Advances in Gaucher Disease 17 mulation, even patients with Parkinson disease without GBA1 mutations may benefit from this therapy, especially as pharmacokinetic studies indicate reasonable levels in the brain. In collaboration with Drs Juan Marugan, Christopher Aus-tin and Wei Zheng at the new National Center for Advancing Translational Sciences at the NIH, we initially used quantitative high-throughput screening (qHTS) to rapidly evaluate ~60,000 compounds at multiple concentrations simultaneously, and this enabled us to successfully identify inhibitors that could serve as potential pharmacologic chaperones to treat Gaucher disease. However, we then redirected our studies toward the identification of non-inhibitory compounds. To accomplish this, we introduced a new strategy using “We demonstrated that lysosomal dysfunction leads to impaired autophagy in Gaucher macrophages, which prevents the delivery of inflammasomes and leads to increased secretion of the inflammatory cytokine IL-1. We showed that elevated p62 levels play a pivotal role in the polarization of Gaucher macrophages and that these macrophages manifest inflammatory phenotypes because of impaired patient spleen as the source of mutant enzyme to screen over 250,000 compounds. We identified and confirmed potential compounds active at nanomolar concentrations. We then focused our atten- tion on optimizing two different series of non-inhibitory small molecule chaperones. However, as the screens were completed, we faced an enormous challenge. The two promising lead non-inhibitory chaperone classes identified through our screens sucessfully improved translocation of the enzyme to the lysosome and enhanced enzymatic activity. However, we did not have an appropriate model for validation that the compounds actually worked to reverse the lysosomal storage, as fibroblasts do not store the substrates glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). We also lacked a neuronal model to test the effect of the compounds on neuronopathic Gaucher disease or GBA1-associated parkinsonism. Therefore, much of our efforts in the past few years focused on the development and use of new models of Gaucher disease. As mentioned above, we cultured peripheral monocytes from twenty patients with type 1 GD which were then differentiated into macrophages. Storage of GlcCer and GlcSph was demonstrated. When the cells were fed lipid-laden red cell ghosts from patients with Gaucher disease (tagged with fluorescent GlcSph), Gaucher macrophages stored dramatic quantities of lipid. Imaging with the lysosomal marker Lamp-2 confirmed that this storage was lysosomal. In contrast, little labeled GlcSph was seen in control macrophages. We then showed that these small molecules increased the glucocerebrosidase activity in Gaucher macrophages by several-fold, and reduced GlcCer storage. We found that the compounds increased the phagocytic index significantly and improved intracellular levels of ROS. Evaluations of autophagy in Gaucher macrophages revealed that the failure of fusion of auto-phagosomes with lysosomes to autophagosomes and ac-tivation of the inflammasome could be reversed with the lead chaperone. We next used the Gaucher iPSCs to generate dopaminergic neurons and found that these cells also exhibited deficient enzymatic activity, reduced levels of glucocerebrosidase and stored GlcCer and GlcSph. The lines generated from patients with Gaucher disease and Parkinsonism or type 2 Gaucher disease also demonstrated elevated levels of ��-synuclein. We treated these dopaminergic neurons with NCGC607, our novel non-inhibitory glucocerebrosidase chaperone, and successfully restored enzyme activity and protein levels and reduced glycolipid storage in the Gaucher dopaminergic neurons. This work demonstrated the potential of these chaperones as a treatment for Gaucher disease, including its neuronopathic forms. In addition, the treatment reduced ��-synuclein levels in dopaminergic neurons from patients with Gaucher disease and parkinsonism, suggesting that chaperoning glucocerebrosidase might be useful in Parkinson disease. AGD autophagy.”
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