Astrocytes display cell autonomous and diverse early reactive states in familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a rapidly progressive and fatal disease. Although astrocytes are increasingly recognized contributors to the underlying pathogenesis, the cellular autonomy and uniformity of astrocyte reactive transformation in different genetic forms of amyotrophic lateral sclerosis remain unresolved. Here we systematically examine these issues by using highly enriched and human induced pluripotent stem cell-derived astrocytes from patients with VCP and SOD1 mutations. We show that VCP mutant astrocytes undergo cell-autonomous reactive transformation characterized by increased expression of complement component 3 (C3) in addition to several characteristic gene expression changes. We then demonstrate that isochronic SOD1 mutant astrocytes also undergo a cell-autonomous reactive transformation, but that this is molecularly distinct from VCP mutant astrocytes. This is shown through transcriptome-wide analyses, identifying divergent gene expression profiles and activation of different key transcription factors in SOD1 and VCP mutant human induced pluripotent stem cell-derived astrocytes. Finally, we show functional differences in the basal cytokine secretome between VCP and SOD1 mutant human induced pluripotent stem cell-derived astrocytes. Our data therefore reveal that reactive transformation can occur cell autonomously in human amyotrophic lateral sclerosis astrocytes and with a striking degree of early molecular and functional heterogeneity when comparing different disease-causing mutations. These insights may be important when considering astrocyte reactivity as a putative therapeutic target in familial amyotrophic lateral sclerosis

A major genetic locus in <i>Trypanosoma brucei</i> is a determinant of host pathology

The progression and variation of pathology during infections can be due to components from both host or pathogen, and/or the interaction between them. The influence of host genetic variation on disease pathology during infections with trypanosomes has been well studied in recent years, but the role of parasite genetic variation has not been extensively studied. We have shown that there is parasite strain-specific variation in the level of splenomegaly and hepatomegaly in infected mice and used a forward genetic approach to identify the parasite loci that determine this variation. This approach allowed us to dissect and identify the parasite loci that determine the complex phenotypes induced by infection. Using the available trypanosome genetic map, a major quantitative trait locus (QTL) was identified on T. brucei chromosome 3 (LOD = 7.2) that accounted for approximately two thirds of the variance observed in each of two correlated phenotypes, splenomegaly and hepatomegaly, in the infected mice (named &lt;i&gt;TbOrg1&lt;/i&gt;). In addition, a second locus was identified that contributed to splenomegaly, hepatomegaly and reticulocytosis (&lt;i&gt;TbOrg2&lt;/i&gt;). This is the first use of quantitative trait locus mapping in a diploid protozoan and shows that there are trypanosome genes that directly contribute to the progression of pathology during infections and, therefore, that parasite genetic variation can be a critical factor in disease outcome. The identification of parasite loci is a first step towards identifying the genes that are responsible for these important traits and shows the power of genetic analysis as a tool for dissecting complex quantitative phenotypic traits

Effect of pre-contraction on β-adrenoceptor-mediated relaxation of rat urinary bladder

Purpose The human physiological bladder contraction is largely mediated by acetylcholine acting on muscarinic receptors, but in pathophysiological settings the relative role of non-cholinergic stimuli gains importance. beta-Adrenoceptor agonists are currently in clinical development as treatments for the overactive bladder syndrome. Therefore, we have explored the ability of the beta-adrenoceptor agonist isoprenaline to induce rat isolated bladder strip relaxation on pre-contraction with the muscarinic agonist carbachol as compared to bladder tone induced by several non-cholinergic stimuli. Methods Bladder tone was induced by passive tension, receptor independently by KCl, carbachol, bradykinin or serotonin. Concentration-response curves were generated for relaxation by isoprenaline, and a single concentration of the receptor-independent relaxant forskolin was also tested. Results The various contractile stimuli induced different degrees of bladder tone, but the ability of isoprenaline or forskolin to relax rat bladder was not correlated with the degree of tone. Isoprenaline was significantly less potent and effective in causing relaxation against carbachol-induced tone than against any other stimulus, whereas no such relationship was observed for forskolin. Conclusions We conclude that beta-adrenoceptor agonists can induce rat bladder relaxation against a wide range of contractile stimuli and are more potent and/or effective against non-cholinergic stimuli than against muscarinic agonism. This profile appears desirable for agents intended for the treatment of overactive bladde

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

A neuroprotective astrocyte state is induced by neuronal signal EphB1 but fails in ALS models

Astrocyte responses to neuronal injury may be beneficial or detrimental to neuronal recovery, but the mechanisms that determine these different responses are poorly understood. Here we show that ephrin type-B receptor 1 (EphB1) is upregulated in injured motor neurons, which in turn can activate astrocytes through ephrin-B1-mediated stimulation of signal transducer and activator of transcription-3 (STAT3). Transcriptional analysis shows that EphB1 induces a protective and anti-inflammatory signature in astrocytes, partially linked to the STAT3 network. This is distinct from the response evoked by interleukin (IL)-6 that is known to induce both pro inflammatory and anti-inflammatory processes. Finally, we demonstrate that the EphB1–ephrin-B1 pathway is disrupted in human stem cell derived astrocyte and mouse models of amyotrophic lateral sclerosis (ALS). Our work identifies an early neuronal help-me signal that activates a neuroprotective astrocytic response, which fails in ALS, and therefore represents an attractive therapeutic target.This work has been funded by Medical Research Council (MR/P008658/1 for A.L.), the Walker Fund (A.L.), John van Geest Fund (A.L.) and the Wellcome Trust (101149/Z/13/A for R.P.). We also acknowledge the DPUK/MRC platform for provision of the Opera Phenix for high-throughput iPSC analysis and the support by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. We are grateful for the support from the Grant Agency of the Czech Republic (GACR 17-21146 S, S.F.). Dr Christopher Sibley is supported by the Edmond Lilly Safra Fellowship, Dr Rickie Patani holds a Wellcome Trust Clinician Scientist Fellowship and Dr András Lakatos holds an MRC Clinician Scientist Fellowship

The British Lexicon Project: Lexical decision data for 28,730 monosyllabic and disyllabic English words

We present a new database of lexical decision times for English words and nonwords, for which two groups of British participants each responded to 14,365 monosyllabic and disyllabic words and the same number of nonwords for a total duration of 16 h (divided over multiple sessions). This database, called the British Lexicon Project (BLP), fills an important gap between the Dutch Lexicon Project (DLP; Keuleers, Diependaele, & Brysbaert, Frontiers in Language Sciences. Psychology, 1, 174, 2010) and the English Lexicon Project (ELP; Balota et al., 2007), because it applies the repeated measures design of the DLP to the English language. The high correlation between the BLP and ELP data indicates that a high percentage of variance in lexical decision data sets is systematic variance, rather than noise, and that the results of megastudies are rather robust with respect to the selection and presentation of the stimuli. Because of its design, the BLP makes the same analyses possible as the DLP, offering researchers with a new interesting data set of word-processing times for mixed effects analyses and mathematical modeling. The BLP data are available at http://crr.ugent.be/blp and as Electronic Supplementary Materials

Basic mechanisms of urgency: roles and benefits of pharmacotherapy

Introduction Since urgency is key to the overactive bladder syndrome, we have reviewed the mechanisms underlying how bladder filling and urgency are sensed, what causes urgency and how this relates to medical therapy. Materials and methods Review of published literature. Results As urgency can only be assessed in cognitively intact humans, mechanistic studies of urgency often rely on proxy or surrogate parameters, such as detrusor overactivity, but these may not necessarily be reliable. There is an increasing evidence base to suggest that the sensation of ‘urgency’ differs from the normal physiological urge to void upon bladder filling. While the relative roles of alterations in afferent processes, central nervous processing, efferent mechanisms and in intrinsic bladder smooth muscle function remain unclear, and not necessarily mutually exclusive, several lines of evidence support an important role for the latter. Conclusions A better understanding of urgency and its causes may help to develop more effective treatments for voiding dysfunction

Identification of Allele-Specific RNAi Effectors Targeting Genetic Forms of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurological disorder affecting an estimated 5–10 million people worldwide. Recent evidence has implicated several genes that directly cause or increase susceptibility to PD. As well as advancing understanding of the genetic aetiology of PD these findings suggest new ways to modify the disease course, in some cases through genetic manipulation. Here we generated a ‘walk-through’ series of RNA Pol III-expressed shRNAs targeting both the α-synuclein A30P and LRRK2 G2019S PD-associated mutations. Allele-specific discrimination of the α-synuclein A30P mutation was achieved with alignments at position 10, 13 and 14 in two model systems, including a heterozygous model mimicking the disease setting, whilst 5′RACE was used to confirm stated alignments. Discrimination of the most common PD-linked LRRK2 G2019S mutation was assessed in hemizygous dual-luciferase assays and showed that alignment of the mutation opposite position 4 of the antisense species produced robust discrimination of alleles at all time points studied. Discrimination at this position was subsequently confirmed using siRNAs, where up to 10-fold discrimination was seen. The results suggest that RNAi-mediated silencing of PD-associated autosomal dominant genes could be a novel therapeutic approach for the treatment of the relevant clinical cases of PD in future

Risk factors for incident falls in older men and women:The English longitudinal study of ageing

Background: falls are a major cause of disability and death in older people, particularly women. Cross-sectional surveys suggest that some risk factors associated with a history of falls may be sex-specific, but whether risk factors for incident falls differ between the sexes is unclear. We investigated whether risk factors for incident falls differ between men and women.Methods: participants were 3298 people aged ≥60 who took part in the Waves 4-6 surveys of the English Longitudinal Study of Ageing. At Wave 4, they provided information about sociodemographic, lifestyle, behavioural and medical factors and had their physical and cognitive function assessed. Data on incident falls during the four-year follow-up period was collected from them at Waves 5 and 6. Poisson regression with robust variance estimation was used to derive relative risks (RR) for the association between baseline characteristics and incident falls.Results: in multivariable-adjusted models that also controlled for history of falls, older age was the only factor associated with increased risk of incident falls in both sexes. Some factors were only predictive of falls in one sex, namely more depressive symptoms (RR (95% CI) 1.03 (1.01,1.06)), incontinence (1.12 (1.00,1.24)) and never having married in women (1.26 (1.03,1.53)), and greater comorbidity (1.04 (1.00,1.08)), higher levels of pain (1.10 (1.04,1.17) and poorer balance, as indicated by inability to attempt a full-tandem stand, (1.23 (1.04,1.47)) in men. Of these, only the relationships between pain, balance and comorbidity and falls risk differed significantly by sex.Conclusions: there were some differences between the sexes in risk factors for incident falls. Our observation that associations between pain, balance and comorbidity and incident falls risk varied by sex needs further investigation in other cohorts. <br/