Hippocampal aggregation signatures of pathogenic UBQLN2 in amyotrophic lateral sclerosis and frontotemporal dementia.

Pathogenic variants in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis and/or frontotemporal dementia characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial amyotrophic lateral sclerosis and/or frontotemporal dementia cases not caused by UBQLN2 pathogenic variants, particularly C9orf72-linked cases. This makes the mechanistic role of mutant ubiquilin 2 protein and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 44 genotypically diverse amyotrophic lateral sclerosis cases with or without frontotemporal dementia, including eight cases with UBQLN2 variants (resulting in p.S222G, p.P497H, p.P506S, p.T487I (two cases), and p.P497L (three cases)). Using multiplexed (5-label) fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43, dipeptide repeat aggregates, and p62, in the hippocampus of controls (n = 6), or amyotrophic lateral sclerosis with or without frontotemporal dementia in sporadic (n = 20), unknown familial (n = 3), SOD1-linked (n = 1), FUS-linked (n = 1), C9orf72-linked (n = 5), and UBQLN2-linked (n = 8) cases. We differentiate between i) ubiquilin 2 aggregation together with phosphorylated TDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation promoted by UBQLN2 pathogenic variants that cause amyotrophic lateral sclerosis/and frontotemporal dementia. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in amyotrophic lateral sclerosis with or without frontotemporal dementia, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene variants and to understand the mechanisms of UBQLN2-linked disease.

Common Genetic Variants Contribute to Risk of Transposition of the Great Arteries.

RATIONALE: Dextro-transposition of the great arteries (D-TGA) is a severe congenital heart defect which affects approximately 1 in 4,000 live births. While there are several reports of D-TGA patients with rare variants in individual genes, the majority of D-TGA cases remain genetically elusive. Familial recurrence patterns and the observation that most cases with D-TGA are sporadic suggest a polygenic inheritance for the disorder, yet this remains unexplored. OBJECTIVE: We sought to study the role of common single nucleotide polymorphisms (SNPs) in risk for D-TGA. METHODS AND RESULTS: We conducted a genome-wide association study in an international set of 1,237 patients with D-TGA and identified a genome-wide significant susceptibility locus on chromosome 3p14.3, which was subsequently replicated in an independent case-control set (rs56219800, meta-analysis P=8.6x10-10, OR=0.69 per C allele). SNP-based heritability analysis showed that 25% of variance in susceptibility to D-TGA may be explained by common variants. A genome-wide polygenic risk score derived from the discovery set was significantly associated to D-TGA in the replication set (P=4x10-5). The genome-wide significant locus (3p14.3) co-localizes with a putative regulatory element that interacts with the promoter of WNT5A, which encodes the Wnt Family Member 5A protein known for its role in cardiac development in mice. We show that this element drives reporter gene activity in the developing heart of mice and zebrafish and is bound by the developmental transcription factor TBX20. We further demonstrate that TBX20 attenuates Wnt5a expression levels in the developing mouse heart. CONCLUSIONS: This work provides support for a polygenic architecture in D-TGA and identifies a susceptibility locus on chromosome 3p14.3 near WNT5A. Genomic and functional data support a causal role of WNT5A at the locus.

Prehospital Ground and Helicopter-Based Extracorporeal Cardiopulmonary Resuscitation (ECPR) Reduce Barriers to ECPR: A GIS Model.

INTRODUCTION: Evidence suggests that Extracorporeal Cardiopulmonary Resuscitation (ECPR) can improve survival rates for nontraumatic out-of-hospital cardiac arrest (OHCA). However, when ECPR is indicated over 50% of potential candidates are unable to qualify in the current hospital-based system due to geographic limitations. This study employs a Geographic Information System (GIS) model to estimate the number of ECPR eligible patients within the United States in the current hospital-based system, a prehospital ECPR ground-based system, and a prehospital ECPR Helicopter Emergency Medical Services (HEMS)-based system. METHODS: We constructed a GIS model to estimate ground and helicopter transport times. Time-dependent rates of ECPR eligibility were derived from the Resuscitation Outcome Consortium (ROC) database, while the Cardiac Arrest Registry to Enhance Survival (CARES) registry determined the number of OHCA patients meeting ECPR criteria within designated transportation times. Emergency Medical Services (EMS) response time, ECPR candidacy determination time, and on-scene time were modeled based on data from the EROCA trial. The combined model was used to estimate the total ECPR eligibility in each system. RESULTS: The CARES registry recorded 736,066 OHCA patients from 2013 to 2021. After applying clinical criteria, 24,661 (3.4%) ECPR-indicated OHCA were identified. When considering overall ECPR eligibility within 45 min from OHCA to initiation, only 11.76% of OHCA where ECPR was indicated were eligible in the current hospital-based system. The prehospital ECPR HEMS-based system exhibited a four-fold increase in ECPR eligibility (49.3%), while the prehospital ground-based system showed a more than two-fold increase (28.4%). CONCLUSIONS: The study demonstrates a two-fold increase in ECPR eligibility for a prehospital ECPR ground-based system and a four-fold increase for a prehospital ECPR HEMS-based system compared to the current hospital-based ECPR system. This novel GIS model can inform future ECPR implementation strategies, optimizing systems of care.

Autophagy and Exercise: Current Insights and Future Research Directions.

Autophagy is a cellular process by which proteins and organelles are degraded inside the lysosome. Exercise is known to influence the regulation of autophagy in skeletal muscle. However, as gold standard techniques to assess autophagy flux in vivo are restricted to animal research, important gaps remain in our understanding of how exercise influences autophagy activity in humans. Using available datasets, we show how the gene expression profile of autophagy receptors and ATG8 family members differ between human and mouse skeletal muscle, providing a potential explanation for their differing exercise-induced autophagy responses. Furthermore, we provide a comprehensive view of autophagy regulation following exercise in humans by summarizing human transcriptomic and phosphoproteomic datasets that provide novel targets of potential relevance. These newly identified phosphorylation sites may provide an explanation as to why both endurance and resistance exercise lead to an exercise-induced reduction in LC3B-II, while possibly divergently regulating autophagy receptors, and, potentially, autophagy flux. We also provide recommendations to use ex vivo autophagy flux assays to better understand the influence of exercise, and other stimuli, on autophagy regulation in humans. This review provides a critical overview of the field and directs researchers towards novel research areas that will improve our understanding of autophagy regulation following exercise in humans.

The contribution of Neanderthal introgression and natural selection to neurodegenerative diseases.

Humans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions. We used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease. We found no evidence for enrichment of positively-selected SNPs in the heritability of Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease, suggesting that common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk. These findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.

Cytoplasmic TDP-43 is involved in cell fate during stress recovery.

Transactive response DNA binding protein 43 (TDP-43) is an RNA processing protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Nuclear TDP-43 mislocalizes in patients to the cytoplasm, where it forms ubiquitin-positive inclusions in affected neurons and glia. Physiologically, cytoplasmic TDP-43 is associated with stress granules (SGs). Here, we explored TDP-43 cytoplasmic accumulation and stress granule formation following osmotic and oxidative stress. We show that sorbitol drives TDP-43 redistribution to the cytoplasm, while arsenite induces the recruitment of cytoplasmic TDP-43 to TIA-1 positive SGs. We demonstrate that inducing acute oxidative stress after TDP-43 cytoplasmic relocalization by osmotic shock induces poly (ADP-ribose) polymerase (PARP) cleavage, which triggers cellular toxicity. Recruitment of cytoplasmic TDP-43 to polyribosomes occurs in an SH-SY5Y cellular stress model and is observed in FTD brain lysate. Moreover, the processing body (P-body) marker DCP1a is detected in TDP-43 granules during recovery from stress. Overall, this study supports a central role for cytoplasmic TDP-43 in controlling protein translation in stressed cells.

Altered SOD1 maturation and post-translational modification in amyotrophic lateral sclerosis spinal cord.

Aberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro and in transgenic animal models of amyotrophic lateral sclerosis. Detailed examination of the protein in disease-affected tissues from amyotrophic lateral sclerosis patients, however, remains scarce. We used histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from amyotrophic lateral sclerosis cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration. We provide evidence of the mislocalization and accumulation of structurally disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial amyotrophic lateral sclerosis cases, and sporadic amyotrophic lateral sclerosis cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in amyotrophic lateral sclerosis cases, and clearly differentiated all forms of amyotrophic lateral sclerosis from controls. Substantial heterogeneity in the presence of these changes was also observed between amyotrophic lateral sclerosis cases. Our data demonstrate that varying forms of SOD1 proteinopathy are a common feature of all forms of amyotrophic lateral sclerosis, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in amyotrophic lateral sclerosis. Most of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development.

ALS/FTD mutations in UBQLN2 impede autophagy by reducing autophagosome acidification through loss of function.

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.

Regulation of Synapse Weakening through Interactions of the Microtubule Associated Protein Tau with PACSIN1.

Hyperphosphorylation of the microtubule associated protein tau (tau) is inextricably linked to several neurodegenerative diseases, collectively termed tauopathies, in which synapse dysfunction occurs through largely unidentified mechanisms. Our research aimed to uncover molecular mechanisms by which phosphorylation of tau (pTau) affects synapse function. Using combined molecular and electrophysiological analysis with in vitro genetic knock-in of phosphorylation mutant human tau in male rat CA1 hippocampal neurons, we show an interplay between tau and protein kinase C and casein kinase substrate in neurons protein 1 (PACSIN1) that regulates synapse function. pTau at serine residues 396/404 decreases tau:PACSIN1 binding and evokes PACSIN1-dependent functional and structural synapse weakening. Knock-down of tau or PACSIN1 increases AMPA receptor (AMPAR)-mediated current at extrasynaptic regions, supporting a role for these proteins in affecting AMPAR trafficking. The pTau-induced PACSIN1 dissociation may represent a pathophysiological regulator of synapse function that underlies tauopathy-associated synapse defects.SIGNIFICANCE STATEMENT Knowledge is still lacking for how hyperphosphorylation of tau and its effectors lead to synaptic and neuronal dysfunction. Our results provide crucial insight for this mechanistic understanding; we show that specific tau phosphorylation events modulate its protein interaction with PACSIN1 and thus elicits synapse weakening likely through PACSIN1-dependent regulation of AMPA receptor (AMPAR) trafficking. These findings develop our understanding of molecular events that may be relevant to cellular changes underpinning tauopathy-associated neurodegenerative diseases.

Muscle fiber type-specific autophagy responses following an overnight fast and mixed meal ingestion in human skeletal muscle.

The aim of the present study was to investigate the fiber type-specific abundance of autophagy-related proteins after an overnight fast and following ingestion of a mixed meal in human skeletal muscle. Twelve overweight, healthy young male volunteers underwent a 3-h mixed meal tolerance test following an overnight fast. Blood samples were collected in the overnight-fasted state and throughout the 180-min postmeal period. Skeletal muscle biopsies were collected in the fasted state, and at 30 and 90 min after meal ingestion. Protein content of key autophagy markers and upstream signaling responses were measured in whole muscle and pooled single fibers using immunoblotting. In the fasted state, type I fibers displayed lower LC3B-I but higher LC3B-II abundance and higher LC3B-II/LC3B-I ratio compared with type II fibers (P < 0.05). However, there were no fiber type differences in p62/SQSTM1, unc-51 like autophagy activating kinase (ULK1), ATG5, or ATG12 (P > 0.05). Compared with the fasted state, there was a reduction in LC3B-II abundance, indicative of lower autophagosome content, in whole muscle and in both type I and type II fibers following meal ingestion (P < 0.05). This reduction in autophagosome content occurred alongside similar increases in p-AktS473 and p-mTORS2448 in both type I and type II muscle fibers (P < 0.05). In human skeletal muscle, type I fibers have a greater autophagosome content than type II fibers in the overnight-fasted state despite comparable abundance of other key upstream autophagy proteins. Autophagy is rapidly inhibited in both fiber types following the ingestion of a mixed meal.NEW & NOTEWORTHY This study examined the fiber type-specific content of key autophagy proteins in human muscle. We showed that markers of autophagosome content are higher in type I fibers in the overnight-fasted state, whereas autophagy is rapidly inhibited in both type I and type II fibers after the ingestion of a mixed meal.

Elucidating the Role of Cerebellar Synaptic Dysfunction in C9orf72-ALS/FTD - a Systematic Review and Meta-Analysis.

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) with synaptic dysfunction identified as an early pathological hallmark. Although TDP-43 pathology and overt neurodegeneration are largely absent from the cerebellum, the pathological hallmarks of RNA foci and dipeptide repeat protein (DPR) inclusions are most abundant. Here, we present a systematic literature search in the databases of PubMed, Scopus, Embase, Web of Science and Science Direct up until March 5, 2021, which yielded 19,515 publications. Following the exclusion criteria, 72 articles were included having referred to C9orf72, synapses and the cerebellum. Meta-analyses were conducted on studies which reported experimental and control groups with means and standard deviations extracted from figures using the online tool PlotDigitizer. This revealed dendritic defects (P = 0.03), reduced C9orf72 in human patients (P = 0.005) and DPR-related neuronal loss (P = 0.0006) but no neuromuscular junction abnormalities (P = 0.29) or cerebellar neuronal loss (P = 0.23). Our results suggest that dendritic arborisation defects, synaptic gene dysregulation and altered synaptic neurotransmission may drive cerebellar synaptic dysfunction in C9-ALS/FTD. In this review, we discuss how the chronological appearance of the different pathological hallmarks alters synaptic integrity which may have profound implications for disease progression. We conclude that a reduction in C9orf72 protein levels combined with the accumulation of RNA foci and DPRs act synergistically to drive C9 synaptopathy in the cerebellum of C9-ALS/FTD patients.

A shortened surface electromyography recording is sufficient to facilitate home fasciculation assessment.

INTRODUCTION/AIMS: Fasciculations are an early clinical hallmark of amyotrophic lateral sclerosis (ALS), amenable to detection by high-density surface electromyography (HDSEMG). In conjunction with the Surface Potential Quantification Engine (SPiQE), HDSEMG offers improved spatial resolution for the analysis of fasciculations. This study aims to establish an optimal recording duration to enable longitudinal remote monitoring in the home. METHODS: Twenty patients with ALS and five patients with benign fasciculation syndrome (BFS) underwent serial 30 min HDSEMG recordings from biceps brachii and gastrocnemii. SPiQE was independently applied to abbreviated epochs within each 30-min recording (0-5, 0-10, 0-15, 0-20, and 0-25 min), outputting fasciculation frequency, amplitude median and amplitude interquartile range. Bland-Altman plots and intraclass correlation coefficients (ICC) were used to assess agreement with the validated 30-min recording. RESULTS: In total, 506 full recordings were included. The 5 min recordings demonstrated diverse and relatively poor agreement with the 30 min baselines across all parameters, muscles and patient groups (ICC = 0.32-0.86). The 15-min recordings provided more acceptable and stable agreement (ICC = 0.78-0.98), which did not substantially improve in longer recordings. DISCUSSION: For the detection and quantification of fasciculations in patients with ALS and BFS, HDSEMG recordings can be halved from 30 to 15 min without significantly compromising the primary outputs. Reliance on a shorter recording duration should lead to improved tolerability and repeatability among patients, facilitating longitudinal remote monitoring in patients' homes.

Impella in Transport: Physiology, Mechanics, Complications, and Transport Considerations.

Cardiogenic shock (CS) represents a spectrum of hemodynamic deficits in which the cardiac output is insufficient to provide adequate tissue perfusion. The Impella (Abiomed Inc, Danvers, MA) device, a contemporary percutaneous ventricular support, is most often indicated for classic, deteriorating, and extremis Society for Coronary Angiography and Intervention stages of CS, which describe CS that is not responsive to optimal medical management and conventional treatment measures. Impella devices are an evolving field of mechanical support that is used with increasing frequency. Critical care transport medicine crews are required to transport patient support by the Impella device with increasing frequency. It is important that critical care transport medicine crews are familiar with the Impella device and are able to troubleshoot complications that may arise in the transport environment. This article reviews many aspects of the Impella device critical to the transport of this complex patient population.

Prior exercise enhances skeletal muscle microvascular blood flow and mitigates microvascular flow impairments induced by a high-glucose mixed meal in healthy young men.

KEY POINTS: Exercise, insulin-infusion and low-glucose mixed-nutrient meal ingestion increases muscle microvascular blood flow which in part facilitates glucose delivery and disposal. In contrast, high-glucose ingestion impairs muscle microvascular blood flow which may contribute to impaired postprandial metabolism. We investigated the effects of prior cycling exercise on postprandial muscle microvascular blood flow responses to a high-glucose mixed-nutrient meal ingested 3 and 24 h post-exercise. Prior exercise enhanced muscle microvascular blood flow and mitigated microvascular impairments induced by a high-glucose mixed meal ingested 3 h post-exercise, and to a lesser extent 24 h post-exercise. High-glucose ingestion 3 h post-exercise leads to greater postprandial blood glucose, non-esterified fatty acids, and fat oxidation, and a delay in the insulin response to the meal compared to control. Effects of acute exercise on muscle microvascular blood flow persist well after the cessation of exercise which may be beneficial for conditions characterized by microvascular and glycaemic dysfunction. ABSTRACT: Exercise, insulin-infusion and low-glucose mixed-nutrient meal ingestion lead to increased muscle microvascular blood flow (MBF), whereas high-glucose ingestion impairs MBF. We investigated whether prior cycling exercise could enhance postprandial muscle MBF and prevent MBF impairments induced by high-glucose mixed-nutrient meal ingestion. In a randomized cross-over design, eight healthy young men ingested a high-glucose mixed-nutrient meal (1.1 g glucose/kg body weight; 45% carbohydrate, 20% protein and 35% fat) after an overnight fast (no-exercise control) and 3 h and 24 h after moderate-intensity cycling exercise (1 h at 70-75% V̇O2peak ). Skeletal muscle MBF, measured directly by contrast-enhanced ultrasound, was lower at 60 min and 120 min postprandially compared to baseline in all conditions (P < 0.05), with a greater decrease occurring from 60 min to 120 min in the control (no-exercise) condition only (P < 0.001). Despite this meal-induced decrease, MBF was still markedly higher compared to control in the 3 h post-exercise condition at 0 min (pre-meal; 74%, P = 0.004), 60 min (112%, P = 0.002) and 120 min (223%, P < 0.001), and in the 24 h post-exercise condition at 120 min postprandially (132%, P < 0.001). We also report that in the 3 h post-exercise condition postprandial blood glucose, non-esterified fatty acids (NEFAs), and fat oxidation were substantially elevated, and the insulin response to the meal delayed compared to control. This probably reflects a combination of increased post-exercise exogenous glucose appearance, substrate competition, and NEFA-induced insulin resistance. We conclude that prior cycling exercise elicits long-lasting effects on muscle MBF and partially mitigates MBF impairments induced by high-glucose mixed-nutrient meal ingestion.

Transethnic Genome-Wide Association Study Provides Insights in the Genetic Architecture and Heritability of Long QT Syndrome.

BACKGROUND: Long QT syndrome (LQTS) is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. A causal rare genetic variant with large effect size is identified in up to 80% of probands (genotype positive) and cascade family screening shows incomplete penetrance of genetic variants. Furthermore, a proportion of cases meeting diagnostic criteria for LQTS remain genetically elusive despite genetic testing of established genes (genotype negative). These observations raise the possibility that common genetic variants with small effect size contribute to the clinical picture of LQTS. This study aimed to characterize and quantify the contribution of common genetic variation to LQTS disease susceptibility. METHODS: We conducted genome-wide association studies followed by transethnic meta-analysis in 1656 unrelated patients with LQTS of European or Japanese ancestry and 9890 controls to identify susceptibility single nucleotide polymorphisms. We estimated the common variant heritability of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 single nucleotide polymorphisms previously associated with the QT-interval in the general population using a polygenic risk score. RESULTS: Genome-wide association analysis identified 3 loci associated with LQTS at genome-wide statistical significance (P<5×10-8) near NOS1AP, KCNQ1, and KLF12, and 1 missense variant in KCNE1(p.Asp85Asn) at the suggestive threshold (P<10-6). Heritability analyses showed that ≈15% of variance in overall LQTS susceptibility was attributable to common genetic variation (h2SNP 0.148; standard error 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT-interval in the general population (rg=0.40; P=3.2×10-3). The polygenic risk score comprising common variants previously associated with the QT-interval in the general population was greater in LQTS cases compared with controls (P<10-13), and it is notable that, among patients with LQTS, this polygenic risk score was greater in patients who were genotype negative compared with those who were genotype positive (P<0.005). CONCLUSIONS: This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT-interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT-interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS.

Nuclear RNA foci from C9ORF72 expansion mutation form paraspeckle-like bodies.

The GGGGCC (G4C2) repeat expansion mutation in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (G4C2) n repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA hLinc-p21. As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (G4C2) n RNA foci. Overexpression of (G4C2)72 RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (G4C2)72 RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA NEAT1 Moreover, the knockdown of SFPQ protein in C9ORF72 expansion mutation-positive fibroblasts significantly reduces the number of (G4C2) n RNA foci. In conclusion, (G4C2) n RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation.

Autophagy is not involved in lipid accumulation and the development of insulin resistance in skeletal muscle.

OBJECTIVE: To investigate the effect of high fat diet-induced insulin resistance on autophagy markers in the liver and skeletal muscle of mice in the fasted state and following an oral glucose bolus. METHODS: Forty C57BL/6J male mice were fed either a high fat, high sucrose (HFSD, n = 20) or standard chow control (CON, n = 20) diet for 16 weeks. Upon trial completion, mice were gavaged with water or glucose and skeletal muscle and liver were collected 15 min post gavage. Protein abundance and gene expression of autophagy markers and activation of related signalling pathways were assessed. RESULTS: Compared to CON, the HFSD intervention increased LC3B-II and p62/SQSTM1 protein abundance in the liver which is indicative of elevated autophagosome content via reduced clearance. These changes coincided with inhibitory autophagy signalling through elevated p-mTOR S2448 and p-ULK1S758. HFSD did not alter autophagy markers in skeletal muscle. Administration of an oral glucose bolus had no effect on autophagy markers or upstream signalling responses in either tissue regardless of diet. CONCLUSION: HFSD induces tissue-specific autophagy impairments, with autophagosome accumulation indicating reduced lysosomal clearance in the liver. In contrast, autophagy markers were unchanged in skeletal muscle, indicating that autophagy is not involved in the development of skeletal muscle insulin resistance.

CYLD is a causative gene for frontotemporal dementia - amyotrophic lateral sclerosis.

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.

A hospital-wide initiative to redesign substance use disorder care: Impact on pharmacotherapy initiation.

Background: Substance use disorder (SUD) treatment in general medical settings remains underutilized. We evaluated 5 years of a hospital-wide SUD initiative which included an inpatient addiction consult team (ACT), low-threshold Bridge Clinic, recovery coaches, and office-based addiction treatment (OBAT) nurses. Methods: Naturalistic registry study. We calculated frequencies of patient contacts, types of substance use diagnoses, and medication treatment initiation and duration. Results: From 2014 to 2019, 7,036 unique patients were seen, including 4,959 by ACT, 1,197 in Bridge Clinic, 2,250 by a recovery coach, and 979 by an OBAT nurse. The median age was 47, 31% were women, 80% were white, 7% were black, 6% were Hispanic/Latinx, and 25% were experiencing homelessness. Alcohol use disorder was seen in 62%, opioid use disorder in 54%, cocaine use disorder in 29%, benzodiazepine use disorder in 14%, and stimulant use disorder in 7%. Co-occurring medical and psychiatric illnesses were common; 35% had hepatitis C, 59% depression, 66% anxiety, and 13% schizophrenia. 1,623 patients received a prescription for buprenorphine during the study period (42% of patients with OUD), 877 for oral naltrexone, and 163 for extended-release naltrexone. The mean length of continuous treatment was 178.4 days for buprenorphine, 47.7 days for oral naltrexone, and 1.29 injections for extended-release naltrexone. Conclusion: A hospital SUD initiative effectively initiated SUD pharmacotherapy with naltrexone and buprenorphine. Medication treatment episodes were longer with buprenorphine.

Mutant C9orf72 human iPSC-derived astrocytes cause non-cell autonomous motor neuron pathophysiology.

Mutations in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS). Accumulating evidence implicates astrocytes as important non-cell autonomous contributors to ALS pathogenesis, although the potential deleterious effects of astrocytes on the function of motor neurons remains to be determined in a completely humanized model of C9orf72-mediated ALS. Here, we use a human iPSC-based model to study the cell autonomous and non-autonomous consequences of mutant C9orf72 expression by astrocytes. We show that mutant astrocytes both recapitulate key aspects of C9orf72-related ALS pathology and, upon co-culture, cause motor neurons to undergo a progressive loss of action potential output due to decreases in the magnitude of voltage-activated Na+ and K+ currents. Importantly, CRISPR/Cas-9 mediated excision of the C9orf72 repeat expansion reverses these phenotypes, confirming that the C9orf72 mutation is responsible for both cell-autonomous astrocyte pathology and non-cell autonomous motor neuron pathophysiology.