Conservation of the glucan phosphatase laforin is linked to rates of molecular evolution and the glucan metabolism of the organism

<p>Abstract</p> <p>Background</p> <p>Lafora disease (LD) is a fatal autosomal recessive neurodegenerative disease. A hallmark of LD is cytoplasmic accumulation of insoluble glucans, called Lafora bodies (LBs). Mutations in the gene encoding the phosphatase laforin account for ~50% of LD cases, and this gene is conserved in all vertebrates. We recently demonstrated that laforin is the founding member of a unique class of phosphatases that dephosphorylate glucans.</p> <p>Results</p> <p>Herein, we identify laforin orthologs in a protist and two invertebrate genomes, and report that laforin is absent in the vast majority of protozoan genomes and it is lacking in all other invertebrate genomes sequenced to date. We biochemically characterized recombinant proteins from the sea anemone <it>Nematostella vectensis </it>and the amphioxus <it>Branchiostoma floridae </it>to demonstrate that they are laforin orthologs. We demonstrate that the laforin gene has a unique evolutionary lineage; it is conserved in all vertebrates, a subclass of protists that metabolize insoluble glucans resembling LBs, and two invertebrates. We analyzed the intron-exon boundaries of the laforin genes in each organism and determine, based on recently published reports describing rates of molecular evolution in <it>Branchiostoma </it>and <it>Nematostella</it>, that the conservation of laforin is linked to the molecular rate of evolution and the glucan metabolism of an organism.</p> <p>Conclusion</p> <p>Our results alter the existing view of glucan phosphorylation/dephosphorylation and strongly suggest that glucan phosphorylation is a multi-Kingdom regulatory mechanism, encompassing at least some invertebrates. These results establish boundaries concerning which organisms contain laforin. Laforin is conserved in all vertebrates, it has been lost in the vast majority of lower organisms, and yet it is an ancient gene that is conserved in a subset of protists and invertebrates that have undergone slower rates of molecular evolution and/or metabolize a carbohydrate similar to LBs. Thus, the laforin gene holds a unique place in evolutionary biology and has yielded insights into glucan metabolism and the molecular etiology of Lafora disease.</p

Glucan Phosphatase Variants for Starch Phosphorylation

Glucan phosphatase nucleotide or polypeptide variants of the presently-disclosed subject matter can alter the biophysical properties of starch in vitro or in planta, as well as the total starch biomass production in planta as compared to plants expressing wild-type glucan phosphatases. Plants producing the polypeptide variants of the presently-disclosed subject matter can have increased starch accumulation, increased starched biomass, and/or starch having desired biophysical properties. A method of the presently-disclosed subject matter for producing altered starch includes providing a plant that produces a glucan phosphatase polypeptide variant that comprises an amino acid mutation and collecting starch from the plant

Serotonergic Therapy in Epilepsy

Purpose of review The serotonergic system is implicated in multiple aspects of epilepsy, including seizure susceptibility, sudden unexpected death in epilepsy (SUDEP), and comorbid depression. Despite the complexity of serotonin’s effects on various neuronal networks, ongoing research provides considerable insight into the role of serotonin in human epilepsy. This review explores the potential roles of serotonergic therapies to improve clinical outcomes in epilepsy. Recent findings In recent decades research has markedly increased our knowledge of the diverse effects of serotonin on brain function. Animal models of epilepsy have identified the influence of serotonin on seizure threshold in specific brain regions, serotoninergic augmentation’s protective effects on terminal apnea and mortality in SUDEP, and mechanisms underlying behavioral improvement in some models of comorbid depression. Human clinical studies are largely consistent with animal data, but the translation into definitive treatment decisions has moved less rapidly. Summary Evidence for serotonergic therapy is promising for improvement in seizure control and prevention of SUDEP. For some epilepsies, such as Dravet syndrome, basic research on serotonin receptor agonists has translated into a positive clinical trial for fenfluramine. The cumulative results of safety and efficacy studies support the routine use of SSRIs for comorbid depression in epilepsy

Identification of proteins potentially involved in the formation of Lafora bodies, a hallmark of Lafora disease

Lafora Disease (LD) is a fatal teenage-onset progressive myoclonus epilepsy. It is characterized by the formation of Lafora bodies (LBs), deposits of abnormally branched, insoluble, hyperphosphorylated glycogen-like polymers that are generally believed to trigger the development of the clinical symptoms of LD. 58% and 35% of the LD cases are caused by mutations in EPM2A (laforin) and EPM2B (malin), respectively. However, little is known about their function in LB formation. Two different mechanisms have been proposed to explain the accumulation of insoluble LBs: first, excessive glycogen phosphorylation and, second, an imbalance between glycogen synthesizing enzymes. The present study aims at the identification of proteins involved in the molecular mechanisms leading to LB formation and appearance of LD and the phosphorylation of glycogen

Thermophilic Phosphatases and Methods for Processing Starch Using the Same

The presently-disclosed subject matter includes thermophilic glucan phosphatase polypeptides. In some embodiments the polypeptide includes non-native laforin polypeptides, or fragments and/or variants thereof, and in some instances the polypeptide can alter the biophysical properties of starch in vitro or in planta. The presently-disclosed subject matter also includes isolated polynucleotides encoding the present polypeptides, methods for processing starch by exposing starch to the present polypeptides, and methods for making the present polypeptides

Lafora disease offers a unique window into neuronal glycogen metabolism

Lafora disease (LD) is a fatal, autosomal recessive, glycogen-storage disorder that manifests as severe epilepsy. LD results from mutations in the gene encoding either the glycogen phosphatase laforin or the E3 ubiquitin ligase malin. Individuals with LD develop cytoplasmic, aberrant glycogen inclusions in nearly all tissues that more closely resemble plant starch than human glycogen. This Minireview discusses the unique window into glycogen metabolism that LD research offers. It also highlights recent discoveries, including that glycogen contains covalently bound phosphate and that neurons synthesize glycogen and express both glycogen synthase and glycogen phosphorylase

Frontotemporal Dementia Nonsense Mutation of Progranulin Rescued by Aminoglycosides

Frontotemporal dementia (FTD) is an early onset dementia and is characterized by progressive atrophy of the frontal and/or temporal lobes. FTD is highly heritable with mutations in progranulin accounting for 5-26% of cases in different populations. Progranulin is involved in endocytosis, secretion and lysosomal processes, but its function under physiological and pathological conditions remains to be defined. Many FTD-causing nonsense progranulin mutations contain a premature termination codon (PTC), thus progranulin haploinsufficiency has been proposed as a major disease mechanism. Currently, there is no effective FTD treatment or therapy. Aminoglycosides are a class of antibiotics that possess a less known function to induce eukaryotic ribosomal readthrough of PTCs to produce a full-length protein. The aminoglycoside-induced readthrough strategy has been utilized to treat multiple human diseases caused by PTCs. In this study, we tested the only clinically approved readthrough small molecule PTC124 and eleven aminoglycosides in a cell culture system on four PTCs responsible for FTD or a related neurodegenerative disease amyotrophic lateral sclerosis. We found that the aminoglycosides G418 and gentamicin B1 rescued the expression of the progranulin R493X mutation. G418 was more effective than gentamicin B1 (~50% rescue vs \u3c 10%), and the effect was dose and time-dependent. The proganulin readthrough protein displayed similar subcellular localization as the wild-type proganulin protein. These data provide an exciting proof-of-concept that aminoglycosides or other readthrough-promoting compounds are a therapeutic avenue for familial FTD caused by proganulin PTC mutations

Expression, Purification and Characterization of Soluble Red Rooster Laforin as a Fusion Protein in Escherichia Coli

BACKGROUND: The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue. RESULTS: In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin. CONCLUSIONS: Gg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease

Increasing Access to Natural Areas: Connecting Physical and Social Dimensions

Report of the 2015 Berkley Workshop Held at the Asticou Inn, Northeast Harbor, Maine - July 201