Spinal and bulbar muscular atrophy : new insights into the disease mechanism and prospects for pharmacological therapy

Expansion of a polyglutamine-encoding trinucleotide CAG repeat in the androgen receptor (AR) gene causes spinal and bulbar muscular atrophy (SBMA, or Kennedy’s disease). SBMA is an adult-onset disease characterized by progressive muscle weakness and atrophy due to the degeneration of lower motor neurons in the brainstem and spinal cord. At present, effective disease-modifying treatment is not available for this disorder. Neuronal dysfunction in SBMA is at least in part due to a toxic gain of function of the mutant AR, however, the underlying mechanism in the pathogenesis is not known. Work in this thesis identified new disease features of SBMA (Study I), investigated the effect of the polyglutamine expansion in the context of normal AR function (Study II), and explored pharmacological strategies for reducing mutant AR as potential treatments (Studies III and IV). In Study I, we describe a 29-year old SBMA patient with a 68 CAG repeat, the largest reported to date. The patient had an unusually early onset and novel clinical features, including developmental defects and autonomic dysfunction. In Study II, we examined the effect of the polyglutamine expansion in the AR on androgen-induced differentiation of neuronal cells. We show that mutant AR expression in this model leads to aberrant neurite outgrowth and reduced cell cycle arrest. The expanded polyglutamine tract in the AR interferes with the activity of the ubiquitin ligase APC/C-Cdh1, a critical regulator of cell cycle exit and neuronal architecture. These findings suggest that cellular abnormalities due to the stabilization of APC/C-Cdh1-dependent substrates may contribute to the pathogenic mechanism in SBMA. Augmentation of insulin-like growth factor (IGF)-1/Akt signaling was previously shown to promote the degradation of polyglutamine-expanded AR. In Study III, we tested the efficacy of exogenous IGF-1 administration in a transgenic mouse model of SBMA. We report that systemic delivery of IGF-1 reduces mutant AR accumulation and ameliorates disease manifestations in SBMA mice. We also tested a novel curcumin analog in cell and animal models of SBMA and investigated its mechanism of action. We show in Study IV that this compound enhances the clearance of mutant AR and mitigates the SBMA phenotype in Drosophila melanogaster and mice. The protective effect of the compound on mutant ARinduced degeneration in Drosophila is mediated through the Nrf1/Nrf2-dependent antioxidant response. Our results establish IGF-1 and curcumin analogs as candidates for therapeutic intervention in SBMA. In summary, our findings extend the known phenotype of SBMA. We also provide evidence that the mutant AR alters ubiquitin-dependent degradation pathways that are necessary for neuronal differentiation and function. Lastly, our results demonstrate the preclinical efficacy of IGF-1 and curcumin analogs in SBMA and warrant further investigation of these compounds in clinical studies

Lipids From Trypanosoma cruzi Amastigotes of RA and K98 Strains Generate a Pro-inflammatory Response via TLR2/6

Lipids from microorganisms are ligands of Toll like receptors (TLRs) and modulate the innate immune response. Herein, we analyze in vitro the effect of total lipid extracts from Trypanosoma cruzi amastigotes of RA and K98 strains (with polar biological behavior) on the induction of the inflammatory response and the involvement of TLRs in this process. We demonstrated that total lipid extracts from both strains induced lipid body formation, cyclooxygenase-2 expression and TNF-a and nitric oxide release in macrophages, as well as NF-κB activation and IL-8 release in HEK cells specifically through a TLR2/6 dependent pathway. We also evaluated the inflammatory response induced by total lipid extracts obtained from lysed parasites that were overnight incubated to allow the action of parasite hydrolytic enzymes, such as Phospholipase A1, over endogenous phospholipids. After incubation, these total lipid extracts showed a significantly reduced pro-inflammatory response, which could be attributed to the changes in the content of known bioactive lipid molecules like lysophospholipids and fatty acids, here reported. Moreover, analyses of total fatty acids in each lipid extract were performed by gas chromatography-mass spectrometry. Our results indicate a relevant role of T. cruzi lipids in the induction of a pro-inflammatory response through the TLR2/6 pathway that could contribute to the modulation of the immune response and host survival.Fil: Bott, Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Carneiro, Alan B.. Instituto Oswaldo Cruz; BrasilFil: Gimenez, Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: López, María Gabriela. Instituto Nacional de Tecnología Agropecuaria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lammel, Estela María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Atella, Georgia C.. Universidade Federal do Rio de Janeiro; BrasilFil: Bozza, Patricia T.. Instituto Oswaldo Cruz; BrasilFil: Belaunzarán, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

Variants in ATP6V0A1 cause progressive myoclonus epilepsy and developmental and epileptic encephalopathy

Bott et al. here reports that de novo and biallelic variants in ATP6V0A1 gene affect the ability of the V-ATPase complex to translocate protons and acidify the endolysosomal compartment in neurons, causing a severe neurological phenotype ranging from developmental and epileptic encephalopathy to progressive myoclonus epilepsy. The vacuolar H+-ATPase is a large multi-subunit proton pump, composed of an integral membrane V0 domain, involved in proton translocation, and a peripheral V1 domain, catalysing ATP hydrolysis. This complex is widely distributed on the membrane of various subcellular organelles, such as endosomes and lysosomes, and plays a critical role in cellular processes ranging from autophagy to protein trafficking and endocytosis. Variants in ATP6V0A1, the brain-enriched isoform in the V0 domain, have been recently associated with developmental delay and epilepsy in four individuals. Here, we identified 17 individuals from 14 unrelated families with both with new and previously characterized variants in this gene, representing the largest cohort to date. Five affected subjects with biallelic variants in this gene presented with a phenotype of early-onset progressive myoclonus epilepsy with ataxia, while 12 individuals carried de novo missense variants and showed severe developmental and epileptic encephalopathy. The R740Q mutation, which alone accounts for almost 50% of the mutations identified among our cases, leads to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment, causing autophagic dysfunction and severe developmental defect in Caenorhabditis elegans. Altogether, our findings further expand the neurological phenotype associated with variants in this gene and provide a direct link with endolysosomal acidification in the pathophysiology of ATP6V0A1-related conditions.Peer reviewe

Early onset and novel features in a spinal and bulbar muscular atrophy patient with a 68 CAG repeat

AbstractSpinal and bulbar muscular atrophy (SBMA) is an X-linked neuromuscular disease caused by a trinucleotide (CAG) repeat expansion in the androgen receptor gene. Patients with SBMA have weakness, atrophy, and fasciculations in the bulbar and extremity muscles. Individuals with CAG repeat lengths greater than 62 have not previously been reported. We evaluated a 29year old SBMA patient with 68 CAGs who had unusually early onset and findings not seen in others with the disease. Analysis of the androgen receptor gene confirmed the repeat length of 68 CAGs in both peripheral blood and fibroblasts. Evaluation of muscle and sensory function showed deficits typical of SBMA, and in addition the patient had manifestations of autonomic dysfunction and abnormal sexual development. These findings extend the known phenotype associated with SBMA and shed new insight into the effects of the mutated androgen receptor

Defining Natural History: Assessment of the Ability of College Students to Aid in Characterizing Clinical Progression of Niemann-Pick Disease, Type C

Niemann-Pick Disease, type C (NPC) is a fatal, neurodegenerative, lysosomal storage disorder. It is a rare disease with broad phenotypic spectrum and variable age of onset. These issues make it difficult to develop a universally accepted clinical outcome measure to assess urgently needed therapies. To this end, clinical investigators have defined emerging, disease severity scales. The average time from initial symptom to diagnosis is approximately 4 years. Further, some patients may not travel to specialized clinical centers even after diagnosis. We were therefore interested in investigating whether appropriately trained, community-based assessment of patient records could assist in defining disease progression using clinical severity scores. In this study we evolved a secure, step wise process to show that pre-existing medical records may be correctly assessed by non-clinical practitioners trained to quantify disease progression. Sixty-four undergraduate students at the University of Notre Dame were expertly trained in clinical disease assessment and recognition of major and minor symptoms of NPC. Seven clinical records, randomly selected from a total of thirty seven used to establish a leading clinical severity scale, were correctly assessed to show expected characteristics of linear disease progression. Student assessment of two new records donated by NPC families to our study also revealed linear progression of disease, but both showed accelerated disease progression, relative to the current severity scale, especially at the later stages. Together, these data suggest that college students may be trained in assessment of patient records, and thus provide insight into the natural history of a disease

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

A blood atlas of COVID-19 defines hallmarks of disease severity and specificity.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19

Effectiveness of a national quality improvement programme to improve survival after emergency abdominal surgery (EPOCH): a stepped-wedge cluster-randomised trial

BACKGROUND: Emergency abdominal surgery is associated with poor patient outcomes. We studied the effectiveness of a national quality improvement (QI) programme to implement a care pathway to improve survival for these patients. METHODS: We did a stepped-wedge cluster-randomised trial of patients aged 40 years or older undergoing emergency open major abdominal surgery. Eligible UK National Health Service (NHS) hospitals (those that had an emergency general surgical service, a substantial volume of emergency abdominal surgery cases, and contributed data to the National Emergency Laparotomy Audit) were organised into 15 geographical clusters and commenced the QI programme in a random order, based on a computer-generated random sequence, over an 85-week period with one geographical cluster commencing the intervention every 5 weeks from the second to the 16th time period. Patients were masked to the study group, but it was not possible to mask hospital staff or investigators. The primary outcome measure was mortality within 90 days of surgery. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN80682973. FINDINGS: Treatment took place between March 3, 2014, and Oct 19, 2015. 22 754 patients were assessed for elegibility. Of 15 873 eligible patients from 93 NHS hospitals, primary outcome data were analysed for 8482 patients in the usual care group and 7374 in the QI group. Eight patients in the usual care group and nine patients in the QI group were not included in the analysis because of missing primary outcome data. The primary outcome of 90-day mortality occurred in 1210 (16%) patients in the QI group compared with 1393 (16%) patients in the usual care group (HR 1·11, 0·96-1·28). INTERPRETATION: No survival benefit was observed from this QI programme to implement a care pathway for patients undergoing emergency abdominal surgery. Future QI programmes should ensure that teams have both the time and resources needed to improve patient care. FUNDING: National Institute for Health Research Health Services and Delivery Research Programme