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Gaucher's disease
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Drosophila genes associated with Gaucher's disease

CG31148 (Glycoside hydrolase)
CG31414 (Glycoside hydrolase)
X box binding protein-1 (XBP1)
Heat shock 70-kDa protein cognate 3 (BiP)
Related terms


ER stress
Parkinson's disease
Overview of the disease

Gaucher disease (GD) is a lysosomal storage disease, caused by mutations in the gene encoding lysosomal acid β-glucocerebrosidase (GCase), designated GBA (glucosidase, beta, acid). As a result, glucosylceramide (GlcCer) is not properly degraded and accumulates primarily in cells of mononuclear phagocyte origin. More than 300 mutations have been identified in the GBA gene. A large fraction of them are missense mutations, though premature termination, splice site mutations, deletions and recombinant alleles have been recognized as well. There are several abundant mutations. The N370S mutation is the most prevalent among type 1 GD patients, while the L444P mutation is most common among the neuronopathic types of GD. The majority of patients homozygous for this mutation develop type 3 GD. The 84GG mutation is an insertion of a guanine 84 nucleotides downstream from the first initiator methionine of the GBA mRNA, resulting in premature protein termination. There are two GBA homologs in Drosophila, designated CG31414 and CG31148, both encoding proteins showing ~31% identity and ~49% similarity to the human GCase (Maor, 2013 and references therein).

As a lysosomal enzyme, GCase is synthesized on endoplasmic reticulum (ER) bound polyribosomes. Upon its entry into the ER, it undergoes N-linked glycosylation on four asparagines, after which it is subject to ER quality control (ERQC). When correctly folded it shuttles to the Golgi compartment for further modifications on the N-glycans and finally it traffics to the lysosomes. Mutant GCase variants are recognized as misfolded proteins and undergo various degrees of ER associated degradation (ERAD). The accumulation of misfolded molecules in the ER, activate signaling events known as the unfolded protein response (UPR). UPR monitors the conditions in the ER, by sensing insufficiency in protein folding capacity and translates this information into gene expression (Maor, 2013 and references therein).

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Relevant studies of Gaucher's disease

Maor, G., Rencus-Lazar, S., Filocamo, M., Steller, H., Segal, D. and Horowitz, M. (2013). Unfolded protein response in Gaucher disease: from human to Drosophila. Orphanet J Rare Dis 8: 140. PubMed ID: 24020503

Abstract
In Gaucher disease (GD), resulting from mutations in the GBA gene, mutant β-glucocerebrosidase (GCase) molecules are recognized as misfolded in the endoplasmic reticulum (ER). They are retrotranslocated to the cytoplasm, where they are ubiquitinated and undergo proteasomal degradation in a process known as the ER Associated Degradation (ERAD). It has been shown earlier that the degree of ERAD of mutant GCase correlates with GD severity. Persistent presence of mutant, misfolded protein molecules in the ER leads to ER stress and evokes the unfolded protein response (UPR). This study investigated the presence of UPR in several GD models, using molecular and behavioral assays. UPR was found to operate in skin fibroblasts from GD patients and carriers of GD mutations. UPR was also recapitulated in two different Drosophila models for carriers of GD mutations: flies heterozygous for the endogenous mutant GBA orthologs and flies expressing the human N370S or L444P mutant GCase variants. Both fly models exhibit early death, indicating the deleterious effect of mutant GCase during development. The double heterozygous flies, and the transgenic flies, expressing mutant GCase in dopaminergic/serotonergic cells develop locomotion deficit. These results strongly suggest that mutant GCase induces the UPR in GD patients as well as in carriers of GD mutations and leads to development of locomotion deficit in flies heterozygous for GD mutations (Maor, 2013).

Highlights

  • Activation of UPR in GD derived fibroblasts.
  • Splicing of Xbp1 as a UPR marker in GD derived fibroblasts.
  • Elevation in phosphorylation of eIF2α in GD derived fibroblasts.
  • Activation of UPR in carriers of GD mutations.
  • Activation of UPR in Drosophila GD-like carriers.
  • Locomotor dysfunction in flies expressing human mutant GCase variant.

Discussion
This study shows that persistent presence of mutant GCase activates the UPR, in humans and in Drosophila.  Several earlier studies have already noticed the existence of UPR in GD derived skin fibroblasts. In one N370S/N370S Type 1 GD derived skin fibroblast line, a significant increase in mRNA level of the UPR related genes ATF6, BiP and Xbp1 has been documented. This is accompanied by a concomitant increase in the protein level of active ATF6, BiP and phosphorylated eIF2α. UPR activation has also been reported in cell lines derived from GD patients homozygous for the N370S and the L444P mutations. Further, the efficacy of catechin in alleviating UPR in one GD derived line (N370S/N370S) has been documented (Maor, 2013).

This study shows UPR activation in skin fibroblasts derived from GD patients manifests by an increase in mRNA and protein levels of BiP and CHOP, splicing of Xbp1 and phosphorylation of eIF2α. It was also shown that in fibroblast lines derived from carriers of GD mutations there is UPR as well. Interestingly, the 84GG mutation is not expected to culminate in a mature protein. Since there is UPR in carriers of this mutation, one has to assume that either the 84GG mutant RNA participates in translation of a very short peptide (26 amino acids long), shorter than the full size leader of GCase (38 amino acids long), thereby blocking ER entrance of newly synthesized proteins, or the 84GG mRNA-bound polyribosomes block ER entrance, thereby leading to development of ER stress and, as a result, to UPR. These hypotheses will have to be further tested (Maor, 2013).

ERAD and UPR are well conserved across species and Drosophila has become an important tool in studying these phenomena. The short life span of the fly with the sophisticated molecular and genetic tools for fast establishment of transgenic lines, and availability of deletions and mutations in any chosen gene have made it an attractive model, which allows fast screening and analyses of large populations. This study could recapitulate UPR in two fly models: one corresponding to carriers of GD mutations and the other involving transgenic flies expressing the human N370S or the L444P mutant proteins (Maor, 2013).

All lysosomal enzymes are synthesized on ER bound polyribosomes and upon their entry into the ER undergo N-linked glycosylation and quality control, after which they shuttle to the Golgi apparatus. Following further modifications there, they are trafficked to the lysosomes. Therefore, all mutant lysosomal enzymes are expected to undergo ERAD and induce the UPR machinery. UPR has already been documented in other lysosomal diseases. Thus, in Fabry disease which results from mutation in the α-galactosidase-A encoding gene (α-Gal-A) and accumulation of the globotrioside Gb3, mutant variants undergo ERAD, which induces the UPR. UPR has also been documented in skin fibroblasts from patients suffering from ceroid lupofusinosis (CLN) 1, 2, 3, 6 and 8, as well as in cells of patients with GM1 gangliosidosis (suffering from reduced activity of β-galactosidase), Tay Sachs disease (reduced activity of β-hexosaminidase A) and Niemann Pick type C2 (mutations in the NPC2 gene). Interestingly, in some model systems for lysosomal diseases UPR has not been not recapitulated. For instance, UPR was not demonstrated in neuronal cells derived from knockout mice lacking the GBA gene, or in animals or cells treated with the GCase non-competitive inhibitor CBE. Lack of UPR in both of these cases is likely due to the absence of mutant GCase in the ER. Likewise, in NPC1-deficient mice and in an NPC1 cell-based model, created by knocking down the expression of NPC1 using RNA interference, UPR was not found to operate. Again, in both cases, no mutant protein was present in the ER to induce the UPR machinery (Maor, 2013).

In recent years, association has been demonstrated between GD and Parkinson's disease (PD), a neurodegenerative disease affecting 1% of individuals over 60 years old. Thus, there is a higher propensity among Type 1 GD patients and among carriers of GD mutations to develop PD in comparison to the non-GD population. Brains of carriers of GD mutations who develop PD display Lewy bodies (LB) and loss of substantia nigra neurons. Carriers of GBA mutations tend to have more cortical LBs than those of non-carriers (82% versus 43%, respectively), suggesting that mutant GCase variants promote α-synuclein aggregation directly. It has been shown that expression of mutant human GCase, but not that of the normal counterpart, leads to increase in α-synuclein accumulation in MES23.5, PC12, and HEK293 cell lines, arguing that mutant GCase has a direct role in α-synuclein accumulation, and most probably, aggregation (Maor, 2013).

Since carriers of GD mutations do not accumulate GlcCer in their brain and its build-up has not been demonstrated in brains of Type 1 GD patients, this study speculates that ERAD of mutant GCase contributes to the development of PD among GD patients and carriers of GD mutations. It has been shown that parkin interacts with mutant GCase and mediates its Lysine 48 ubiquitination and proteasomal degradation. The study proposes that this interaction between parkin and mutant GCase leads to deleterious effect in dopaminergic cells caused by the accumulation of parkin substrates, which are potentially toxic. It has also been shown that mutant GCase competes with two known substrates of parkin, PARIS and ARTS, whose accumulation in cells leads to apoptosis. PARIS is a transcription repressor of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α (PGC-1α) expression. PGC-1α is a master regulator of mitochondrial biogenesis. Thus, PARIS accumulation impedes mitochondria biogenesis. Interestingly, PARIS accumulates in mouse models of parkin inactivation and in PD patients’ brains. ARTS is a mitochondrial protein that initiates caspase activation, upstream of cytochrome c release in the mitochondrial apoptotic pathway (Maor, 2013).

This study now extends the hypothesis for the role of mutant GCase in the development of PD. The study proposes that ERAD of mutant GCase leads to accumulation of parkin substrates, some of which are deleterious, like PARIS and ARTS. This accumulation leads to cell death. Also UPR, as part of the cellular ER stress, induced by the persistent presence of mutant GCase in the ER, leads to cellular death. Therefore, both, ERAD of mutant GCase and UPR contribute to dopaminergic cell death and development of PD. It still remains to be tested whether and how mutant GCase leads to aggregation of α-synuclein (Maor, 2013).

It was shown that expression of the mutant fly orthologs of GBA or expression of human mutant N370S or L444P proteins leads to death at early stages of the fly development. The flies expressing mutant GCase in the dopaminergic/serotonergic cells develop locomotion dysfunction, reminiscent of PD. This is the first animal model in which carriers of GD mutations develop parkisonian signs. Parkin insolubility has been associated with lack of degradation of ubiquitinated proteins and accumulation of α-synuclein and parkin in autophagosomes, suggesting autophagic defects in PD. To test parkin’s role in mediating autophagic clearance, lentiviral gene transfer was used to express human wild type or mutant parkin (T240R) with α-synuclein in the rat striatum. Lentiviral expression of α-synuclein leads to accumulation of autophagic vacuoles, while co-expression of parkin with α-synuclein facilitates autophagic clearance. Expression of parkin loss-of-function mutation does not affect autophagic clearance. Taken together, the data suggest that functional parkin regulates autophagosome clearance. It is possible, and remains to be proven, that the interaction between parkin and mutant GCase variants in dopaminergic cells attenuates normal autophagy, which leads to α-synuclein aggregation (Maor, 2013).

Results from this study strongly indicate a direct association between mutant GCase and development of Parkinsonian signs in the fly. However, there is another paradigm, arguing that insufficient lysosomal mutant GCase activity leads to substrate accumulation (GlcCer or glucosylsphingosine), α-synuclein aggregation, block in trafficking of GCase to lysosomes and development of PD [60,62,68-70]. It has been shown that that in brain sections derived from 12 months old D409V homozygous mice (but not from D409V heterozygous animals) there are α-synuclein and ubiquitin aggregates in the hippocampus, cerebral cortex and cerebellum. Memory deficits are present in these mice at 6 months of age. Administration of normal enzyme to the brain using gene therapy with an AAV derived vector, expressing a normal human GBA cDNA, significantly reduces the aggregation of ubiquitin and α-synuclein and ameliorated the memory deficit (Maor, 2013).

Development of PD in carriers of GD mutations implies that the presence of a mutant GBA allele is a dominant predisposing factor. This is a unique case of an autosomal recessive metabolic disease with a dominant element, namely the tendency of carriers of GD mutations to develop PD. Dominance results either from haploinsufficiency or from gain of function. If haploinsufficiency accounts for the development of PD in carriers of GD mutations, it implies insufficient GCase activity in the dopaminergic neurons. Why is it not manifested in macrophages, in which case the disease would have been dominant? If, alternatively, the dominance results from gain of function, then its development depends on accumulation of enough deleterious product (mutant GCase, in our case), as in the case of Alzheimer disease, which displays age dependent accumulation of β-amyloid and tau, or Huntington disease, which exhibits accumulation of huntingtin; results from this study suggest the gain of function alternative (Maor, 2013).

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Dasari, S. K., Schejter, E., Bialik, S., Shkedy, A., Levin-Salomon, V., Levin-Zaidman, S. and Kimchi, A. (2017). Death by over-eating: The Gaucher Disease associated gene GBA1, identified in a screen for mediators of autophagic cell death, is necessary for developmental cell death in Drosophila midgut. Cell Cycle [Epub ahead of print] PubMed ID: 28933588

Abstract

Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. A thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells has been reported. To gain mechanistic insight into this death program, a signalome-wide RNAi screen has been performed for genes whose functions are necessary for resveratrol-induced death. The screen identified GBA1a, the gene encoding the lysosomal enzyme glucocerebrosidase, as an important mediator of autophagic cell death. This study further showed the physiological relevance of GBA1a to developmental cell death in midgut regression during Drosophila metamorphosis. A delay was observed in midgut cell death in two independent Gba1a RNAi lines, indicating the critical importance of Gba1a for midgut development. Interestingly, loss-of-function GBA1 mutations lead to Gaucher Disease and are a significant risk factor for Parkinson Disease, which have been associated with defective autophagy. Thus GBA1a is a conserved element critical for maintaining proper levels of autophagy, with high levels leading to autophagic cell death (Dasari, 2017).

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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: e69147. PubMed ID: 23936319

Abstract
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 using 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).

Highlights

  • Generation of transgenic flies carrying hGBA variants.
  • Expression of hGBA carrying the RecNciI mutation causes neurodevelopmental defects in the Drosophila eye.
  • Endoplasmic reticulum (ER) stress is detected in hGBR transgenic flies.
  • Ambroxol can recover the morphological defects and decrease ER stress in hGBA transgenic flies.

Discussion
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 is 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).

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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

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 homologues 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).

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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: 11654-11670. PubMed ID: 27852774

Abstract

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).

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Reviews

Nagral, A. (2014). Gaucher disease. J Clin Exp Hepatol. 4(1):37-50. PubMed ID: 25755533

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ER stress and unfolded protein response (UPR) associated diseases

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Date revised: 18 August 2015

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