Impaired malin expression and interaction with partner proteins in Lafora disease.

Lafora disease (LD) is an autosomal recessive myoclonus epilepsy with onset in the teenage years leading to death within a decade of onset. LD is characterized by the overaccumulation of hyperphosphorylated, poorly branched, insoluble, glycogen-like polymers called Lafora bodies. The disease is caused by mutations in either EPM2A, encoding laforin, a dual specificity phosphatase that dephosphorylates glycogen, or EMP2B, encoding malin, an E3-ubiquitin ligase. While glycogen is a widely accepted laforin substrate, substrates for malin have been difficult to identify partly due to the lack of malin antibodies able to detect malin in vivo. Here we describe a mouse model in which the malin gene is modified at the C-terminus to contain the c-myc tag sequence, making an expression of malin-myc readily detectable. Mass spectrometry analyses of immunoprecipitates using c-myc tag antibodies demonstrate that malin interacts with laforin and several glycogen-metabolizing enzymes. To investigate the role of laforin in these interactions we analyzed two additional mouse models: malin-myc/laforin knockout and malin-myc/LaforinCS, where laforin was either absent or the catalytic Cys was genomically mutated to Ser, respectively. The interaction of malin with partner proteins requires laforin but is not dependent on its catalytic activity or the presence of glycogen. Overall, the results demonstrate that laforin and malin form a complex in vivo, which stabilizes malin and enhances interaction with partner proteins to facilitate normal glycogen metabolism. They also provide insights into the development of LD and the rescue of the disease by the catalytically inactive phosphatase.

Lack of liver glycogen causes hepatic insulin resistance and steatosis in mice.

Disruption of the Gys2 gene encoding the liver isoform of glycogen synthase generates a mouse strain (LGSKO) that almost completely lacks hepatic glycogen, has impaired glucose disposal, and is pre-disposed to entering the fasted state. This study investigated how the lack of liver glycogen increases fat accumulation and the development of liver insulin resistance. Insulin signaling in LGSKO mice was reduced in liver, but not muscle, suggesting an organ-specific defect. Phosphorylation of components of the hepatic insulin-signaling pathway, namely IRS1, Akt, and GSK3, was decreased in LGSKO mice. Moreover, insulin stimulation of their phosphorylation was significantly suppressed, both temporally and in an insulin dose response. Phosphorylation of the insulin-regulated transcription factor FoxO1 was somewhat reduced and insulin treatment did not elicit normal translocation of FoxO1 out of the nucleus. Fat overaccumulated in LGSKO livers, showing an aberrant distribution in the acinus, an increase not explained by a reduction in hepatic triglyceride export. Rather, when administered orally to fasted mice, glucose was directed toward hepatic lipogenesis as judged by the activity, protein levels, and expression of several fatty acid synthesis genes, namely, acetyl-CoA carboxylase, fatty acid synthase, SREBP1c, chREBP, glucokinase, and pyruvate kinase. Furthermore, using cultured primary hepatocytes, we found that lipogenesis was increased by 40% in LGSKO cells compared with controls. Of note, the hepatic insulin resistance was not associated with increased levels of pro-inflammatory markers. Our results suggest that loss of liver glycogen synthesis diverts glucose toward fat synthesis, correlating with impaired hepatic insulin signaling and glucose disposal.

Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation.

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.

Novel method for detection of glycogen in cells.

y: Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing g lutathione S-transferase, a cM c eptitope and the tbd1 BM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions.

Muscle glycogen remodeling and glycogen phosphate metabolism following exhaustive exercise of wild type and laforin knockout mice.

Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease.

Glycogen phosphomonoester distribution in mouse models of the progressive myoclonic epilepsy, Lafora disease.

Glycogen is a branched polymer of glucose that acts as an energy reserve in many cell types. Glycogen contains trace amounts of covalent phosphate, in the range of 1 phosphate per 500-2000 glucose residues depending on the source. The function, if any, is unknown, but in at least one genetic disease, the progressive myoclonic epilepsy Lafora disease, excessive phosphorylation of glycogen has been implicated in the pathology by disturbing glycogen structure. Some 90% of Lafora cases are attributed to mutations of the EPM2A or EPM2B genes, and mice with either gene disrupted accumulate hyperphosphorylated glycogen. It is, therefore, of importance to understand the chemistry of glycogen phosphorylation. Rabbit skeletal muscle glycogen contained covalent phosphate as monoesters of C2, C3, and C6 carbons of glucose residues based on analyses of phospho-oligosaccharides by NMR. Furthermore, using a sensitive assay for glucose 6-P in hydrolysates of glycogen coupled with measurement of total phosphate, we determined the proportion of C6 phosphorylation in rabbit muscle glycogen to be ∼20%. C6 phosphorylation also accounted for ∼20% of the covalent phosphate in wild type mouse muscle glycogen. Glycogen phosphorylation in Epm2a(-/-) and Epm2b(-/-) mice was increased 8- and 4-fold compared with wild type mice, but the proportion of C6 phosphorylation remained unchanged at ∼20%. Therefore, our results suggest that C2, C3, and/or C6 phosphate could all contribute to abnormal glycogen structure or to Lafora disease.

Incorporation of phosphate into glycogen by glycogen synthase.

The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of (32)P from [ß-(32)P]UDP-glucose to glycogen by glycogen synthase. The (32)P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The (32)P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [ß-(32)P]UDP generated in the reaction. Furthermore, [(32)P]UDP did not bind non-covalently to glycogen. The (32)P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation.

Protein degradation and quality control in cells from laforin and malin knockout mice.

Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforinmalin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/ quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a(-/-), Epm2b(-/-), and Epm2a(-/-) Epm2b(-/-) mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b(-/-) and Epm2a(-/-) Epm2b(-/-) cells) but not laforin (Epm2a(-/-) cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal.

Laforin and malin knockout mice have normal glucose disposal and insulin sensitivity.

Lafora disease is a fatal, progressive myoclonus epilepsy caused in ~90% of cases by mutations in the EPM2A or EPM2B genes. Characteristic of the disease is the formation of Lafora bodies, insoluble deposits containing abnormal glycogen-like material in many tissues, including neurons, muscle, heart and liver. Because glycogen is important for glucose homeostasis, the aberrant glycogen metabolism in Lafora disease might disturb whole-body glucose handling. Indeed, Vernia et al. [Vernia, S., Heredia, M., Criado, O., Rodriguez de Cordoba, S., Garcia-Roves, P.M., Cansell, C., Denis, R., Luquet, S., Foufelle, F., Ferre, P. et al. (2011) Laforin, a dual-specificity phosphatase involved in Lafora disease, regulates insulin response and whole-body energy balance in mice. Hum. Mol. Genet., 20, 2571-2584] reported that Epm2a-/- mice had enhanced glucose disposal and insulin sensitivity, leading them to suggest that laforin, the Epm2a gene product, is involved in insulin signaling. We analyzed 3-month- and 6-7-month-old Epm2a-/- mice and observed no differences in glucose tolerance tests (GTTs) or insulin tolerance tests (ITTs) compared with wild-type mice of matched genetic background. At 3 months, Epm2b-/- mice also showed no differences in GTTs and ITTs. In the 6-7-month-old Epm2a-/- mice, there was no evidence for increased insulin stimulation of the phosphorylation of Akt, GSK-3 or S6 in skeletal muscle, liver and heart. From metabolic analyses, these animals were normal with regard to food intake, oxygen consumption, energy expenditure and respiratory exchange ratio. By dual-energy X-ray absorptiometry scan, body composition was unaltered at 3 or 6-7 months of age. Echocardiography showed no defects of cardiac function in Epm2a-/- or Epm2b-/- mice. We conclude that laforin and malin have no effect on whole-body glucose metabolism and insulin sensitivity, and that laforin is not involved in insulin signaling.

PTG depletion removes Lafora bodies and rescues the fatal epilepsy of Lafora disease.

Lafora disease is the most common teenage-onset neurodegenerative disease, the main teenage-onset form of progressive myoclonus epilepsy (PME), and one of the severest epilepsies. Pathologically, a starch-like compound, polyglucosan, accumulates in neuronal cell bodies and overtakes neuronal small processes, mainly dendrites. Polyglucosan formation is catalyzed by glycogen synthase, which is activated through dephosphorylation by glycogen-associated protein phosphatase-1 (PP1). Here we remove PTG, one of the proteins that target PP1 to glycogen, from mice with Lafora disease. This results in near-complete disappearance of polyglucosans and in resolution of neurodegeneration and myoclonic epilepsy. This work discloses an entryway to treating this fatal epilepsy and potentially other glycogen storage diseases.