis through neutral sphingomyelinase (nSMase), an enzyme important for EV release and ceramide metabolism, enhanced protein aggregation when knocked down in muscle, but did not modify Gba1b mutant protein aggregation when knocked down in neurons. Lipidomic analysis of nSMase knockdown on ceramide and glucosylceramide levels suggested that Gba1b mutant protein aggregation may depend on relative depletion of specific ceramide species often enriched in EVs. Finally, ectopically expressed GCase was identified within isolated EVs. Together, these findings suggest that GCase deficiency promotes accelerated protein aggregate spread between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread (Jewett, 2021).

Many genetic influences of PD have now been identified, and much work has been focused on how these genes lead to protein aggregation through mechanisms such as protein misfolding and autophagy defects. However, none of these genes have been implicated in cell-to-cell spread of pathogenic protein aggregates, which closely correlates with clinical disease progression. Proteomic analysis and non-cell-autonomous rescue of protein aggregation in Gba1b mutants has led to the hypothesize that GBA mutations may influence the rate of propagation of protein aggregates between neurons. This work suggests a link between GBA mutations and faster spread of intracellular protein aggregates via a novel EV-mediated mechanism, possibly explaining the recent clinical finding that GBA mutations accelerate the progression of clinical disease. Using a Drosophila model of GBA deficiency that manifests accelerated protein aggregation, this study found that expressing WT GCase in specific tissues of a GBA-deficient fly can not only rescue protein aggregation cell-autonomously and in distant tissues, but also rescue alterations in protein cargo observed in EVs isolated from Gba1b mutant hemolymph. Interestingly, ectopically expressed WT GCase itself was found within EVs of GBA-deficient flies, suggesting that the non-cell-autonomous rescue due to GCase expression is mediated by both reduction in aggregated proteins in EVs and trafficking of GCase via EVs to distant cells and tissues. Perturbing EV biogenesis through decreased expression of ESCRT-independent nSMase affected protein aggregation in local tissues in a tissue-dependent manner, and further decreased a subset of Cer species already reduced in Gba1b mutants. Interestingly, this subset of Cer species is known to be enriched in EV membranes. Together, these findings suggest that mutations in GBA result in the accelerated spread of protein aggregates through changes in cellular lipid composition and dysregulation of proteins trafficked by EVs (Jewett, 2021).

Although the model of GBA mutations promoting spread of protein aggregates via EVs is novel, the idea that proteostasis can be maintained in a non-cell-autonomous fashion is well supported in the literature. For example, in C. elegans, misfolded α-synuclein accumulating in endo-lysosomal vesicles was found to be transmitted from muscle to the hypodermis, a nearby tissue, for degradation. It is possible that a non-cell-autonomous mechanism is necessary because certain tissues may be more efficient in reducing protein aggregation. This has been previously described, where overexpression of FOXO in Drosophila muscle decreased aging-related protein aggregates in muscle as well as brain and other distant tissues, but FOXO overexpression in adipose tissue was unable to prevent protein aggregation in muscle. In the current model, overexpressing dGba1b in Drosophila muscle or neuronal tissue prevented accumulation of protein aggregates throughout the organism, however overexpression of WT GCase in midgut and fat body did not significantly reduce protein aggregation in the brain. These discrepancies could be due to tissue-specific biogenesis of EVs, which could depend on factors such as metabolic rate or endovesicular trafficking. Although dGba1b is expressed in all tissues, a second homologue of human GBA1, dGba1a, is expressed only in the midgut. Gba1b mutants retain ~25% expression of dGba1a. Deficiency of dGba1a was found to extend lifespan and does not result in significant accumulation of GlcCer, suggesting that there can be significant tissue-specific differences in function for GCase that could influence EV biogenesis (Jewett, 2021).

The unexpected results from perturbation of EV biogenesis suggest that the EV-mediated regulation of protein aggregation is tissue-specific and complex. Because an increase in EV-intrinsic proteins and alteration of protein cargo were observed in Gba1b mutants (Thomas, 2018), it is anticipated that genetic perturbations decreasing the biogenesis of EVs might rescue protein aggregation non-cell-autonomously by reducing the production of dysregulated EVs. However, decreased expression of ESCRT-independent nSMase in muscle did not rescue protein aggregation in heads, suggesting that a tissue-specific decrease in biogenesis of dysregulated EVs is not sufficient to reduce protein aggregation in the rest of the organism, and the cargo of EVs may need to be corrected to reduce spread of protein aggregation. In contrast, decreased expression of nSMase in the nervous system had no effect on protein aggregation in the head. This difference in outcome in perturbation of EV biogenesis in muscle and neurons could be due to cell-specific compensatory mechanisms or intrinsic metabolic demands and solicits further investigation (Jewett, 2021).

A possible explanation for why decreased muscle expression of nSMase enhanced cell-autonomous protein aggregation and EV protein cargo alterations is that both GCase and nSMase enzymatically produce Cer. If GCase-deficient phenotypes are dependent on a relative reduction in Cer, decreased nSMase expression could exacerbate Gba1b mutant phenotypes. Indeed, lipidomic analysis of alterations in Cer metabolism due to nSMase knockdown revealed a further decrease in a subset of Cer species that were already significantly decreased in Gba1b mutants compared to controls. The further reduction in Cer species due to nSMase knockdown correlates with enhancement of cell-autonomous protein aggregation and EV cargo alterations, suggesting that accelerated protein aggregation in Gba1b mutants is mediated by Cer deficiency rather than GlcCer accumulation, as nSMase knockdown had a much more modest effect on the significantly increased levels of GlcCer species in Gba1b mutants compared to controls (Jewett, 2021).

Cer has been implicated in the composition and biogenesis of EVs, and nSMase knockdown further altered EV cargo in Gba1b mutants, suggesting that decreased Cer levels may directly influence EV biogenesis in Gba1b mutants. However, Cer species were not globally decreased, suggesting that the regulation of Cer metabolism is complex and may be more dependent on specific Cer species. Interestingly, only 1 of the 9 Cer species significantly increased in Gba1b mutants versus controls had a monounsaturated fatty acyl group, while all 5 of the Cer species significantly decreased in Gba1b mutants versus controls had a monounsaturated fatty acyl group, suggesting GBA influences the metabolism of specific subset of Cer species that may be implicated in Gba1b mutant phenotypes. This subset of Cer species is enriched in species with long chain monounsaturated fatty acyl chains. Interestingly, lipids with monounsaturated fatty acyl groups are an abundant component in mammalian exosome membranes. Investigating the alterations in lipid composition of EVs resulting from GCase deficiency and nSMase knockdown will be important in elucidating the role of Cer metabolism in Gba1b mutant phenotypes (Jewett, 2021).

This work suggests that GCase deficiency influences EV biogenesis to promote faster propagation of pathogenic protein aggregates throughout the tissues of an organism, which may be a compensatory response to cell-autonomous lysosomal stress. In the initial characterization of the Drosophila GBA-deficient model, accelerated insoluble ubiquitinated protein aggregates, accumulation of Ref(2)P, and oligomerization of ectopically expressed human α-synuclein was found in Gba1b mutants, suggesting an impairment in lysosomal degradation. A similar GBA-deficient Drosophila model also found evidence of lysosomal dysfunction, including enlarged lysosomes in GBA-deficient brains. However, proteomic analysis of Gba1b mutants did not support a profound impairment in autophagy, but instead suggested dysregulation of EVs with altered protein cargo that could be suppressed locally with knockdown of genes encoding ESCRT machinery important for EV biogenesis. Based on these results, it is believed that the initial observations of increased insoluble ubiquitinated proteins and Ref(2)P in Gba1b mutants are due to lysosomal stress. One possible explanation for the proteomic findings is that there may be a compensatory increase in EV biogenesis and packaging of autophagy substrates within EVs for discard outside of the cell in Gba1b mutants. Such an increase may have prevented detection of defects in autophagy. Upregulation of EV biogenesis may be cell-autonomously neuroprotective in the setting of lysosomal stress, particularly in aggregation-prone neurodegenerative diseases such as PD. It was recently demonstrated in a human neuronal cell culture model of PD that inhibiting macroautophagy protects against α-synuclein-induced cell death by promoting the release of α-synuclein-containing EVs. However, it remains possible that upregulating EV biogenesis may relieve lysosomal stress within cells containing aggregate-prone proteins, while simultaneously promoting the spread of protein aggregates between cells and throughout the organism (Jewett, 2021).

This work suggests a novel mechanism for GBA in reducing the spread of pathogenic protein aggregation from cell-to-cell via regulation of EV protein cargo, but many key questions remain. To better understand the progression of neurodegenerative diseases, it is important to uncover the mechanisms by which GCase deficiency alters EV protein content and biogenesis, identify the specific changes in EVs facilitating propagation of pathogenic protein aggregates, and determine how these changes influence recipient cells internalizing dysregulated EVs. GCase is a critical enzyme in ceramide metabolism, hydrolyzing glucosylceramide into glucose and ceramide. Ceramides are a key component of EV membranes, and alterations in ceramide metabolism due to GCase deficiency may directly influence EV biogenesis and protein cargo trafficked via EVs. Further studies using this Drosophila model and mammalian cell culture models should better elucidate how GCase deficiency alters the protein cargo of EVs to induce propagation of pathogenic protein aggregates, as well as whether endogenous GCase is enzymatically functional when trafficked to distant tissues via EVs. Understanding this mechanism could have broad implications in understanding the pathogenesis of aggregate-prone neurodegenerative diseases and reveal new therapeutic targets to slow or halt disease progression (Jewett, 2021).

The effect of mutant GBA1 on accumulation and aggregation of alpha-synuclein

Gaucher disease (GD) patients and carriers of GD mutations have a higher propensity to develop Parkinson disease (PD) in comparison to the non-GD population. This implies that the mutant GBA1 allele is a predisposing factor for the development of PD. One of the major characteristics of PD is the presence of oligomeric α-synuclein-positive inclusions known as Lewy bodies in the dopaminergic neurons localized to the substantia nigra pars compacta. This study tested whether presence of human mutant GCase leads to accumulation and aggregation of α-synuclein in two models: in SHSY5Y neuroblastoma cells endogenously expressing α-synuclein and stably transfected with human GCase variants, and in Drosophila melanogaster co-expressing normal human α-synuclein and mutant human GCase. The results showed that heterologous expression of mutant, but not WT, human GCase in SHSY5Y cells, led to a significant stabilization of α-synuclein and to its aggregation. In parallel, there was also a significant stabilization of mutant, but not WT, GCase. Co-expression of human α-synuclein and human mutant GCase in the dopaminergic cells of flies initiated α-synuclein aggregation, earlier death of these cells and significantly shorter life span, compared to flies expressing α-synuclein or mutant GCase alone. Taken together, these results strongly indicate that human mutant GCase contributes to accumulation and aggregation of α-synuclein. In the fly, this aggregation leads to development of more severe parkinsonian signs in comparison to flies expressing either mutant GCase or α-synuclein alone (Maor, 2019).

Drosophila melanogaster mutated in its GBA1b ortholog recapitulates neuronopathic Gaucher disease

Gaucher disease (GD) results from mutations in the GBA1 gene, that encodes lysosomal glucocerebrosidase (GCase). The two fly GBA1 orthologs, GBA1a and GBA1b each contains a Minos element insertion that truncates its coding sequence. In the GBA1a(m/m) flies, which express a mutant protein, missing 33 C-terminal amino acids, there was no decrease in GCase activity or substrate accumulation. However, GBA1b(m/m) mutant flies presented a significant decrease in GCase activity with concomitant substrate accumulation, that included C14:1 glucosylceramide and C14:0 glucosylsphingosine. GBA1b(m/m) mutant flies showed activation of the Unfolded Protein Response (UPR) and presented inflammation and neuroinflammation that culminated in development of a neuronopathic disease. Treatment with ambroxol did not rescue GCase activity or reduce substrate accumulation; however, it ameliorated UPR, inflammation and neuroinflammation, and increased life span. These results highlight the resemblance between the phenotype of the GBA1b(m/m) mutant fly and neuronopathic GD and underlie relevance of mutant flies in further GD studies as well as a model to test possible therapeutic modalities (Cabasso, 2019).

Glucocerebrosidase deficiency promotes protein aggregation through dysregulation of extracellular vesicles

Mutations in the glucosylceramidase beta (GBA) gene are strongly associated with neurodegenerative diseases marked by protein aggregation. GBA encodes the lysosomal enzyme glucocerebrosidase that breaks down glucosylceramide. A common explanation for the link between GBA mutations and protein aggregation is that lysosomal accumulation of glucosylceramide causes impaired autophagy. This study tested this hypothesis directly by measuring protein turnover and abundance in Drosophila mutants with deletions in the GBA ortholog Gba1b. Proteomic analyses revealed that known autophagy substrates, which had severely impaired turnover in autophagy-deficient Atg7 mutants, showed little to no overall slowing of turnover or increase in abundance in Gba1b mutants. Striking changes were found in the turnover and abundance of proteins associated with extracellular vesicles (EVs), that have been proposed as vehicles for the spread of protein aggregates in neurodegenerative disease. Western blotting of isolated EVs confirmed the increased abundance of EV proteins in Gba1b mutants, and nanoparticle tracking analysis revealed that Gba1b mutants had six times as many EVs as controls. Genetic perturbations of EV production in Gba1b mutants suppressed protein aggregation, demonstrating that the increase in EV abundance contributed to the accumulation of protein aggregates. Together, these findings indicate that glucocerebrosidase deficiency causes pathogenic changes in EV metabolism and may promote the spread of protein aggregates through extracellular vesicles (Thomas, 2018).

Mutations in the gene encoding the lysosomal enzyme glucocerebrosidase, glucosylceramidase beta (GBA), are associated with neurodegeneration and brain protein aggregation (Gegg, 2018; Nalls, 2013). Homozygous mutations in GBA cause the lysosomal storage disorder Gaucher disease, which in some cases includes devastating neurological symptoms, while heterozygous GBA mutations are the strongest risk factor for both Parkinson disease (PD) and the related disorder dementia with Lewy bodies. Up to 10% of individuals with nonfamilial PD carry a GBA mutation. In addition, PD patients with a GBA mutation have faster progression of both motor and cognitive symptoms. To study the mechanisms underlying the association between GBA mutations and neurodegeneration, this study created a Drosophila model of glucocerebrosidase (GCase) deficiency. Drosophila has two GBA homologs, designated Gba1a and Gba1b. The Gba1a gene is expressed exclusively in the midgut, and deletion of this gene does not appear to confer deleterious phenotypes. By contrast, the Gba1b gene is ubiquitously expressed, and Gba1b deletion causes marked abnormalities. Previously work has shown that Gba1b null mutants exhibit phenotypes including shortened lifespan, locomotor and memory deficits, neurodegeneration, accumulation of the autophagy adaptor Ref(2)P (p62/SQSTM1), and accumulation of ubiquitinated protein aggregates (Davis, 2016). Similar phenotypes were subsequently seen in an independently generated Gba1b null mutant (Kinghorn, 2016; Thomas, 2018 and references therein).

The protein aggregation and elevated Ref(2)P levels in Gba1b mutants suggested that they had impaired autophagy, as did morphological changes in the autolysosomal system noted by Kinghorn (2016). These findings are consistent with previous reports of autolysosomal impairment upon loss of GCase activity. Based on such findings, it has been hypothesized that lysosomal accumulation of glucosylceramide, the normal substrate of GCase, leads to impairment of autophagy. However, none of the work implicating autophagy in the pathogenic effects of GCase deficiency has yet established that GCase loss of function causes global impairment of autophagic degradation (Thomas, 2018).

To investigate the autophagy failure model of GBA pathogenesis, this study used proteomics-based techniques to measure protein turnover and abundance in Gba1b mutants and controls, as well as in flies with mutations in key autophagy (Atg7) or mitophagy (parkin) genes. While Atg7 mutants showed marked and widespread slowing of autophagy substrate turnover, Gba1b mutants did not. The effects of Gba1b mutation on the turnover and abundance of autophagy substrates also failed to correlate with those of Atg7 or parkin mutations. Moreover, no deficits were detected in turnover mediated by the proteasome, microautophagy, or endocytosis. However, high incidences were found of faster turnover and increased abundance among proteins associated with extracellular vesicles (EVs), which have been previously suggested as a mechanism for the spread of protein aggregates in neurodegenerative disease. Biochemical studies confirmed increased abundance of EV marker proteins in isolated EVs from Gba1b mutants, and nanoparticle tracking analysis showed that the mutants had markedly increased numbers of EVs. Genetic manipulations to reduce EV production decreased the accumulation of ubiquitinated protein aggregates and Ref(2)P in Gba1b mutants, supporting the model that excessive EV abundance promotes the accumulation of protein aggregates. Together, these findings suggest that the most important pathological consequence of Gba1b loss of function is not failure of autophagic protein degradation but excessive production of extracellular vesicles (Thomas, 2018).

Impairment of autolysosomal degradation is widely thought to explain the increased risk of neurodegeneration associated with mutations in GBA, which encodes the lysosomal enzyme glucocerebrosidase (GCase), and multiple studies have found hallmarks of impaired autophagy associated with GCase loss of function. These hallmarks have included accumulation of ubiquitinated protein aggregates, increased abundance of autophagic flux markers such as p62/SQSTM1 and LC3-II, impairment of autophagosome-lysosome fusion, and changes in the size and number of autophagosomes and lysosomes. These indications that GCase deficiency leads to autophagy impairment have been found in diverse experimental systems, including multiple animal models, cultured cells, iPSC-derived human neuronal models, and postmortem patient samples. Initial characterization of Drosophila Gba1b mutants, which revealed extensive ubiquitinated protein aggregates and markedly elevated levels of the p62 ortholog Ref(2)P, also appeared to support the model that GCase deficiency impairs autophagic degradation. In the current work, however, proteomic measurement of protein turnover and abundance showed no evidence that degradation of autophagy substrates was globally impaired in Gba1b mutants. The mutants also showed no evidence of failure in other protein degradation pathways. Instead, faster turnover and increased abundance of proteins associated with extracellular vesicles (EVs) was found. Followup experiments on isolated EVs confirmed increased abundance of EV marker proteins and revealed a strikingly increased number of EVs. Furthermore, genetic manipulations that reduced EV formation suppressed both the increased protein aggregation and the increased Ref(2)P abundance observed in Gba1b mutants. These findings suggest that dysregulation of extracellular vesicles, rather than failure of autophagic degradation, may be the primary mechanism by which GCase deficiency leads to protein aggregation and neurodegeneration (Thomas, 2018).

Although the many previous reports of autophagy impairment in GCase-deficient organisms appear incompatible with the current protein turnover findings, it is not believed that the current findings contradict previous work. When common markers of autolysosomal function such as Ref(2)P/p62 and insoluble ubiquitinated protein were measured, Drosophila Gba1b mutants show results comparable to those seen in vertebrate models of GCase deficiency. Proteomic measurements of protein abundance in the current study are also consistent with previous reports of increased lysosomal mass in GCase deficiency. The abundance of the lysosomal marker Lamp1 was nearly tripled in Gba1b mutants, and 41% of lysosomal proteins were significantly increased in abundance. Nevertheless, protein turnover measurements reveal that the overall rates of degradation through lysosomal processes are not grossly altered. Thus, one possible explanation of the current findings is that the efficiency of autolysosomal degradation is decreased, with lower throughput per unit of autolysosomal mass, but that the organism has compensated by increasing the amount of autolysosomal machinery available. Because this compensation is sufficient to maintain degradation rates, Gba1b mutants could be described as being under autolysosomal stress rather than in autolysosomal failure. Over time, the degree of stress may exceed the capacity to compensate, and aged Gba1b mutants may show overt failure of lysosomal degradation. Even if this is the case, late failure of autolysosomal degradation cannot explain the behavioral and biochemical abnormalities that begin in early adulthood (Thomas, 2018).

Another explanation for the apparent discrepancy between the current findings of normal autophagic substrate turnover and previous reports of impaired autophagy is that commonly used autophagy markers are not solely representative of autophagic flux. This is especially true of Ref(2)P, or p62, which has multiple nonautophagic functions and is transcriptionally upregulated by stress. In addition, p62 and LC3 have recently been detected in mammalian EVs, and this study found increased levels of oligomeric Ref(2)P in EVs from Gba1b mutants. It is therefore possible that the increased Ref(2)P levels detected in Gba1b mutants result from a combination of stress response and EV dysregulation (Thomas, 2018).

This work leaves unanswered the question of how GCase deficiency results in increased EV abundance, but does suggest two possible explanations. Increased production of EVs could be caused either by lysosomal stress or by changes in membrane lipid composition. Lysosomal stress has been shown in cultured cells to promote the release of exosomes, a major type of EV. Exosomes are generated when a multivesicular endosome (MVE) fuses with the plasma membrane rather than the lysosome, releasing its intraluminal vesicles into extracellular space. Lysosomal blockade increases the probability that an MVE will fuse with the plasma membrane. If lysosomal stress rather than outright failure is sufficient to trigger increased exosome release, it could account for the overabundance of EVs in Gba1b mutants (Thomas, 2018).

A second explanation for increased EVs in GCase-deficient animals is that abnormal membrane lipid composition may directly alter EV biogenesis. Lipid composition determines membrane fluidity and curvature, and thus controls the size, shape, and fusion kinetics of EVs. In fact, lipid rafts, particularly those enriched in ceramide, are required for formation of at least one type of EV. Membrane changes such as those caused by GCase deficiency, including accumulation of glucosylceramide and altered ceramide levels could alter EV functioning at any stage from formation to internalization by a recipient cell. Either increased or decreased probability of ceramide-dependent EV formation could lead to increased overall EV production, as suppression of one type of EV has been shown to cause overproduction of another type (Thomas, 2018).

While understanding the mechanism by which GCase deficiency causes increased EV release is an important goal of future work, an equally important question is how increased EV abundance in Gba1b mutants promotes the accumulation of protein aggregates. EVs have been increasingly implicated in the pathogenesis of neurodegenerative disease. Many disease-associated proteins, including prion protein, α-synuclein, β-amyloid, and tau, are detected in EVs, which have been proposed as vehicles for the well-documented progressive spread of protein aggregates from one brain region to another. In support of this model, toxic forms of these disease-associated proteins are more abundant in EVs from humans with neurodegenerative diseases such as Alzheimer disease, dementia with Lewy bodies, and Parkinson disease (PD), and EVs from these patients can induce protein aggregation in recipient cells under experimental conditions. However, progression of these diseases has not yet been conclusively demonstrated to be mediated by EVs. Perhaps the strongest evidence that EVs promote the spread of protein aggregates has been found for prion protein. Stimulating the release of EVs increased the cell-to-cell spread of misfolded prion protein, and decreasing EV release reduced the spread. The current findings appear to follow the same pattern: genetic interference with EV production suppressed protein aggregation in Gba1b mutants. If the same holds true for other aggregation-prone proteins, conditions that increase EV release could promote the spread of protein aggregates and thus be risk factors for neurodegenerative disease (Thomas, 2018).

This model is illustrated in the paper (see Increased EV production promotes the spread of protein aggregates in glucocerebrosidase-deficient organisms). When GCase activity is normal, EVs travel between cells, carrying both factors that promote protein aggregation (e.g., disease-associated proteins such as α-synuclein) and factors that oppose it (e.g., chaperones). Some cells likely generate more aggregates than others, and may therefore release more aggregate-promoting factors, including small aggregate 'seeds.' Quality control mechanisms in recipient cells successfully combat protein aggregation, and aggregates accumulate only slowly with age. If GCase activity is absent or reduced, however, more EVs are generated: this results in greater cell-to-cell transfer of aggregate-prone proteins, perhaps simply because these proteins are normally part of EV cargo. In particular, they may be normal cargo of ESCRT-dependent EVs, given the finding that knockdown of ESCRTs in Gba1b mutants ameliorated the mutants' protein aggregation phenotype. Alternatively, GCase deficiency may alter cargo selection so that more aggregation-prone proteins are loaded into EVs. The net effect of the EV changes is transfer of aggregation-producing factors in quantities that overwhelm quality control mechanisms, leading to excessive accumulation of ubiquitin-protein aggregates in recipient cells (Thomas, 2018).

The NAD+ precursor nicotinamide riboside rescues mitochondrial defects and neuronal loss in iPSC and fly models of Parkinson's disease

While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. These questions were addressed using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. This study reports that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. These findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases (Schondorf, 2018).

Minos-insertion mutant of the Drosophila GBA gene homologue showed abnormal phenotypes of climbing ability, sleep and life span with accumulation of hydroxy-glucocerebroside

Gaucher's disease in humans is considered a deficiency of glucocerebrosidase (GlcCerase) that results in the accumulation of its substrate, glucocerebroside (GlcCer). Although mouse models of Gaucher's disease have been reported from several laboratories, these models are limited due to the perinatal lethality of GlcCerase gene. This study examined phenotypes of Drosophila melanogaster homologous genes of the human Gaucher's disease gene by using Minos insertion. One of two Minos insertion mutants to unknown function gene (CG31414; Glucocerebrosidase 1b) accumulates the hydroxy-GlcCer in whole body of Drosophila melanogaster. This mutant showed abnormal phenotypes of climbing ability and sleep, and short lifespan. These abnormal phenotypes are very similar to that of Gaucher's disease in human. In contrast, another Minos insertion mutant (CG31148; Glucocerebrosidase 1a) and its RNAi line did not show such severe phenotype as observed in CG31414 gene mutation. The data suggests that Drosophila CG31414 gene mutation might be useful for unraveling the molecular mechanism of Gaucher's disease (Kawasaki, 2017).

A Drosophila model of neuronopathic Gaucher disease demonstrates lysosomal-autophagic defects and altered mTOR signalling and is functionally rescued by rapamycin

Glucocerebrosidase (GBA1) mutations are associated with Gaucher disease (GD), an autosomal recessive disorder caused by functional deficiency of glucocerebrosidase (GBA), a lysosomal enzyme that hydrolyzes glucosylceramide to ceramide and glucose. Neuronopathic forms of GD can be associated with rapid neurological decline (Type II) or manifest as a chronic form (Type III) with a wide spectrum of neurological signs. Furthermore, there is now a well-established link between GBA1 mutations and Parkinson's disease (PD), with heterozygote mutations in GBA1 considered the commonest genetic defect in PD. This study describes a novel Drosophila model of GD that lacks the two fly GBA1 orthologs (Gba1a and Gba1b). This knock-out model recapitulates the main features of GD at the cellular level with severe lysosomal defects and accumulation of glucosylceramide in the fly brain. A block in autophagy flux was demonstrated in association with reduced lifespan, age-dependent locomotor deficits and accumulation of autophagy substrates in dGBA-deficient fly brains. Furthermore, mechanistic target of rapamycin (mTOR) signaling is downregulated in dGBA knock-out flies, with a concomitant upregulation of Mitf gene expression, the fly ortholog of mammalian TFEB, likely as a compensatory response to the autophagy block. Moreover, the mTOR inhibitor rapamycin is able to partially ameliorate the lifespan, locomotor, and oxidative stress phenotypes. Together, these results demonstrate that this dGBA1-deficient fly model is a useful platform for the further study of the role of lysosomal-autophagic impairment and the potential therapeutic benefits of rapamycin in neuronopathic GD. These results also have important implications for the role of autophagy and mTOR signaling in GBA1-associated PD (Kinghorn, 2016).

This study generated a Drosophila model of neuronopathic GD. Simple genetics could not be used to precisely excise both Drosophila GBA1 genes, dGBA1a and dGBA1b, due to the presence of the CG31413 gene between them. Therefore, serial homologous recombination was used to knock out both dGBA1 genes either separately or together in the fly. Of the two fly GBA orthologs, only dGBA1b is expressed in the fly brain (Flyatlas). dGBA1b^-/- fly heads, lacking dGBA1b expression, displayed accumulation of the dGBA substrate glucosylceramide. Furthermore, the glucosylceramide isoforms (C16:0, C18:0, C22:0) were similar to those found in human spleen tissue (with the addition of hydroxylated or longer chain isoforms including C18:0-OH, C20:0-OH, C24:1, and C24:0 in the human tissue. Moreover, this is consistent with data showing accumulation of glucosylceramide and glucosylsyphingosine in Types II and III GD patient brains (Kinghorn, 2016).

Abnormally engorged lysosomes in dGBA1b^-/- fly brains were visualized with LysoTracker, confirming the validity of this neuronopathic GD fly model. Lysosomal integrity is necessary for normal functioning of the autophagy degradation machinery, and accordingly \autophagy dysfunction was observed in dGBA1b^-/- brains, with accumulation of the autophagolysosomal protein Atg8/LC3. Thus, this study demonstrated autophagy defects at the level of the autophagic machinery in vivo in a GD Drosophila model, following on from in vitro studies in GD macrophages and iPSCs showing impaired autophagy (Kinghorn, 2016).

Neuronal-specific autophagy deficits, through loss of the mouse autophagy genes atg7 or atg5, result in accumulation of polyubiquitinated proteins, behavioral defects, and reduced longevity. Consistent with this, dGBA1b^-/- flies were shown to display reduced survival and locomotor ability and brain accumulation of p62/Ref(2)P and polyubiquitinated proteins. Both p62 and ubiquitin accumulate in neurodegenerative diseases, such as Alzheimer's disease and PD, and multiple pathogenic proteins, including p62 and β-amyloid, have been identified in neuronopathic GD mouse brains (Kinghorn, 2016).

Surprisingly, knock-out of dGBA1a, which is predominantly expressed in the digestive system, significantly increased survival. This finding parallels recent research in Drosophila and Caenorhabditis elegans demonstrating that the intestine is an important target organ for mediating lifespan extension at the organismal level, with single-gene manipulations specifically in the gut prolonging lifespan. The role of dGBA in the gut in regulating lifespan was beyond the scope of this study but is an area that warrants further investigation. This study demonstrated, however, that the lifespan extension seen in dGBA1a-/- flies is not associated with any improvement in locomotor ability (Kinghorn, 2016).

Mitochondrial integrity is necessary for normal autophagy-lysosomal system functioning. Indeed, mitochondrial dysfunction occurs in GD mouse models, likely due to defects in the autophagic removal of damaged mitochondria. In keeping with this, the deficiency of the Drosophila autophagy gene atg7 is associated with sensitivity to oxidative stressors. Accordingly, this study demonstrated mitochondrial abnormalities in dGBA1b^-/- flies, with giant mitochondria, reduced ATP and hypersensitivity to oxidative stress. Enlarged mitochondria and decreased ATP occur in neuronopathic GD mice, and giant mitochondria are directly linked to Drosophila autophagy defects. Therefore, the accumulation of autophagic substrates and giant mitochondria in dGBA1b^-/- flies is consistent with autophagy block (Kinghorn, 2016).

Furthermore, it was demonstrated that the neuropathological defects in dGBA1b^-/- flies occur independently of α-synuclein, confirming that dGBA deficiency is sufficient to cause neurodegeneration. A recent study using a deletion mutant harboring imprecise removal of the dGBA1b gene, as well as the incomplete removal of the dGBA1a gene and excision of the interjacent CG31413 gene, provides further validation of this model for studying GBA1-associated neurodegeneration. Loss of 60% of GCase activity was also associated with reduced survival and climbing ability in addition to accumulation of autophagy substrates. GCase deficiency was also shown to enhance α-synuclein pathology in the fly. The current model has the advantage over existing models, as it allows the precise and complete knock-out of each of the dGBA genes, and the dissection of the role of dGBA in different tissues. The ability to knock out only brain-specific dGBA1b is particularly relevant given the finding that loss of dGBA in the gut causes prolongevity effects. This model also avoids the production of truncated, possibly pathogenic, variants of dGBA (Kinghorn, 2016).

The nutrient-sensing mTOR is localized to the lysosomal surface, where it modulates autophagy. It has thus been speculated that abnormal lysosomal function leads to activation of autophagy by mTOR downregulation. Indeed, a recent study reported increased expression of mTOR signaling pathway genes in neuronopathic GD mice. In view of the autophagy defect in dGBA1b^-/- flies, this study probed mTOR signaling by analyzing S6K phosphorylation downstream of mTORC1. Interestingly, a decrease in S6K phosphorylation was detected and hence downregulation of mTORC1 compared with controls. Furthermore, although not statistically significant, there was a trend for mTOR to be further downregulated in dGBA1b^-/- flies treated with rapamycin, a known mTORC1 inhibitor. Because rapamycin has protective effects in animal models of PD and other neurodegenerative diseases, dGBA1b^-/- flies were treated with rapamycin, and it was found that, despite the autophagy block, it was able to partially ameliorate many neurotoxic phenotypes. Rapamycin treatment led to an improvement in the lifespan and climbing phenotypes, in addition to the hypersensitivity to oxidative and starvation stressors seen in dGBA1b^-/- flies. These results are somewhat surprising given that rapamycin treatment of iPSC-derived neuronal cells from GD patients caused cell death, demonstrating that drug effects in vitro vary from those at the organismal level. Furthermore, although rapamycin increases the lifespan and protects against starvation conditions in control flies, the rescue of age-related climbing defects and oxidative stress appeared to be specific to dGBA-deficient flies. Indeed, rapamycin had no effect on the climbing ability of control flies, and rapamycin-treated control flies were more sensitive to H2O2 than untreated flies. The reason for the opposing effects of rapamycin on the oxidative stress responses of dGBA1b^-/- and control flies is unclear, but may be related to the differential activation of oxidative stress pathways between dGBA1b^-/- and healthy controls (Kinghorn, 2016).

Lastly, this study demonstrated that knock-out of dGBA in the brain results in an upregulation of Mitf and its downstream target, Atg8a, suggesting a compensatory response to the autophagy block. This is in contrast to findings in a recent study showing downregulation of TFEB expression in GD iPSC-derived cells. The reasons for these differing results is not clear, but possible explanations may relate to variations in autophagy block between models, and the fact that neuronal cells are newly differentiated and may reflect early disease compared with adult flies. Mitf gene expression may also vary in the presence of mutant dGBA. The fly model displays very little dGBA expression compared with iPSC cells that are derived from mutant carriers, and therefore, despite showing very low levels of GCase activity, express mutant GBA. Mutant GCase is known to exert gain-of-function toxic effects, including in Drosophila, with upregulation of the unfolded protein response. Knockdown of Mitf in Drosophila leads to similar phenotypes to those in dGBA1b^-/- flies, including autophagy substrate accumulation, upregulation of Atg8-II, giant mitochondria, as well as enlarged lysosomes. Thus, the autophagy defects seen in dGBA deficiency appear to phenocopy Mitf downregulation in the fly. This suggests that upregulating Mitf as a potential therapeutic strategy in GD and GBA-linked PD may be limited in its efficacy due to the inability of GBA-deficient neuronal tissue to stimulate autophagy. Further studies are warranted to explore the complex interaction between mTORC1 and TFEB/Mitf in GD (Kinghorn, 2016).

Rapamycin treatment of dGBA1b^-/- flies did not upregulate Mitf gene expression, suggesting that rapamycin does not act through Mitf signaling to rescue the neurotoxic phenotypes of dGBA-deficient flies. It is hypothesized, therefore, that mTOR is downregulated in dGBA-deficient flies as a compensatory response to lysosomal-autophagy block. Possibly as a consequence of lysosomal dysfunction in dGBA-deficient flies, the normal interaction between mTORC1 and TFEB/Mitf at the surface of the lysosome is disrupted, leading to changes in TFEB/Mitf gene expression. Therefore, by exerting small additional effects on mTORC1 signaling, rapamycin may mediate its beneficial effects. Furthermore, Mitf also plays a critical role in lipid metabolism and recapitulates the function of TFEB in mammals in this regard. Mitf expression may therefore contribute to the reduced triacylglyceride (TAG) that it seen in dGBA1b^-/- flies (Kinghorn, 2016).

In conclusion, the autophagy defects seen in GD flies, likely as a result of failure of the fusion of autophagosomes and lysosomes, may also be relevant to PD linked to GBA1 mutations. The results raise the possibility that therapies aimed at ameliorating not only lysosomal dysfunction, but also autophagic abnormalities, including lowering mTOR activity, may be effective in treating GBA1-associated disease. They also suggest that rapamycin may offer significant health benefits in neuronopathic GD and in GBA1-related synucleinopathies. Together, these data demonstrate that these Drosophila models of dGBA-deficiency are a useful platform to further study the downstream effects of lysosomal-autophagic dysfunction and to identify genetic modifiers and new therapeutic targets in neuronopathic GD and GBA1-associated PD (Kinghorn, 2016).

Glucocerebrosidase deficiency in Drosophila results in alpha-Synuclein-independent protein aggregation and neurodegeneration

Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher's disease, and are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because alpha-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing alpha-synuclein toxicity. To explore this hypothesis, the Drosophila GBA1 homolog, dGBA1b, was deleted, and the phenotypes of dGBA1b mutants were compared in the presence and absence of alpha-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human alpha-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased alpha-synuclein aggregation relative to controls. However, alpha-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. These findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of alpha-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity (Davis, 2016).

To gain insight into the molecular mechanisms underlying PD, DLB and neuronopathic forms of GD, a Drosophila model of glucocerebrosidase deficiency was developed. Glucocerebrosidase deficiency in Drosophila results in shortened lifespan, a variety of age-dependent behavioral phenotypes, neurodegeneration and the accumulation of insoluble proteins that are normally degraded through an autophagic mechanism. While these phenotypes are reminiscent of α-synucleinopathies, glucocerebrosidase deficiency only mildly influenced the neuronal toxicity and aggregation of α-synuclein, and ectopic expression of α-synuclein did not significantly enhance the glucocerebrosidase deficient phenotypes. Together, these findings indicate that the pathological consequences of glucocerebrosidase deficiency in Drosophila are largely independent of α-synuclein, and that glucocerebrosidase deficiency is the major contributor to pathology in diseases associated with GBA1 mutations (Davis, 2016).

The finding that α-synuclein is not a central participant in the pathogenesis associated with glucocerebrosidase deficiency is consistent with recent studies in two different fish species. The inverse correlation between glucocerebrosidase activity and α-synuclein aggregation in Drosophila is also consistent with previous studies in rodent models, vertebrate cell culture, post-mortem brain tissues from PD patients, and a recent study in Drosophila. Although it was only possible to observe an influence of glucocerebrosidase deficiency on the aggregation of the p.A53T variant of α-synuclein, this finding may simply reflect the fact that this variant is more aggregation-prone, thus allowing increased sensitivity to detect aggregation. However, this work showing the dGBA1b gene is the predominant Drosophila GBA1 homolog expressed in the fly head contrasts with a recent report showing that neuronal inactivation of the Drosophila dGBA1a gene exacerbated the toxicity of α-synuclein in dopaminergic neurons and in the fly eye (Suzuki, 2015). Additional studies will be required to fully address the role of dGBA1a, which appears to remain largely functional in the available mutant, and to definitively rule out a role for the CG31413 gene situated between dGBA1a and dGBA1b on the phenotypes of GBA1^ΔTT homozygotes (Davis, 2016).

Although this work indicates that glucocerebrosidase deficiency has little influence on the toxicity of α-synuclein, the association of GBA1 mutations with PD and DLB frequently involves heterozygous carriers of GBA1 missense alleles. This finding has led to the suggestion that the GBA1 mutations act through a dominant toxic gain-of-function mechanism to cause PD and DLB, perhaps by seeding α-synuclein aggregates via a prion-like mechanism. Because this work involved a putative null allele of dGBA1b, this potential model of pathogenesis could not be addressed. Previous work also indicates that ectopic expression of human α-synuclein in Drosophila confers only mild phenotypic consequences, so it is also possible that the influence of glucocerebrosidase deficiency on α-synuclein toxicity is not readily evident in Drosophila. While these potential confounds are fully acknowledged, several compelling observations suggest that a loss-of-function mechanism best explains the influence of GBA1 mutations on PD and DLB. For example, many different GBA1 mutations are associated with α-synucleinopathies, including putative null alleles, and the molecular severity of a GBA1 allele correlates with the risk of developing an α-synucleinopathy in heterozygous carriers. Perhaps the strongest evidence for a loss-of-function mechanism is the finding that individuals with biallelic GBA1 mutations have a substantially elevated risk for developing PD relative to heterozygous GBA1 mutation carriers. Together these findings offer support for the relevance of animal models bearing null alleles of the GBA1 gene, including this fly model of glucocerebrosidase deficiency, on understanding of the influence of GBA1 mutations in PD and DLB (Davis, 2016).

The glucocerebrosidase deficient fly model should be a valuable tool in future work aimed at understanding the mechanisms underlying the neurodegenerative diseases associated with mutations in GBA1. Although glucocerebrosidase deficiency does not result in dopaminergic neuron degeneration in Drosophila, this finding does not necessarily challenge the utility of the fly model to understand the role of glucocerebrosidase deficiency in PD. Previous work has established that mutational inactivation of Drosophila homologs of genes involved in heritable forms of PD often results in phenotypes that appear discordant with those seen in humans. For example, null mutations of the PINK1 or parkin genes in Drosophila result in dramatic muscle degeneration and germ line defects that are not evident in humans bearing null mutations in these genes. However, substantial insight into the roles of PINK1 and Parkin in mitochondrial quality control derived directly from studies of PINK1 and Parkin in the Drosophila musculature and germ line. It is anticipated that similarly important insight into the mechanisms underlying neuronopathic GD, PD and DLB will come from studies of the phenotypes of the fly model of glucocerebrosidase deficiency (Davis, 2016).

This work suggests at least two general mechanisms by which glucocerebrosidase deficiency triggers neuropathology. First, glucocerebrosidase deficiency may impair autophagy, resulting in increased protein aggregation. This work in GBA1b mutant flies showing accumulation of Ref(2)P, HMW α-synuclein aggregates, and protein aggregates that are normally degraded through an autophagic mechanism supports this model. Glucocerebrosidase is an important lysosomal enzyme in lipid metabolism, and a deficiency in this enzyme could influence lysosome membrane fluidity, vesicular dynamics, and the biogenesis of lysosomes. These effects could impair the trafficking of misfolded proteins to the lysosome and/or fusion of autophagic vacuoles. As no decrease was observed in Cathepsin D activity in GBA1b mutant flies, lysosomal function may not be impaired by glucocerebrosidase deficiency. Alternatively, glucocerebrosidase deficiency may promote the formation of protein aggregates, rather than impair their degradation. Lipid composition has been shown to influence the kinetics of formation of protein aggregates and α-synuclein fibrilization, suggesting that an alteration in lipid composition resulting from glucocerebrosidase deficiency could accelerate the accumulation of protein aggregates. These aggregates might subsequently seed further aggregation in a prion-like mechanism. In support of this model, lipid composition has been shown to affect the kinetics of amyloid-β aggregation, and recent studies suggest that non-autonomous spreading of α-synuclein fibrils may contribute to PD pathogenesis. While it remains unclear whether the increased protein aggregates that were observed in GBA1b mutant flies are due to impaired degradation or accelerated formation of misfolded proteins, α-synuclein expression did not enhance the abundance of protein aggregates, arguing against an additive influence of α-synuclein on protein aggregation metabolism. Future experiments will be required to distinguish between these models and reveal the underlying mechanism of GBA1-mediated accumulation of protein aggregates (Davis, 2016).

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. This study 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 (Sanchez-Martinez, 2016).

The contribution of mutant GBA to the development of Parkinson disease in Drosophila

Gaucher disease (GD) results from mutations in the acid beta-glucocerebrosidase (GCase) encoding gene, GBA, which leads to accumulation of glucosylceramides. GD patients and carriers of GD mutations have a significantly higher propensity to develop Parkinson disease (PD) in comparison to the non-GD population. This study used Drosophila to show that development of PD in carriers of GD mutations results from the presence of mutant GBA alleles. Drosophila has two GBA orthologs (CG31148 and CG31414), each of which has a minos insertion, which creates C-terminal deletion in the encoded GCase. Flies double heterozygous for the endogenous mutant GBA orthologs presented Unfolded Protein Response (UPR) and developed parkinsonian signs, manifested by death of dopaminergic cells, defective locomotion and a shorter life span. Transgenic flies carrying the mutant human N370S, L444P and the 84GG variants were established. UPR activation and development of parkinsonian signs could be recapitulated in flies expressing these three mutant variants. UPR and parkinsonian signs could be partially rescued by growing the double heterozygous flies, or flies expressing the N370S or the L444P human mutant GCase variants, in the presence of the pharmacological chaperone ambroxol, which binds and removes mutant GCase from the ER. However flies expressing the 84GG mutant, that does not express mature GCase, did not exhibit rescue by ambroxol. These results strongly suggest that the presence of a mutant GBA allele in dopaminergic cells leads to ER stress and to their death, and contributes to development of Parkinson disease (Maor, 2016).

Glucocerebrosidase deficiency accelerates the accumulation of proteinase K-resistant alpha-synuclein and aggravates neurodegeneration in a Drosophila model of Parkinson's disease

Alpha-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Recent multicenter genetic studies have revealed that mutations in the glucocerebrosidase 1 (GBA1) gene, that are responsible for Gaucher's disease, are strong risk factors for PD and DLB. However, the mechanistic link between the functional loss of glucocerebrosidase (GCase) and the toxicity of αSyn in vivo is not fully understood. This study employed Drosophila models to examine the effect of GCase deficiency on the neurotoxicity of αSyn and its molecular mechanism. Behavioral and histological analyses show that knockdown of the Drosophila homolog of GBA1 (dGBA1) exacerbates the locomotor dysfunction, loss of dopaminergic neurons and retinal degeneration of αSyn-expressing flies. This phenotypic aggravation is associated with the accumulation of proteinase K (PK)-resistant αSyn, rather than with changes in the total amount of αSyn, raising the possibility that glucosylceramide (GlcCer), a substrate of GCase, accelerates the misfolding of αSyn. Indeed, in vitro experiments reveal that GlcCer directly promotes the conversion of recombinant αSyn into the PK-resistant form, representing a toxic conformational change. Similar to dGBA1 knockdown, knockdown of the Drosophila homolog of β-galactosidase (β-Gal) also aggravates locomotor dysfunction of the αSyn flies, and its substrate GM1 ganglioside accelerates the formation of PK-resistant αSyn. These findings suggest that the functional loss of GCase or β-Gal promotes the toxic conversion of αSyn via aberrant interactions between αSyn and their substrate glycolipids, leading to the aggravation of αSyn-mediated neurodegeneration (Suzuki, 2015).

This study investigated the molecular mechanisms underlying the effect of GCase deficiency on αSyn toxicity. It was demonstrated that loss of GCase function exacerbates αSyn neurotoxicity in vivo and that this aggravation is associated with the accelerated accumulation of PK-resistant αSyn. In addition, GlcCer, which  accumulates in the brain of dGBA1 knockdown flies, directly promotes the formation of PK-resistant αSyn, suggesting that the accumulation of GlcCer by GCase deficiency promotes the toxic conversion of αSyn, leading to exacerbation of its neurotoxicity (Suzuki, 2015).

The phenotypic aggravation by GCase deficiency in αSyn flies was associated with the accumulation of PK-resistant αSyn, rather than with changes in the total amount of αSyn, suggesting that the production of this PK-resistant αSyn species might play a key role in the neurotoxicity. Although the toxicity of PK-resistant αSyn was not directly demonstrated, there was a tight association between the neurotoxicity of αSyn and its PK resistance. PK-resistant αSyn oligomers that are formed as an intermediate conformer in the course of in vitro αSyn fibrillization have been shown to cause oxidative stress in primary neurons at much higher levels than non-PK-resistant oligomers. It has been shown that two kinds of αSyn fibrils exhibiting different vulnerabilities to PK digestion can be isolated from repetitive seeded fibrillization, and the αSyn strain more resistant to PK digestion is more toxic to neurons. In addition, αSyn fibril strains produced using different buffers show different vulnerabilities to PK digestion, and their toxicities are associated with their resistance to PK digestion. Interestingly, αSyn fibrils with different levels of PK resistance have different structures, cross-seeding abilities and propagation properties both in vitro and in vivo, all of which are reminiscent of the properties of prions. Therefore, it is possible that the accelerated formation of PK-resistant αSyn that was observed in the GCase-deficient flies represents the ‘prion-like conversion’ of αSyn and that this toxic species leads to phenotypic aggravation by promoting the prion-like seeding and propagation of αSyn (Suzuki, 2015).

The idea that αSyn is degraded in lysosomes has led to several studies on the basis of the hypothesis that loss of GCase activity compromises the αSyn-degrading function of lysosomes, resulting in αSyn accumulation. Several groups have demonstrated that decreased GCase activity results in increased amounts of αSyn, using cultured neurons, human iPSC-derived neurons from GBA1 mutation carriers and mice treated with a GCase inhibitor. In contrast, two other groups have reported that GCase activity does not correlate with the amount of αSyn in neuronal cells, whereas the expression of a mutant GCase that maintains its enzyme activity increases the amount of αSyn, favoring a gain-of-function mechanism in the pathogenesis of GBA1-associated PD. In the fly model, the amount of total αSyn was not significantly increased by GCase deficiency, despite the phenotypic aggravation. However, a recent study using PD model mice with a GBA1 mutation has shown that the total amount of αSyn in the brain lysates is not increased, but the rate of αSyn degradation assessed by pulse-chase experiments is decreased in primary neurons from the same mice. Thus, the possibility that αSyn degradation is compromised by lysosomal dysfunction can not be completely excluded, even though changes in the total amount of αSyn are not detected (Suzuki, 2015).

In addition to the fly model experiments, it was demonstrated by in vitro experiments that GlcCer directly promotes the formation of PK-resistant αSyn, as a mechanism for the increased accumulation of PK-resistant αSyn in the dGBA1a-RNAi flies. These results are consistent with a previous report showing a direct effect of GlcCer on the stability of αSyn oligomers. Moreover, a significant increase in αSyn dimers by the incubation of αSyn with GlcCer-containing liposomes was also found, which is consistent with the finding that the amount of αSyn dimers is significantly increased in GD patients. It was also shown that β-Gal knockdown exacerbates the locomotor dysfunction of αSyn flies, and GM1 directly promotes the PK resistance of αSyn, supporting the hypothesis that aberrant interactions of αSyn with glycolipids trigger the toxic conversion of αSyn, resulting in increased neurotoxicity in vivo. It has been demonstrated that GM1 specifically binds to αSyn and induces its oligomerization, thereby inhibiting its fibrillation. Interestingly, a recent report shows that iPSC-derived neurons from GBA1-associated PD patients exhibit not only decreased GCase activity, but also decreased β-Gal activity, which can be rescued by zinc-finger nuclease-mediated gene correction, implying a crosstalk between GCase and β-Gal activities. Taken together, it is possible that a loss of β-Gal activity also contributes to the acceleration of αSyn toxicity in GBA1-associated PD. It is noted that the direct binding of GM1 to the amyloid β protein also triggers its toxic conversion, implying a common or similar role of glycolipids in the conversion of neurodegenerative disease-related proteins from their non-toxic to toxic forms (Suzuki, 2015).

Then, where in a cell does the accumulated GlcCer interact with αSyn to convert it into a PK-resistant form? One possibility is that αSyn is transported into lysosomes via macroautophagy or chaperone-mediated autophagy, where it interacts with accumulated GlcCer. Then, GlcCer-associated αSyn is secreted from the cells, taken up by itself or by the surrounding cells and accumulates in the cytosol. The other possibility is that the accumulated GlcCer in the lysosome leaks into the cytosol and interacts with αSyn in the cytosol, as the leakage of GlcCer into the cytosol has been reported in both GD patients and GD model mice. There have been no reports to date of the level of GlcCer in the brain of PD patients with a GBA1 mutation. However, in iPSC-derived neurons from two PD patients with a heterozygous GBA1 mutation (RecNcil/wt and N370S/wt), that causes an approximately 50% decrease in GCase activity, the amount of GlcCer has been reported to be about 2-fold higher than that of isogenic gene-corrected iPSC-derived neurons. Furthermore, GlcCer has been reported to accumulate in the brains of GD patients, in which GCase activity decreases (by 80–90%). GCase activity has also been found to be moderately decreased in the brains of GBA1 mutant carrier PD patients (58% decrease in the substantia nigra). Collectively, these data suggest that GlcCer accumulates in the brains of GBA1 mutation carrier PD patients (Suzuki, 2015).

This study focused on the loss-of-function aspect of GBA1 mutations, but there is another possibility arguing the gain-of-function toxicity of mutant GCase, because most mutant GCases are prone to misfold in the endoplasmic reticulum (ER). Human skin fibroblasts derived from GD patients and carriers are reported to induce the unfolded protein response, which is also observed in Drosophila models of GD expressing human mutant GCase. Ambroxol, a potential pharmacological chaperone for mutant GCase, has been shown to ameliorate both ER stress and the phenotypes of these Drosophila models. Interestingly, ambroxol treatment also suppresses the misfolding of mutant GCase, subsequently resulting in an enhancement of cellular GCase activity. Therefore, chemical chaperone therapy can be expected to exert beneficial effects against GD, via the amelioration of both the gain-of-function aspect through ER stress and the loss-of-function aspect through decreased GCase activity. As ER stress has been suggested to be involved in the neurodegeneration that occurs in PD, the synergistic effects of chemical chaperone therapy would also be effective for GBA1-associated PD patients, through the suppression of both ER stress and the toxic conversion of αSyn by GlcCer accumulation (Suzuki, 2015).

Expression of human Gaucher disease gene GBA generates neurodevelopmental defects and ER stress in Drosophila eye

Gaucher disease (GD) is the most common of the lysosomal storage disorders and is caused by defects in the GBA gene encoding glucocerebrosidase (GlcCerase). The accumulation of its substrate, glucocylceramide (GlcCer) is considered the main cause of GD. This study shows that the expression of human mutated GlcCerase gene (hGBA) that is associated with neuronopathy in GD patients causes neurodevelopmental defects in Drosophila eyes. The data indicate that endoplasmic reticulum (ER) stress is elevated in Drosophila eye carrying mutated hGBAs by use of the ER stress markers dXBP1 (X box binding protein-1) and dBiP (Heat shock 70-kDa protein cognate 3). It was also found that Ambroxol, a potential pharmacological chaperone for mutated hGBAs, can alleviate the neuronopathic phenotype through reducing ER stress. The study demonstrates a novel mechanism of neurodevelopmental defects mediated by ER stress through expression of mutants of human GBA gene in the eye of Drosophila (Suzuki, 2013).

This study shows that hGBA with the RecNciI mutation, which causes type 2 GD (acute neurological abnormalities in humans), shows severe neurodevelopmental defects in Drosophila eyes. The primary defect in GD is an obvious deficiency in the activity of the lysosomal enzyme GlcCerase. Deficiencies in GlcCerase result in the accumulation of its lipid substrate GlcCer in the lysosomal compartment of macrophages. The defects associated with GD are thought to be caused by GlcCer accumulation. In fact, mouse models of GD base the study on the notion that GD phenotypes are caused by accumulated stored GlcCer. Therefore, mutations or deletions have been constructed from the endogenous homologous genes of mouse genome. In some cases, GlcCerase variants are retained to various degrees in the ER as seen in cells of patients with GD. These findings suggest that mutated GlcCerase itself is toxic, but this has yet to be confirmed at molecular level. Drosophila transgenic lines generated in this study can serve as a powerful tool for investigating molecular mechanisms of neurodegeneration as well as novel therapeutic targets of GD, because data suggest that ER stress, due to misfolding of the GlcCer protein, may be a contributory factor in the pathology of GD (Suzuki, 2013).

It was found that mutated hGBAs cause ER stress as well as neurodevelopmental defects in Drosophila eyes, which suggest that protein products of GlcCerase might be toxic to the ER. This finding suggests that mutated GlcCerase could serve as a new therapeutic target for type 2 GD. ER stress contributes to neurodegeneration across a range of neurodegenerative disorders and it might also be responsible for neurodegeneration in the eyes of Drosophila transfected with hGBAs, especially when they harbor the RecNciI mutation that is associated with acute neurological abnormalities in GD patients. Previous reports indicate that ER stress is a common mediator of apoptosis in both neurodegenerative and non-neurodegenerative lysosomal storage disorders including GD. Unfolded protein response activation observed in fibroblast cells from neuronopathic GD patients might be a common mediator of apoptosis in neurodegenerative lysosomal storage disorders. This suggests that mutated hGBAs may cause apoptosis through ER stress in Drosophila eyes (Suzuki, 2013).

Ambroxol is known as a pharmacological chaperone for mutant glucocerebrosidase including the L444P point mutation. It was found that Ambroxol can decrease ER stress and ameliorate neurodevelopmental defects in Drosophila with the RecNciI mutation. The complex allele RecNciI also includes L444P point mutation. The data suggests that Ambroxol acts as a pharmacological chaperone for the RecNciI GlcCerase variant in Drosophila eye. As ER stress contributes to neurodegeneration across a range of neurodegenerative disorders, Ambroxol may have an important use in ameliorating neurodegeneration in GD patients (Suzuki, 2013).

Search PubMed for articles about Drosophila

Cabasso, O., Paul, S., Dorot, O., Maor, G., Krivoruk, O., Pasmanik-Chor, M., Mirzaian, M., Ferraz, M., Aerts, J. and Horowitz, M. (2019). Drosophila melanogaster Mutated in its GBA1b Ortholog Recapitulates Neuronopathic Gaucher Disease. J Clin Med 8(9). PubMed ID: 31505865

Davis, M. Y., Trinh, K., Thomas, R. E., Yu, S., Germanos, A. A., Whitley, B. N., Sardi, S. P., Montine, T. J. and Pallanck, L. J. (2016). Glucocerebrosidase Deficiency in Drosophila Results in alpha-Synuclein-Independent Protein Aggregation and Neurodegeneration. PLoS Genet 12(3): e1005944. PubMed ID: 27019408

Gegg, M. E. and Schapira, A. H. V. (2018). The role of glucocerebrosidase in Parkinson disease pathogenesis. FEBS J 285(19): 3591-3603. PubMed ID: 29385658

Jewett, K. A., Thomas, R. E., Phan, C. Q., Lin, B., Milstein, G., Yu, S., Bettcher, L. F., Neto, F. C., Djukovic, D., Raftery, D., Pallanck, L. J. and Davis, M. Y. (2021). Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles. PLoS Genet 17(2): e1008859. PubMed ID: 33539341

Kawasaki, H., Suzuki, T., Ito, K., Takahara, T., Goto-Inoue, N., Setou, M., Sakata, K. and Ishida, N. (2017). Minos-insertion mutant of the Drosophila GBA gene homologue showed abnormal phenotypes of climbing ability, sleep and life span with accumulation of hydroxy-glucocerebroside. Gene [Epub ahead of print]. PubMed ID: 28286087

Kinghorn, K. J., Gronke, S., Castillo-Quan, J. I., Woodling, N. S., Li, L., Sirka, E., Gegg, M., Mills, K., Hardy, J., Bjedov, I. and Partridge, L. (2016). A Drosophila Model of Neuronopathic Gaucher Disease Demonstrates Lysosomal-Autophagic Defects and Altered mTOR Signalling and Is Functionally Rescued by Rapamycin. J Neurosci 36(46): 11654-11670. PubMed ID: 27852774

Maor, G., Cabasso, O., Krivoruk, O., Rodriguez, J., Steller, H., Segal, D. and Horowitz, M. (2016). The contribution of mutant GBA to the development of parkinson disease in Drosophila. Hum Mol Genet [Epub ahead of print]. PubMed ID: 27162249

Maor, G., Rapaport, D. and Horowitz, M. (2019). The effect of mutant GBA1 on accumulation and aggregation of alpha-synuclein. Hum Mol Genet 28(11): 1768-1781. PubMed ID: 30615125

Nalls, M. A., Duran, R., Lopez, G., Kurzawa-Akanbi, M., McKeith, I. G., Chinnery, P. F., Morris, C. M., Theuns, J., Crosiers, D., Cras, P., Engelborghs, S., De Deyn, P. P., Van Broeckhoven, C., Mann, D. M., Snowden, J., Pickering-Brown, S., Halliwell, N., Davidson, Y., Gibbons, L., Harris, J., Sheerin, U. M., Bras, J., Hardy, J., Clark, L., Marder, K., Honig, L. S., Berg, D., Maetzler, W., Brockmann, K., Gasser, T., Novellino, F., Quattrone, A., Annesi, G., De Marco, E. V., Rogaeva, E., Masellis, M., Black, S. E., Bilbao, J. M., Foroud, T., Ghetti, B., Nichols, W. C., Pankratz, N., Halliday, G., Lesage, S., Klebe, S., Durr, A., Duyckaerts, C., Brice, A., Giasson, B. I., Trojanowski, J. Q., Hurtig, H. I., Tayebi, N., Landazabal, C., Knight, M. A., Keller, M., Singleton, A. B., Wolfsberg, T. G. and Sidransky, E. (2013). A multicenter study of glucocerebrosidase mutations in dementia with Lewy bodies. JAMA Neurol 70(6): 727-735. PubMed ID: 23588557

Schondorf, D. C., Ivanyuk, D., Baden, P., Sanchez-Martinez, A., De Cicco, S., Yu, C., Giunta, I., Schwarz, L. K., Di Napoli, G., Panagiotakopoulou, V., Nestel, S., Keatinge, M., Pruszak, J., Bandmann, O., Heimrich, B., Gasser, T., Whitworth, A. J. and Deleidi, M. (2018). The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC and Fly Models of Parkinson's Disease . Cell Rep 23(10): 2976-2988. PubMed ID: 29874584

Sanchez-Martinez, A., Beavan, M., Gegg, M. E., Chau, K. Y., Whitworth, A. J. and Schapira, A. H. (2016). Parkinson disease-linked GBA mutation effects reversed by molecular chaperones in human cell and fly models. Sci Rep 6: 31380. PubMed ID: 27539639

Suzuki, M., Fujikake, N., Takeuchi, T., Kohyama-Koganeya, A., Nakajima, K., Hirabayashi, Y., Wada, K. and Nagai, Y. (2015). Glucocerebrosidase deficiency accelerates the accumulation of proteinase K-resistant alpha-synuclein and aggravates neurodegeneration in a Drosophila model of Parkinson's disease. Hum Mol Genet 24(23): 6675-6686. PubMed ID: 26362253

Suzuki, T., Shimoda, M., Ito, K., Hanai, S., Aizawa, H., Kato, T., Kawasaki, K., Yamaguchi, T., Ryoo, H. D., Goto-Inoue, N., Setou, M., Tsuji, S. and Ishida, N. (2013). Expression of human Gaucher disease gene GBA generates neurodevelopmental defects and ER stress in Drosophila eye. PLoS One 8(8): e69147. PubMed ID: 23936319

Thomas, R. E., Vincow, E. S., Merrihew, G. E., MacCoss, M. J., Davis, M. Y. and Pallanck, L. J. (2018). Glucocerebrosidase deficiency promotes protein aggregation through dysregulation of extracellular vesicles. PLoS Genet 14(9): e1007694. PubMed ID: 30256786

date revised: 10 October 2021

Home page: The Interactive Fly © 2021 Thomas Brody, Ph.D.