Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity.Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients.

BackgroundIn 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). We have shown in the past 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).MethodsWe investigated the presence of UPR in several GD models, using molecular and behavioral assays.ResultsOur results show the existence of UPR in skin fibroblasts from GD patients and carriers of GD mutations. We could recapitulate UPR 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. We encountered early death in both fly models, indicating the deleterious effect of mutant GCase during development. The double heterozygous flies, and the transgenic flies, expressing mutant GCase in dopaminergic/serotonergic cells developed locomotion deficit.ConclusionOur 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.

Gaucher disease (GD) results from mutations in the acid β-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. In this study, we used the fruit fly Drosophila melanogaster 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. We also established transgenic flies carrying the mutant human N370S, L444P and the 84GG variants. 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 endoplasmic reticulum (ER). However flies expressing the 84GG mutant, that does not express mature GCase, did not exhibit rescue by ambroxol. Our 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 PD.

Gaucher disease (GD) results from mutations in the GBA1 gene, which encodes lysosomal glucocerebrosidase (GCase). The large number of mutations known to date in the gene lead to a heterogeneous disorder, which is divided into a non-neuronopathic, type 1 GD, and two neurological, type 2 and type 3, forms. We studied the two fly GBA1 orthologs, GBA1a and GBA1b. Each contains a Minos element insertion, which truncates its coding sequence. In the GBA1am/m flies, which express a mutant protein, missing 33 C-terminal amino acids, there was no decrease in GCase activity or substrate accumulation. However, GBA1bm/m mutant flies presented a significant decrease in GCase activity with concomitant substrate accumulation, which included C14:1 glucosylceramide and C14:0 glucosylsphingosine. GBA1bm/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. Our results highlight the resemblance between the phenotype of the GBA1bm/m mutant fly and neuronopathic GD and underlie its relevance in further GD studies as well as a model to test possible therapeutic modalities.

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Inability to properly degrade unfolded or misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress and unfolded protein response. This is particularly important in cases of diseases in which the mutant proteins undergo ER-associated degradation (ERAD), as in Gaucher disease (GD). GD is a genetic, autosomal recessive disease that results from mutations in the GBA1 gene, encoding the lysosomal enzyme acid β-glucocerebrosidase (GCase). We have shown that mutant GCase variants undergo ERAD, the degree of which is a major determinant of disease severity. Most ERAD substrates undergo polyubiquitination and proteasomal degradation. Therefore, one expects that mutant GCase variants are substrates for several E3 ubiquitin ligases in different cells. We tested the possibility that ITCH, a known E3 ubiquitin ligase, with a pivotal role in proliferation and differentiation of the skin, recognizes mutant GCase variants and mediates their polyubiquitination and degradation. Our results strongly suggest that ITCH interacts with mutant GCase variants and mediates their lysine 48 polyubiquitination and degradation.

Fabry disease, an X-linked recessive lysosomal disease, results from mutations in the GLA gene encoding lysosomal α-galactosidase A (α-Gal A). Due to these mutations, there is accumulation of globotriaosylceramide (GL-3) in plasma and in a wide range of cells throughout the body. Like other lysosomal enzymes, α-Gal A is synthesized on endoplasmic reticulum (ER) bound polyribosomes, and upon entry into the ER it undergoes glycosylation and folding. It was previously suggested that α-Gal A variants are recognized as misfolded in the ER and undergo ER-associated degradation (ERAD). In the present study, we used Drosophila melanogaster to model misfolding of α-Gal A mutants. We did so by creating transgenic flies expressing mutant α-Gal A variants and assessing development of ER stress, activation of the ER stress response and their relief with a known α-Gal A chaperone, migalastat. Our results showed that the A156V and the A285D α-Gal A mutants underwent ER retention, which led to activation of unfolded protein response (UPR) and ERAD. UPR could be alleviated by migalastat. When expressed in the fly’s dopaminergic cells, misfolding of α-Gal A and UPR activation led to death of these cells and to a shorter life span, which could be improved, in a mutation-dependent manner, by migalastat.