• Identifying pathogenic mechanisms and new therapeutic targets for Gaucher disease using induced pluripotent stem cells

      Srikanth, Manasa; Feldman, Ricardo A. (2020)
      Gaucher Disease (GD), the most common lysosomal storage disorder, is caused by mutations in the GBA1 gene, which codes for the lysosomal enzyme β-glucocerebrosidase (GCase). GCase breaks down sphingolipids but when it is mutated, it causes the accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). The common manifestations of GD include hepatosplenomegaly, anemia, thrombocytopenia, skeletal disease, and in case of severe mutations, there are also fatal neurological manifestations. The conventional treatment is not effective in managing the skeletal or neurological manifestations. Hence, a better understanding of the underlying mechanisms that cause GD pathology is required for development of effective therapeutic strategies. The goal of this thesis was to identify the molecular mechanisms responsible for phenotypic alterations in osteoblasts and neuronal cells from GD patients, thereby pinpoint molecular targets for therapeutic intervention. Our laboratory utilizes patient-specific induced pluripotent stem cells (iPSCs) harboring GBA1 mutations to model GD. We have previously differentiated these iPSCs to various cell types and have shown that we can recapitulate the pathologic hallmarks of GD. Thus, in this study, we generated GD-iPSC derived osteoblasts and neuronal cells and found that mutations in GBA1 disrupt the canonical Wnt signaling and lysosomal compartment in these cell types. The phenotypic consequence of this was observed in the form of defective osteoblast differentiation and maturation as well as loss of midbrain/hindbrain neuronal progenitors in the respective cell types. Due to the known lysosomal dysregulation in GD, we then explored the mTOR pathway which is upstream of lysosomal biogenesis. We found hyperactivation of mTOR in GD neuronal cells was mediated by the significant accumulation of GlcSph, a lysolipid of GlcCer. In addition, when we blocked the conversion of GlcCer to GlcSph using acid ceramidase inhibitors, we were able to reverse mTOR hyperactivation and restore lysosomal expression, suggesting that GlcSph is partly, if not fully, responsible for the lysosomal abnormalities observed in GD. In conclusion, our study reveals that activation of canonical Wnt pathway or suppression of mTOR pathway ameliorates the phenotypic abnormalities in GD and identifies b-catenin, mTOR and acid ceramidase as potential therapeutic targets for GD.
    • Using Disease-Specific Induced Pluripotent Stem Cells to Understand the Etiology of the Hematopoietic Defects in Gaucher Disease

      Sgambato, Judi Azevedo; Feldman, Ricardo A. (2014)
      Gaucher disease (GD) is the most common lysosomal storage disease, resulting from mutations in the gene encoding the lysosomal enzyme, glucocerebrosidase (GCase). GCase deficiency results in the lysosomal accumulation of sphingolipids, primarily in macrophages and neurons leading to multi-organ pathology including hepatosplenomegaly, bone disease, neurological symptoms, and cytopenias. It is currently unclear whether the hematopoietic abnormalities of GD are due to an inherent defect in the hematopoietic stem cell population or are a consequence of the other pathological manifestations. To investigate the potential effects of GCase deficiency on hematopoietic progenitor cells (HPCs), we generated GD-HPCs from induced pluripotent stem cells (GD-iPSCs) representing all 3 clinical subtypes of GD. We found no differences in the efficiency of GD-HPC generation or the kinetics of hematopoietic marker expression compared to controls, suggesting that the consequences of enzyme deficiency lie downstream of the generation of the HPC pool. We assessed the multipotency of GD-HPCs through colony formation assays, liquid differentiation protocols, and flow cytometry. Examination of the lineage potential of GD-HPCs by these methods revealed skewed commitment to the myelo-erythroid lineage with increased myeloid differentiation and decreased erythroid differentiation. This decreased erythroid differentiation could account for the anemia present in patients. The increased myeloid differentiation may represent a mechanism to compensate for the accumulation of dysfunctional lipid-laden macrophages. Supplementation of the mutant HPCs with recombinant enzyme restored GD-HPC myelo-erythroid multipotency, demonstrating the direct role of GCase enzyme activity in HPC lineage specification. Enzyme deficiency also impaired megakaryocytepoiesis, reflecting the thrombocytopenia found in patients. Interestingly, GD-HPCs also gave rise to conspicuous lipid-laden macrophage cells. These cells are reminiscent of hallmark Gaucher cells found infiltrating patient tissues, for the first time suggesting the direct derivation of Gaucher cells from HPCs. Together, these findings offer unique insights into the importance of GCase enzymatic activity in HPC multipotency and suggest that enzyme deficiency within this population could account for the cytopenias present in GD patients. Additionally, they suggest a unique origin for the lipid-laden Gaucher cells central to GD pathology.