Gaucher iPSC-derived macrophages produce elevated levels of inflammatory mediators and serve as a new platform for therapeutic development.
ABSTRACT: Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid ?-glucocerebrosidase (GCase; GBA) gene. The hallmark of GD is the presence of lipid-laden Gaucher macrophages, which infiltrate bone marrow and other organs. These pathological macrophages are believed to be the sources of elevated levels of inflammatory mediators present in the serum of GD patients. The alteration in the immune environment caused by GD is believed to play a role in the increased risk of developing multiple myeloma and other malignancies in GD patients. To determine directly whether Gaucher macrophages are abnormally activated and whether their functional defects can be reversed by pharmacological intervention, we generated GD macrophages by directed differentiation of human induced pluripotent stem cells (hiPSC) derived from patients with types 1, 2, and 3 GD. GD hiPSC-derived macrophages expressed higher levels of tumor necrosis factor ?, IL-6, and IL-1? than control cells, and this phenotype was exacerbated by treatment with lipopolysaccharide. In addition, GD hiPSC macrophages exhibited a striking delay in clearance of phagocytosed red blood cells, recapitulating the presence of red blood cell remnants in Gaucher macrophages from bone marrow aspirates. Incubation of GD hiPSC macrophages with recombinant GCase, or with the chaperones isofagomine and ambroxol, corrected the abnormal phenotypes of GD macrophages to an extent that reflected their known clinical efficacies. We conclude that Gaucher macrophages are the likely source of the elevated levels of inflammatory mediators in the serum of GD patients and that GD hiPSC are valuable new tools for studying disease mechanisms and drug discovery.
Project description:Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid ?-glucocerebrosidase gene. To model GD, we generated human induced pluripotent stem cells (hiPSC), by reprogramming skin fibroblasts from patients with type 1 (N370S/N370S), type 2 (L444P/RecNciI), and type 3 (L444P/L444P) GD. Pluripotency was demonstrated by the ability of GD hiPSC to differentiate to all three germ layers and to form teratomas in vivo. GD hiPSC differentiated efficiently to the cell types most affected in GD, i.e., macrophages and neuronal cells. GD hiPSC-macrophages expressed macrophage-specific markers, were phagocytic, and were capable of releasing inflammatory mediators in response to LPS. Moreover, GD hiPSC-macrophages recapitulated the phenotypic hallmarks of the disease. They exhibited low glucocerebrosidase (GC) enzymatic activity and accumulated sphingolipids, and their lysosomal functions were severely compromised. GD hiPSC-macrophages had a defect in their ability to clear phagocytosed RBC, a phenotype of tissue-infiltrating GD macrophages. The kinetics of RBC clearance by types 1, 2, and 3 GD hiPSC-macrophages correlated with the severity of the mutations. Incubation with recombinant GC completely reversed the delay in RBC clearance from all three types of GD hiPSC-macrophages, indicating that their functional defects were indeed caused by GC deficiency. However, treatment of induced macrophages with the chaperone isofagomine restored phagocytosed RBC clearance only partially, regardless of genotype. These findings are consistent with the known clinical efficacies of recombinant GC and isofagomine. We conclude that cell types derived from GD hiPSC can effectively recapitulate pathologic hallmarks of the disease.
Project description:The knowledge of individual response to a therapy, which can be assesed by in vitro screening, is essential for the development of therapeutics. Chaperone therapy is based on the ability of small molecules to fold the mutant protein to recover its function. As a novel approach for the treatment of Gaucher disease (GD), ambroxol was recently identified as a chaperone for GD, caused by the pathogenic variants in GBA gene, resulting in lysosomal enzyme glucocerebrosidase (GCase) deficiency. Since ambroxol activity is mutation-dependent, the assessment of the chaperone action requires adaptation of a cell model with genetic format identical to the patient. We compared the chaperone activity of ambroxol using different primary cells derived from GD patients with different GBA genotypes. Ambroxol enhanced GCase activity in cells with wild type GBA and in those, compound heterozygous for N370S, but was ineffective in cell lines with complex GBA alleles. In cells from patients with neuropathic GD and L444P/L444P genotype, the response to ambroxol was varied. We conclude that chaperone activity depends on diverse factors in addition to a particular GBA genotype. We showed that PBMCs and macrophages are the most relevant cell-based methods to screen the efficacy of ambroxol therapy. For pediatric patients, a non-invasive source of primary cells, urine derived kidney epithelial cells, have a vast potential for drug screening in GD. These findings demonstrate the importance of personalized screening to evaluate efficacy of chaperone therapy, especially in patients with neuronopathic GD.
Project description:Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase ?-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.
Project description:Point mutations in beta-glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lysosomes thereby causing Gaucher Disease (GD). Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs), increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), and allow increased lysosomal transport of the enzyme. A high-throughput screen of the 50,000-compound Maybridge library identified two, non-carbohydrate-based inhibitory molecules, a 2,4-diamino-5-substituted quinazoline (IC(50) 5 microM) and a 5-substituted pyridinyl-2-furamide (IC(50) 8 microM). They raised the levels of functional GCase 1.5-2.5-fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium-exchange mass spectrometry, identified a domain III active-site loop (residues 243-249) as being significantly stabilized upon binding of isofagomine or either of these two new compounds; this suggests a common mechanism for PC enhancement of intracellular transport.
Project description:Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid ?-glucosidase encoding gene (<i>GBA1</i>), resulting in the deficient activity of acid ?-glucosidase (GCase). To date, there is no approved treatment for the neurological manifestations of the disease. The role of Ambroxol as a chaperone for mutant GCase has been extensively demonstrated <i>in vitro</i>. Furthermore, different authors have reported beneficial effects of high doses of Ambroxol on neurological manifestations in patients affected by GD. In this report, we describe the <i>in vitro</i> and <i>in vivo</i> effects of Ambroxol in two patients (P1 and P2) affected by the neurological form of GD and epilepsy, carrying mutations already reported as responsive to the chaperone. Indeed, P1 presented the N188S mutation in compound heterozygous with a null allele (IVS2 + 1G > A) and P2 was homozygous for the L444P mutation. As expected, a beneficial effect of Ambroxol was observed in cultured fibroblasts as well as <i>in vivo</i>, both on epilepsy and on biomarkers of GD, in P1. However, Ambroxol was completely undefective in P2, suggesting that other factors besides the <i>GBA1</i> mutation itself would be involved in the response therapy which would be difficult to predict based on the patient genotype. The present report expands the experience of Ambroxol treatment in neurological GD patients and highlights the need to <i>in vitro</i> test the individual response to Ambroxol even in patients carrying mutations already classified as responsive to the chaperone.
Project description:Structurally destabilizing mutations in acid beta-glucosidase (GCase) can result in Gaucher disease (GD). The iminosugar isofagomine (IFG), a competitive inhibitor and a potential pharmacological chaperone of GCase, is currently undergoing clinical evaluation for the treatment of GD. An X-ray crystallographic study of the GCase-IFG complex revealed a hydrogen bonding network between IFG and certain active site residues. It was suggested that this network may translate into greater global stability. Here it is demonstrated that IFG does increase the global stability of wild-type GCase, shifting its melting curve by approximately 15 degrees C and that it enhances mutant GCase activity in pre-treated N370S/N370S and F213I/L444P patient fibroblasts. Additionally, amide hydrogen/deuterium exchange mass spectroscopy (H/D-Ex) was employed to identify regions within GCase that undergo stabilization upon IFG-binding. H/D-Ex data indicate that the binding of IFG not only restricts the local protein dynamics of the active site, but also propagates this effect into surrounding regions.
Project description:Gaucher disease (GD) is an inherited lysosomal disorder, originating from deficient activity of the lysosomal enzyme glucocerebrosidase (GCase). Normally, GCase hydrolyzes glucocerebroside (GC) to glucose and ceramide; however, impaired activity of this enzyme leads to the accumulation of GC in macrophages, termed "Gaucher cells." Gaucher disease is associated with hepatosplenomegaly, cytopenias, skeletal complications and in some forms involves the central nervous system. Coagulation abnormalities are common among GD patients due to impaired production and chronic consumption of coagulation factors. Bleeding phenomena are variable (as are other symptoms of GD) and include mucosal and surgical hemorrhages. FOUR MAIN ETIOLOGICAL FACTORS ACCOUNT FOR THE HEMOSTATIC DEFECT IN GD: thrombocytopenia, abnormal platelet function, reduced production of coagulation factors, and activation of fibrinolysis. Thrombocytopenia relates not only to hypersplenism and decreased megakaryopoiesis by the infiltrated bone marrow but also to immune thrombocytopenia. Autoimmunity, especially the induction of platelet antibody production, might cause persistent thrombocytopenia. Enzyme replacement therapy reverses only part of the impaired coagulation system in Gaucher disease. Other therapeutic and supportive measures should be considered to prevent and/or treat bleeding in GD. Gaucher patients should be evaluated routinely for coagulation abnormalities especially prior to surgery and dental and obstetric procedures.
Project description:Gaucher disease (GD) is the most common lysosomal storage disease resulting from mutations in the lysosomal enzyme glucocerebrosidase (GCase). The hematopoietic abnormalities in GD include the presence of characteristic Gaucher macrophages that infiltrate patient tissues and cytopenias. At present, it is not clear whether these cytopenias are secondary to the pathological activity of Gaucher cells or a direct effect of GCase deficiency on hematopoietic development. To address this question, we differentiated induced pluripotent stem cells (iPSCs) derived from patients with types 1, 2, and 3 GD to CD34(+)/CD45(+)/CD43(+)/CD143(+) hematopoietic progenitor cells (HPCs) and examined their developmental potential. The formation of GD-HPCs was unaffected. However, these progenitors demonstrated a skewed lineage commitment, with increased myeloid differentiation and decreased erythroid differentiation and maturation. Interestingly, myeloid colony-formation assays revealed that GD-HPCs, but not control-HPCs, gave rise to adherent, macrophage-like cells, another indication of abnormal myelopoiesis. The extent of these hematologic abnormalities correlated with the severity of the GCase mutations. All the phenotypic abnormalities of GD-HPCs observed were reversed by incubation with recombinant GCase, indicating that these developmental defects were caused by the mutated GCase. Our results show that GCase deficiency directly impairs hematopoietic development. Additionally, our results suggest that aberrant myelopoiesis might contribute to the pathological properties of Gaucher macrophages, which are central to GD manifestations. The hematopoietic developmental defects we observed reflect hematologic abnormalities in patients with GD, demonstrating the utility of GD-iPSCs for modeling this disease.
Project description: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.
Project description:Gaucher disease (GD), the most common lysosomal storage disorder of humans, is caused by mutations in the gene coding for the enzyme glucocerebrosidase (GCase). Clinical manifestations vary among patients with the three types of GD, and phenotypic heterogeneity occurs even among patients with identical mutations. To gain insight into why phenotypic heterogeneity occurs in GD, we investigated mechanisms underlying the net loss of GCase catalytic activity in cultured skin fibroblasts derived from patients with the three types of GD. The findings indicate that the loss of catalytic activity of GCase correlates with its quantitative reduction, rather than a decrease in functional capacity of mutant enzyme. Use of a proteasome inhibitor, lactacystin, resulted in increased expression of GCase, suggesting a mechanism of protein degradation in GD. Furthermore, reduced binding of GCase to TCP1 ring complex (TRiC), a regulator of correct protein folding, may result in defective maturation of nascent GCase in GD cells. Additionally, increased interaction between GCase and c-Cbl, an E3 ubiquitin ligase, may be involved in the degradation and loss of GCase in GD. The findings suggest that specific molecular mediators involved in GCase maturation and degradation could be responsible for phenotypic variation among patients with the same genotypes and that these mediators could be therapeutically targeted to increase GCase activity in patients with GD.