Meta-analysis of human and mouse ALS astrocytes reveals multi-omic signatures of inflammatory reactive states

Astrocytes contribute to motor neuron death in amyotrophic lateral sclerosis (ALS), but whether they adopt deleterious features consistent with inflammatory reactive states remains incompletely resolved. To identify inflammatory reactive features in ALS human induced pluripotent stem cell (hiPSC)–derived astrocytes, we examined transcriptomics, proteomics, and glutamate uptake in VCP-mutant astrocytes. We complemented this by examining other ALS mutations and models using a systematic meta-analysis of all publicly-available ALS astrocyte sequencing data, which included hiPSC-derived astrocytes carrying SOD1, C9orf72, and FUS gene mutations as well as mouse ALS astrocyte models with SOD1^{G93A} mutation, Tardbp deletion, and Tmem259 (also known as membralin) deletion. ALS astrocytes were characterized by up-regulation of genes involved in the extracellular matrix, endoplasmic reticulum stress, and the immune response and down-regulation of synaptic integrity, glutamate uptake, and other neuronal support processes. We identify activation of the TGFB, Wnt, and hypoxia signaling pathways in both hiPSC and mouse ALS astrocytes. ALS changes positively correlate with TNF, IL1A, and complement pathway component C1q-treated inflammatory reactive astrocytes, with significant overlap of differentially expressed genes. By contrasting ALS changes with models of protective reactive astrocytes, including middle cerebral artery occlusion and spinal cord injury, we uncover a cluster of genes changing in opposing directions, which may represent down-regulated homeostatic genes and up-regulated deleterious genes in ALS astrocytes. These observations indicate that ALS astrocytes augment inflammatory processes while concomitantly suppressing neuronal supporting mechanisms, thus resembling inflammatory reactive states and offering potential therapeutic targets

Rigorous confidence intervals for critical probabilities

We use the method of Balister, Bollobas and Walters to give rigorous 99.9999% confidence intervals for the critical probabilities for site and bond percolation on the 11 Archimedean lattices. In our computer calculations, the emphasis is on simplicity and ease of verification, rather than obtaining the best possible results. Nevertheless, we obtain intervals of width at most 0.0005 in all cases

Nucleocytoplasmic mRNA redistribution accompanies RNA binding protein mislocalization in ALS motor neurons and is restored by VCP ATPase inhibition

Amyotrophic lateral sclerosis (ALS) is characterized by nucleocytoplasmic mislocalization of the RNA-binding protein (RBP) TDP-43. However, emerging evidence suggests more widespread mRNA and protein mislocalization. Here, we employed nucleocytoplasmic fractionation, RNA sequencing, and mass spectrometry to investigate the localization of mRNA and protein in induced pluripotent stem cell-derived motor neurons (iPSMNs) from ALS patients with TARDBP and VCP mutations. ALS mutant iPSMNs exhibited extensive nucleocytoplasmic mRNA redistribution, RBP mislocalization, and splicing alterations. Mislocalized proteins exhibited a greater affinity for redistributed transcripts, suggesting a link between RBP mislocalization and mRNA redistribution. Notably, treatment with ML240, a VCP ATPase inhibitor, partially restored mRNA and protein localization in ALS mutant iPSMNs. ML240 induced changes in the VCP interactome and lysosomal localization and reduced oxidative stress and DNA damage. These findings emphasize the link between RBP mislocalization and mRNA redistribution in ALS motor neurons and highlight the therapeutic potential of VCP inhibition

Integrated transcriptome landscape of ALS identifies genome instability linked to TDP-43 pathology

Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibiting TDP-43 proteinopathy. Elucidating pathomechanisms has been hampered by disease heterogeneity and difficulties accessing motor neurons. Human induced pluripotent stem cell-derived motor neurons (iPSMNs) offer a solution; however, studies have typically been limited to underpowered cohorts. Here, we present a comprehensive compendium of 429 iPSMNs from 15 datasets, and 271 post-mortem spinal cord samples. Using reproducible bioinformatic workflows, we identify robust upregulation of p53 signalling in ALS in both iPSMNs and post-mortem spinal cord. p53 activation is greatest with C9orf72 repeat expansions but is weakest with SOD1 and FUS mutations. TDP-43 depletion potentiates p53 activation in both post-mortem neuronal nuclei and cell culture, thereby functionally linking p53 activation with TDP-43 depletion. ALS iPSMNs and post-mortem tissue display enrichment of splicing alterations, somatic mutations, and gene fusions, possibly contributing to the DNA damage response

Safety and efficacy of digoxin:systematic review and meta-analysis of observational and controlled trial data

Objective To clarify the impact of digoxin on death and clinical outcomes across all observational and randomised controlled trials, accounting for study designs and methods. Data sources and study selection Comprehensive literature search of Medline, Embase, the Cochrane Library, reference lists, and ongoing studies according to a prospectively registered design (PROSPERO: CRD42014010783), including all studies published from 1960 to July 2014 that examined treatment with digoxin compared with control (placebo or no treatment). Data extraction and synthesis Unadjusted and adjusted data pooled according to study design, analysis method, and risk of bias. Main outcome measures Primary outcome (all cause mortality) and secondary outcomes (including admission to hospital) were meta-analysed with random effects modelling. Results 52 studies were systematically reviewed, comprising 621 845 patients. Digoxin users were 2.4 years older than control (weighted difference 95% confidence interval 1.3 to 3.6), with lower ejection fraction (33% v 42%), more diabetes, and greater use of diuretics and anti-arrhythmic drugs. Meta-analysis included 75 study analyses, with a combined total of 4 006 210 patient years of follow-up. Compared with control, the pooled risk ratio for death with digoxin was 1.76 in unadjusted analyses (1.57 to 1.97), 1.61 in adjusted analyses (1.31 to 1.97), 1.18 in propensity matched studies (1.09 to 1.26), and 0.99 in randomised controlled trials (0.93 to 1.05). Meta-regression confirmed that baseline differences between treatment groups had a significant impact on mortality associated with digoxin, including markers of heart failure severity such as use of diuretics (P=0.004). Studies with better methods and lower risk of bias were more likely to report a neutral association of digoxin with mortality (P<0.001). Across all study types, digoxin led to a small but significant reduction in all cause hospital admission (risk ratio 0.92, 0.89 to 0.95; P<0.001; n=29 525). Conclusions Digoxin is associated with a neutral effect on mortality in randomised trials and a lower rate of admissions to hospital across all study types. Regardless of statistical analysis, prescription biases limit the value of observational data

Reactive astrocytes in ALS display diminished intron retention

Reactive astrocytes are implicated in amyotrophic lateral sclerosis (ALS), although the mechanisms controlling reactive transformation are unknown. We show that decreased intron retention (IR) is common to human-induced pluripotent stem cell (hiPSC)-derived astrocytes carrying ALS-causing mutations in VCP, SOD1 and C9orf72. Notably, transcripts with decreased IR and increased expression are overrepresented in reactivity processes including cell adhesion, stress response and immune activation. This was recapitulated in public-datasets for (i) hiPSC-derived astrocytes stimulated with cytokines to undergo reactive transformation and (ii) in vivo astrocytes following selective deletion of TDP-43. We also re-examined public translatome sequencing (TRAP-seq) of astrocytes from a SOD1 mouse model, which revealed that transcripts upregulated in translation significantly overlap with transcripts exhibiting decreased IR. Using nucleocytoplasmic fractionation of VCP mutant astrocytes coupled with mRNA sequencing and proteomics, we identify that decreased IR in nuclear transcripts is associated with enhanced nonsense mediated decay and increased cytoplasmic expression of transcripts and proteins regulating reactive transformation. These findings are consistent with a molecular model for reactive transformation in astrocytes whereby poised nuclear reactivity-related IR transcripts are spliced, undergo nuclear-to-cytoplasmic translocation and translation. Our study therefore provides new insights into the molecular regulation of reactive transformation in astrocytes

Serum and cerebrospinal fluid biomarker profiles in acute SARS-CoV-2-associated neurological syndromes.

Preliminary pathological and biomarker data suggest that SARS-CoV-2 infection can damage the nervous system. To understand what, where and how damage occurs, we collected serum and CSF from patients with COVID-19 and characterized neurological syndromes involving the PNS and CNS (n = 34). We measured biomarkers of neuronal damage and neuroinflammation, and compared these with non-neurological control groups, which included patients with (n = 94) and without (n = 24) COVID-19. We detected increased concentrations of neurofilament light, a dynamic biomarker of neuronal damage, in the CSF of those with CNS inflammation (encephalitis and acute disseminated encephalomyelitis) [14 800 pg/ml (400, 32 400)], compared to those with encephalopathy [1410 pg/ml (756, 1446)], peripheral syndromes (Guillain-Barré syndrome) [740 pg/ml (507, 881)] and controls [872 pg/ml (654, 1200)]. Serum neurofilament light levels were elevated across patients hospitalized with COVID-19, irrespective of neurological manifestations. There was not the usual close correlation between CSF and serum neurofilament light, suggesting serum neurofilament light elevation in the non-neurological patients may reflect peripheral nerve damage in response to severe illness. We did not find significantly elevated levels of serum neurofilament light in community cases of COVID-19 arguing against significant neurological damage. Glial fibrillary acidic protein, a marker of astrocytic activation, was not elevated in the CSF or serum of any group, suggesting astrocytic activation is not a major mediator of neuronal damage in COVID-19

Aspects of mental health dysfunction among survivors of childhood cancer

BACKGROUND: Some previous studies have reported that survivors of childhood cancer are at an increased risk of developing long-term mental health morbidity, whilst others have reported that this is not the case. Therefore, we analysed 5-year survivors of childhood cancer using the British Childhood Cancer Survivor Study (BCCSS) to determine the risks of aspects of long-term mental health dysfunction. PROCEDURE: Within the BCCSS, 10 488 survivors completed a questionnaire that ascertained mental health-related information via 10 questions from the Short Form-36 survey. Internal analyses were conducted using multivariable logistic regression to determine risk factors for mental health dysfunction. External analyses were undertaken using direct standardisation to compare mental health dysfunction in survivors with UK norms. RESULTS: This study has shown that overall, childhood cancer survivors had a significantly higher prevalence of mental health dysfunction for 6/10 questions analysed compared to UK norms. Central nervous system (CNS) and bone sarcoma survivors reported the greatest dysfunction, compared to expected, with significant excess dysfunction in 10 and 6 questions, respectively; the excess ranged from 4.4–22.3% in CNS survivors and 6.9–15.9% in bone sarcoma survivors. Compared to expected, excess mental health dysfunction increased with attained age; this increase was greatest for reporting ‘limitations in social activities due to health', where the excess rose from 4.5% to 12.8% in those aged 16–24 and 45+, respectively. Within the internal analyses, higher levels of educational attainment and socio-economic classification were protective against mental health dysfunction. CONCLUSIONS: Based upon the findings of this large population-based study, childhood cancer survivors report significantly higher levels of mental health dysfunction than those in the general population, where deficits were observed particularly among CNS and bone sarcoma survivors. Limitations were also observed to increase with age, and thus it is important to emphasise the need for mental health evaluation and services across the entire lifespan. There is evidence that low educational attainment and being unemployed or having never worked adversely impacts long-term mental health. These findings provide an evidence base for risk stratification and planning interventions

Repeat liver resection after a hepatic or extended hepatic trisectionectomy for colorectal liver metastasis

AbstractObjectiveA right and left hepatic trisectionectomy and an extended trisectionectomy are the largest liver resections performed for malignancy. This report analyses a series of 23 patients who had at least one repeat resection after a hepatic trisectionectomy for colorectal liver metastasis (CRLM).MethodsA retrospective analysis of a single-centre prospective liver resection database from May 1996 to April 2009 was used for patient identification. Full notes, radiology and patient reviews were analysed for a variety of factors with respect to survival.ResultsTwenty-three patients underwent up to 3 repeat hepatic resections after 20 right and 3 left hepatic trisectionectomies. In 18 patients the initial surgery was an extended trisectionectomy. Overall 1-, 3- and 5-year survival rates after a repeat resection were 100%, 46% and 32%, respectively. No factors predictive for survival were identified.ConclusionA repeat resection after a hepatic trisectionectomy for CRLM can offer extended survival and should be considered where appropriate