Laforin-malin complex degrades polyglucosan bodies in concert with glycogen debranching enzyme and brain isoform glycogen phosphorylase

Abstract

In Lafora disease (LD), the deficiency of either EPM2A or NHLRC1, the genes encoding the phosphatase laforin and E3 ligase, respectively, causes massive accumulation of less-branched glycogen inclusions, known as Lafora bodies, also called polyglucosan bodies (PBs), in several types of cells including neurons. The biochemical mechanism underlying the PB accumulation, however, remains undefined. We recently demonstrated that laforin is a phosphatase of muscle glycogen synthase (GS1) in PBs, and that laforin recruits malin, together reducing PBs. We show here that accomplishment of PB degradation requires a protein assembly consisting of at least four key enzymes: laforin and malin in a complex, and the glycogenolytic enzymes, glycogen debranching enzyme 1 (AGL1) and brain isoform glycogen phosphorylase (GPBB). Once GS1-synthesized polyglucosan accumulates into PBs, laforin recruits malin to the PBs where laforin dephosphorylates, and malin degrades the GS1 in concert with GPBB and AGL1, resulting in a breakdown of polyglucosan. Without fountional laforin-malin complex assembled on PBs, GPBB and AGL1 together are unable to efficiently breakdown polyglucosan. All these events take place on PBs and in cytoplasm. Deficiency of each of the four enzymes causes PB accumulation in the cytoplasm of affected cells. Demonstration of the molecular mechanisms underlying PB degradation lays a substantial biochemical foundation that may lead to understanding how PB metabolizes and why mutations of either EPM2A or NHLRC1 in humans cause LD. Mutations in AGL1 or GPBB may cause diseases related to PB accumulation.

Fig. 1

Proteasomal inhibition increases glycogen-positive bodies that intertwine with proteins GS1, AGL1 and GPBB. a Proteasomal inhibition by MG132 generates PBs. HEK293 cells grown on cover-glass were treated with or without 2.5μM MG132 in the culture medium for 8 hrs before methanol fixation and PAS staining. The staining was carried out after the fixed cells were pretreated with or without 0.2U/ml α-amylase for 15 min. b Short-term, low-dose treatment with MG132 increases insoluble glycogen without apoptotic induction. After treatment of HEK293 with 2.5 μM MG132 for 8 hrs or 10 μM for 20 hrs, insoluble glycogen was isolated, quantified and expressed as a value divided by protein content in the amyloglucosidase/amylase-digested solution. Vehicle DMSO treatment of the cells for 8 or 20 hrs gave a similar glycogen content and the 8-hr treatment shown as control. The cells after treated with 5 μM MG132 for 8 or 10 hrs were subjected to apoptotic analysis by flow cytometry after being stained with Annexin V (positive for apoptosis) and nuclear dye DAPI (positive for death). c,d MG132-induced PBs contain GS1, p-GS1, AGL1 and GPBB. Untransfected or transfected HEK293 cells with AGL1-Flag or GPBB-Flag were or were not treated with 2.5μM MG132 for 8 hrs before being fixed for immunofluorescent staining with antibodies to endogenous GS1, pS641/645 GS1, GPBB or glycogen, or to exogenous Flag-tagged proteins. Only merged fluorescent photos are shown. Scale bar, 10μm. e Western-blot detected these components in the isolated PBs from HEK293 cells, treated as in c.

Fig. 2

Functional laforin-malin complex prevents PB accumulation. a,b Dysfunctional laforin-malin complex accumulates PBs that are stained positively for GS1, p-GS1, AGL1, GPBB and glycogen. HEK293 cells were transfected with combined plasmids for 36 hr as indicated above the photos. After transfection, the cells were fixed by methanol and doubly stained with antibodies to tags (above the photos) or endogenous glycogen, GS1 or pS641/645 GS1 (in the photos). c Laforin-L87P complex-generated PBs are resistant to α-amylase hydrolysis. Transfected HEK293 cells, as in a, were pretreated with or without 0.2U/ml α-amylase for 30 min before being stained with PAS reagents. d Laforin-L87P complex-generated PBs were located in the cytoplasm but not in lysosomes and autophagosomes. The transfected HEK293 cells were fixed by methanol or by 4% paraformaldehyde (for the LC3-EGFP transfection) and doubly stained, either with antibodies to tags or LAMP1; the LC3-EGFP was not stained with antibody. Scale bar, 10μm.

Fig. 3

Laforin-malin complex degrades GS1 in PB pool. a Functional laforin-malin complex degrades GS1 in PB deposit. HEK293 cells were transfected for 36 hrs with the indicated tagged plasmids, and were lysed by 0.55% NP40 lysis buffer to separate cytosol and polyglucosan pellet by serial centrifugation. Ubiquitination was detected in the two fractions after the pellet was digested completely with amyloglucosidase and amylase. b Both laforin and malin are required for the degradations of GS1 and PBs. The transfected HEK293 cells, as in a, were fixed and doubly stained with antibodies to tags or endogenous p-GS1 or K48-linked polyubiquitin. c,d Laforin-malin complex did not degrade the GS1, which was incapable of binding to and synthesizing glycogen. HEK293 cells were cotransfected with laforin-L87P or laforin-malin complex, together with GS1, its catalytically inactive mutant GS1-EE2A, or its glycogen-binding mutant GS1-B-Mt, for 36 hrs before being fixed for immunostaining (c) or PB isolation (d). IP-Western blotted on PBs after completely digested with amyloglucosidase and amylase. Scale bars in b, 10μm; in c, 20μm

Fig. 4

Functional laforin-malin complex, in concert with AGL1 and GPBB, degrades PBs in vivo. a,b Laforin-malin complex efficiently removes PBs synthesized by GS1 or its active truncated forms in vivo. HEK293 cells were transfected alone or in combinations with the indicated plasmids for 36 hrs before being fixed for double staining by using antibodies to tags (out of the photos) or glycogen (in the photos). c PAS staining shows PB removal by the laforin–malin complex in the cotransfected HEK293 cells as indicated. d AGL1 and GPBB are required for PB removal by laforin-malin complex. HEK293 cells were cotransfected with plasmids of GS1-ΔN10, Laforin (L), Malin (M), shAGL1, shGPBB, empty vector (V) or Scrambled sh control (Sr) as indicated for 36 hrs, and then fixed with methanol for PB staining by using antibody to glycogen. Scale bars: a,b, 10 μm; c,d, 50 μm .

Fig. 4

Functional laforin-malin complex, in concert with AGL1 and GPBB, degrades PBs in vivo. a,b Laforin-malin complex efficiently removes PBs synthesized by GS1 or its active truncated forms in vivo. HEK293 cells were transfected alone or in combinations with the indicated plasmids for 36 hrs before being fixed for double staining by using antibodies to tags (out of the photos) or glycogen (in the photos). c PAS staining shows PB removal by the laforin–malin complex in the cotransfected HEK293 cells as indicated. d AGL1 and GPBB are required for PB removal by laforin-malin complex. HEK293 cells were cotransfected with plasmids of GS1-ΔN10, Laforin (L), Malin (M), shAGL1, shGPBB, empty vector (V) or Scrambled sh control (Sr) as indicated for 36 hrs, and then fixed with methanol for PB staining by using antibody to glycogen. Scale bars: a,b, 10 μm; c,d, 50 μm .

Fig. 5

Functional laforin-malin complex, together with AGL1 and GPBB, degrades PBs in vitro. a Sequential transfection shows PB removal by the functional laforin-malin complex. HEK293 cells were transfected first with GS1 along with laforin or C265S for 24 hrs to allow PB formation, then transfected a second time with malin or its mutant L87P for another 24 hrs. After the sequential transfection, the cells were fixed for double staining to detect PBs. b,c Functional laforin-malin complex disrupts PBs in vitro. The isolated PBs of GS1-ΔN10 were digested with the isolated functional or dysfunctional laforin-malin complex, which were all isolated from transfected HEK293 cells, in glycogen hydrolysis buffer for 24 hrs (b) or 1 hr (c) at 37°C. After completing the digestion and following a 18,000g, 30-min centrifugation, the supernatant of PBs was subjected to Western-blot (b) or G1P determination (c). The parallel, isolated components from empty vector-transfected HEK293 cells served as the control for the laforin-malin complex. GSK3β acts as loading control of GS1-ΔN10 polyglucosan. The G6P was determined after conversion of G1P to G6P by phosphoglucomutase (PGM). G6P content in the digested supernatant of GS1-ΔN10 polyglucosan with amyloglucosidase and α-amylase served as a negative control for G1P (c). Scale bars, 10μm.

Fig. 6

The laforin-malin complex in concert with AGL1 and GPBB degrades PBs in neuronal cells. a AGL1 and GPBB are accumulated in the PB deposits isolated from N2A cells that stably express the silencer of small hairpin (sh) RNA of Epm2a (shL) or Nhlrc1 (shM). AGL1 or GPBB was transfected into shL or shM or scrambled control (Sr) cells for 36 hrs before PB isolation. Western-blot detected the distribution of AGL1, GPBB, GS1 and p-GS1 in the cytosolic portions and PB deposits. The densitometry quantitation relative to GAPDH was shown in the bottom of each of the western blotting. b Knockdown efficiency of shRNA of Agl (shA) or Pygb (shG). N2A cells were lentivirally infected with shA or shG silencer for 48 hrs, and then selected with 6.5 μg/ml blasticidin for one week (about 70% cells positive for the silencer marker EGFP) before being lysed for detection of endogenous Agl1 and Gpbb. c,d Knockdown of Agl1 or Gygb increases the accumulation of laforin and malin in PB deposits and reduced PB degradation by laforin-malin complex. N2A cells stably expressing shA or shG were transiently transfected with plasmids of malin, laforin or both for 36 hrs. The accumulation of laforin and malin in shA or shG N2A cells was detected by Western-blot (c). Quantitation of glycogen content in PB deposits was determined after complete digestion (d). e,f AGL1 and GPBB are accumulated in neurons of Epm2a KO mice. LBs isolated from brains of 5-month-old Epm2a KO mice were digested and subjected to analysis of AGL1 and GPBB by western-blot (e). Insoluble glycogen isolated from age-matched WT mice acted as a control. Quantitation of the accumulation of indicated components in the insoluble glycogen pools of the two strains is shown (f). g GPBB accumulation in LBs of Epm2a KO and malin KO mice. Hippocampal sections of 5-month-old mice of the two strains were stained with antibodies to GPBB or glycogen or the astrocyte marker GFAP. Scale bars, 50 μm.

Fig. 6

The laforin-malin complex in concert with AGL1 and GPBB degrades PBs in neuronal cells. a AGL1 and GPBB are accumulated in the PB deposits isolated from N2A cells that stably express the silencer of small hairpin (sh) RNA of Epm2a (shL) or Nhlrc1 (shM). AGL1 or GPBB was transfected into shL or shM or scrambled control (Sr) cells for 36 hrs before PB isolation. Western-blot detected the distribution of AGL1, GPBB, GS1 and p-GS1 in the cytosolic portions and PB deposits. The densitometry quantitation relative to GAPDH was shown in the bottom of each of the western blotting. b Knockdown efficiency of shRNA of Agl (shA) or Pygb (shG). N2A cells were lentivirally infected with shA or shG silencer for 48 hrs, and then selected with 6.5 μg/ml blasticidin for one week (about 70% cells positive for the silencer marker EGFP) before being lysed for detection of endogenous Agl1 and Gpbb. c,d Knockdown of Agl1 or Gygb increases the accumulation of laforin and malin in PB deposits and reduced PB degradation by laforin-malin complex. N2A cells stably expressing shA or shG were transiently transfected with plasmids of malin, laforin or both for 36 hrs. The accumulation of laforin and malin in shA or shG N2A cells was detected by Western-blot (c). Quantitation of glycogen content in PB deposits was determined after complete digestion (d). e,f AGL1 and GPBB are accumulated in neurons of Epm2a KO mice. LBs isolated from brains of 5-month-old Epm2a KO mice were digested and subjected to analysis of AGL1 and GPBB by western-blot (e). Insoluble glycogen isolated from age-matched WT mice acted as a control. Quantitation of the accumulation of indicated components in the insoluble glycogen pools of the two strains is shown (f). g GPBB accumulation in LBs of Epm2a KO and malin KO mice. Hippocampal sections of 5-month-old mice of the two strains were stained with antibodies to GPBB or glycogen or the astrocyte marker GFAP. Scale bars, 50 μm.