Lafora Disease: Mechanisms Involved in Pathogenesis

Indiana University-Purdue University Indianapolis (IUPUI)Lafora disease is a neurodegenerative disorder caused by mutations in either the EPM2A or the EPM2B gene that encode a glycogen phosphatase, laforin and an E3 ubiquitin ligase, malin, respectively. A hallmark of the disease is accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle and heart. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein degradation/quality control. We evaluated three arms of protein quality control (the autophagolysosomal pathway, the ubiquitin-proteasomal pathway, and ER stress response) in embryonic fibroblasts from Epm2a-/-, Epm2b-/- and Epm2a-/- Epm2b-/- mice. There was an mTOR-dependent impairment in autophagy, decreased proteasomal activity but an uncompromised ER stress response in the knockout cells. These defects may be secondary to the glycogen overaccumulation. The absence of malin, but not laforin, decreased the level of LAMP1, a marker of lysosomes, suggesting a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. To understand the physiological role of malin, an unbiased diGly proteomics approach was developed to search for malin substrates. Ubiquitin forms an isopeptide bond with lysine of the protein upon ubiquitination. Proteolysis by trypsin cleaves the C-terminal Arg-Gly-Gly residues in ubiquitin and yields a diGly remnant on the peptides. These diGly peptides were immunoaffinity purified using anti-diGly antibody and then analyzed by mass spectrometry. The mouse skeletal muscle ubiquitylome was studied using diGly proteomics and we identified 244 nonredundant ubiquitination sites in 142 proteins. An approach for differential dimethyl labeling of proteins with diGly immunoaffinity purification was also developed. diGly peptides from skeletal muscle of wild type and Epm2b-/- mice were immunoaffinity purified followed by differential dimethyl labeling and analyzed by mass spectrometry. About 70 proteins were identified that were present in the wild type and absent in the Epm2b-/- muscle tissue. The initial results identified 14 proteins as potential malin substrates, which would need validation in future studies

The malin-laforin complex suppresses the cellular toxicity of misfolded proteins by promoting their degradation through the ubiquitin-proteasome system

Lafora disease (LD), a progressive form of inherited epilepsy, is associated with widespread neurodegeneration and the formation of polyglucosan bodies in the neurons. Laforin, a protein phosphatase, and malin, an E3 ubiquitin ligase, are two of the proteins that are defective in LD. We have shown recently that laforin and malin (referred together as LD proteins) are recruited to aggresome upon proteasomal blockade, possibly to clear misfolded proteins through the ubiquitin–proteasome system (UPS). Here we test this possibility using a variety of cytotoxic misfolded proteins, including the expanded polyglutamine protein, as potential substrates. Laforin and malin, together with Hsp70 as a functional complex, suppress the cellular toxicity of misfolded proteins, and all the three members of this complex are required for this function. Laforin and malin interact with misfolded proteins and promote their degradation through the UPS. LD proteins are recruited to the polyglutamine aggregates and reduce the frequency of aggregate-positive cells. Taken together, our results suggest that the malin–laforin complex is a novel player in the neuronal response to misfolded proteins and could be potential therapeutic targets for neurodegenerative disorders associated with cytotoxic proteins

Novel mutation in the NHLRC1 gene in a Malian family with a severe phenotype of Lafora disease

We studied a Malian family with parental consanguinity and two of eight siblings affected with late-childhood-onset progressive myoclonus epilepsy and cognitive decline, consistent with the diagnosis of Lafora disease. Genetic analysis showed a novel homozygous single-nucleotide variant in the NHLRC1 gene, c.560A>C, producing the missense change H187P. The changed amino acid is highly conserved, and the mutation impairs malin's ability to degrade laforin in vitro. Pathological evaluation showed manifestations of Lafora disease in the entire brain, with particularly severe involvement of the pallidum, thalamus, and cerebellum. Our findings document Lafora disease with severe manifestations in the West African population

Laforin, a Dual Specificity Phosphatase Involved in Lafora Disease, Is Present Mainly as Monomeric Form with Full Phosphatase Activity

Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both in vitro and in vivo. Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity

On Best-Effort Utility Accrual Real-Time Scheduling on Multiprocessors

We consider the problem of scheduling real-time tasks on a multiprocessor system. Our primary focus is scheduling on multiprocessor systems where the total task utilization demand, U, is greater than m, the number of processors on a multiprocessor system---i.e., the total available processing capacity of the system. When U > m, the system is said to be overloaded; otherwise, the system is said to be underloaded. While significant literature exists on multiprocessor real-time scheduling during underloads, little is known about scheduling during overloads, in particular, in the presence of task dependencies---e.g., due to synchronization constraints. We consider real-time tasks that are subject to time/utility function (or TUF) time constraints, which allow task urgency to be expressed independently of task importance---e.g., the most urgent task being the least important. The urgency/importance decoupling allowed by TUFs is especially important during overloads, when not all tasks can be optimally completed. We consider the timeliness optimization objective of maximizing the total accrued utility and the number of deadlines satisfied during overloads, while ensuring task mutual exclusion constraints and freedom from deadlocks. This problem is NP-hard. We develop a class of polynomial-time heuristic algorithms, called the Global Utility Accrual (or GUA) class of algorithms. The algorithms construct a directed acyclic graph representation of the task dependency relationship, and build a global multiprocessor schedule of the zero in-degree tasks to heuristically maximize the total accrued utility and ensure mutual exclusion. Potential deadlocks are detected through a cycle-detection algorithm, and resolved by aborting a task in the deadlock cycle. The GUA class of algorithms include two algorithms, namely, the Non-Greedy Global Utility Accrual (or NG-GUA) and Greedy Global Utility Accrual (or G-GUA) algorithms. NG-GUA and G-GUA differ in the way schedules are constructed towards meeting all task deadlines, when possible to do so. We establish several properties of the algorithms including conditions under which all task deadlines are met, satisfaction of mutual exclusion constraints, and deadlock-freedom. We create a Linux-based real-time kernel called ChronOS for multiprocessors. ChronOS is extended from the PREEMPT_RT real-time Linux patch, which provides optimized interrupt service latencies and real-time locking primitives. ChronOS provides a scheduling framework for the implementation of a broad range of real-time scheduling algorithms, including utility accrual, non-utility accrual, global, and partitioned scheduling algorithms. We implement the GUA class of algorithms and their competitors in ChronOS and conduct experimental studies. The competitors include G-EDF, G-NP-EDF, G-FIFO, gMUA, P-EDF and P-DASA. Our study reveals that the GUA class of algorithms accrue higher utility and satisfy greater number of deadlines than the deadline-based scheduling algorithms by as much as 750% and 600%, respectively. In addition, we observe that G-GUA accrues higher utility than NG-GUA during overloads by as much as 25% while NG-GUA satisfies greater number of deadlines than G-GUA by as much as 5% during underloads.Master of Scienc

Deciphering the role of malin in the Lafora progressive myoclonus epilepsy

8 páginas, 3 figuras.Lafora disease (LD) is a fatal, autosomal recessive neurodegenerative disorder that results in progressive myoclonus epilepsy. A hallmark of LD is the accumulation of insoluble, aberrant glycogen-like structures called Lafora bodies. LD is caused by mutations in the gene encoding the E3 ubiquitin ligase malin or the glucan phosphatase laforin. Although LD was first described in 1911, its symptoms are still lacking a consistent molecular explanation and, consequently, a cure is far from being achieved. Some data suggest that malin forms a functional complex with laforin. This complex promotes the ubiquitination of proteins involved in glycogen metabolism and misregulation of pathways involved in this process results in Lafora body formation. In addition, recent results obtained from both cell culture and LD mouse models have highlighted a role of the laforin-malin complex in the regulation of endoplasmic reticulum-stress and protein clearance pathways. These results suggest that LD should be considered as a novel member of the group of protein clearance diseases such as Parkinson's, Huntington's, or Alzheimer's, in addition to being a glycogen metabolism disease. Herein, we review the latest results concerning the role of malin in LD and attempt to decipher its function. © 2012 IUBMB IUBMB Life, 64(10): 801-808, 2012.This work was supported by a grant from the Spanish Ministry of Education and Science (SAF2011-27442), a grant from la Fundació La Marato de TV3 (ref. 100130) and a grant from Generalitat Valenciana (Prometeo 2009/051) to P.S. and the National Institutes of Health grants R00NS061803, P20RR020171, R01NS070899 and University of Kentucky College of Medicine startup funds to M.S.G.Peer reviewe

ChronOS Linux: a best-effort real-time multiprocessor Linux kernel,”

ABSTRACT We present ChronOS Linux, a best-effort real-time Linux kernel for chip multiprocessors (CMPs). ChronOS addresses the intersection of three problem spaces: a) OS-support for obtaining best-effort timing assurances, b) real-time Linux kernel augmented with the PREEMPT_RT patch, and c) OS support for CMP-aware real-time scheduling. While each of these spaces have been studied in the past, their intersection, which has strong problem motivations, was previously empty. Best-effort timeliness targets real-time applications with run-time uncertainties and resource overloads, and optimizes collective application timeliness -as specified by the application. ChronOS directly supports the implementation of best-effort real-time schedulers on CMPs, in addition to others, in the global and partitioned scheduling disciplines. ChronOS extends the PREEMPT RT Linux patch, and thus provides full kernel preemptibility and retains stock Linux features. We validate our claims by reporting on the implementation of a suite of best-effort and non-best-effort CMP schedulers on a quad-core AMD Phenom platform