This work employs adult polyglucosan body disease (APBD) models to explore the efficacy and mechanism of action of the polyglucosan‐reducing compound 144DG11. APBD is a glycogen storage disorder (GSD) caused by glycogen branching enzyme (GBE) deficiency causing accumulation of poorly branched glycogen inclusions called polyglucosans. 144DG11 improved survival and motor parameters in a GBE knockin (Gbeys/ys) APBD mouse model. 144DG11 reduced polyglucosan and glycogen in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic polyglucosan. At the cellular level, 144DG11 increased glycolytic, mitochondrial, and total ATP production. The molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. 144DG11 also enhanced mitochondrial activity and modulated lysosomal features as revealed by bioenergetic, image‐based phenotyping and proteomics analyses. As an effective lysosomal targeting therapy in a GSD model, 144DG11 could be developed into a safe and efficacious glycogen and lysosomal storage disease therapy.

Primary fibroblasts from patient’s skin biopsies are directly isolated without any alteration in the genome, retaining in culture conditions their endogenous cellular characteristics and biochemical properties. The aim of this study was to identify a distinctive cell phenotype for potential drug evaluation in fibroblasts from Huntington’s Disease (HD) patients, using image-based high content analysis. We show that HD fibroblasts have a distinctive nuclear morphology associated with a nuclear actin cap deficiency. This in turn affects cell motility in a similar manner to fibroblasts from Hutchinson-Gilford progeria syndrome (HGPS) patients used as known actin cap deficient cells. Moreover, treatment of the HD cells with either Latrunculin B, used to disrupt actin cap formation, or the antioxidant agent Mitoquinone, used to improve mitochondrial activity, show expected opposite effects on actin cap associated morphological features and cell motility. Deep data analysis allows strong cluster classification within HD cells according to patients’ disease severity score which is distinct from HGPS and matching controls supporting that actin cap is a biomarker in HD patients’ cells correlated with HD severity status that could be modulated by pharmacological agents as tool for personalized drug evaluation.

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-Sirt1-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 K27 histone acetylation and global DNA hypomethylation suggesting that in some way the disease imprinted a distinctive cell phenotype in these 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 these cells express a strong and reversible disease phenotype without expressing the causal G6PC1 gene.

This work employs Adult Polyglucosan Body Disease (APBD) models to explore the efficacy and mechanism of action of 144DG11, a new polyglucosan-reducing lead compound discovered by a high-throughput screen (HTS). APBD is an adult onset glycogen storage disorder (GSD) manifesting as a debilitating progressive axonopathic leukodystrophy. APBD is caused by glycogen branching enzyme (GBE) deficiency leading to poorly branched and insoluble glycogen inclusions, which precipitate as neuropathogenic polyglucosans (PG). 144DG11 led to prolonged survival and improved motor parameters in a GBE knockin (Gbeys/ys) APBD mouse model. Histopathologically, 144DG11 reduced PG and glycogen levels in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic PG. These results were also reflected at the cellular level by increased glycolytic, mitochondrial and total ATP production. Mechanistically, we show that the molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. Enhanced mitochondrial activity and lysosomal modifications were also the most pronounced effects of 144DG11 in APBD patient fibroblasts as discovered by image-based multiparametric phenotyping analysis and corroborated by proteomics. In summary, this work presents a broad mechanistic and target-based characterization of 144DG11 in in vivo and cell models of the prototypical GSD APBD. This investigation warrants development of 144DG11 into a safe and efficacious GSD therapy.