Abstract
Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforinmalin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/ quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a(-/-), Epm2b(-/-), and Epm2a(-/-) Epm2b(-/-) mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b(-/-) and Epm2a(-/-) Epm2b(-/-) cells) but not laforin (Epm2a(-/-) cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal.
Highlights
Lafora disease involves impaired glycogen metabolism and protein degradation
Glycogen Levels in mouse primary embryonic fibroblasts (MEFs) from Mouse Models of Lafora Disease—Because Lafora disease is characterized by the accumulation of glycogen in patients and the different mouse models (10 –13), glycogen content was analyzed in MEFs from wild type (WT), Epm2aϪ/Ϫ (LKO), Epm2bϪ/Ϫ (MKO), and double Epm2aϪ/Ϫ Epm2bϪ/Ϫ (DKO) mice
Recent studies on Lafora disease indicate that the Lafora disease proteins, laforin and malin, may be involved in the control of a number of cellular functions and/or pathways, including glycogen metabolism and protein quality control
Summary
Lafora disease involves impaired glycogen metabolism and protein degradation. Results: Impaired proteasomal degradation and mTOR-dependent autophagy in the absence of laforin or malin but decreased LAMP1 and GABARAPL1, perhaps indicative of defective lysosomal glycogen trafficking, only in malin deficiency. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents Both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. We analyzed the effect of defects in laforin and malin on protein degradation and quality control by investigating the three different arms of the cellular quality control process (the ubiquitin-proteasomal pathway, the autophago-lysosomal pathway, and the ER stress response) in mouse primary embryonic fibroblasts (MEFs) derived from Epm2aϪ/Ϫ, Epm2bϪ/Ϫ, and double Epm2aϪ/Ϫ Epm2bϪ/Ϫ mice. Loss of laforin or malin led generally to similar phenotypes, we found a novel potential function of malin on lysosomes not shared by laforin
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