Altered TFEB-mediated lysosomal biogenesis in Gaucher disease iPSC-derived neuronal cells.

Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase). The severe forms of GD are associated with neurodegeneration with either rapid (Type 2) or slow progression (Type 3). Although the neurodegenerative process in GD has been linked to lysosomal dysfunction, the mechanisms involved are largely unknown. To identify the lysosomal alterations in GD neurons and uncover the mechanisms involved, we used induced pluripotent stem cells (iPSCs) derived from patients with GD. In GD iPSC-derived neuronal cells (iPSC-NCs), GBA1 mutations caused widespread lysosomal depletion, and a block in autophagic flux due to defective lysosomal clearance of autophagosomes. Autophagy induction by rapamycin treatment in GD iPSC-NCs led to cell death. Further analysis showed that in GD iPSC-NCs, expression of the transcription factor EB (TFEB), the master regulator of lysosomal genes, and lysosomal gene expression, were significantly downregulated. There was also reduced stability of the TFEB protein and altered lysosomal protein biosynthesis. Treatment of mutant iPSC-NCs with recombinant GCase (rGCase) reverted the lysosomal depletion and autophagy block. The effect of rGCase on restoring lysosomal numbers in mutant cells was enhanced in the presence of overexpressed TFEB, but TFEB overexpression alone did not reverse the lysosomal depletion phenotype. Our results suggest that GBA1 mutations interfere with TFEB-mediated lysosomal biogenesis, and that the action of GCase in maintaining a functioning pool of lysosomes is exerted in part through TFEB. The lysosomal alterations described here are likely to be a major determinant in GBA1-associated neurodegeneration.

Gaucher iPSC-derived macrophages produce elevated levels of inflammatory mediators and serve as a new platform for therapeutic development.

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid ß-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 α, IL-6, and IL-1ß 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.

Gaucher Disease-Induced Pluripotent Stem Cells Display Decreased Erythroid Potential and Aberrant Myelopoiesis.

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.