Glycogen storage disease type III (GSDIII) is a hereditary glycogenosis caused by deficiency of the glycogen debranching enzyme (GDE), an enzyme, encoded by Agl, enabling glycogen degradation by catalyzing alpha-1,4-oligosaccharide side chain transfer and alpha-1,6-glucose cleavage. GDE deficiency causes accumulation of phosphorylase-limited dextrin, leading to liver disorder followed by fatal myopathy. Here, we tested the capacity of the new autophagosomal activator GHF-201 to alleviate disease burden by clearing pathogenic glycogen surcharge in the GSDIII mouse model Agl^−/−. We used open field, grip strength, and rotarod tests for evaluating GHF-201’s effects on locomotion, a biochemistry panel to quantify hematological biomarkers, indirect calorimetry to quantify in vivo metabolism, transmission electron microscopy to quantify glycogen in muscle, and fibroblast image analysis to determine cellular features affected by GHF-201. GHF-201 was able to improve all locomotion parameters and partially reversed hypoglycemia, hyperlipidemia and liver and muscle malfunction in Agl^−/− mice. Treated mice burnt carbohydrates more efficiently and showed significant improvement of aberrant ultrastructural muscle features. In GSDIII patient fibroblasts, GHF-201 restored mitochondrial membrane polarization and corrected lysosomal swelling. In conclusion, GHF-201 is a viable candidate for treating GSDIII as it recovered a wide range of its pathologies in vivo, in vitro, and ex vivo.

Summary: Glycogen storage disorder type 1a (GSD1a) is caused by loss-of-function mutations in the catalytic subunit of glucose-6-phosphatase enzyme ( G6PC1 ) in the liver, kidney and intestine exclusively. Here we show the surprising results that while not expressing G6PC1 , primary skin fibroblasts isolated from GSD1a patients’ skin biopsies preserve a distinctive disease phenotype irrespective of the different culture conditions under which they grow. This discovery was initially made by phenotypic image-based high content analysis (HCA). Deeper analysis into this disease phenotype, revealed impaired lysosomal and mitochondrial functions in GSD1a cells, which were driven by a transcriptional dysregulation of the NAD ⁺ /NADH-Sirt-1-TFEB regulatory axis. This dysregulation impacts the normal balance between mitochondrial biogenesis and mitophagy in the patients’ cells. The distinctive GSD1a fibroblasts phenotype involves elevated H3 histone acetylation, global DNA hypomethylation, differences in the chromatin accessibility and different RNA-seq and metabolomic profiles, all of which suggesting that in some way a distinctive disease cell phenotype is programmed in these cells in vivo and that this phenotype is maintained in vitro . Supporting this notion, reversing H3 acetylation in these cells erased the original cellular phenotype in GSD1a cells. Remarkably, GHF201, an established glycogen reducing molecule, which ameliorated GSD1a pathology in a liver-targeted inducible L.G6pc ^- knockout mouse model, also reversed impaired cellular functions in GSD1a patients’ fibroblasts. Altogether, this experimental evidence strongly suggests that GSD1a fibroblasts express a strong and reversible disease phenotype without expressing the causal G6PC1 gene.

The Target of Rapamycin (TOR) kinase is a core component of two evolutionarily conserved complexes, TORC1 and TORC2, which regulate growth, metabolism, and stress responses. In Schizosaccharomyces pombe, TORC2 is dispensable for proliferation under optimal conditions but is essential for survival and adaptation to a variety of stress conditions, including DNA damage and replication stress. The MluI-binding factor (MBF) transcription complex regulates G1/S progression and the DNA replication stress response. Previously, we demonstrated that TORC2-Gad8 is required for the upregulation of MBF-dependent gene transcription in response to replication stress. Here, we show that in response to replication stress TORC2 is necessary for the accumulation of the initiating form of RNA polymerase II (Pol II) at MBF promoters. In contrast, the elongating form of Pol II aberrantly accumulates at MBF coding regions in TORC2-deficient cells under both induced and non-induced conditions, suggesting a defect in balancing Pol II initiation and elongation that leads to impaired MBF gene induction. Unexpectedly, TORC2-deficient cells also exhibit aberrant upregulation of stress-activated genes during replication stress, including a distinct subset of Pap1-dependent oxidative stress genes. Consistent with this, TORC2 mutant cells accumulate reactive oxygen species in response to replication stress. Together, our findings suggest that TORC2 is required to ensure proper upregulation of MBF-dependent gene transcription during replication stress, and to suppress inappropriate activation of oxidative stress response pathways.

Glycogen storage disease type III (GSDIII) is a hereditary glycogenosis caused by deficiency of the glycogen debranching enzyme (GDE), an enzyme, encoded by Agl, enabling glycogen degradation by catalyzing alpha-1,4-oligosaccharide side chain transfer and alpha-1,6-glucose cleavage. GDE deficiency causes accumulation of phosphorylase-limited dextrin, leading to liver disorder followed by fatal myopathy. We tested here the capacity of the new autophagosomal activator GHF-201 to alleviate disease burden by clearing pathogenic glycogen surcharge in the GSDIII mouse model Agl-/-. We used open field, grip strength and rotarod tests for evaluating GHF-201’s effects on locomotion, biochemistry panel to quantify hematological biomarkers, indirect calorimetry to quantify in vivo metabolism, transmission electron microscopy to quantify glycogen in muscle, and fibroblast image analysis to determine cellular features affected by GHF-201. GHF-201 was able to improve all locomotion parameters and partially reversed hypoglycemia, hyperlipidemia and liver and muscle malfunction in Agl-/- mice. Treated mice burnt carbohydrates more efficiently and showed significant improvement of aberrant ultrastructural muscle features. In GSDIII patient fibroblasts, GHF-201 restored mitochondrial membrane polarization and corrected lysosomal swelling. In conclusion, GHF-201 is a viable candidate for treating GSDIII as it recovered a wide range of its pathologies in vivo, in vitro, and ex vivo.

Mucopolysaccharidosis type I (MPS-I) is a hereditary lysosomal storage disorder caused by deficiency in the lysosomal enzyme α-L-iduronidase (IDUA) leading to pathogenic over-accumulation of glycosaminoglycans (GAGs), major constituent of connective tissue and mediators of cell signaling. We tested the therapeutic efficacy of the autolysosomal activator GHF201 in the Idua ^−/− mouse model of MPS-I. We show that GHF201 reduced GAGs and increased cathepsin activity in Idua ^−/− mouse embryonic fibroblasts (MEFs) and brain cells. In vivo GHF201 improved aberrant locomotion and behavior, reduced interstitial cathepsins in joints and liver, increased systemic catabolism, corrected bone dysplasia, increased autophagic flux, and ameliorated CNS neurodegeneration and neuroinflammation. GHF201 also reduced brain N-glycans, which are degraded in lysosomes. In conclusion, the lysosomal and autophagic activator GHF201 was demonstrated to improve various pathophysiological parameters in MPS-I–modeling Idua ^−/− female mice. Our results suggest that GHF201 can complement the inadequate Aldurazyme enzyme replacement therapy as an add-on therapy.