Evaluating the Glycogenic Activity and Therapeutic Capacity of PPP1R3D in a Mouse Model of Lafora Disease

Lafora disease (LD) is an intractable, neurodegenerative epilepsy caused by loss-of-function mutations in the EPM2A or EPM2B genes. Central to LD is the accumulation of malstructured glycogen (Lafora bodies; LB) in neurons, a consequence of dysregulated glycogen synthesis. Glycogen synthase catalyzes glycogen formation and is activated by dephosphorylation. The latter is mediated by glycogen-targeting subunits of protein phosphatase 1, including PTG (R5) and R6, known formally as PPP1R3D, both abundantly expressed in brain. PTG knockout in LD mice rescues LD, including near-complete disappearance of LB and neurodegeneration. I examined whether the same could be achieved with R6 knockout. Despite significant brain glycogen and LB reductions in R6-deficient-Epm2a-/- mice, substantial amounts of LB remained and neurodegeneration was not rescued. This partial effect remains unexplained. Future experiments to resolve the difference with PTG will shed important light on both LB formation and Lafora disease, and brain glycogen metabolism.M.Sc

AMG 510: the kryptonite of mutant KRASG12C

Over three decades of cancer therapy research have been dedicated to investigating the most frequently mutated oncogene: KRAS. Approximately one million cancer deaths per year worldwide are traced to mutations in KRAS, which promote tumour formation and survival. Countless failed anti-KRAS therapies have deemed KRAS “undruggable”, as traditional medicinal chemistry seemed ill-equipped to design drugs against proteins, such as KRAS, with no obvious binding sites or “pockets”. Recently, the clinical development of a covalently binding small molecule known as AMG 510 has suggested that it may be the most promising anti-KRAS therapy. KRAS resides within the RAS family of GTPase proteins described as on/off switches for cell growth and proliferation. The desire to specifically target mutant KRASG12C stems from its presence in some of the deadliest cancers, such as colorectal, pancreatic, and lung adenocarcinomas. The substitution of guanine with nucleophilic cysteine disrupts the GTPase activity of KRAS so that it is persistently active, forcing cells into a hyperproliferative state that increases susceptibility to mutation.</p

AMG 510: the kryptonite of mutant KRASG12C

Over three decades of cancer therapy research have been dedicated to investigating the most frequently mutated oncogene: KRAS. Approximately one million cancer deaths per year worldwide are traced to mutations in KRAS, which promote tumour formation and survival. Countless failed anti-KRAS therapies have deemed KRAS “undruggable”, as traditional medicinal chemistry seemed ill-equipped to design drugs against proteins, such as KRAS, with no obvious binding sites or “pockets”. Recently, the clinical development of a covalently binding small molecule known as AMG 510 has suggested that it may be the most promising anti-KRAS therapy. KRAS resides within the RAS family of GTPase proteins described as on/off switches for cell growth and proliferation. The desire to specifically target mutant KRASG12C stems from its presence in some of the deadliest cancers, such as colorectal, pancreatic, and lung adenocarcinomas. The substitution of guanine with nucleophilic cysteine disrupts the GTPase activity of KRAS so that it is persistently active, forcing cells into a hyperproliferative state that increases susceptibility to mutation.</p

The promise and potential of the adeno-associated viral vector in gene therapy

The adeno-associated virus (AAV) vector system has emerged as one of the most attractive methods of gene therapy, namely for its favourable safety profile, non-pathogenicity in humans, and efficient delivery of genetic material. As a vehicle of gene delivery, it can deliver a gene or modify an existing one by infecting and transducing cells, most notably those that are post mitotic. Conveniently, the adeno-associated virus’s capsid can be manipulated and serotypes harnessed, so as to more efficiently target tissue-specific diseases. Development and maturation of the AAV system has seen its integration with other gene therapy technologies such as the CRISPR-Cas system, and RNA interference, further enhancing the power and variety of its therapeutic applications. Several clinical trials involving the AAV system are underway. Its use in the treatment of a progressive motor neuron disease, spinal muscular atrophy type 1, is just one of several examples of its translational success. However, the AAV system has limitations that must be circumvented in order to maximise its effectiveness in humans; host anti-viral responses and the restricting carrying capacity of the vector are examples of such barriers that are being actively tackled by multidisciplinary teams in hopes of optimising and perfecting its therapeutic prowess </p

SGK1 (glucose transport), dishevelled2 (wnt signaling), LC3/p62 (autophagy) and p53 (apoptosis) proteins are unaltered in Lafora disease

Glycogen forms through the concerted actions of glycogen synthase (GS) which elongates glycogen strands, and glycogen branching enzyme (GBE).  Lafora disease (LD) is a fatal neurodegenerative epilepsy that results from neuronal accumulation of hyperphosphorylated glycogen with excessively long strands (called polyglucosans).  There is no GBE deficiency in LD.  Instead, the disease is caused by loss-of-function mutations in the EPM2A or EPM2B genes, encoding, respectively, a phosphatase, laforin, and an E3 ubiquiting ligase, malin.   A number of experimentally derived hypotheses have been published to explain LD, including:  The SGK1 hypothesis - Phosphorylated SGK1 (pSGK1) raises cellular glucose uptake and levels, which would activate GS.  Based on observing increased pSGK1 in LD mice it was proposed that raised pSGK1 leads to polyglucosan generation through GS hyperactivation.  The Dishevelled2 hypothesis - Downregulating malin in cell culture was reported to increase levels of dishevelled2, which through the wnt/glycogen synthase kinase-3 pathway would likewise overactivate GS.  The Autophagic defect hypothesis - Polyglucosans may be natural byproducts of normal glycogen metabolism.  LD mice were reported to be autophagy-defective.  LD would arise from failed autophagy leading to failed polyglucosan clearance.  Finally, the p53 hypothesis - laforin and malin were reported to downregulate p53, their absence leading to increased p53, which would activate apoptosis, leading to the neurodegeneration of LD.  In the present work we repeat key experiments that underlie these four hypotheses.  We are unable to confirm increased pSGK1, dishevelled2, or p53 in LD mice, nor the reported autophagic defects.  Our work does not support the above hypotheses in understanding this unique and severe form of epilepsy

Abnormal glycogen chain length pattern, not hyperphosphorylation, is critical in Lafora disease

12 p.-7 fig. Nitschke, Felix et al.Lafora disease (LD) is a fatal progressive epilepsy essentially caused by loss-of-function mutations in the glycogen phosphatase laforin or the ubiquitin E3 ligase malin. Glycogen in LD is hyperphosphorylated and poorly hydrosoluble. It precipitates and accumulates into neurotoxic Lafora bodies (LBs). The leading LD hypothesis that hyperphosphorylation causes the insolubility was recently challenged by the observation that phosphatase-inactive laforin rescues the laforin-deficient LD mouse model, apparently through correction of a general autophagy impairment. We were for the first time able to quantify brain glycogen phosphate. We also measured glycogen content and chain lengths, LBs, and autophagy markers in several laforin- or malin-deficient mouse lines expressing phosphatase-inactive laforin. We find that: (i) in laforindeficient mice, phosphatase-inactive laforin corrects glycogen chain lengths, and not hyperphosphorylation, which leads to correction of glycogen amounts and prevention of LBs; (ii) in malin-deficient mice, phosphatase-inactive laforin confers no correction; (iii) general impairment of autophagy is not necessary in LD. We conclude that laforin’s principle function is to control glycogen chain lengths, in a malin-dependent fashion, and that loss of this control underlies LD.This work was supported by families and friends of the Chelsea’s Hope Lafora Disease Research Fund, Associazione Italiana Lafora (AILA), France-Lafora, the Milana and Tatjana Gajic Lafora Disease Foundation, Genome Canada, the Ontario Brain Institute (OBI) and The National Institute of Neurological Disorders and Stroke of the National Institutes of Health (NIH) under award number P01 NS097197. Mitchell A. Sullivan was supported by an NHMRC CJ Martin Fellowship (GNT1092451).Peer reviewe

Adverse Effects Related to Corticosteroid Use in Sepsis, Acute Respiratory Distress Syndrome, and Community-Acquired Pneumonia: A Systematic Review and Meta-Analysis

We postulate that corticosteroid-related side effects in critically ill patients are similar across sepsis, acute respiratory distress syndrome (ARDS), and community-acquired pneumonia (CAP). By pooling data across all trials that have examined corticosteroids in these three acute conditions, we aim to examine the side effects of corticosteroid use in critical illness. We performed a comprehensive search of MEDLINE, Embase, Centers for Disease Control and Prevention library of COVID research, CINAHL, and Cochrane center for trials. We included randomized controlled trials (RCTs) that compared corticosteroids to no corticosteroids or placebo in patients with sepsis, ARDS, and CAP. We summarized data addressing the most described side effects of corticosteroid use in critical care: gastrointestinal bleeding, hyperglycemia, hypernatremia, superinfections/secondary infections, neuropsychiatric effects, and neuromuscular weakness. We included 47 RCTs ( = 13,893 patients). Corticosteroids probably have no effect on gastrointestinal bleeding (relative risk [RR], 1.08; 95% CI, 0.87-1.34; absolute risk increase [ARI], 0.3%; moderate certainty) or secondary infections (RR, 0.97; 95% CI, 0.89-1.05; absolute risk reduction, 0.5%; moderate certainty) and may have no effect on neuromuscular weakness (RR, 1.22; 95% CI, 1.03-1.45; ARI, 1.4%; low certainty) or neuropsychiatric events (RR, 1.19; 95% CI, 0.82-1.74; ARI, 0.5%; low certainty). Conversely, they increase the risk of hyperglycemia (RR, 1.21; 95% CI, 1.11-1.31; ARI, 5.4%; high certainty) and probably increase the risk of hypernatremia (RR, 1.59; 95% CI, 1.29-1.96; ARI, 2.3%; moderate certainty). In ARDS, sepsis, and CAP, corticosteroids are associated with hyperglycemia and probably with hypernatremia but likely have no effect on gastrointestinal bleeding or secondary infections. More data examining effects of corticosteroids, particularly on neuropsychiatric outcomes and neuromuscular weakness, would clarify the safety of this class of drugs in critical illness