Lafora disease (LD) is a fatal neurodegenerative epilepsy that is autosomal recessive and is characterized by rapid neurological deterioration. LD is one of five diseases that represent the inherited progressive myoclonus epilepsy (PME) family (Berkovic et al. 1986, 1991; Girard et al. 2013). Due to the progressive nature of neurodegeneration, the frequency of myoclonic seizures, and the variety of seizure types that can present, PMEs are often difficult to diagnose at onset (reviewed in Minassian 2001).
LD is characterized by myoclonic, tonic-clonic, and focal-occipital seizures among other seizure types that increase in frequency and severity with age, with the first tonic-clonic epileptic presentation occurring in late childhood or early adolescence. Other symptoms include dementia, visual hallucinations, ataxia, absence, and grand mal seizures along with epilepsy triggered by stimulus (reviewed in Minassian 2001). Death occurs approximately 10 years following initial onset due to complications such as status epilepticus, aspiration pneumonia, or respiratory failure. There is significant neuronal loss across the CNS upon autopsy (reviewed in Minassian 2001; Ganesh et al. 2006).
Approximately 60% of LD cases result from missense mutations, nonsense mutations, deletions, or insertions in the EPM2A gene encoding laforin. LD-associated missense mutations are scattered throughout the protein, and multiple studies have shown that these mutations disrupt phosphatase activity, glycogen binding, dimerization, protein-protein interactions, or localization (Fig. 1) (Wang et al. 2002; Gentry et al. 2007; Roma-Mateo et al. 2011; Wang and Roach 2004; Fernandez-Sanchez et al. 2003; Tagliabracci et al. 2007; Raththagala et al. 2015; Ganesh et al. 2004). LD also results from mutations in the EPM2B (epilepsy, progressive myoclonic type 2B) gene encoding the E3 ubiquitin ligase malin (Chan et al. 2003).
A hallmark of LD is the presence of insoluble polyglucosan bodies termed Lafora bodies (LBs) in the cytoplasm of cells from most tissues. These tissues, including neurons, cardiac muscle, and liver, are also tissues that display highest laforin expression in normal individuals (reviewed in Ganesh et al. 2006). The spherical LB inclusions are 3–40 μm in size and can grow large enough to occupy the entire cytoplasm. The inclusions found in LD contain only a small amount of ubiquitinated protein and are instead composed of improperly branched and hyperphosphorylated glucose moieties. Despite the presence of LBs in a variety of tissues throughout the body, neurological symptoms predominate (reviewed in Minassian 2001).
Laforin Expression, Isoforms, and Localization
Laforin mRNA is widely expressed in the human body. All tissues examined contain laforin mRNA, including: brain, heart, skeletal muscle, liver, lung, placenta, kidney, testis, spleen, thymus, prostate, ovary, small intestine, and pancreas (Minassian et al. 1998). Various brain regions such as the cerebellum, cortex, medulla, putamen, and spinal cord also express laforin transcript at similar levels, demonstrating the ubiquitous expression of laforin in the brain (Ganesh et al. 2001).
The sequences of nine splice variants of the EPM2A gene are available in the Universal Protein Resource (www.uniprot.org), and five isoforms have been biochemically characterized (Dubey et al. 2012; Dubey and Ganesh 2008). The dominant splice variant codes for a 331 amino acid product which has phosphatase activity, binds to glycogen, dimerizes, and interacts with malin; other isoforms, which possess various combinations of these characteristics, may function to regulate the activity or localization of the dominant isoform (Dubey and Ganesh 2008; Ganesh et al. 2002). The dominant 331 amino acid isoform is the best characterized and the one discussed throughout this review.
It was initially observed that overexpressed laforin was largely found in the cytosol with some signs of endoplasmic reticulum (ER) localization, and lower levels are found in the nucleus (Ganesh et al. 2000, 2002; Minassian et al. 2001). Later it was shown that laforin interacts with glycogen in vitro and in vivo, and overexpressed laforin colocalizes with sites of glycogen synthesis in cells, which are cytosolic and associated with the ER (Wang et al. 2002; Tiberia et al. 2012). Mutations in the CBM disrupt glycogen colocalization, and are found in the nucleus (Wang et al. 2002; Singh et al. 2012). Laforin localization appears to be determined by the presence of glycogen. One study reported that laforin translocates from the nucleus to the cytoplasm upon glucose uptake and glycogen synthesis in cell culture (Singh et al. 2012). They showed that cytoplasmic translocation of laforin is suppressed in the presence of forskolin, which promotes glycogen degradation despite high glucose, and maintained in the presence of glucosamine, which prevents glycogen breakdown despite the absence of glucose (Singh et al. 2012). Other studies show that overexpressed and endogenous laforin sediments with microsomal/glycogen particle fractions during high-speed centrifugation (Fernandez-Sanchez et al. 2003; Tiberia et al. 2012). Studies of laforin in mouse models have confirmed that glycogen and laforin levels are positively correlated (see “Regulation of Laforin Activity and Concentration”). Furthermore, glycogen metabolic enzymes such as glycogen synthase (GS) and protein targeting to glycogen (PTG/R5) also localize to glycogen granules, and in addition to colocalizing with them, laforin has been shown to interact with these enzymes using yeast two-hybrid screening and immunoprecipitation, and may participate in their regulation (See “Interaction and Signaling”) (Fernandez-Sanchez et al. 2003). These studies strongly suggest laforin is part of a complex of enzymes on the granule that regulate its synthesis and degradation.
Laforin Phosphatase Activity
Consistent with its classification as a DSP, laforin exhibits in vitro phosphatase activity against pSer, pThr, and pTyr residues and can utilize the artificial substrates 3-O-methylflourescein phosphate (OMFP) and para-nitrophenyl phosphate (p-NPP) (Dukhande et al. 2010; Sherwood et al. 2013; Roma-Mateo et al. 2016). Although binding partners for laforin were identified, laforin’s substrate remained elusive at first. Dixon and colleagues first identified that laforin contains a CBM and demonstrated that this domain allows laforin to bind glucans (Wang et al. 2002). Out of 65 dual specificity phosphatases, laforin is the only DSP with a CBM (Tonks 2006). This fact coupled with a finding from the 1960s that LBs have increased phosphate prompted Gentry and colleagues to test if laforin could liberate phosphate from glucans. Laforin does indeed dephosphorylate glucans, while other DSPs and phosphatases from other families lack this activity (Sherwood et al. 2013; Worby et al. 2006). This was the first description of an enzyme that released phosphate from carbohydrates, establishing laforin as the founding member of the glucan phosphatase family. It was subsequently demonstrated that laforin can bind and dephosphorylate glycogen, its glucan activity is unique to laforin among vertebrate DSPs, and laforin orthologs from other organisms also possess phosphatase activity (Gentry and Pace 2009; Tagliabracci et al. 2007, Raththagala et al. 2015; Sherwood et al. 2013; Brewer et al. 2014). There are two other glucan phosphatases in plants, Starch Excess 4 (SEX4) and Like SEX Four 2 (LSF2) (Santelia et al. 2011; Kotting et al. 2009; Meekins et al. 2016). SEX4, LSF2, and laforin make up the glucan phosphatase family (reviewed in Emanuelle et al. 2016; Gentry et al. 2016). SEX4 also contains a CBM and DSP, though in the reverse orientation, and laforin and SEX4 are functional homologs since SEX4 deficiency in plants can be partially complemented by laforin (Gentry et al. 2007).
The identification of laforin as a glucan phosphatase explains how loss of laforin leads to increased phosphate content in glycogen (Tagliabracci et al. 2007; Sakai et al. 1970). But why does increased phosphate lead to glycogen accumulation and LB formation? Work from the Roach lab demonstrated that as phosphate levels increase in glycogen, glycogen becomes insoluble (Tagliabracci et al. 2008). Minassian and colleagues isolated glycogen from cells transfected with laforin and showed that LD-associated laforin mutations caused glycogen to transform into poorly branched, amylopectin-like aggregates (Tiberia et al. 2012). Furthermore, in mice, in the absence of laforin or malin, not only is glycogen accumulated, the structure, branching and chain length distribution of glycogen granules are altered (Nitschke et al. 2013; Valles-Ortega et al. 2011). Like plant starch, the aberrant glycogen that eventually forms LBs has less frequent branching, longer chain lengths, and higher phosphate levels, consistent with what was reported in the early histological studies of LD patient tissues in the 1960s (Gentry et al. 2009; Yokoi et al. 1968; Schnabel and Seitelberger 1968).
Regulation of Laforin Activity and Concentration
Multiple groups have shown that mutations in the CBM and DSP domain lead to a loss of phosphatase activity, while only mutations in the CBM lead to a loss of glycogen binding. It has now been established that laforin binding to glycogen is largely mediated by the CBM and dephosphorylation by the DSP domain (Raththagala et al. 2015; Dias et al. 2015). Our lab recently determined the X-ray crystal structure of laforin bound to phosphate and maltohexaose, a six-unit glucan that models the linear chains found in glycogen (Fig. 2c) (Raththagala et al. 2015). The structure revealed an antiparallel homodimer, with a dimer interface mediated exclusively by the DSP domains and an architecture that is unique among the glucan phosphatases (reviewed in Emanuelle et al. 2016; Gentry et al. 2016). The CBM and DSP domain of each subunit are tightly integrated, and LD mutations at this interface lead to increased interdomain dynamics and a loss of glycogen phosphatase activity, indicating the CBM-DSP interface is important for function (Raththagala et al. 2015). We also demonstrated that binding affinity increases with chain length, that dimerization is important for cooperative, high-affinity binding of long oligosaccharides, and that LD mutations have differential effects on laforin binding and phosphatase activity. These results support the notion that laforin preferentially targets long glucan chains like those found in LBs. Dimerization of laforin has been a matter of debate in the field, but the extended dimer interface in the crystal structure and solution-based experiments from multiple groups have confirmed that laforin is dimeric (Raththagala et al. 2015; Dukhande et al. 2011; Sanchez-Martin et al. 2013; Sankhala et al. 2014). Another group proposed the laforin structure with a CBM-CBM dimer interface, but this arrangement was determined using a partial structure containing only the DSP domain and homology modeling (Sankhala et al. 2014).
The RING-type E3 ubiquitin ligase malin binds and ubiquitinates laforin in cell culture (Gentry et al. 2005). The interaction between laforin and malin is mediated by the protein-interaction domain of malin containing six NHL (NCL-1, HT2A, and LIN-41 proteins) repeats (Gentry et al. 2005; Lohi et al. 2005). In overexpression systems, coexpression of laforin and malin leads to a decrease in laforin levels and inhibition of proteasome activity by MG132 results in increased laforin levels, indicating that laforin levels are regulated by the proteasome (Gentry et al. 2005; Mittal et al. 2007). In addition, malin ubiquitination of laforin was recapitulated using purified components in vitro (Gentry et al. 2005). In support of this finding, malin knockout (KO) mice display increased levels of laforin (Tiberia et al. 2012; Valles-Ortega et al. 2011; DePaoli-Roach et al. 2010). However, this presents a paradox since mutations in EPM2B, the gene encoding malin, also result in LD. If malin negatively regulates laforin, why do mutations in either gene lead to LB formation and LD? One hypothesis proposed by multiple groups in the field is that laforin and malin work in tandem: laforin first binds to glycogen and dephosphorylates it, and then laforin must be ubiquitinated by malin and removed before the construction of glycogen proceeds, preventing its entrapment in the glycogen macromolecule (Tiberia et al. 2012; Gentry et al. 2005). Another group proposed that laforin and malin are interdependent enzymes, laforin presenting itself as a substrate to prevent the autoubiquitination of malin, and malin ubiquitinating the phosphatase-inactive laforin monomer (Mittal et al. 2015). The answer to this question is still not clear, but there is evidence that in addition to their independent functions, laforin and malin may function as a complex (further discussed in “Interactions and Signaling”).
It is also important to note that laforin protein levels correlate with glycogen stores. Various transgenic mouse models with increased or decreased glycogen levels display parallel increases and decreases in laforin protein levels while laforin transcript levels are unchanged (Wang et al. 2006a). Glycogen synthase levels are increased in malin KO mice, and both glycogen synthase and laforin are associated with insoluble, accumulated glycogen (Tiberia et al. 2012; Valles-Ortega et al. 2011; DePaoli-Roach et al. 2010). Furthermore, laforin sediments with LBs via a low speed centrifugation step and colocalizes with LBs in histological sections from LD mouse models, suggesting laforin preferentially binds to LBs over glycogen (Tiberia et al. 2012; Criado et al. 2012; Chan et al. 2004).
Interactions and Signaling
Interestingly, the D146N malin mutation has been associated with a mild phenotype in LD patients, and this mutation has been reported to disrupt the interaction between laforin and malin (Vilchez et al. 2007). Based on these data, it is plausible that there are two ways in which laforin regulates glycogen metabolism: (1) by removing covalent phosphate and (2) by acting as a scaffold/adaptor protein for malin-mediated K63 ubiquitination. Perhaps K63 polyubiquitination affects the localization and/or activity of GS, PTG, GDE, and AMPK, but not protein levels; this would explain how malin could regulate these enzymes without promoting their degradation. Furthermore, malin is unique among E3 ubiquitin ligases in that it contains only a six-NHL domain in addition to its RING domain. Other RING-containing E3s typically contain additional substrate binding domains, such as the B-box or coiled-coil domain (Roma-Mateo et al. 2011). Laforin may function in place of these additional domains as an adaptor for malin substrates. This model would unify the present data, supporting both the presence of a complex and the independent roles of laforin as a glycogen phosphatase.
Multiple studies have described a link between laforin and autophagy, oxidative stress, glutamate transport and inflammation. A number of important studies were published describing autophagy defects in laforin- and malin-deficient mice, including lower levels of LC3-II, fewer autophagosomes, and higher levels of p62 (Criado et al. 2012; Aguado et al. 2010; Puri et al. 2012; Jain et al. 2016). These defects were observed as early as postnatal day 16 in mice, before the appearance of LBs. Interestingly, an increase in mTOR activation, which negatively regulates autophagy, was observed in the laforin KO mice, but not malin KO mice, and overexpression of laforin in cell culture increased autophagy and decreased mTOR activity (Aguado et al. 2010). However, subsequent studies have shown that the autophagy impairment in LD models is likely a secondary effect of glycogen accumulation as autophagic defects are reversed when glycogen synthesis is ablated (Garyali et al. 2014; Duran et al. 2014). Mouse models also indicate the absence of laforin or malin leads to increased numbers of reactive glia and microglia and increased expression of cytokines, decreased mitochondrial function and antioxidant enzymes, and altered glutamate transport (Roma-Mateo et al. 2014; Lopez-Gonzalez et al. 2016; Munoz-Ballester et al. 2016). These effects could be directly related to the absence of laforin or malin, or they could be secondary effects of aberrant glycogen metabolism and/or a loss of metabolic control in the cell.
In addition to dephosphorylating glycogen, some studies have reported that laforin interacts with and dephosphorylates glycogen synthase kinase3β (GSK3β) at serine-9 and modifies the function of GSK3β in Wnt signaling (Lohi et al. 2005; Wang et al. 2006). However, other groups have not observed interaction with GSK3β or its dephosphorylation by laforin (Wang et al. 2007; Worby et al. 2006). Additionally, one group showed that laforin dephosphorylates the microtubule-stabilizing protein tau, but this result has not been confirmed in vitro using purified components and could be an indirect effect (Puri et al. 2009).
The Role of Laforin in Lafora Disease
A number of recent studies from the Minassian, Roach and Guinovart labs have elegantly shown that glycogen accumulation is the pathogenic cause in LD (Fig. 2a). Protein targeting to glycogen (PTG) is a regulatory subunit of Protein Phosphatase 1 (PP1), which dephosphorylates glycogen synthase (GS) and glycogen phosphorylase (GP). GS promotes synthesis and GP promotes the breakdown of glycogen, and these enzymes are down and upregulated by phosphorylation, respectively. Thus, PTG acts through PP1 to promote glycogen synthesis. The Roach and Minassian lab generated mice lacking both laforin and PTG and showed that the double KO animals had normal glycogen levels and no signs of neurodegeneration, indicating they had rescued the disease (Turnbull et al. 2011). The same rescue was achieved by knocking out PTG and malin, or when glycogen synthesis was completely eliminated by knocking out GS and laforin or GS and malin; these mice have no LBs and no neurological impairments (Duran et al. 2014; Pederson et al. 2013; Turnbull et al. 2014). Furthermore, in cell culture, glycogen accumulation leads to neuronal cell death, and constitutive activation of GS in flies and mice leads to decreased lifespan, locomotion problems, and neuronal loss, indicating glycogen accumulation drives neurodegeneration (Vilchez et al. 2007; Duran et al. 2012). Thus, it is clear that the loss of laforin or malin causes LD due to a loss of control over glycogen metabolism.
How laforin and malin promote normal glycogen metabolism is still unclear, but laforin and malin may function both independently and as a complex to regulate glycogen metabolic enzymes (See “Interactions and Signaling”). The importance of the phosphatase activity of laforin has been challenged by a recent study in which a catalytically inactive laforin was ectopically expressed in laforin KO mice (Gayarre et al. 2014). However, this protein was grossly overexpressed, almost 100-fold more than endogenous laforin in the wild-type mice. If laforin regulates glycogen metabolism both via dephosphorylation and as an adaptor protein for malin, gross overexpression even of a catalytically inactive form may compensate for the loss of phosphatase activity through its role as an adaptor protein.
Studies from the 1990s recognized that glycogen contains trace amounts of phosphate (Lomako et al. 1994, 1993). One study proposed that the phosphate content of glycogen may be related to the age of the granule, and increased phosphate may be a metabolic marker that promoted lysosomal degradation of glycogen (Lomako et al. 1994). Glycogen is degraded via two pathways: by the action of GP and GDE and also via the lysosome in a process recently termed “glycophagy” (Jiang et al. 2011). Phosphate may be a marker to distinguish these two pathways. Further, a recent study reported that laforin and glycogen phosphate play a role in glycogen remodeling following exhausting exercise, since phosphate content remained suppressed in WT mice following glycogen depletion, and glycogen remodeling was delayed in laforin KO mice (Irimia et al. 2015).
In plants, glucose is stored in the form of starch; the long glucan chains form double helices that exclude water, rendering starch a densely packed, insoluble macromolecule that is inaccessible to the enzymes that break it down (Bertoft 2015). Phosphorylation of the outer chains at C6 and C3 positions by kinases promotes solubilization, providing access to starch hydrolytic enzymes (Blennow 2015; Smirnova et al. 2015; Pfister and Zeeman 2016). However, phosphorylation can also block the action of these enzymes, so the phosphatases SEX4 and LSF2 are also required to dephosphorylate the chains (Silver et al. 2014). The absence of a kinase or phosphatase in plants leads to a starch-excess phenotype, much like the absence of laforin leads to LB formation in humans and mice. Phosphate in glycogen is likely to play an analogous, although distinct, role in regulating its metabolism.
Recent studies have shown that glycogen plays an important and dynamic role in the brain (Brown 2004; Gibbs et al. 2007; Swanson et al. 1990; Sickmann et al. 2012; Dinuzzo et al. 2014a, b; Duran et al. 2013). Contrary to the long-held belief that only astrocytes can produce glycogen, Guinovart and coworkers have shown that neurons possess glycogen metabolic enzymes, but glycogen must be tightly regulated in neurons: neuronal glycogen promotes cell survival in hypoxic conditions while its accumulation leads to cell death (Valles-Ortega et al. 2011; Vilchez et al. 2007; Duran et al. 2014; Saez et al. 2014). Glycogen regulation is complex: the enzymes that directly remodel glycogen are controlled by reversible phosphorylation and allosteric modulators, and glycogen exists in distinct subcellular pools that vary in size and metabolic role. It is likely that there is a spatiotemporal element to its regulation by metabolic enzymes that may involve laforin and malin (Graham et al. 2010; Obel et al. 2012).
Laforin is a bimodular protein that contains an amino-terminal CBM followed by a DSP domain and is encoded by the EPM2A gene. Mutations in EPM2A result in LD. Laforin has a number of potential functions: binding to glycogen, dephosphorylating glycogen, acting as a scaffold on the glycogen molecule, and mediating ubiquitination by malin. With substantial in vitro biochemical data, mouse models, and studies in human patients, laforin is undisputedly a glucan phosphatase, and this enzyme class is conserved in many organisms, from protists to plants and humans, justifying its importance in many forms of life (Gentry et al. 2007, 2009; Gentry and Pace 2009). There are also numerous studies from many labs supporting the interaction of laforin with malin and various glycogen metabolic enzymes (Fig. 3). Although it is not yet clear how laforin is involved in the regulation of these enzymes, it is clear that LD centers on glycogen metabolism, in which laforin plays a critical role.
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