Protein Laforin Research Articles

Genome-wide association study of pancreatic fat: The Multiethnic Cohort Adiposity Phenotype Study.

Several studies have found associations between higher pancreatic fat content and adverse health outcomes, such as diabetes and the metabolic syndrome, but investigations into the genetic contributions to pancreatic fat are limited. This genome-wide association study, comprised of 804 participants with MRI-assessed pancreatic fat measurements, was conducted in the ethnically diverse Multiethnic Cohort-Adiposity Phenotype Study (MEC-APS). Two genetic variants reaching genome-wide significance, rs73449607 on chromosome 13q21.2 (Beta = -0.67, P = 4.50x10-8) and rs7996760 on chromosome 6q14 (Beta = -0.90, P = 4.91x10-8) were associated with percent pancreatic fat on the log scale. Rs73449607 was most common in the African American population (13%) and rs79967607 was most common in the European American population (6%). Rs73449607 was also associated with lower risk of type 2 diabetes (OR = 0.95, 95% CI = 0.89–1.00, P = 0.047) in the Population Architecture Genomics and Epidemiology (PAGE) Study and the DIAbetes Genetics Replication and Meta-analysis (DIAGRAM), which included substantial numbers of non-European ancestry participants (53,102 cases and 193,679 controls). Rs73449607 is located in an intergenic region between GSX1 and PLUTO, and rs79967607 is in intron 1 of EPM2A. PLUTO, a lncRNA, regulates transcription of an adjacent gene, PDX1, that controls beta-cell function in the mature pancreas, and EPM2A encodes the protein laforin, which plays a critical role in regulating glycogen production. If validated, these variants may suggest a genetic component for pancreatic fat and a common etiologic link between pancreatic fat and type 2 diabetes.

Personalized Diagnosis of Lafora Disease through Biochemical Characterization of SNPs

Lafora disease (LD) is an autosomal recessive neurodegenerative disease. Patients develop normally as children, and are typically diagnosed during adolescence, when the disease presents as a form of progressive myoclonus epilepsy. The disease then progresses rapidly, with patients experiencing seizures of increasing severity and frequency, along with loss of muscle control and neurodegeneration, leading to death within 10 years. Multiple therapies are in development for LD patients, however, recent studies suggest that early detection will be critical for effective treatment of LD patients. It is known that patients with Lafora Disease have mutations in either Epilepsy, Progressive Mycolonus 2A (EPM2A), or Epilepsy, Progressive Mycolonus 2B (EPM2B), which code for the proteins laforin and malin, respectively. Laforin is the only known mammalian phosphatase that can remove phosphate from glycogen, and malin is an E3 ligase that has been shown to ubiquitinate enzymes involved in glycogen synthesis and degradation. Together, these proteins play critical roles in regulating glycogen metabolism. Disease causing mutations occur throughout each protein, suggesting multiple roles for both laforin and malin in glycogen regulation. Given the diversity of disease-causing mutations in these proteins, it is challenging to determine if a mutation will be deleterious prior to the onset of epilepsy in the patient. In order to effectively treat LD patients, disease-causing mutations will need to be identified prior to the onset of symptoms, while the patient still shows normal development. Therefore, physicians need a reliable method for determining mutation pathogenicity. Here, we present a biochemical pipeline for laforin to characterize newly identified SNPs and determine pathogenicity. Through the integration of multiple techniques including differential scanning fluorimetry, phosphatase activity assays, and HDX analysis, our lab has characterized the impact of SNPs on laforin function. This pipeline will allow physicians and patients to obtain personalized diagnoses of Lafora Disease prior to the onset of symptoms.

Hormonal Regulation of Novel Glycogen Metabolic Proteins Hari Priya Vemana, Ehab M. Abo‐Ali, John L. Croft, Shraddha Bhutkar, Michael Woolford, Vikas V. DukhandeDepartment of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, USA

Lafora disease (LD) is a rare autosomal recessive disorder manifesting as a progressive myoclonic epilepsy. LD is characterized by the formation of Lafora bodies, insoluble deposits containing abnormal glycogen‐like material in many tissues. LD is caused by mutations in the genes encoding dual‐specificity phosphatase laforin (EPM2A) or the E3 ubiquitin ligase malin (EPM2B / NHLRC1). Several research studies on LD have shown that laforin functions as a phosphatase for glycogen. Malin forms a functional complex with laforin and either malin alone or the laforin‐malin complex is known to ubiquitinate multiple proteins involved in glycogen metabolism. It is well established that hormones such as insulin regulates glycogen metabolism but hormonal regulation of laforin and malin is not completely elucidated. In type‐2 diabetes, defects in insulin responsiveness leads to a state called insulin resistance. Our objective is to study the effect of hormones and nutrients on novel glycogen metabolic proteins laforin and malin in physiology and diabetes pathophysiology. We used liver cells in culture and a rodent model of diabetes using high‐fat diet and low dose streptozotocin (HF‐STZ). We measured gene expression, protein expression, protein posttranslational modifications, and functional activities of laforin and malin. Our preliminary data indicates that both diabetic conditions and insulin modulate the expression of LD proteins. Our ongoing studies are directed towards delineating the molecular mechanisms of these alterations. This study can serve to find novel potential targets for the pharmacotherapy of type‐2 diabetes.Support or Funding InformationThis study is supported by an award from National Institute of General Medical Sciences of the National Institute of Health, Award number: SC2GM125550.

Lafora Disease: Differential Metabolic Disturbances in Neurons and Astrocytes

The brain is an incredible energy‐demanding organ and even small disturbances in brain energetics may have fatal consequences. Lafora disease (LD) is a rare metabolic and neurodegenerative disorder clinically presenting with tonic‐clonic epileptic seizures and myoclonic jerks. LD patients experience rapid neurodegeneration starting in early adolescence and the disease is indisputable fatal typically within 10 years of onset. LD is caused by mutations in the genes EPM2A or EPM2B encoding the proteins Laforin and Malin, both implicated in glycogen metabolism. However, the mechanism of disease is poorly understood, though emerging evidence suggests that neurons and glial cells are affected differently in LD. This study aims to elucidate the effect of laforin on central carbon metabolism in different cellular compartments. We employed a systematic characterization of glucose metabolism in cerebral brain slices from 3‐months old Epm2a−/− and wildtype animals in addition to primary cultures of neurons and astrocytes utilizing [U‐13C]glucose and subsequent analysis by gas chromatography‐mass spectroscopy. Furthermore, slices were incubated with [U‐13C]glutamine and [U‐13C]glutamate to assess neuronal and astrocytic metabolic compartmentation, since glutamine is predominantly taken up and metabolized by neurons, and glutamate by astrocytes. The results of this study revealed complex disturbances of central carbon metabolism in the LD brain with clear metabolic distinctions between cerebral cell types. Disturbed oxidative glucose metabolism was observed in both primary cultures of neurons and astrocytes and in acutely isolated brain slices. Interestingly, the results indicate a compartmentalized glucose metabolism where neuronal glucose metabolism is downregulated, and astrocytic glucose metabolism upregulated in Epm2A−/− mice. Furthermore, neuronal glutamine metabolism was increased whereas glial glutamate metabolism was unaffected. Overall, this study provides evidence of altered glucose and neurotransmitter metabolism in LD related to loss of laforin and hereby dysfunctional glycogen metabolism. Further investigation of metabolic alterations in cerebral cell types may provide important insights into the mechanism of disease and targeted treatment.

Mystery Case: Lafora periodic acid–Schiff inclusion bodies

A 16-year-old boy presented with a 5-year history of progressive cognitive decline and behavioral change followed by generalized tonic-clonic and myoclonic seizures refractory to many anticonvulsants (valproic acid, phenobarbital, clonazepam, and topiramate) and cerebellar ataxia a year later. The son of consanguineous parents, he had a family history of 2 cousins who both had epilepsy and died at the ages of 15 and 25 years. Laboratory screening tests, including lactic acid, fundus examination, brain neuroimaging, and CSF, were normal. Skin biopsy (figure) revealed periodic acid–Schiff-positive intracellular polyglucosan inclusion bodies in myoepithelial and sweat gland duct cells. These findings are typical of Lafora disease, a fatal autosomal recessive disorder caused by mutations in one of 2 known genes, both located at chromosome 6: EPM2A , which encodes the protein laforin, and EPM2B , which encodes the protein malin. Differential diagnosis must be made with other causes of progressive myoclonic epilepsies, most commonly Unverricht-Lundborg disease (Baltic myoclonus), myoclonus epilepsy and ragged-red fibers syndrome, neuronal ceroid lipofuscinosis, and type 1 sialidosis. There is no treatment, and therapy is mainly supportive and symptomatic.1,2

Lafora disease proteins laforin and malin negatively regulate the HIPK2-p53 cell death pathway

Lafora disease (LD) is an autosomal recessive, progressive, and fatal form of a neurodegenerative disorder characterized by the presence of Lafora polyglucosan bodies. LD is caused by defects in either the laforin protein phosphatase or the malin E3 ubiquitin ligase. Laforin and malin were shown play key roles in proteolytic processes, unfolded stress response, and glycogen metabolism. Therefore, the LD proteins laforin and malin are thought to function as pro-survival factors and their loss thus could result in neurodegeneration. To understand the molecular pathway leading to the cell death in LD, in the present study, we investigated the possible role of LD proteins in the p53-mediated cell death pathway. We show that loss of laforin or malin results in the increased level and activity of p53, both in cellular and animal models of LD, and that this is primarily due to the increased levels of Hipk2, a proapoptotic activator of p53. Overexpression of laforin or malin confers protection against Hipk2-mediated cell death by targeting the Hipk2 to the cytoplasmic compartment. Taken together, our study strengthens the notion that laforin and malin are pro-survival factors, and that the activation of Hipk2-p53 cell death pathway might underlie neurodegeneration in LD.

Determining the Role of Lafora Disease Patient Mutations on Glycogen Dephosphorylation.

Lafora Disease (LD) is fatal, congenital neurodegenerative epilepsy caused by mutations in the gene that encodes the protein laforin. These mutations disrupt glycogen metabolism in cells causing accumulations of malformed glycogen inclusions called Lafora Bodies (LB). LBs inhibit normal neuronal functions, leading to epilepsy, dementia, and death by age 25. Laforin contains a carbohydrate binding module (CBM) and a dual specificity phosphatase (DSP) domain. To date, more than 40 laforin point mutations have been identified in LD patients. However, how each of these mutations leads to LB formation and/or how they impact glycogen dephosphorylation is not known. Our lab recently determined the 3‐dimensional structure of laforin using X‐ray crystallography. The laforin X‐ray crystal structure reveals that these patient point mutations localize in four different regions within the DSP and CBM domains suggesting multiple possible disease mechanisms. To determine the role of each of these mutations on glycogen dephosphorylation, we employed a generic pNPP phosphatase activity assay and a biologically relevant assay utilizing glycogen to measure glycogen phosphatase activity. The glycogen phosphatase assay was very specific and faithfully reported both wildtype and mutant laforin phosphatase activity. Mutations that are located in the CBM‐DSP interface and the DSP‐DSP dimer interface resulted in 50‐80% decrease, suggesting the importance of these regions in optimal laforin activity. We found a 70‐90% decrease in glycogen phosphatase activity for mutations that are localized in the CBM binding site and the DSP active site.

Exploring the Structural Insights on Human Laforin Mutation K87A in Lafora Disease—A Molecular Dynamics Study

Lafora disease (LD) is an autosomal recessive, progressive form of myoclonus epilepsy which affects worldwide. LD occurs mainly in countries like southern Europe, northern Africa, South India, and in the Middle East. LD occurs with its onset mainly in teenagers and leads to decline and death within 2 to 10 years. The genes EPM2A and EPM2B are commonly involved in 90 % of LD cases. EPM2A codes for protein laforin which contains an amino terminal carbohydrate binding module (CBM) belonging to the CBM20 family and a carboxy terminal dual specificity phosphatase domain. Mutations in laforin are found to abolish glycogen binding and have been reported in wet lab methods. In order to investigate on structural insights on laforin mutation K81A, we performed molecular dynamics (MD) simulation studies for native and mutant protein. MD simulation results showed loss of stability due to mutation K87A which confirmed the structural reason for conformational changes observed in laforin. The conformational change of mutant laforin was confirmed by analysis using root mean square deviation, root mean square fluctuation, solvent accessibility surface area, radius of gyration, hydrogen bond, and principle component analysis. Our results identified that the flexibility of K87A mutated laforin structure, with replacement of acidic amino acid to aliphatic amino acid in functional CBM domain, have more impact in abolishing glycogen binding that favors LD.

Interaction Between DUSP8 and the Polyglutamine Protein Ataxin-1

Polyglutamine (polyQ) diseases are a group of inherited neurodegenerative disorders caused by the expansion of polyQ repeats in the disease proteins. Expansion of polyQ repeats presumably leads to misfolding and aggregation of polyQ proteins. Although details of the molecular mechanisms are still elusive, transcriptional dysfunction and oxidative damage are suggested as underlying events responsible for polyQ pathogenesis. Misfolded polyQ proteins are frequently concentrated into intranuclear inclusions, which increase generation of reactive oxygen species (ROS) and activate stress signaling pathways. In cultured cells expressing expanded polyQ proteins, mitogen-activated protein kinases (MAPK), e.g., p38 and JNK, are activated and the cytotoxicity induced by polyQ proteins, partially through sustained activation of MAP kinases, can be reduced by administration of a p38 inhibitor. MAPK are activated by MAPK kinase (MEK)-mediated phosphorylation of threonine and tyrosine residues located in the signature sequence (TxY) and MEK is in turn activated by MAPK kinase kinase (MEKK). Since MAPK are involved in a wide variety of cellular processes including cell survival and apoptosis, cells must tightly regulate the magnitude and duration of MAPK activation. The negative regulation of MAPK is often achieved by phosphatase-mediated dephosphorylation of MAPK. A group of protein phosphatases referred as type I cysteinebased protein tyrosine phosphatases including DUSPs (dualspecificity phosphatases) dephosphorylate both tyrosine and serine/threonine residues within the same substrate and as a result counteract MAPK. Involvement of protein tyrosine phosphatases (PTPs) in polyQ-induced cell death has been reported in previous studies. Several PTP proteins including MKP-1 are up-regulated by over-expression of cytotoxic polyQ-expanded huntingtin (Htt) fragment. Moreover, polyQ-expanded Htt fragment activates JNK pathway by reducing solubility of JNK phosphatase M3/6 (also known as hVH5 or DUSP8). A recent study demonstrated that a DUSP protein laforin can suppress the cytotoxicity caused by misfolded proteins and hinted that phosphatases can be potential targets for pharmacological intervention of neurodegenerative disorders. The polyQ protein ataxin-1 is a soluble protein of about 816 amino acids, which varies depending on the length of polyQ repeats, and located both in the cytoplasm and nucleus. Although the exact functions are not completely understood, ataxin-1 is apparently involved in transcription regulation through its ability to interact with several transcription factors as well as RNA. While it is generally assumed that expansion of polyQ repeats causes misfolding of ataxin-1 and leads to neuronal death in spinocerebellar ataxia 1 (SCA1), contribution of other domains to pathogenesis also becomes increasingly clear. Besides N-terminal polyQ region, AXH domain (570-689; for interaction with transcription factors and RNA), endogenous phosphorylation site (776) and nuclear localization signal (795-798) are present in the Cterminus of ataxin-1. Phosphorylation of ataxin-1 is particularly important since it is implicated in pathogenicity of SCA1. Phosphorylation at Ser776 is critical for the interaction of ataxin-1 with 14-3-3 proteins that regulate its entry into the nucleus. Previously we demonstrated that ataxin-1, capable of activating JNK by itself, does not directly interact with this MAPK. Such observations led us to suspect that misfolded polyQ proteins, while not directly binding to JNK, could physically associate with PTPs (e.g., DUSP proteins) and thereby regulate JNK activation. To explore this possibility, we first investigated if ataxin-1 actually interacts with MAPK phosphatases in vivo. Upon screening for PTPs that physically interact with normal ataxin-1[30Q], we found out that a number of human PTPs including DUSP2, 8, 13 and 24 strongly associated with ataxin-1[30Q] (Table 1).

Laforin Prevents Stress-Induced Polyglucosan Body Formation and Lafora Disease Progression in Neurons

Glycogen, the largest cytosolic macromolecule, is soluble because of intricate construction generating perfect hydrophilic-surfaced spheres. Little is known about neuronal glycogen function and metabolism, though progress is accruing through the neurodegenerative epilepsy Lafora disease (LD) proteins laforin and malin. Neurons in LD exhibit Lafora bodies (LBs), large accumulations of malconstructed insoluble glycogen (polyglucosans). We demonstrated that the laforin-malin complex reduces LBs and protects neuronal cells against endoplasmic reticulum stress-induced apoptosis. We now show that stress induces polyglucosan formation in normal neurons in culture and in the brain. This is mediated by increased glucose-6-phosphate allosterically hyperactivating muscle glycogen synthase (GS1) and is followed by activation of the glycogen digesting enzyme glycogen phosphorylase. In the absence of laforin, stress-induced polyglucosans are undigested and accumulate into massive LBs, and in laforin-deficient mice, stress drastically accelerates LB accumulation and LD. The mechanism through which laforin-malin mediates polyglucosan degradation remains unclear but involves GS1 dephosphorylation by laforin. Our work uncovers the presence of rapid polyglucosan metabolism as part of the normal physiology of neuroprotection. We propose that deficiency in the degradative phase of this metabolism, leading to LB accumulation and resultant seizure predisposition and neurodegeneration, underlies LD.

Lafora Progressive Myoclonus Epilepsy: Recent Insights into Cell Degeneration

Lafora disease (LD) is a fatal autosomal recessive form of progressive myoclonus epilepsy. Patients manifest myoclonus and tonic-clonic seizures, visual hallucinations, intellectual, and progressive neurologic deterioration beginning in adolescence. The two genes known to be involved in Lafora disease are EPM2A and NHLRC1 (EPM2B). The EPM2A gene encodes laforin, a dual-specificity protein phosphatase, and the NHLRC1 gene encodes malin, an E3-ubiquitin ligase. The two proteins interact with each other and, as a complex, are thought to regulate glycogen synthesis. It may also be considered as a disorder of carbohydrate metabolism because of the formation of polyglucosan inclusion bodies in neural and other tissues due to abnormalities of the proteins laforin or malin. The review also outlines important patents related to Lafora disease.

Lafora Disease

Lafora disease is a rare, fatal, autosomal recessive, progressive myoclonic epilepsy. It may also be considered as a disorder of carbohydrate metabolism because of the formation of polyglucosan inclusion bodies in neural and other tissues due to abnormalities of the proteins laforin or malin. The condition is characterized by epilepsy, myoclonus and dementia. Diagnostic findings on MRI and neurophysiological testing are not definitive and biopsy or genetic studies may be required. Therapy in Lafora disease is currently limited to symptomatic management of the epilepsy, myoclonus and intercurrent complications. With a greater understanding of the pathophysiological processes involved, there is justified hope for future therapies.

Laforin, the most common protein mutated in Lafora disease, regulates autophagy.

Lafora disease (LD) is an autosomal recessive, progressive myoclonus epilepsy, which is characterized by the accumulation of polyglucosan inclusion bodies, called Lafora bodies, in the cytoplasm of cells in the central nervous system and in many other organs. However, it is unclear at the moment whether Lafora bodies are the cause of the disease, or whether they are secondary consequences of a primary metabolic alteration. Here we describe that the major genetic lesion that causes LD, loss-of-function of the protein laforin, impairs autophagy. This phenomenon is confirmed in cell lines from human patients, mouse embryonic fibroblasts from laforin knockout mice and in tissues from such mice. Conversely, laforin expression stimulates autophagy. Laforin regulates autophagy via the mammalian target of rapamycin kinase-dependent pathway. The changes in autophagy mediated by laforin regulate the accumulation of diverse autophagy substrates and would be predicted to impact on the Lafora body accumulation and the cell stress seen in this disease that may eventually contribute to cell death.

Loss of function of the cytoplasmic isoform of the protein laforin (EPM2A) causes Lafora progressive myoclonus epilepsy.

Lafora disease is the most severe teenage-onset progressive epilepsy, a unique form of glycogenosis with perikaryal accumulation of an abnormal form of glycogen, and a neurodegenerative disorder exhibiting an unusual generalized organellar disintegration. The disease is caused by mutations of the EPM2A gene, which encodes two isoforms of the laforin protein tyrosine phosphatase, having alternate carboxyl termini, one localized in the cytoplasm (endoplasmic reticulum) and the other in the nucleus. To date, all documented disease mutations, including the knockout mouse model deletion, have been in the segment of the protein common to both isoforms. It is therefore not known whether dysfunction of the cytoplasmic, nuclear, or both isoforms leads to the disease. In the present work, we identify six novel mutations, one of which, c.950insT (Q319fs), is the first mutation specific to the cytoplasmic laforin isoform, implicating this isoform in disease pathogenesis. To confirm this mutation's deleterious effect on laforin, we studied the resultant protein's subcellular localization and function and show a drastic reduction in its phosphatase activity, despite maintenance of its location at the endoplasmic reticulum.