Increased laforin and laforin binding to glycogen underlie Lafora body formation in malin-deficient Lafora disease

Abstract

The solubility of glycogen, essential to its metabolism, is a property of its shape, a sphere generated through extensive branching during synthesis. Lafora disease (LD) is a severe teenage-onset neurodegenerative epilepsy and results from multiorgan accumulations, termed Lafora bodies (LB), of abnormally structured aggregation-prone and digestion-resistant glycogen. LD is caused by loss-of-function mutations in the EPM2A or EPM2B gene, encoding the interacting laforin phosphatase and malin E3 ubiquitin ligase enzymes, respectively. The substrate and function of malin are unknown; an early counterintuitive observation in cell culture experiments that it targets laforin to proteasomal degradation was not pursued until now. The substrate and function of laforin have recently been elucidated. Laforin dephosphorylates glycogen during synthesis, without which phosphate ions interfere with and distort glycogen construction, leading to LB. We hypothesized that laforin in excess or not removed following its action on glycogen also interferes with glycogen formation. We show in malin-deficient mice that the absence of malin results in massively increased laforin preceding the appearance of LB and that laforin gradually accumulates in glycogen, which corresponds to progressive LB generation. We show that increasing the amounts of laforin in cell culture causes LB formation and that this occurs only with glycogen binding-competent laforin. In summary, malin deficiency causes increased laforin, increased laforin binding to glycogen, and LB formation. Furthermore, increased levels of laforin, when it can bind glycogen, causes LB. We conclude that malin functions to regulate laforin and that malin deficiency at least in part causes LB and LD through increased laforin binding to glycogen.

FIGURE 1.

Brain LB and glycogen quantity and quality in young and old malin-deficient (Epm2b^−/−) mice. A–C, representative brain sections (hippocampal region) from Epm2b^−/− mice stained with PASD. LB, examples of which are shown by arrows, were absent at 1 month (1M; A), present by 3 months (3M; B), and profuse by 12 months (12M; C) of age. D, equivalent section from an Epm2a^−/− mouse at 12 months. Scale bars = 50 μm. E, total brain glycogen (including normal and abnormal (polyglucosan) glycogen) at 1 and 12 months in Epm2b^−/− and Epm2a^−/− mice (n = five to six animals per genotype). Data are reported in micromoles of glucose from glycogen/g of tissue. WT, wild-type; KO, knock-out. F, branching degree of glycogen from skeletal muscle in 1-month-old Epm2b^−/− mice is the same as in wild-type mice (n = three animals per genotype). Iodine intercalates polysaccharide helical chains to generate characteristic spectra of visible light absorption, and the degree of branching is inversely proportional to the wavelength at which maximal amount of light is absorbed (33).

FIGURE 2.

GS quantity and activity in 1-month-old malin-deficient (Epm2b^−/−) mice. A, GS quantity in total brain extract (n = three mice per genotype). B, GS quantity in the LSS (10,000 × g) and LSP of brain extracts (n = three mice per genotype). C, GS activity in skeletal muscle (activity ratio = GS activity in the absence of any added Glc-6-P (G6P) divided by that at 8 mm Glc-6-P; n = five mice per genotype). WT, wild-type; KO, knock-out.

FIGURE 3.

Glycogen phosphate in skeletal muscle (nanomoles of phosphate/μmol of glucose from glycogen). WT, wild-type; KO, knock-out.

FIGURE 4.

Laforin in brain tissue of malin-deficient (Epm2b^−/−) mice. A, total laforin levels. B, laforin levels in the LSS (10,000 × g) and LSP. C, left and middle panels, laforin immunogold electron microscopy. Note the increase in laforin levels (gold particles (black dots)) in knock-out (KO) mice. The asterisks indicate post-synaptic density. Right panel, gold particle density quantification. D, Epm2a quantitative RT-PCR in Epm2b^−/− brain. Quantity is relative to levels of the control gene hydroxymethylbilane synthase. E, laforin levels in high speed (200,000 × g) supernatants (HSS) and high speed glycogen-containing pellets (HSP).

FIGURE 5.

Glycogen binding-competent laforin overexpression results in formation of LB-like structures. First row, empty Myc vector; second and third rows, glycogen binding-competent Myc-tagged wild-type and C265S mutant laforin; fourth and fifth rows, Myc-tagged W32G and F83L mutant laforin, which did not bind glycogen. Green, Myc immunostaining; red, glycogen; blue, DAPI. Scale bars = 10 μm. See also supplemental Fig. 1 for a lower magnification showing a wider field of cells.

FIGURE 6.

LB-like aggregates resulting from laforin overexpression resist amylase digestion. See the legend to Fig. 5 for description of rows. Left panels, staining with PAS; right panels, staining with PASD. Scale bars = 10 μm.

FIGURE 7.

Glycogen is poorly branched in LB-like aggregates. A, iodine spectra of glycogen preparations enriched for the insoluble glycogen aggregates from cells transfected as described for Figs. 5 and 6. Note that the wavelengths of maximal visible light absorption by glycogen in cells overexpressing glycogen-binding laforin (red curves) are shifted close to that of amylopectin (AP; green curve), whereas those of non-glycogen-binding laforin (black curves) are close to that of glycogen from cells transfected with empty vector (blue curve). B, same experiment as in A repeated three times (three separate transfections per clone). The average wavelength at maximal absorption is shown for each clone. C, glycogen quantities in the cells from B.

FIGURE 8.

Model of LB pathogenesis. In normal glycogen metabolism, a recurring GS enzymatic error incorporates phosphate into glycogen. Laforin (laf) removes this phosphate and then is itself removed by malin (mal)-mediated degradation. In laforin deficiency, phosphate cannot be removed and accumulates. In malin deficiency, laforin binds glycogen to remove the phosphate but then cannot be removed, itself accumulating in glycogen. In laforin or malin deficiency, non-removal of phosphate or laforin disrupts the intricate spherical structure of glycogen, leading it to precipitate and accumulate in LB. Ub, ubiquitin.