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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by progressive loss of upper and lower motor neurons. ALS is on a pathogenetic disease spectrum with frontotemporal dementia (FTD), with patients sometimes experiencing elements of both conditions (ALS-FTSD). For mutations associated with ALS-FTSD, such as theC9orf72hexanucleotide repeat expansion (HRE), the factors influencing where an individual may lie on this spectrum require further characterisation. Here, using NanoString molecular barcoding with a panel of 770 neuroinflammatory genes, we interrogate inflammatory dysregulation at the level of gene expression. We identified 20 dysregulated neuroinflammatory genes in the motor cortex of deeply clinically phenotyped C9-ALSpost-mortemcases, with enrichment of microglial and inflammatory response gene sets. Our analyses also revealed two distinct ALS-related neuroinflammatory panel signatures (NPS), NPS1 and NPS2, delineated by the direction of expression of proinflammatory, axonal transport and synaptic signalling pathways. Two genes with significant correlations to available clinical metrics were selected for validation:FKBP5andBDNF. FKBP5 and its signalling partner, NF-κB, appeared to have a cell-type-specific staining distribution, with activated (i.e., nuclear) NF-κB immunoreactivity in C9-ALS. Expression ofBDNF, a correlate of disease duration, was confirmed to be higher in individuals with long compared to short disease duration using BaseScope™in situhybridisation. Finally, we compared NPS between C9-ALS cases and those from deeply clinically phenotyped sporadic ALS (sALS) and SOD1-ALS cohorts, with NPS1 and NPS2 appearing across all cohorts. A subset of these signatures was also detected in publicly available RNA-sequencing data from independent C9-ALS and sALS cohorts, underscoring the relevance of these pathways across cohorts. Our findings highlight the importance of tailoring therapeutic approaches based on distinct molecular signatures that exist between and within genetic and sporadic cohorts.

Neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) are traditionally considered strictly neurological disorders. However, clinical presentation is not restricted to neurological systems, and non-central nervous system (CNS) manifestations, particularly gastrointestinal (GI) symptoms, are common. Our objective was to understand the systemic distribution of pathology in archived non-CNS tissues, taken as part of routine clinical practice during life from people with ALS. We examined tissue from 13 people who went on to develop ALS; including sporadic ALS (n = 12) and C9orf72 hexanucleotide repeat expansion (n = 1). The tissue cohort consisted of 68 formalin-fixed paraffin embedded samples from 21 surgical cases (some patients having more than one case over their lifetimes), from 8 organ systems, which we examined for evidence of phosphorylated TDP-43 (pTDP-43) pathology. We identified pTDP-43 aggregates in multiple cell types of the GI tract, including macrophages and dendritic cells within the lamina propria; as well as ganglion/neuronal and glial cells of the myenteric plexus. Aggregates were also noted within lymph node parenchyma, blood vessel endothelial cells, and chondrocytes. We note that in all cases with non-CNS pTDP-43 pathology, aggregates were present prior to ALS diagnosis and in some instances preceded neurological symptom onset by more than 10 years. These data imply that patients with microscopically unexplained non-CNS symptoms could have occult protein aggregation that could be detected many years prior to neurological involvement.

Dietary restriction (DR), limiting calories or specific nutrients without malnutrition, extends lifespan across diverse taxa. Traditionally, this lifespan extension has been explained as a result of diet-mediated changes in the trade-off between lifespan and reproduction, with survival favoured when resources are scarce. However, a recently proposed alternative suggests that the selective benefit of the response to DR is the maintenance of reproduction. This hypothesis predicts that lifespan extension is a side effect of benign laboratory conditions, and DR individuals would be frailer and unable to deal with additional stressors, and thus lifespan extension should disappear under more stressful conditions. We tested this by rearing outbred female fruit flies (Drosophila melanogaster) on 10 different protein:carbohydrate diets. Flies were either infected with a bacterial pathogen (Pseudomonas entomophila), injured with a sterile pinprick or unstressed. We monitored lifespan, fecundity and measures of ageing. DR extended lifespan and reduced reproduction irrespective of injury and infection. Infected flies on lower protein diets had particularly poor survival. Exposure to infection and injury did not substantially alter the relationship between diet and ageing patterns. These results do not provide support for lifespan extension under DR being a side effect of benign laboratory conditions. This article is protected by copyright. All rights reserved.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease with no cure and limited treatment options. There is therefore an urgent need for effective therapeutic interventions in this disease. This protocol outlines the strategy for a systematic review and meta-analysis to identify, from in vivo animal studies, potential therapeutic interventions for ALS cases in which an interferon response is induced. Our aim is to perform a comprehensive review of the ALS literature to compile a list of (i) interventions affecting interferon signalling pathways and (ii) specific interferon signalling pathway targets that may be manipulated for therapeutic benefit in patients with ALS. We will do this by assessing the pre-clinical literature for the effects of interferon signalling pathway manipulation on the following primary and secondary outcome measures comparing models of ALS/FTD to controls; the primary outcome measure being survival, and secondary outcome measures including histological, biochemical, and behavioural metrics. We will also carry out a structured quality assessment of the literature to provide recommendations for future studies.

ObjectiveNeurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) are traditionally considered strictly neurological disorders. However, clinical presentation is not restricted to neurological systems, and non-central nervous system (CNS) manifestations, particularly gastrointestinal (GI) symptoms, are common. Our objective was to understand the systemic distribution of pathology in archived non-CNS tissues, taken as part of routine clinical practice during life from people with ALS.DesignWe requested all surgical specimens of non-CNS tissue taken during life from 48 people with ALS, for whom evidence of the characteristic proteinopathy associated with ALS had been identified in the CNS after death (i.e., the pathological cytoplasmic accumulation of phosphorylated TDP-43 (pTDP-43) aggregates). Of the 48 patients, 13 had sufficient tissue for evaluation: 12 patients with sporadic ALS and 1 patient with a C9orf72 hexanucleotide repeat expansion. The final cohort consisted of 68 formalin-fixed paraffin embedded tissue samples from 22 surgical cases (some patients having more than one case over their lifetimes), representing 8 organ systems, which we examined for evidence of pTDP-43 pathology. The median age of tissue removal was 62.4 years old and median tissue removal to death was 6.3 years.ResultsWe identified pTDP-43 aggregates in multiple cell types of the GI tract (i.e., colon and gallbladder), including macrophages and dendritic cells within the lamina propria, as well as neuronal and glial cells of the myenteric plexus. Aggregates were also noted within lymph node parenchyma, blood vessel endothelial cells, and chondrocytes. We note that in all cases with non-CNS pTDP-43 pathology, aggregates were present prior to ALS diagnosis (median=3years) and, in some instances, preceded neurological symptom onset by more than 10 years.ConclusionThese data imply that patients with non-CNS symptoms may have occult protein aggregation that could be detected many years prior to neurological involvement.SummaryNeurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) are traditionally considered strictly neurological disorders. However, clinical presentation is not restricted to neurological systems, and non-central nervous system (CNS) manifestations, particularly gastrointestinal (GI) symptoms, are common. Our objective was to understand the systemic distribution of TDP-43 pathology in archived non-CNS tissues, taken as part of routine clinical practice during life from people with ALS. We identified pTDP-43 aggregates in multiple cell types of the GI tract (i.e., colon and gallbladder), and within lymph node parenchyma, blood vessel endothelial cells, and chondrocytes. We note that in all cases with non-CNS pTDP-43 pathology, aggregates were present prior to ALS diagnosis (median=24months) and, in some instances, preceded neurological symptom onset by more than 10years. These data imply that patients with non-CNS symptoms may have occult protein aggregation tha could be detected many years prior to neurological involvement.Graphical AbstractGraphical Abstract.Ante-mortem tissue cohort comprised of tissue taken from people with ALS demonstrates non-CNS accumulation of pTDP-43 aggregates prior to symptom onset.Schematic of workflow to identify pTDP-43 aggregates indicative of non-central nervous system (CNS) manifestations of ALS. Lower panel left: cartoon depicting organs and cell types that had evidence of pTDP-43 aggregation in ALS patient non-CNS ante-mortem tissue. Lower panel right: cartoon depicting organs with no evidence of pTDP-43 aggregation in ALS patient non-CNS ante-mortem tissue.

ObjectivesMotor neuron disease (MND) is an incurable progressive neurodegenerative disease with limited treatment options. There is a pressing need for innovation in identifying therapies to take to clinical trial. Here, we detail a systematic and structured evidence-based approach to inform consensus decision making to select the first two drugs for evaluation in Motor Neuron Disease-Systematic Multi-arm Adaptive Randomised Trial (MND-SMART:NCT04302870), an adaptive platform trial. We aim to identify and prioritise candidate drugs which have the best available evidence for efficacy, acceptable safety profiles and are feasible for evaluation within the trial protocol.MethodsWe conducted a two-stage systematic review to identify potential neuroprotective interventions. First, we reviewed clinical studies in MND, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease and multiple sclerosis, identifying drugs described in at least one MND publication or publications in two or more other diseases. We scored and ranked drugs using a metric evaluating safety, efficacy, study size and study quality. In stage two, we reviewed efficacy of drugs in MND animal models, multicellular eukaryotic models and human induced pluripotent stem cell (iPSC) studies. An expert panel reviewed candidate drugs over two shortlisting rounds and a final selection round, considering the systematic review findings, late breaking evidence, mechanistic plausibility, safety, tolerability and feasibility of evaluation in MND-SMART.ResultsFrom the clinical review, we identified 595 interventions. 66 drugs met our drug/disease logic. Of these, 22 drugs with supportive clinical and preclinical evidence were shortlisted at round 1. Seven drugs proceeded to round 2. The panel reached a consensus to evaluate memantine and trazodone as the first two arms of MND-SMART.DiscussionFor future drug selection, we will incorporate automation tools, text-mining and machine learning techniques to the systematic reviews and consider data generated from other domains, including high-throughput phenotypic screening of human iPSCs.

Dietary restriction (DR), limiting calories or specific nutrients, extends lifespan across diverse taxa. This lifespan extension has been explained as diet-mediated changes in the trade-off between lifespan and reproduction, with survival favoured with scarce resources. Another evolutionary hypothesis suggests the selective benefit of the response is the maintenance of reproduction. This hypothesis predicts that lifespan extension is a side effect of benign laboratory conditions, where DR individuals are frailer and unable to deal with additional stressors, and thus lifespan extension should disappear under more stressful conditions. We tested this by rearing outbred female Drosophila melanogaster on 10 different protein:carbohydrate diets. Flies were either infected with a bacterial pathogen (Pseudomonas entomophila), injured or unstressed. We monitored lifespan, fecundity and ageing measures. DR extended lifespan and reduced reproduction irrespective of injury and infection. These results do not support lifespan extension under DR being a side effect of benign laboratory conditions.

Interventions targeting mitochondrial dysfunction have the potential to extend survival in preclinical models of amyotrophic lateral sclerosis. The aim of this systematic review was to assess the efficacy of targeting mitochondria as a potential therapeutic target in amyotrophic lateral sclerosis. Preclinical studies written in the English language were identified with no restrictions on publication date from PubMed, Medline and EMBASE databases. All studies adopting interventions targeting mitochondria to treat amyotrophic lateral sclerosis in genetic or drug-induced organism models were considered for inclusion. A total of 76 studies were included in the analysis. Survival data were extracted, and the meta-analysis was completed in RevMan 5 software. We show that targeting mitochondrial dysfunction in amyotrophic lateral sclerosis results in a statistically significant improvement in survival (Z = 5.31; P

Drosophila suzukii (Matsumura) is one of the most damaging and costly pests to invade temperate horticultural regions in recent history. Conventional control of this pest is challenging, and an environmentally benign microbial biopesticide is highly desirable. A thorough exploration of the pathogens infecting this pest is not only the first step on the road to the development of an effective biopesticide, but also provides a valuable comparative dataset for the study of viruses in the model family Drosophilidae. Here we use a metatransciptomic approach to identify viruses infecting this fly in both its native (Japanese) and invasive (British and French) ranges. We describe eighteen new RNA viruses, including members of the Picornavirales, Mononegavirales, Bunyavirales, Chuviruses, Nodaviridae, Tombusviridae, Reoviridae, and Nidovirales, and discuss their phylogenetic relationships with previously known viruses. We also detect 18 previously described viruses of other Drosophila species that appear to be associated with D. suzukii in the wild.

Drosophila suzukii(Matsumura) is one of the most damaging and costly pests to invade temperate horticultural regions in recent history. Conventional control of this pest is challenging, and an environmentally benign microbial biopesticide is highly desirable. A thorough exploration of the pathogens infecting this pest is not only the first step on the road to the development of an effective biopesticide, but also provides a valuable comparative dataset for the study of viruses in the model familyDrosophilidae.Here we use a metatransciptomic approach to identify viruses infecting this fly in both its native (Japanese) and invasive (British and French) ranges. We describe 18 new RNA viruses, including members of the Picornavirales, Mononegavirales, Bunyavirales, Chuviruses,Nodaviridae, Tombusviridae, Reoviridae,and Nidovirales, and discuss their phylogenetic relationships with previously known viruses. We also detect 18 previously described viruses of otherDrosophilaspecies that appear to be associated withD. suzukiiin the wild.

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host–virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research., Sequencing of metagenomic RNA and small RNA identifies more than 20 new viruses associated with the fruit fly Drosophila melanogaster, and large-scale surveys show that many are common in the lab and in the field., Author Summary The fruit fly Drosophila melanogaster is extensively used as a model species for molecular biology and genetics. It is also widely studied for its evolutionary history, helping us understand how natural selection has shaped the genome. Drosophila research has been particularly valuable in determining how the insect immune system interacts with viruses and how co-evolution between hosts and viruses can shape the host immune system. Understanding insect–virus coevolution is important because some viruses—such as those which cause dengue and yellow fever in humans—also infect their insect vectors, and because the viruses of bees and other pollinators are implicated in pollinator decline. Although we have an increasingly good idea of how flies recognise and combat viral pathogens, we still have much to learn about the viruses they encounter and interact with in the wild. In this paper, we sequence all of the genetic material from a large collection of wild fruit flies and use it to identify more than 20 new viruses. We then survey individual wild flies and laboratory stocks to find out which viruses are common, which are rare, and which species of fruit fly they infect. Our results provide valuable tools and an evolutionary and ecological perspective that will help to improve Drosophila as a model for host–virus biology in the future.

The recombinational environment influences patterns of molecular evolution through the effects of Hill–Robertson interference. Here, we examine genome-wide patterns of gene expression with respect to recombinational environment inDrosophila melanogaster. We find that regions of the genome lacking crossing over exhibit elevated levels of expression, and this is most pronounced for genes on the entirely non-crossing over fourth chromosome. We find no evidence for differences in the patterns of gene expression between regions of high, intermediate and low crossover frequencies. These results suggest that, in the absence of crossing over, selection to maintain control of expression may be compromised, perhaps due to the accumulation of deleterious mutations in regulatory regions. Alternatively, higher gene expression may be evolving to compensate for defective protein products or reduced translational efficiency.