Hyperphosphorylation and Aggregation of Tau in Laforin-deficient Mice, an Animal Model for Lafora Disease

Lafora progressive myoclonous epilepsy (Lafora disease; LD) is caused by mutations in the EPM2A gene encoding a dual specificity protein phosphatase named laforin. Our analyses on the Epm2a gene knock-out mice, which developed most of the symptoms of LD, reveal the presence of hyperphosphorylated Tau protein (Ser³⁹⁶ and Ser²⁰²) as neurofibrillary tangles (NFTs) in the brain. Intriguingly, NFTs were also observed in the skeletal muscle tissues of the knock-out mice. The hyperphosphorylation of Tau was associated with increased levels of the active form of GSK3β. The observations on Tau protein were replicated in cell lines using laforin overexpression and knockdown approaches. We also show here that laforin and Tau proteins physically interact and that the interaction was limited to the phosphatase domain of laforin. Finally, our in vitro and in vivo assays demonstrate that laforin dephosphorylates Tau, and therefore laforin is a novel Tau phosphatase. Taken together, our study suggests that laforin is one of the critical regulators of Tau protein, that the NFTs could underlie some of the symptoms seen in LD, and that laforin can contribute to the NFT formation in Alzheimer disease and other tauopathies.Lafora disease (LD)² is an autosomal recessive and a fatal form of progressive myoclonus epilepsy characterized by the presence of Lafora polyglucosan bodies in the affected tissues (1). The symptoms of LD include stimulus-sensitive epilepsy, dementia, ataxia, and rapid neurologic deterioration (1, 2). LD is caused by mutations in the EPM2A gene encoding laforin, a dual specificity protein phosphatase, or in the NHLRC1 gene encoding malin, an E3 ubiquitin ligase (3–7). Both laforin and malin are ubiquitously expressed (3, 5), associated with the endoplasmic reticulum (4, 7), form aggresome upon proteasomal blockade (7), and clear misfolded protein through ubiquitin-proteasome (8). Laforin has two functional domains: a phosphatase domain (dual specificity phosphatase domain; DSPD) and a carbohydrate binding domain (CBD) (9). The CBD helps laforin to target to the glycogen particle and to the Lafora bodies (9, 10), and the DSPD of laforin dephosphorylates carbohydrate moieties (11). Recent studies have further shown that laforin and malin together regulate the cellular levels of PTG, the adaptor protein targeting to glycogen, and that the loss of either malin or laforin results in increased levels of PTG that eventually lead to excessive glycogen deposition (12–14). Although this model explains the genesis of Lafora bodies, the molecular etiology of LD is yet to be understood. For example, unlike this cell line study (12), the presence of Lafora bodies does not lead to neuronal cell death in the two murine models of LD (10, 15), and no difference in the level of PTG was seen in laforin-deficient mice (16).³ However, widespread degeneration of neurons was seen in laforin-deficient mouse with the absence of Lafora bodies, suggesting that the polyglucosan bodies may not play a primary role in the epileptogenesis (15). The laforin dominant-negative transgenic mice line also developed Lafora bodies but had no signs of neurodegeneration or epileptic seizures (10). Thus, the neurodegenerative changes are likely to underlie the etiology of some of the LD symptoms (1). The mouse model developed by the knockdown of the Epm2a gene exhibited a majority of the symptoms known in LD, including the ataxia, spontaneous myoclonic seizures, EEG epileptiform activity, and impaired behavioral responses (15). The knock-out animals showed a number of degenerative changes that include swelling and/or loss of morphological features of mitochondria, endoplasmic reticulum, Golgi apparatus, and the neuronal processes (15). Preliminary histochemical investigations have also suggested the possible presence of neurofibrillary tangles (NFTs) in the knock-out mice (17). In this study, we have characterized the biochemical properties of Tau protein in the animal model of LD and identified laforin as an interacting partner of Tau. Our study identifies laforin to be one of the critical regulators of Tau protein and suggests that the Tau pathology might underlie some of the symptoms seen in LD.