A novel GSK3 inhibitor that promotes self-renewal in mouse embryonic stem cells.

Small molecules that regulate cell stemness have the potential to make a major contribution to regenerative medicine. In the course of screening for small molecules that affect stemness in mouse embryonic stem cells (mESCs), we discovered that NPD13432, an aurone derivative, promoted self-renewal of mESCs. Normally, mESCs start to differentiate upon withdrawal of 2i/LIF. However, cells treated with the compound continued to express endogenous Nanog, a pluripotency marker protein essential for sustaining the undifferentiated state, even in the absence of 2i/LIF. Biochemical characterization revealed that NPD13432 inhibited GSK3α and GSK3ß with IC50 values of 92 nM and 310 nM, respectively, suggesting that the compound promotes self-renewal in mESCs by inhibiting GSK3. The chemical structure of the compound is unique among known molecules with this activity, providing an opportunity to develop new inhibitors of GSK3, as well as chemical tools for investigating cell stemness.

Discovery and Development of Small-Molecule Inhibitors of Glycogen Synthase.

The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure-activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.

Norepinephrine control of ventromedial hypothalamic nucleus glucoregulatory neurotransmitter expression in the female rat: Role of monocarboxylate transporter function.

The ventromedial hypothalamic nucleus (VMN) is a critical component of the neural circuitry that regulates glucostasis. Astrocyte glycogen is a vital reserve of glucose and its oxidizable metabolite L-lactate. In hypoglycemic female rats, estradiol-dependent augmentation of VMN glycogen phosphorylase (GP) protein requires hindbrain catecholamine input. Research here investigated the premise that norepinephrine (NE) regulation of VMN astrocyte metabolism shapes local glucoregulatory neurotransmitter signaling in this sex. Estradiol-implanted ovariectomized rats were pretreated by intra-VMN administration of the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxy-cinnamic acid (4CIN) or vehicle before NE delivery to that site. NE caused 4CIN-reversible reduction or augmentation of VMN glycogen synthase and phosphorylase expression. 4CIN prevented NE stimulation of gluco-inhibitory (glutamate decarboxylase65/67) and suppression of gluco-stimulatory (neuronal nitric oxide synthase) neuron marker proteins. These outcomes imply that effects of noradrenergic stimulation of VMN astrocyte glycogen depletion on glucoregulatory transmitter signaling may be mediated, in part, by glycogen-derived substrate fuel provision. NE control of astrocyte glycogen metabolism may involve down-regulated adrenoreceptor (AR), e.g. alpha1 and alpha2, alongside amplified beta1 AR and estrogen receptor-beta signaling. Noradrenergic hypoglycemia was refractory to 4CIN, implying that additional NE-sensitive VMN glucoregulatory neurochemicals may be insensitive to monocarboxylate uptake. Augmentation of circulating free fatty acids by combinatory NE and 4CIN, but not NE alone implies that acute hypoglycemia induced here is an insufficient stimulus for mobilization of these fuels, but is adequate when paired with diminished brain monocarboxylate fuel availability.

Inhibitory properties of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives acting on glycogen metabolising enzymes.

Glycogen synthase (GS) and glycogen phosphorylase (GP) are the key enzymes that control, respectively, the synthesis and degradation of glycogen, a multi-branched glucose polymer that serves as a form of energy storage in bacteria, fungi and animals. An abnormal glycogen metabolism is associated with several human diseases. Thus, GS and GP constitute adequate pharmacological targets to modulate cellular glycogen levels by means of their selective inhibition. The compound 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) is a known potent inhibitor of GP. We studied the inhibitory effect of DAB, its enantiomer LAB, and 29 DAB derivatives on the activity of rat muscle glycogen phosphorylase (RMGP) and E. coli glycogen synthase (EcGS). The isoform 4 of sucrose synthase (SuSy4) from Solanum tuberosum L. was also included in the study for comparative purposes. Although these three enzymes possess highly conserved catalytic site architectures, the DAB derivatives analysed showed extremely diverse inhibitory potential. Subtle changes in the positions of crucial residues in their active sites are sufficient to discriminate among the structural differences of the tested inhibitors. For the two Leloir-type enzymes, EcGS and SuSy4, which use sugar nucleotides as donors, the inhibitory potency of the compounds analysed was synergistically enhanced by more than three orders of magnitude in the presence of ADP and UDP, respectively. Our results are consistent with a model in which these compounds bind to the subsite in the active centre of the enzymes that is normally occupied by the glucosyl residue which is transferred between donor and acceptor substrates. The ability to selectively inhibit the catalytic activity of the key enzymes of the glycogen metabolism may represent a new approach for the treatment of disorders of the glycogen metabolism.

Selective photoregulation of the activity of glycogen synthase and glycogen phosphorylase, two key enzymes in glycogen metabolism.

Glycogen is a polymer of α-1,4- and α-1,6-linked glucose units that provides a readily available source of energy in living organisms. Glycogen synthase (GS) and glycogen phosphorylase (GP) are the two enzymes that control, respectively, the synthesis and degradation of this polysaccharide and constitute adequate pharmacological targets to modulate cellular glycogen levels, by means of inhibition of their catalytic activity. Here we report on the synthesis and biological evaluation of a selective inhibitor that consists of an azobenzene moiety glycosidically linked to the anomeric carbon of a glucose molecule. In the ground state, the more stable (E)-isomer of the azobenzene glucoside had a slight inhibitory effect on rat muscle GP (RMGP, IC50 = 4.9 mM) and Escherichia coli GS (EcGS, IC50 = 1.6 mM). After irradiation and subsequent conversion to the (Z)-form, the inhibitory potency of the azobenzene glucoside did not significantly change for RMGP (IC50 = 2.4 mM), while its effect on EcGS increased 50-fold (IC50 = 32 µM). Sucrose synthase 4 from potatoes, a glycosyltransferase that does not operate on glycogen, was only slightly inhibited by the (E)-isomer (IC50 = 0.73 mM). These findings could be rationalized on the basis of kinetic and computer-aided docking analysis, which indicated that both isomers of the azobenzene glucoside mimic the EcGS acceptor substrate and exert their inhibitory effect by binding to the glycogen subsite in the active center of the enzyme. The ability to selectively photoregulate the catalytic activity of key enzymes of glycogen metabolism may represent a new approach for the treatment of glycogen metabolism disorders.

Pathological glycogenesis through glycogen synthase 1 and suppression of excessive AMP kinase activity in myeloid leukemia cells.

The rapid proliferation of myeloid leukemia cells is highly dependent on increased glucose metabolism. Through an unbiased metabolomics analysis of leukemia cells, we found that the glycogenic precursor UDP-D-glucose is pervasively upregulated, despite low glycogen levels. Targeting the rate-limiting glycogen synthase 1 (GYS1) not only decreased glycolytic flux but also increased activation of the glycogen-responsive AMP kinase (AMPK), leading to significant growth suppression. Further, genetic and pharmacological hyper-activation of AMPK was sufficient to induce the changes observed with GYS1 targeting. Cancer genomics data also indicate that elevated levels of the glycogenic enzymes GYS1/2 or GBE1 (glycogen branching enzyme 1) are associated with poor survival in AML. These results suggest a novel mechanism whereby leukemic cells sustain aberrant proliferation by suppressing excess AMPK activity through elevated glycogenic flux and provide a therapeutic entry point for targeting leukemia cell metabolism.

Insulin induces an increase in cytosolic glucose levels in 3T3-L1 cells with inhibited glycogen synthase activation.

Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

Polyglucosan neurotoxicity caused by glycogen branching enzyme deficiency can be reversed by inhibition of glycogen synthase.

Uncontrolled elongation of glycogen chains, not adequately balanced by their branching, leads to the formation of an insoluble, presumably neurotoxic, form of glycogen called polyglucosan. To test the suspected pathogenicity of polyglucosans in neurological glycogenoses, we have modeled the typical glycogenosis Adult Polyglucosan Body Disease (APBD) by suppressing glycogen branching enzyme 1 (GBE1, EC 2.4.1.18) expression using lentiviruses harboring short hairpin RNA (shRNA). GBE1 suppression in embryonic cortical neurons led to polyglucosan accumulation and associated apoptosis, which were reversible by rapamycin or starvation treatments. Further analysis revealed that rapamycin and starvation led to phosphorylation and inactivation of glycogen synthase (GS, EC 2.4.1.11), dephosphorylated and activated in the GBE1-suppressed neurons. These protective effects of rapamycin and starvation were reversed by overexpression of phosphorylation site mutant GS only if its glycogen binding site was intact. While rapamycin and starvation induce autophagy, autophagic maturation was not required for their corrective effects, which prevailed even if autophagic flux was inhibited by vinblastine. Furthermore, polyglucosans were not observed in any compartment along the autophagic pathway. Our data suggest that glycogen branching enzyme repression in glycogenoses can cause pathogenic polyglucosan buildup, which might be corrected by GS inhibition.

Design and synthesis of 2-N-substituted indazolone derivatives as non-carboxylic acid glycogen synthase activators.

Glycogen synthase (GS) catalyzes the transfer of glucose residues from UDP-glucose to a glycogen polymer chain, a critical step for glucose storage. Patients with type 2 diabetes normally exhibit low glycogen levels and decreased muscle glucose uptake is the major defect in whole body glucose disposal. Therefore, activating GS may provide a potential approach for the treatment of type 2 diabetes. In order to identify non-carboxylic acids GS activators, we designed and synthesized a series of 2-N-alkyl- and 2-N-aryl-indazolone derivatives and studied their activity in activating human GS.

MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism.

Corneal epithelium relies on abundant glycogen stores as its primary energy source. MicroRNA-31 (miR-31), a corneal epithelial-preferred miRNA, negatively regulates factor inhibiting hypoxia-inducible factor-1 (FIH-1). Since HIF-1α is involved in anaerobic energy production, we investigated the role that miR-31 and FIH-1 play in regulating corneal epithelial glycogen. We used antagomirs (antago) to reduce the level of miR-31 in primary human corneal epithelial keratinocytes (HCEKs), and a miR-31-resistant FIH-1 to increase FIH-1 levels. Antago-31 raised FIH-1 levels and significantly reduced glycogen stores in HCEKs compared to irrelevant-antago treatment. Similarly, HCEKs retrovirally transduced with a miR-31-resistant FIH-1 had markedly reduced glycogen levels compared with empty vector controls. In addition, we observed no change in a HIF-1α reporter or known genes downstream of HIF-1α indicating that the action of FIH-1 and miR-31 on glycogen is HIF-1α-independent. An enzyme-dead FIH-1 mutation failed to restore glycogen stores, indicating that FIH-1 negatively regulates glycogen in a hydroxylase-independent manner. FIH-1 overexpression in HCEKs decreased AKT signaling, activated GSK-3ß, and inactivated glycogen synthase. Treatment of FIH-1-transduced HCEKs with either a myristolated Akt or a GSK-3ß inhibitor restored glycogen stores, confirming the direct involvement of Akt/GSK-3ß signaling. Silencing FIH-1 in HCEKs reversed the observed changes in Akt-signaling. Glycogen regulation in a HIF-1α-independent manner is a novel function for FIH-1 and provides new insight into how the corneal epithelium regulates its energy requirements.

Proteomic analysis of ribosomes: translational control of mRNA populations by glycogen synthase GYS1.

Ribosomes exist as a heterogenous pool of macromolecular complexes composed of ribosomal RNA molecules, ribosomal proteins, and numerous associated "nonribosomal" proteins. To identify nonribosomal proteins that may modulate ribosome activity, we examined the composition of translationally active and inactive ribosomes using a proteomic multidimensional protein identification technology. Notably, the phosphorylated isoform of glycogen synthase, glycogen synthase 1 (GYS1), was preferentially associated with elongating ribosomes. Depletion of GYS1 affected the translation of a subset of cellular mRNAs, some of which encode proteins that modulate protein biosynthesis. These findings argue that GYS1 abundance, by virtue of its ribosomal association, provides a feedback loop between the energy state of the cells and the translation machinery.

Glycogen synthesis in brain and astrocytes is inhibited by chronic lithium treatment.

Lithium is a drug widely used to treat bipolar disorder. It has been shown to inhibit the total activity of phosphoglucomutase (PGM) from rat brains. In this work, we show that lithium inhibits in vitro PGM activity in the cortex, hippocampus, striatum, brainstem and cerebellum. As a compensatory effect, chronic lithium treatment of Wistar rats for 6 weeks caused a 1.6-fold upregulation of cortex PGM activity. No difference was observed in the other areas tested. Another effect of chronic lithium administration was a drastic reduction of glycogen content in rat brains, as PGM activity is essential for its synthesis. In a primary culture of astrocytes, which are the main cellular components of the brain that produce glycogen, administration of 1mM lithium for 3 days markedly reduced the steady state of glycogen content. In agreement with this result, lithium did not cause insulin-like effects as previously observed in hepatocytes where lithium activated glycogen synthesis. Reduction of glycogen content was due to inhibition of glycogen synthesis, as incorporation of [(14)U(-)C]-glucose into glycogen was impaired by lithium. Consistent with these results, incubation of glucose-starved astrocytes with lithium did not stimulate dephosphorylation of glycogen synthase, which normally occurs with re-feeding of glucose. Furthermore, in a chronically treated astrocyte culture, glycogen synthase was phosphorylated constitutively. Our results indicate that chronic lithium treatment can inhibit glycogen synthesis in brain suggesting that this effect might contribute to lithium's therapeutic effect.

Development of a quantitative 96-well method to image glycogen storage in primary rat hepatocytes.

Within the liver, hormonal control of glycogen metabolism allows for rapid release and uptake of glucose from the circulation, providing a reserve of glucose that can be utilised by other organs. Traditionally, cellular glycogen storage has been detected using Periodic acid Schiff (PAS) staining of histopathology samples or a biochemical assay. Colorimetric measurement of glycogen content using PAS staining is hard to quantify whilst biochemical techniques give limited information about events such as cytotoxicity or allow analysis of hepatic heterogeneity. Here, we describe the development of an imaging based method to quantify glycogen storage in 96-well cultures of primary rat hepatocytes using the inherent fluorescence properties of the Schiff reagent. PAS-stained hepatocytes were imaged using an automated fluorescent microscope, with the amount of glycogen present in each cell being quantified. Using this technique, we found an increase in glycogen storage in response to insulin (EC50 = 0.31 nM) that was in agreement with that determined using biochemical quantification (EC50 = 0.32 nM). Furthermore, a dose dependent increase in glycogen storage was also seen in response to glycogen synthase kinase inhibitors and glycogen phosphorylase inhibitors. This technique allows rapid assessment of cellular glycogen storage in response to hormones and small molecule inhibitors.

Dysregulation of glycogen synthase COOH- and NH2-terminal phosphorylation by insulin in obesity and type 2 diabetes mellitus.

CONTEXT: Insulin-stimulated glucose disposal is impaired in obesity and type 2 diabetes mellitus (T2DM) and is tightly linked to impaired skeletal muscle glucose uptake and storage. Impaired activation of glycogen synthase (GS) by insulin is a well-established defect in both obesity and T2DM, but the underlying mechanisms remain unclear. DESIGN AND PARTICIPANTS: Insulin action was investigated in a matched cohort of lean healthy, obese nondiabetic, and obese type 2 diabetic subjects by the euglycemic-hyperinsulinemic clamp technique combined with muscle biopsies. Activity, site-specific phosphorylation, and upstream signaling of GS were evaluated in skeletal muscle. RESULTS: GS activity correlated inversely with phosphorylation of GS site 2+2a and 3a. Insulin significantly decreased 2+2a phosphorylation in lean subjects only and induced a larger dephosphorylation at site 3 in lean compared with obese subjects. The exaggerated insulin resistance in T2DM compared with obese subjects was not reflected by differences in site 3 phosphorylation but was accompanied by a significantly higher site 1b phosphorylation during insulin stimulation. Hyperphosphorylation of another Ca(2+)/calmodulin-dependent kinase-II target, phospholamban-Thr17, was also evident in T2DM. Dephosphorylation of GS by phosphatase treatment fully restored GS activity in all groups. CONCLUSIONS: Dysregulation of GS phosphorylation plays a major role in impaired insulin regulation of GS in obesity and T2DM. In obesity, independent of T2DM, this is associated with impaired regulation of site 2+2a and likely site 3, whereas the exaggerated insulin resistance to activate GS in T2DM is linked to hyperphosphorylation of at least site 1b. Thus, T2DM per se seems unrelated to defects in the glycogen synthase kinase-3 regulation of GS.

Modulation of glycogen synthesis by RNA interference: towards a new therapeutic approach for glycogenosis type II.

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by defects in the acid alpha-glucosidase gene, which leads to lysosomal glycogen accumulation and enlargement of the lysosomes mainly in cardiac and muscle tissues, resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severely affected patients. Enzyme replacement therapy has already proven to be beneficial in this disease, but correction of pathology in skeletal muscle still remains a challenge. As substrate deprivation was successfully used to improve the phenotype in other lysosomal storage disorders, we explore here a novel therapeutic approach for GSDII based on a modulation of muscle glycogen synthesis. Short hairpin ribonucleic acids (shRNAs) targeted to the two major enzymes involved in glycogen synthesis, i.e. glycogenin (shGYG) and glycogen synthase (shGYS), were selected. C2C12 cells and primary myoblasts from GSDII mice were stably transduced with lentiviral vectors expressing both the shRNAs and the enhanced green fluorescent protein (EGFP) reporter gene. Efficient and specific inhibition of GYG and GYS was associated not only with a decrease in cytoplasmic and lysosomal glycogen accumulation in transduced cells, but also with a strong reduction in the lysosomal size, as demonstrated by confocal microscopy analysis. A single intramuscular injection of recombinant AAV-1 (adeno-associated virus-1) vectors expressing shGYS into newborn GSDII mice led to a significant reduction in glycogen accumulation, demonstrating the in vivo therapeutic efficiency. These data offer new perspectives for the treatment of GSDII and could be relevant to other muscle glycogenoses.

Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats.

Diabetic nephropathies are characterized by glycogen accumulation in distal tubular cells, which eventually leads to their apoptosis. The present study aims to determine whether adiponectin and AMPK are involved in the regulation of glycogen synthase (GS) in these structures. Western blots of isolated distal tubules revealed the presence of adiponectin receptor ADIPOR1, catalytic AMPK subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding AMPK subunit beta(2). ADIPOR2 was not detected. Expression levels of ADIPOR1, AMPKalpha(1), AMPKalpha(2), and AMPKbeta(2) were increased in streptozotocin-treated diabetic rats, whereas phosphorylated active AMPK levels were strongly decreased. Immunohistochemistry revealed the presence of ADIPOR1 on the luminal portion of distal tubules and thick ascending limb cells. Catalytic subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding subunit beta(2) were also found in the same cells, confirming immunoblot results. In vitro, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR; 2 mM) and globular adiponectin (10 mug/ml) activated catalytic AMPK in distal tubules isolated from kidneys of normal rats but much more weakly in those from diabetic rats. GS inhibition paralleled AMPK activation in both groups of animals: active GS levels were low in control animals and elevated in diabetic ones. Finally, glucose-6-phosphate, an allosteric activator of GS, was also increased in diabetic rats. These results demonstrate that in distal tubular cells, adiponectin through luminal ADIPOR1 activates AMPK, leading to the inhibition of GS. During hyperglycemia, this regulation is altered, which may explain, at least in part, the accumulation of large glycogen deposits.

Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes.

Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = -0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism.

Control of mammalian glycogen synthase by PAS kinase.

The regulation of glycogen metabolism is critical for the maintenance of glucose and energy homeostasis in mammals. Glycogen synthase, the enzyme responsible for glycogen production, is regulated by multisite phosphorylation in yeast and mammals. We have previously identified PAS kinase as a physiological regulator of glycogen synthase in Saccharomyces cerevisiae. We provide evidence here that PAS kinase is an important regulator of mammalian glycogen synthase. Glycogen synthase is efficiently phosphorylated by PAS kinase in vitro at Ser-640, a known regulatory phosphosite. Efficient phosphorylation requires a region of PAS kinase outside the catalytic domain. This region appears to mediate a direct interaction between glycogen synthase and PAS kinase, thereby targeting kinase activity to this substrate specifically. This interaction is regulated by the PAS kinase PAS domain, raising the possibility that this interaction (and phosphorylation event) is modulated by the cellular metabolic state. This mode of regulation provides a mechanism for metabolic status to impinge directly on the cellular decision of whether to store or use available energy.

Novel glycogen synthase kinase 3 and ubiquitination pathways in progressive myoclonus epilepsy.

Lafora progressive myoclonus epilepsy, caused by defective laforin or malin, insidiously present in normal teenagers with cognitive decline, followed by rapidly intractable epilepsy, dementia and death. Pathology reveals neurodegeneration with neurofibrillary tangle formation and Lafora bodies (LBs). LBs are deposits of starch-like polyglucosans, insufficiently branched and hence insoluble glycogen molecules resulting from glycogen synthase (GS) overactivity relative to glycogen branching enzyme activity. We previously made the unexpected observation that laforin, in the absence of which polyglucosans accumulate, specifically binds polyglucosans. This suggested that laforin's role is to detect polyglucosan appearances during glycogen synthesis and to initiate mechanisms to downregulate GS. Glycogen synthase kinase 3 (GSK3) is the principal inhibitor of GS. Dephosphorylation of GSK3 at Ser 9 activates GSK3 to inhibit GS through phosphorylation at multiple sites. Glucose-6-phosphate is a potent allosteric activator of GS. Glucose-6-phosphate levels are high when the amount of glucose increases and its activation of GS overrides any phospho-inhibition. Here, we show that laforin is a GSK3 Ser 9 phosphatase, and therefore capable of inactivating GS through GSK3. We also show that laforin interacts with malin and that malin is an E3 ubiquitin ligase that binds GS. We propose that laforin, in response to appearance of polyglucosans, directs two negative feedback pathways: polyglucosan-laforin-GSK3-GS to inhibit GS activity and polyglucosan-laforin-malin-GS to remove GS through proteasomal degradation.