Animal Models for the Study of Gaucher Disease.

In Gaucher disease (GD), a relatively common sphingolipidosis, the mutant lysosomal enzyme acid ß-glucocerebrosidase (GCase), encoded by the GBA1 gene, fails to properly hydrolyze the sphingolipid glucosylceramide (GlcCer) in lysosomes, particularly of tissue macrophages. As a result, GlcCer accumulates, which, to a certain extent, is converted to its deacylated form, glucosylsphingosine (GlcSph), by lysosomal acid ceramidase. The inability of mutant GCase to degrade GlcSph further promotes its accumulation. The amount of mutant GCase in lysosomes depends on the amount of mutant ER enzyme that shuttles to them. In the case of many mutant GCase forms, the enzyme is largely misfolded in the ER. Only a fraction correctly folds and is subsequently trafficked to the lysosomes, while the rest of the misfolded mutant GCase protein undergoes ER-associated degradation (ERAD). The retention of misfolded mutant GCase in the ER induces ER stress, which evokes a stress response known as the unfolded protein response (UPR). GD is remarkably heterogeneous in clinical manifestation, including the variant without CNS involvement (type 1), and acute and subacute neuronopathic variants (types 2 and 3). The present review discusses animal models developed to study the molecular and cellular mechanisms underlying GD.

The effect of mutant GBA1 on accumulation and aggregation of α-synuclein.

Gaucher disease (GD) patients and carriers of GD mutations have a higher propensity to develop Parkinson's disease (PD) in comparison to the non-GD population. This implies that 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. In the present study we 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. Our 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 with flies expressing α-synuclein or mutant GCase alone. Taken together, our 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.

Misfolding of Lysosomal α-Galactosidase a in a Fly Model and Its Alleviation by the Pharmacological Chaperone Migalastat.

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.

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

Gaucher disease (GD) results from mutations in the acid ß-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.

NEK3-mediated SNAP29 phosphorylation modulates its membrane association and SNARE fusion dependent processes.

Intracellular membrane fusion depends on the presence of specific mediators, the vesicle (v-) and the target (t-) SNAREs (Soluble N-ethylmaleimide-sensitive factor, NSF, attachment protein SNAP receptors), whose interaction brings apposing membranes to close proximity and initiates their fusion. SNAP29 (synaptosomal-associated protein 29), a t-SNARE protein, is involved in multiple fusion events during intracellular transport and affects structure of organelles such as the Golgi apparatus and focal adhesions. Mutations in SNAP29 gene result in CEDNIK (Cerebral dysgenesis, neuropathy, ichthyosis and palmoplantar keratoderma) syndrome. In the present study, we show that NEK3 (NIMA-never in mitosis gene A-related kinase 3)-mediated serine 105 (S105) phosphorylation of SNAP29 directs its membrane association, without which cells present defective focal adhesion formation, impaired Golgi structure and attenuated cellular recycling. In contrast to a phosphorylation-defective serine 105 to alanine (S105A) mutant, wildtype SNAP29, partially rescued the abnormal morphology of a CEDNIK patient derived fibroblasts. Our results highlight the importance of NEK3-mediated S105 phosphorylation of SNAP29 for its membrane localization and for membrane fusion dependent processes.

UPR activation and CHOP mediated induction of GBA1 transcription in Gaucher disease.

Chronic presence of mutant, misfolded proteins in the endoplasmic reticulum (ER) initiates ER stress and induces the Unfolded Protein Response (UPR). In Gaucher disease (GD), resulting from mutations in the GBA1 gene, encoding lysosomal acid ß-glucocerebrosidase (GCase), a certain fraction of the mutant variants is retained in the ER and activates the UPR. We have previously shown UPR activation in GD derived fibroblasts, in fibroblasts that derived from carriers of GD mutations and in Drosophila models of carriers of GD mutations. In the present work we extended our studies to include a large collection of fibroblasts, EBV-transformed B-cells and white blood cells (WBCs) that derived from GD patients. The results showed UPR activation in all tested cells. They also indicated that transcription of the GBA1 gene is upregulated through activation of the UPR-induced CHOP transcription factor. Transcription of the MAN2B gene, encoding alpha-mannosidase and of the ACP gene, encoding acid phosphatase was also elevated presumably through CHOP activation. Our results highlight the existence of chronic stress in GD derived cells due to the presence of ER-retained mutant GCase, which leads to upregulation of GBA1 expression.

New Directions in Gaucher Disease.

In Gaucher disease (GD), mutant lysosomal acid ß-glucocerebrosidase fails to properly hydrolyze its substrate, glucosylceramide, which accumulates in the lysosomes. Due to its phenotypic heterogeneity, GD has been classified into type 1, non-neuronopathic, and types 2 and 3, the neuronopathic forms, based on the primary involvement of the central nervous system. Neuroinflammation and necroptotic death may appear in the neuronopathic forms of GD, whereas type 1 GD patients may develop Parkinson disease (PD), a prototype of protein misfolding disorders of the nervous system. PD is significantly more prevalent among GD carriers and patients than among the non-GD populations. It is apparent that the amount of mutant enzyme present in lysosomes depends on the amount of mutant enzyme recognized as correctly folded in the endoplasmic reticulum (ER) for physiologically correct transport through the Golgi apparatus to the lysosome. Mutant enzyme recognized as misfolded is retained in the ER, inducing the Unfolded Protein Response. In the current review, we present three discrete areas of interest: molecular and cellular mechanisms underlying the association between GD and PD; the clinical and genetic associations between GD and PD; and treatment options for GD. We also discuss the relevance of induced pleuripotent stem cells to the above associations.

ITCH regulates degradation of mutant glucocerebrosidase: implications to Gaucher disease.

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 ß-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.

Short stature, onychodysplasia, facial dysmorphism, and hypotrichosis syndrome is caused by a POC1A mutation.

Disproportionate short stature refers to a heterogeneous group of hereditary disorders that are classified according to their mode of inheritance, clinical skeletal and nonskeletal manifestations, and radiological characteristics. In the present study, we report on an autosomal-recessive osteocutaneous disorder that we termed SOFT (short stature, onychodysplasia, facial dysmorphism, and hypotrichosis) syndrome. We employed homozygosity mapping to locate the disease-causing mutation to region 3p21.1-3p21.31. Using whole-exome-sequencing analysis complemented with Sanger direct sequencing of poorly covered regions, we identified a homozygous point mutation (c.512T>C [p.Leu171Pro]) in POC1A (centriolar protein homolog A). This mutation was found to cosegregate with the disease phenotype in two families. The p.Leu171Pro substitution affects a highly conserved amino acid residue and is predicted to interfere with protein function. Poc1, a POC1A ortholog, was previously found to have a role in centrosome stability in unicellular organisms. Accordingly, although centrosome structure was preserved, the number of centrosomes and their distribution were abnormal in affected cells. In addition, the Golgi apparatus presented a dispersed morphology, cholera-toxin trafficking from the plasma membrane to the Golgi was aberrant, and large vesicles accumulated in the cytosol. Collectively, our data underscore the importance of POC1A for proper bone, hair, and nail formation and highlight the importance of normal centrosomes in Golgi assembly and trafficking from the plasma membrane to the Golgi apparatus.

Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase ß-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.

EHD2 shuttles to the nucleus and represses transcription.

EHD {EH [Eps15 (epidermal growth factor receptor substrate 15) homology]-domain-containing} proteins participate in several endocytic events, such as the internalization and the recycling processes. There are four EHD proteins in mammalian cells, EHD1-EHD4, each with diverse roles in the recycling pathway of endocytosis. EHD2 is a plasma-membrane-associated member of the EHD family that regulates internalization. Since several endocytic proteins have been shown to undergo nucleocytoplasmic shuttling and have been assigned roles in regulation of gene expression, we tested the possibility that EHD proteins also shuttle to the nucleus. Our results showed that, among the three EHD proteins (EHD1-EHD3) that were tested, only EHD2 accumulates in the nucleus under nuclear export inhibition treatment. Moreover, the presence of a NLS (nuclear localization signal) was essential for its entry into the nucleus. Nuclear exit of EHD2 depended partially on its NES (nuclear export signal). Elimination of a potential SUMOylation site in EHD2 resulted in a major accumulation of the protein in the nucleus, indicating the involvement of SUMOylation in the nuclear exit of EHD2. We confirmed the SUMOylation of EHD2 by employing co-immunoprecipitation and the yeast two-hybrid system. Using GAL4-based transactivation assay as well as a KLF7 (Krüppel-like factor 7)-dependent transcription assay of the p21WAF1/Cip1 [CDKN1A (cyclin-dependent kinase inhibitor 1A)] gene, we showed that EHD2 represses transcription. qRT-PCR (quantitative real-time PCR) of RNA from cells overexpressing EHD2 or of RNA from cells knocked down for EHD2 confirmed that EHD2 represses transcription of the p21WAF1/Cip1 gene.

Gaucher disease paradigm: from ERAD to comorbidity.

Mutations in the GBA gene, encoding the lysosomal acid beta-glucocerebrosidase (GCase), lead to deficient activity of the enzyme in the lysosomes, to glucosylceramide accumulation and to development of Gaucher disease (GD). More than 280 mutations in the GBA gene have been directly associated with GD. Mutant GCase variants present variable levels of endoplasmic reticulum (ER) retention, due to their inability to correctly fold, and undergo ER-associated degradation (ERAD) in the proteasomes. The degree of ER retention and proteasomal degradation is one of the factors that determine GD severity. In the present review, we discuss ERAD of mutant GCase variants and its possible consequences in GD patients and in carriers of GD mutations.

Interaction between parkin and mutant glucocerebrosidase variants: a possible link between Parkinson disease and Gaucher disease.

Gaucher disease (GD), a sphingolipidosis characterized by impaired activity of the lysosomal enzyme glucocerebrosidase (GCase), results from mutations in the GCase-encoding gene, GBA. We have shown that mutant GCase variants present variable degrees of endoplasmic reticulum (ER) retention and undergo ER-associated degradation (ERAD) in the proteasome. Furthermore, the degree of ERAD of mutant GCase variants correlates with and is one of the factors that determine GD severity. An association between GD and Parkinson disease (PD) has been demonstrated by the concurrence of PD in GD patients and the identification of GCase mutations in probands with sporadic PD. One of the genes involved in PD is PARK2, encoding the E3 ubiquitin ligase parkin. Parkin functions in the ERAD of misfolded ER proteins, and it is upregulated by unfolded protein response. Loss of parkin function leads to the accumulation of its substrates, which is deleterious to dopaminergic neurons in PD. We, therefore, tested the possibility that the association between GD and PD reflects the fact that parkin acts as an E3 ligase of mutant GCase variants. Our results showed that mutant GCase variants associate with parkin. Normal parkin, but not its RING finger mutants, affects the stability of mutant GCase variants. Parkin also promotes the accumulation of mutant GCase in aggresome-like structures and is involved in K48-mediated polyubiquitination of GCase mutants, indicating its function as its E3 ligase. We suggest that involvement of parkin in the degradation of mutant GCase explains the concurrence of GD and PD.

RIN2 deficiency results in macrocephaly, alopecia, cutis laxa, and scoliosis: MACS syndrome.

Inherited disorders of elastic tissue represent a complex and heterogeneous group of diseases, characterized often by sagging skin and occasionally by life-threatening visceral complications. In the present study, we report on an autosomal-recessive disorder that we have termed MACS syndrome (macrocephaly, alopecia, cutis laxa, and scoliosis). The disorder was mapped to chromosome 20p11.21-p11.23, and a homozygous frameshift mutation in RIN2 was found to segregate with the disease phenotype in a large consanguineous kindred. The mutation identified results in decreased expression of RIN2, a ubiquitously expressed protein that interacts with Rab5 and is involved in the regulation of endocytic trafficking. RIN2 deficiency was found to be associated with paucity of dermal microfibrils and deficiency of fibulin-5, which may underlie the abnormal skin phenotype displayed by the patients.

EHD2 mediates trafficking from the plasma membrane by modulating Rac1 activity.

EHDs [EH (Eps15 homology)-domain-containing proteins] participate in different stages of endocytosis. EHD2 is a plasma-membrane-associated EHD which regulates trafficking from the plasma membrane and recycling. EHD2 has a role in nucleotide-dependent membrane remodelling and its ATP-binding domain is involved in dimerization, which creates a membrane-binding region. Nucleotide binding is important for association of EHD2 with the plasma membrane, since a nucleotide-free mutant (EHD2 T72A) failed to associate. To elucidate the possible function of EHD2 during endocytic trafficking, we attempted to unravel proteins that interact with EHD2, using the yeast two-hybrid system. A novel interaction was found between EHD2 and Nek3 [NIMA (never in mitosis in Aspergillus nidulans)-related kinase 3], a serine/threonine kinase. EHD2 was also found in association with Vav1, a Nek3-regulated GEF (guanine-nucleotide-exchange factor) for Rho GTPases. Since Vav1 regulates Rac1 activity and promotes actin polymerization, the impact of overexpression of EHD2 on Rac1 activity was tested. The results indicated that wt (wild-type) EHD2, but not its P-loop mutants, reduced Rac1 activity. The inhibitory effect of EHD2 overexpression was partially rescued by co-expression of Rac1 as measured using a cholera toxin trafficking assay. The results of the present study strongly indicate that EHD2 regulates trafficking from the plasma membrane by controlling Rac1 activity.

Lysosomal functions and dysfunctions: Molecular and cellular mechanisms underlying Gaucher disease and its association with Parkinson disease.

Lysosomes have a critical role in maintaining normal cellular homeostasis mediated by their involvement in secretion, plasma membrane repair, cell signaling and energy metabolism. Lysosomal storage disorders (LSDs) are a group of approximately 50 rare disorders caused by lysosomal dysfunction that occur due to mutations in a gene of a lysosomal protein. Gaucher disease (GD), an autosomal recessive disorder and one of the most common LSDs, is caused by the deficiency of the lysosomal enzyme acid-ß-glucocerebrosidase (GCase), due to biallelic mutations in the GBA1 gene. Reduced GCase activity leads to the accumulation of glucosylceramide (GlcCer), which is deacylated by lysosomal acid ceramidase to a toxic metabolite, glucosylshpingosine (GlcSph). Most GBA1 variants are recognized as misfolded in the ER, where the retention for refolding attempts initiates stress and activates the stress response known as the Unfolded Protein Response (UPR). The distinct clinical subtypes of GD are based on whether there is primary involvement of the central nervous system. Type 1 GD (GD1) is the nonneuropathic type, however, the recent recognition of the association of GD with the development of parkinsonism defies this classification. Patients with GD1 and carriers of GBA1 mutations are at risk for the development of parkinsonian manifestations. Parkinson disease (PD), the second most prevalent neurodegenerative disease, culminates in a movement disorder with the premature death of the patients. In PD and related disorders, collectively called synucleinopathies, the hallmark pathology is α-synuclein positive aggregates referred to as Lewy bodies or Lewy neurites and the death of dopaminergic neurons. While PD is mostly sporadic, in ∼5-10% of cases, the disease results from pathogenic variants in a growing number of genes. The most common genetic cause of PD is mutations in GBA1. Two mechanisms have been proposed for this link: (A) a "gain of function" mechanism, in which mutant GCase (protein) contributes to aggregate formation and to the development of PD, and the (B) "haploinsufficiency" ("loss of function") model, suggesting that one normal GBA1 allele is insufficient to carry adequate GCase activity and functional deficiency of GCase impedes α-synuclein metabolism. Lysosomal dysfunction, compromised autophagy and mitophagy further enhance the accumulation of α-synuclein, which results in the development of PD pathology. The present review will elaborate on the biology of GD, its association with PD and related disorders, and discuss the possible mechanisms underlying this association.

Characterization of the ERAD process of the L444P mutant glucocerebrosidase variant.

A large number of mutations in the glucocerebrosidase gene (GBA gene), encoding the lysosomal acid hydrolase glucocerebrosidase (GCase), lead to Gaucher disease (GD). The second most prevalent GD causing mutation, carried by 38% of non-Jewish patients, is L444P, resulting from a T to C transition in nucleotide 6092 of the GBA gene. It is a severe mutation that, in homozygosity, leads to neuropathic type 3 GD. We have previously shown that mutant GCase variants present variable degrees of endoplasmic reticulum (ER) retention and undergo ER associated degradation (ERAD). However, ERAD of the L444P mutant variant of GCase has never been tested. In the current study, we present results indicating that the L444P mutant protein undergoes extensive ERAD. In skin fibroblasts, originated from GD patients homozygous for L444P mutation, the level of GCase is 12%-21% of normal and at least 50% of it is in the ER. The mutant protein undergoes polyubiquitination and proteasome-dependent degradation. Recently Ambroxol, a known expectorant, was identified as a pharmacological chaperone for mutant GCase. We tested the effect of Ambroxol on the L444P mutant GCase and found that it enhances the removal of the mutant enzyme from the ER. In some cases, this removal leads to a concomitant increase in enzymatic activity.

The enigma of the E326K mutation in acid ß-glucocerebrosidase.

A large number of mutations, and several polymorphisms, have been characterized in the GBA gene, encoding the lysosomal enzyme glucocerebrosidase, the activity of which is impaired in Gaucher disease. In this communication we summarize published and new data concerning biochemical characterization of the E326K amino acid change (1093G>A in the GBA1 cDNA) in tissue culture and its association with Parkinson disease, suggesting it is a disease causing mutation and not merely a polymorphism in the GBA gene.

The Uncovered Function of the Drosophila GBA1a-Encoded Protein.

Human GBA1 encodes lysosomal acid ß-glucocerebrosidase (GCase), which hydrolyzes cleavage of the beta-glucosidic linkage of glucosylceramide (GlcCer). Mutations in this gene lead to reduced GCase activity, accumulation of glucosylceramide and glucosylsphingosine, and development of Gaucher disease (GD). Drosophila melanogaster has two GBA1 orthologs. Thus far, GBA1b was documented as a bone fide GCase-encoding gene, while the role of GBA1a encoded protein remained unclear. In the present study, we characterized a mutant variant of the fly GBA1a, which underwent ERAD and mildly activated the UPR machinery. RNA-seq analyses of homozygous mutant flies revealed upregulation of inflammation-associated as well as of cell-cycle related genes and reduction in programmed cell death (PCD)-associated genes, which was confirmed by qRT-PCR. We also observed compromised cell death in the midgut of homozygous larvae and a reduction in pupation. Our results strongly indicated that GBA1a-encoded protein plays a role in midgut maturation during larvae development.