Background Lafora progressive myoclonus epilepsy (Lafora disease; LD) is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others have shown that both proteins form a functional complex that regulates glycogen synthesis by a novel mechanism involving ubiquitination and proteasomal degradation of at least two proteins, glycogen synthase and R5/PTG. Since laforin and malin localized at the endoplasmic reticulum (ER) and their regulatory role likely extend to other proteins unrelated to glycogen metabolism, we postulated that their absence may also affect the ER-unfolded protein response pathway. Methodology/Principal Findings Here, we demonstrate that siRNA silencing of laforin in Hek293 and SH-SY5Y cells increases their sensitivity to agents triggering ER-stress, which correlates with impairment of the ubiquitin-proteasomal pathway and increased apoptosis. Consistent with these findings, analysis of tissue samples from a LD patient lacking laforin, and from a laforin knockout (Epm2a-/-) mouse model of LD, demonstrates constitutive high expression levels of ER-stress markers BIP/Grp78, CHOP and PDI, among others. Conclusions/Significance We demonstrate that, in addition to regulating glycogen synthesis, laforin and malin play a role protecting cells from ER-stress, likely contributing to the elimination of unfolded proteins. These data suggest that proteasomal dysfunction and ER-stress play an important role in the pathogenesis of LD, which may offer novel therapeutic approaches for this fatal neurodegenerative disorder.
References
[1]
Ganesh S, Puri R, Singh S, Mittal S, Dubey D (2006) Recent advances in the molecular basis of Lafora's progressive myoclonus epilepsy. J Hum Genet 51: 1–8.
[2]
Delgado-Escueta AV (2007) Advances in lafora progressive myoclonus epilepsy. Curr Neurol Neurosci Rep 7: 428–433.
[3]
Minassian BA, Lee JR, Herbrick JA, Huizenga J, Soder S, et al. (1998) Mutations in a gene encoding a novel protein tyrosine phosphatase cause progressive myoclonus epilepsy. Nat Genet 20: 171–174.
[4]
Serratosa JM, Gomez-Garre P, Gallardo ME, Anta B, de Bernabe DB, et al. (1999) A novel protein tyrosine phosphatase gene is mutated in progressive myoclonus epilepsy of the Lafora type (EPM2). Hum Mol Genet 8: 345–352.
[5]
Chan EM, Young EJ, Ianzano L, Munteanu I, Zhao X, et al. (2003) Mutations in NHLRC1 cause progressive myoclonus epilepsy. Nat Genet 35: 125–127.
[6]
Chan EM, Omer S, Ahmed M, Bridges LR, Bennett C, et al. (2004) Progressive myoclonus epilepsy with polyglucosans (Lafora disease): evidence for a third locus. Neurology 63: 565–567.
[7]
Minassian BA, Ianzano L, Meloche M, Andermann E, Rouleau GA, et al. (2000) Mutation spectrum and predicted function of laforin in Lafora's progressive myoclonus epilepsy. Neurology 55: 341–346.
[8]
Wang J, Stuckey JA, Wishart MJ, Dixon JE (2002) A unique carbohydrate binding domain targets the lafora disease phosphatase to glycogen. J Biol Chem 277: 2377–2380.
[9]
Lohi H, Ianzano L, Zhao XC, Chan EM, Turnbull J, et al. (2005) Novel glycogen synthase kinase 3 and ubiquitination pathways in progressive myoclonus epilepsy. Hum Mol Genet 14: 2727–2736.
[10]
Gentry MS, Worby CA, Dixon JE (2005) Insights into Lafora disease: malin is an E3 ubiquitin ligase that ubiquitinates and promotes the degradation of laforin. Proc Natl Acad Sci U S A 102: 8501–8506.
[11]
Solaz-Fuster MC, Gimeno-Alcaniz JV, Ros S, Fernandez-Sanchez ME, Garcia-Fojeda B, et al. (2008) Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway. Hum Mol Genet 17: 667–678.
[12]
Vilchez D, Ros S, Cifuentes D, Pujadas L, Valles J, et al. (2007) Mechanism suppressing glycogen synthesis in neurons and its demise in progressive myoclonus epilepsy. Nat Neurosci 10: 1407–1413.
[13]
Cheng A, Zhang M, Gentry MS, Worby CA, Dixon JE, et al. (2007) A role for AGL ubiquitination in the glycogen storage disorders of Lafora and Cori's disease. Genes Dev 21: 2399–2409.
[14]
Worby CA, Gentry MS, Dixon JE (2008) Malin decreases glycogen accumulation by promoting the degradation of protein targeting to glycogen (PTG). J Biol Chem 283: 4069–4076.
[15]
Worby CA, Gentry MS, Dixon JE (2006) Laforin, a dual specificity phosphatase that dephosphorylates complex carbohydrates. J Biol Chem 281: 30412–30418.
[16]
Tagliabracci VS, Turnbull J, Wang W, Girard JM, Zhao X, et al. (2007) Laforin is a glycogen phosphatase, deficiency of which leads to elevated phosphorylation of glycogen in vivo. Proc Natl Acad Sci U S A 104: 19262–19266.
[17]
Gentry MS, Dowen RH 3rd, Worby CA, Mattoo S, Ecker JR, et al. (2007) The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease. J Cell Biol 178: 477–488.
[18]
Tagliabracci VS, Girard JM, Segvich D, Meyer C, Turnbull J, et al. (2008) Abnormal metabolism of glycogen phosphate as a cause for Lafora disease. J Biol Chem 283: 33816–33825.
[19]
Sakai M, Austin J, Witmer F, Trueb L (1970) Studies in myoclonus epilepsy (Lafora body form). II. Polyglucosans in the systemic deposits of myoclonus epilepsy and in corpora amylacea. Neurology 20: 160–176.
[20]
Yokoi S, Nakayama H, Negishi T (1975) Biochemical studies on tissues from a patient with Lafora disease. Clin Chim Acta 62: 415–423.
[21]
Ganesh S, Delgado-Escueta AV, Sakamoto T, Avila MR, Machado-Salas J, et al. (2002) Targeted disruption of the Epm2a gene causes formation of Lafora inclusion bodies, neurodegeneration, ataxia, myoclonus epilepsy and impaired behavioral response in mice. Hum Mol Genet 11: 1251–1262.
[22]
Mittal S, Dubey D, Yamakawa K, Ganesh S (2007) Lafora disease proteins malin and laforin are recruited to aggresomes in response to proteasomal impairment. Hum Mol Genet 16: 753–762.
[23]
Grune T, Jung T, Merker K, Davies KJ (2004) Decreased proteolysis caused by protein aggregates, inclusion bodies, plaques, lipofuscin, ceroid, and ‘aggresomes’ during oxidative stress, aging, and disease. Int J Biochem Cell Biol 36: 2519–2530.
[24]
Seki T, Takahashi H, Adachi N, Abe N, Shimahara T, et al. (2007) Aggregate formation of mutant protein kinase C gamma found in spinocerebellar ataxia type 14 impairs ubiquitin-proteasome system and induces endoplasmic reticulum stress. Eur J Neurosci 26: 3126–3140.
[25]
Ron D, Walter P (2007) Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8: 519–529.
[26]
Yoshida H (2007) ER stress and diseases. Febs J 274: 630–658.
[27]
Schroder M (2008) Endoplasmic reticulum stress responses. Cell Mol Life Sci 65: 862–894.
[28]
Lin JH, Walter P, Yen TS (2008) Endoplasmic reticulum stress in disease pathogenesis. Annu Rev Pathol 3: 399–425.
[29]
Ganesh S, Agarwala KL, Ueda K, Akagi T, Shoda K, et al. (2000) Laforin, defective in the progressive myoclonus epilepsy of Lafora type, is a dual-specificity phosphatase associated with polyribosomes. Hum Mol Genet 9: 2251–2261.
[30]
Ianzano L, Young EJ, Zhao XC, Chan EM, Rodriguez MT, et al. (2004) Loss of function of the cytoplasmic isoform of the protein laforin (EPM2A) causes Lafora progressive myoclonus epilepsy. Hum Mutat 23: 170–176.
[31]
Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, et al. (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101: 249–258.
[32]
Paschen W (2003) Endoplasmic reticulum: a primary target in various acute disorders and degenerative diseases of the brain. Cell Calcium 34: 365–383.
[33]
Meusser B, Hirsch C, Jarosch E, Sommer T (2005) ERAD: the long road to destruction. Nat Cell Biol 7: 766–772.
[34]
Carvalho P, Goder V, Rapoport TA (2006) Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126: 361–373.
[35]
Raasi S, Wolf DH (2007) Ubiquitin receptors and ERAD: a network of pathways to the proteasome. Semin Cell Dev Biol 18: 780–791.
[36]
Kostova Z, Tsai YC, Weissman AM (2007) Ubiquitin ligases, critical mediators of endoplasmic reticulum-associated degradation. Semin Cell Dev Biol 18: 770–779.
[37]
Garyali P, Siwach P, Singh PK, Puri R, Mittal S, et al. (2009) The malin-laforin complex suppresses the cellular toxicity of misfolded proteins by promoting their degradation through the ubiquitin-proteasome system. Hum Mol Genet 18: 688–700.
[38]
Gomez-Garre P, Gutierrez-Delicado E, Gomez-Abad C, Morales-Corraliza J, Villanueva VE, et al. (2007) Hepatic disease as the first manifestation of progressive myoclonus epilepsy of Lafora. Neurology 68: 1369–1373.
[39]
Wang Y, Liu Y, Wu C, McNally B, Liu Y, et al. (2008) Laforin confers cancer resistance to energy deprivation-induced apoptosis. Cancer Res 68: 4039–4044.
[40]
Imai Y, Soda M, Takahashi R (2000) Parkin suppresses unfolded protein stress-induced cell death through its E3 ubiquitin-protein ligase activity. J Biol Chem 275: 35661–35664.
[41]
Tsai YC, Fishman PS, Thakor NV, Oyler GA (2003) Parkin facilitates the elimination of expanded polyglutamine proteins and leads to preservation of proteasome function. J Biol Chem 278: 22044–22055.
[42]
Dikshit P, Jana NR (2007) The co-chaperone CHIP is induced in various stresses and confers protection to cells. Biochem Biophys Res Commun 357: 761–765.
