EMP2A gene is located on chromosome 6q24. It consists of four exons coding for a 331 amino acid protein called laforin (10). Laforin has two isoforms, A and B which localize to the ER and to the nucleus, respectively (16, 17). The isoforms differ in their C-termini, and mutations in the unique isoform A’s C-terminus suggests that this is the disease-relevant isoform (17). To date, 40 different mutations and four polymorphisms were identified in this gene (18). These include missense and nonsense mutations, frameshifts and deletions located in the coding region of the gene.

Laforin is a unique protein in that it contains a carbohydrate-binding domain (CBD) of the CBM20 type (19) in its N-terminus and a dual-specificity protein tyrosine phosphatase (DSP) domain in its C-terminus (6, 20). Given the accumulation of polyglycosans in LD and the presence of a CBD, laforin is thought to play an important role in glycogen metabolism (either its synthesis or degradation) (6). Importantly, self-dimerization appears to be necessary for laforin to be functional in vivo (21, 22).

Co-immunoprecipitation studies suggest that full-length laforin binds an uncharacterized protein termed EMP2AIP1 (for EPM2A interacting protein). This protein does not appear to be responsible for LD in those LD families with normal EPM2A and EPM2B genes (23). HIRIP5 is another protein shown to interact with laforin. This protein contains a NifU-like domain and a putative MurD ligase domain. However the role of those domains in HIRIP5 function is not yet clear. Interestingly HIRIP5, like laforin, is ubiquitously expressed in subregions of the brain, but predominantly in the cerebellum and hippocampus. This protein also co-localizes with laforin at the subcellular level. Finally, laforin was able to dephosphorylate HIRIP5 on both tyrosine and serine/threonine residues, suggesting that HIRIP5 is a substrate for laforin (24). A third protein shown to interact with laforin, called PTG, is a regulatory subunit of protein phosphatese-1 (PP1) that enhances glycogen accumulation (21). It was shown that the G240S missense mutation identified in some LD patients disrupts the interaction between laforin and PTG (while glycogen binding and phosphatase activity remain preserved). This observation suggests that PTG is critical for laforin function and that laforin is part of a complex of proteins associated with glycogen and may have a role in regulating its metabolism. Studies using a mammalian two-hybrid system demonstrated that laforin interacts with glycogen synthase kinase-3 (GSK3). Furthermore, laforin reduces GSK3 Ser 9 phosphorylation (25, 26). GSK3 is a potent glycogen synthase (GS) inhibitor. The relationship between GSK3, GS, laforin and LBs is discussed below.

EPM2B gene was identified through genome-wide linkage scan followed by haplotype analysis and homozygosity mapping performed in a cluster of French-Canadian families from Quebec (11, 27). To date, 40 mutations have been found in the EMP2B gene, including insertion, missense and nonsense changes, frameshifts and deletions in both compound heterozygous as well as homozygous states. The EMP2B gene product encodes a 395 amino acid protein named malin which contains a zinc finger of the RING type at the N-terminus and six NHL-repeat motifs at the C-terminus. NHL motifs are likely involved in protein-protein interactions, while the RING-finger motif of malin is typical of E3 ubiquitin ligases. Sub-celllular localization studies showed that MYC-tagged malin, similarly to laforin, also localizes to the cytoplasm at the ER and the nucleus (16, 17, 28).

The canine model was observed in approximately 5% of Miniature Wirehaired Dachshunds (MWHDs) in England. The identified mutation was a dodecamer expansion in the EPM2B gene (32). These animals have a phenotype very similar to the human form of LD, except for the late age of onset (age 6 in dogs, which is equivalent to adulthood in humans).

The transgenic model was created through overexpression of laforin carrying a phosphatase inactivating point mutation (Cys26-6Ser). This dominant-negative model was used to trap the unknown laforin substrate and produce LD pathology. The transgenic LD mouse provided valuable information regarding the laforin protein localization. In brain, this protein localizes to the neuronal soma and dendrites. It was also demonstrated that laforin localizes to the ER, but not to the ribosomes, as initially thought. Importantly, this model allowed the characterization of laforin binding to different polysaccharides, and the preferential binding to polyglucosans over glycogen (33).

Crafting of properly branched and soluble glycogen requires a number of coordinated enzymatic activities. The complex between these enzymes and glycogen has been termed the glycogenosome. A main component of this complex is glycogen synthase (GS), which elongates glycogen strands by adding glucose units. Glycogen branching enzyme (BE) then moves the extended oligosaccharides to branch points, maintaining the globularity and solubility of glycogen. An abnormally high GS to BE ratio results in inadequately branched polysaccharides, namely polyglucosans (36).

Laforin may downregulate GS via PTG-PP1 and via GSK3. PTG serves to target PP1 to the glycogenosome, where PP1 activates GS by dephosphorylation (37). Laforin binds PTG at PTG’s binding site with GS (21). Laforin would therefore downregulate GS by physically outcompeting PTG-PP1 off of GS. GSK3 is the main inhibitor of GS, through phosphorylation of five phosphoregulatory sites on GS (37). Laforin activates GSK3 through dephosphorylation of GSK3 (25, 26). Laforin-activated GSK3 would inactivate GS. In sum, absence of laforin would lead to excess GS activity, GS/BE imbalance, formation of insoluble polyglucosans, and their accumulation into LBs.

The concept of malin and laforin agonistically acting to decrease GS activity in order to promote the right GS/BE balance is in contrast with the observation that malin polyubiquitinates laforin, targeting it for destruction (29). A possible explanation follows: LBs are much more phosphorylated than glycogen, and are in fact more similar to amylopectin than to glycogen. Laforin is able to dephosphorylate amylopectin (38). Therefore, it is possible that laforin could also dephosphorylate LBs, and that the high phosphate content in LBs, compared to normal glycogen, may be a direct consequence of the mutated laforin. Interestingly, glycogen binding appears to inhibit laforin activity (39). Laforin inhibition may be a feedback mechanism to preserve a certain degree of phosphorylation of the glycogen molecule. The role of glycogen dephosphorylation is not clear, but it may be correlated to the maintenance of a properly branched polysaccharide. If laforin activity needs to be kept in check (by glycogen inhibition) to avoid over dephosphorylation of glycogen, it is possible that a mutated malin would lead to lack of ubiquitination and destruction of laforin. Could excess laforin cause such an imbalance of glycogen dephosphorylation to lead to the formation of LBs? Finally, polyglucosans are even more potent inhibitors of laforin DSP activity than normal glycogen. In that case, the initial formation of polyglucosans (either because of mutated laforin, malin or another yet unknown protein) would be aggravated by the further inhibition of any residual laforin activity.