BackgroundLafora 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 FindingsHere, 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/SignificanceWe 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.

The human microbiota correlates closely with the health status of its host. This article analyzes the microbial composition of several subjects under different conditions over time spans that ranged from days to months. Using the Langevin equation as the basis of our mathematical framework to evaluate microbial temporal stability, we proved that stable microbiotas can be distinguished from unstable microbiotas. This initial step will help us to determine how temporal microbiota stability is related to a subject’s health status and to develop a more comprehensive framework that will provide greater insight into this complex system.

R5/PTG is one of the glycogen targeting subunits of type 1 protein phosphatase, a master regulator of glycogen synthesis. R5/PTG recruits the phosphatase to the places where glycogen synthesis occurs, allowing the activation of glycogen synthase and the inactivation of glycogen phosphorylase, thus increasing glycogen synthesis and decreasing its degradation. In this report, we show that the activity of R5/PTG is regulated by AMP-activated protein kinase (AMPK). We demonstrate that AMPK interacts physically with R5/PTG and modifies its basal phosphorylation status. We have also mapped the major phosphorylation sites of R5/PTG by mass spectrometry analysis, observing that phosphorylation of Ser-8 and Ser-268 increased upon activation of AMPK. We have recently described that the activity of R5/PTG is down-regulated by the laforin-malin complex, composed of a dual specificity phosphatase (laforin) and an E3-ubiquitin ligase (malin). We now demonstrate that phosphorylation of R5/PTG at Ser-8 by AMPK accelerates its laforin/ malin-dependent ubiquitination and subsequent proteasomal degradation, which results in a decrease of its glycogenic activity. Thus, our results define a novel role of AMPK in glycogen homeostasis.Glycogen homeostasis depends mainly on the activity of the enzymes involved in its synthesis (glycogen synthase (GS) 2 ) and its degradation (glycogen phosphorylase). These activities are regulated by a complex mechanism involving both allosteric regulation and phosphorylation (1, 2). Interestingly, although there are several kinases (AMPK, PKA, CKI, and glycogen synthase kinase 3) that inhibit glycogen synthesis through the phosphorylation of GS, there is only one known phosphatase (type 1 protein phosphatase (PP1)) that dephosphorylates both GS (leading to its activation) and glycogen phosphorylase (leading to its inactivation), which results in glycogen accumulation (1, 2). PP1 is recruited to glycogen by a family of glycogen targeting proteins, including: G M , G L , R5/PTG, R6, and R3E (3-7). The regulation of the activity of the holoenzyme formed by the PP1 catalytic subunit and one of these glycogen targeting subunits is different in each case. The glycogenic activity of G M is down-regulated by its PKA-dependent phosphorylation; PKA phosphorylates G M at a site in its PP1 binding motif, which leads to its dissociation from PP1. The glycogenic activity of the G L -PP1c holoenzyme is down-regulated by an allosteric mechanism involving phosphorylase-a (8 -10). Little is known about the regulation of the holoenzymes involving R6 or R3E, but in the case of R5/PTG, we and others have recently described that its glycogenic activity is down-regulated by the laforin-malin complex, composed of a dual specificity phosphatase (laforin) and an E3-ubiquitin ligase (malin), which recognizes and ubiquitinates R5/PTG and targets it for proteasomal-dependent degradation (11-13). Laforin and malin are two key proteins related to Lafora disease (LD, OMIM 254780), an autosomal recessive neurodegenerative disorder c...