Inhibiting CSF1R alleviates cerebrovascular white matter disease and cognitive impairment.

White matter abnormalities, related to poor cerebral perfusion, are a core feature of small vessel cerebrovascular disease, and critical determinants of vascular cognitive impairment and dementia. Despite this importance there is a lack of treatment options. Proliferation of microglia producing an expanded, reactive population and associated neuroinflammatory alterations have been implicated in the onset and progression of cerebrovascular white matter disease, in patients and in animal models, suggesting that targeting microglial proliferation may exert protection. Colony-stimulating factor-1 receptor (CSF1R) is a key regulator of microglial proliferation. We found that the expression of CSF1R/Csf1r and other markers indicative of increased microglial abundance are significantly elevated in damaged white matter in human cerebrovascular disease and in a clinically relevant mouse model of chronic cerebral hypoperfusion and vascular cognitive impairment. Using the mouse model, we investigated long-term pharmacological CSF1R inhibition, via GW2580, and demonstrated that the expansion of microglial numbers in chronic hypoperfused white matter is prevented. Transcriptomic analysis of hypoperfused white matter tissue showed enrichment of microglial and inflammatory gene sets, including phagocytic genes that were the predominant expression modules modified by CSF1R inhibition. Further, CSF1R inhibition attenuated hypoperfusion-induced white matter pathology and rescued spatial learning impairments and to a lesser extent cognitive flexibility. Overall, this work suggests that inhibition of CSF1R and microglial proliferation mediates protection against chronic cerebrovascular white matter pathology and cognitive deficits. Our study nominates CSF1R as a target for the treatment of vascular cognitive disorders with broader implications for treatment of other chronic white matter diseases.

Transmembrane protein 97 is a potential synaptic amyloid beta receptor in human Alzheimer's disease.

Synapse loss correlates with cognitive decline in Alzheimer's disease, and soluble oligomeric amyloid beta (Aß) is implicated in synaptic dysfunction and loss. An important knowledge gap is the lack of understanding of how Aß leads to synapse degeneration. In particular, there has been difficulty in determining whether there is a synaptic receptor that binds Aß and mediates toxicity. While many candidates have been observed in model systems, their relevance to human AD brain remains unknown. This is in part due to methodological limitations preventing visualization of Aß binding at individual synapses. To overcome this limitation, we combined two high resolution microscopy techniques: array tomography and Förster resonance energy transfer (FRET) to image over 1 million individual synaptic terminals in temporal cortex from AD (n = 11) and control cases (n = 9). Within presynapses and post-synaptic densities, oligomeric Aß generates a FRET signal with transmembrane protein 97. Further, Aß generates a FRET signal with cellular prion protein, and post-synaptic density 95 within post synapses. Transmembrane protein 97 is also present in a higher proportion of post synapses in Alzheimer's brain compared to controls. We inhibited Aß/transmembrane protein 97 interaction in a mouse model of amyloidopathy by treating with the allosteric modulator CT1812. CT1812 drug concentration correlated negatively with synaptic FRET signal between transmembrane protein 97 and Aß. In human-induced pluripotent stem cell derived neurons, transmembrane protein 97 is present in synapses and colocalizes with Aß when neurons are challenged with human Alzheimer's brain homogenate. Transcriptional changes are induced by Aß including changes in genes involved in neurodegeneration and neuroinflammation. CT1812 treatment of these neurons caused changes in gene sets involved in synaptic function. These data support a role for transmembrane protein 97 in the synaptic binding of Aß in human Alzheimer's disease brain where it may mediate synaptotoxicity.

Distinct neuroinflammatory signatures exist across genetic and sporadic amyotrophic lateral sclerosis cohorts.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. ALS is on a pathogenetic disease spectrum with frontotemporal dementia, referred to as ALS-frontotemporal spectrum disorder (ALS-FTSD). For mutations associated with ALS-FTSD, such as the C9orf72 hexanucleotide repeat expansion, the molecular factors associated with heterogeneity along this spectrum require further characterization. Here, using a targeted NanoString molecular barcoding approach, we interrogate neuroinflammatory dysregulation and heterogeneity at the level of gene expression in post-mortem motor cortex tissue from a cohort of clinically heterogeneous C9-ALS-FTSD cases. We identified 20 dysregulated genes in C9-ALS-FTSD, with enrichment of microglial and inflammatory response gene sets. Two genes with significant correlations to available clinical metrics were selected for validation: FKBP5, a correlate of cognitive function, and brain-derived neurotrophic factor (BDNF), a correlate of disease duration. 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-FTSD. Expression of BDNF, a correlate of disease duration, was confirmed to be higher in individuals with long compared to short disease duration using BaseScope™ in situ hybridization. Our analyses also revealed two distinct neuroinflammatory panel signatures (NPS), NPS1 and NPS2, delineated by the direction of expression of proinflammatory, axonal transport and synaptic signalling pathways. We compared NPS between C9-ALS-FTSD cases and those from sporadic ALS and SOD1-ALS cohorts and identified NPS1 and NPS2 across all cohorts. Moreover, a subset of NPS was also able to separate publicly available RNA sequencing data from independent C9-ALS and sporadic ALS cohorts into two inflammatory subgroups. Importantly, NPS subgroups did not clearly segregate with available demographic, genetic, clinical or pathological features, highlighting the value of molecular stratification in clinical trials for inflammatory subgroup identification. Our findings thus underscore the importance of tailoring therapeutic approaches based on distinct molecular signatures that exist between and within ALS-FTSD cohorts.

Identifying foetal forebrain interneurons as a target for monogenic autism risk factors and the polygenic 16p11.2 microdeletion.

BACKGROUND: Autism spectrum condition or 'autism' is associated with numerous genetic risk factors including the polygenic 16p11.2 microdeletion. The balance between excitatory and inhibitory neurons in the cerebral cortex is hypothesised to be critical for the aetiology of autism making improved understanding of how risk factors impact on the development of these cells an important area of research. In the current study we aim to combine bioinformatics analysis of human foetal cerebral cortex gene expression data with anatomical and electrophysiological analysis of a 16p11.2+/- rat model to investigate how genetic risk factors impact on inhibitory neuron development. METHODS: We performed bioinformatics analysis of single cell transcriptomes from gestational week (GW) 8-26 human foetal prefrontal cortex and anatomical and electrophysiological analysis of 16p11.2+/- rat cerebral cortex and hippocampus at post-natal day (P) 21. RESULTS: We identified a subset of human interneurons (INs) first appearing at GW23 with enriched expression of a large fraction of risk factor transcripts including those expressed from the 16p11.2 locus. This suggests the hypothesis that these foetal INs are vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2+/- rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells. LIMITATIONS: The human foetal gene expression data was acquired from cerebral cortex between gestational week (GW) 8 to 26. We cannot draw inferences about potential vulnerabilities to genetic autism risk factors for cells not present in the developing cerebral cortex at these stages. The analysis 16p11.2+/- rat phenotypes reported in the current study was restricted to 3-week old (P21) animals around the time of weaning and to a single interneuron cell-type while in human 16p11.2 microdeletion carriers symptoms likely involve multiple cell types and manifest in the first few years of life and on into adulthood. CONCLUSIONS: We have identified developing interneurons in human foetal cerebral cortex as potentially vulnerable to monogenic autism risk factors and the 16p11.2 microdeletion and report interneuron phenotypes in post-natal 16p11.2+/- rats.

Synaptic resilience is associated with maintained cognition during ageing.

INTRODUCTION: It remains unclear why age increases risk of Alzheimer's disease and why some people experience age-related cognitive decline in the absence of dementia. Here we test the hypothesis that resilience to molecular changes in synapses contribute to healthy cognitive ageing. METHODS: We examined post-mortem brain tissue from people in mid-life (n = 15), healthy ageing with either maintained cognition (n = 9) or lifetime cognitive decline (n = 8), and Alzheimer's disease (n = 13). Synapses were examined with high resolution imaging, proteomics, and RNA sequencing. Stem cell-derived neurons were challenged with Alzheimer's brain homogenate. RESULTS: Synaptic pathology increased, and expression of genes involved in synaptic signaling decreased between mid-life, healthy ageing and Alzheimer's. In contrast, brain tissue and neurons from people with maintained cognition during ageing exhibited decreases in synaptic signaling genes compared to people with cognitive decline. DISCUSSION: Efficient synaptic networks without pathological protein accumulation may contribute to maintained cognition during ageing.

Recapitulation of the EEF1A2 D252H neurodevelopmental disorder-causing missense mutation in mice reveals a toxic gain of function.

Heterozygous de novo mutations in EEF1A2, encoding the tissue-specific translation elongation factor eEF1A2, have been shown to cause neurodevelopmental disorders including often severe epilepsy and intellectual disability. The mutational profile is unusual; ~50 different missense mutations have been identified but no obvious loss of function mutations, though large heterozygous deletions are known to be compatible with life. A key question is whether the heterozygous missense mutations operate through haploinsufficiency or a gain of function mechanism, an important prerequisite for design of therapeutic strategies. In order both to address this question and to provide a novel model for neurodevelopmental disorders resulting from mutations in EEF1A2, we created a new mouse model of the D252H mutation. This mutation causes the eEF1A2 protein to be expressed at lower levels in brain but higher in muscle in the mice. We compared both heterozygous and homozygous D252H and null mutant mice using behavioural and motor phenotyping alongside molecular modelling and analysis of binding partners. Although the proteomic analysis pointed to a loss of function for the D252H mutant protein, the D252H homozygous mice were more severely affected than null homozygotes on the same genetic background. Mice that are heterozygous for the missense mutation show no behavioural abnormalities but do have sex-specific deficits in body mass and motor function. The phenotyping of our novel mouse lines, together with analysis of molecular modelling and interacting proteins, suggest that the D252H mutation results in a gain of function.

Secondary injury and inflammation after intracerebral haemorrhage: a systematic review and meta-analysis of molecular markers in patient brain tissue.

BACKGROUND: Inflammatory responses to intracerebral haemorrhage (ICH) are potential therapeutic targets. We aimed to quantify molecular markers of inflammation in human brain tissue after ICH compared with controls using meta-analysis. METHODS: We searched OVID MEDLINE (1946-) and Embase (1974-) in June 2020 for studies that reported any measure of a molecular marker of inflammation in brain tissue from five or more adults after ICH. We assessed risk of bias using a modified Newcastle-Ottawa Scale (mNOS; mNOS score 0-9; 9 indicates low bias), extracted aggregate data, and used random effects meta-analysis to pool associations of molecules where more than two independent case-control studies reported the same outcome and Gene Ontology enrichment analysis to identify over-represented biological processes in pooled sets of differentially expressed molecules (International Prospective Register of Systematic Reviews ID: CRD42018110204). RESULTS: Of 7501 studies identified, 44 were included: 6 were case series and 38 were case-control studies (median mNOS score 4, IQR 3-5). We extracted data from 21 491 analyses of 20 951 molecules reported by 38 case-control studies. Only one molecule (interleukin-1ß protein) was quantified in three case-control studies (127 ICH cases vs 41 ICH-free controls), which found increased abundance of interleukin-1ß protein after ICH (corrected standardised mean difference 1.74, 95% CI 0.28 to 3.21, p=0.036, I2=46%). Processes associated with interleukin-1ß signalling were enriched in sets of molecules that were more abundant after ICH. CONCLUSION: Interleukin-1ß abundance is increased after ICH, but analyses of other inflammatory molecules after ICH lack replication. Interleukin-1ß pathway modulators may optimise inflammatory responses to ICH and merit testing in clinical trials.

Mitochondrial bioenergetic deficits in C9orf72 amyotrophic lateral sclerosis motor neurons cause dysfunctional axonal homeostasis.

Axonal dysfunction is a common phenotype in neurodegenerative disorders, including in amyotrophic lateral sclerosis (ALS), where the key pathological cell-type, the motor neuron (MN), has an axon extending up to a metre long. The maintenance of axonal function is a highly energy-demanding process, raising the question of whether MN cellular energetics is perturbed in ALS, and whether its recovery promotes axonal rescue. To address this, we undertook cellular and molecular interrogation of multiple patient-derived induced pluripotent stem cell lines and patient autopsy samples harbouring the most common ALS causing mutation, C9orf72. Using paired mutant and isogenic expansion-corrected controls, we show that C9orf72 MNs have shorter axons, impaired fast axonal transport of mitochondrial cargo, and altered mitochondrial bioenergetic function. RNAseq revealed reduced gene expression of mitochondrially encoded electron transport chain transcripts, with neuropathological analysis of C9orf72-ALS post-mortem tissue importantly confirming selective dysregulation of the mitochondrially encoded transcripts in ventral horn spinal MNs, but not in corresponding dorsal horn sensory neurons, with findings reflected at the protein level. Mitochondrial DNA copy number was unaltered, both in vitro and in human post-mortem tissue. Genetic manipulation of mitochondrial biogenesis in C9orf72 MNs corrected the bioenergetic deficit and also rescued the axonal length and transport phenotypes. Collectively, our data show that loss of mitochondrial function is a key mediator of axonal dysfunction in C9orf72-ALS, and that boosting MN bioenergetics is sufficient to restore axonal homeostasis, opening new potential therapeutic strategies for ALS that target mitochondrial function.

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.

Reversal of proliferation deficits caused by chromosome 16p13.11 microduplication through targeting NFκB signaling: an integrated study of patient-derived neuronal precursor cells, cerebral organoids and in vivo brain imaging.

The molecular basis of how chromosome 16p13.11 microduplication leads to major psychiatric disorders is unknown. Here we have undertaken brain imaging of patients carrying microduplications in chromosome 16p13.11 and unaffected family controls, in parallel with iPS cell-derived cerebral organoid studies of the same patients. Patient MRI revealed reduced cortical volume, and corresponding iPSC studies showed neural precursor cell (NPC) proliferation abnormalities and reduced organoid size, with the NPCs therein displaying altered planes of cell division. Transcriptomic analyses of NPCs uncovered a deficit in the NFκB p65 pathway, confirmed by proteomics. Moreover, both pharmacological and genetic correction of this deficit rescued the proliferation abnormality. Thus, chromosome 16p13.11 microduplication disturbs the normal programme of NPC proliferation to reduce cortical thickness due to a correctable deficit in the NFκB signalling pathway. This is the first study demonstrating a biologically relevant, potentially ameliorable, signalling pathway underlying chromosome 16p13.11 microduplication syndrome in patient-derived neuronal precursor cells.

Familial t(1;11) translocation is associated with disruption of white matter structural integrity and oligodendrocyte-myelin dysfunction.

Although the underlying neurobiology of major mental illness (MMI) remains unknown, emerging evidence implicates a role for oligodendrocyte-myelin abnormalities. Here, we took advantage of a large family carrying a balanced t(1;11) translocation, which substantially increases risk of MMI, to undertake both diffusion tensor imaging and cellular studies to evaluate the consequences of the t(1;11) translocation on white matter structural integrity and oligodendrocyte-myelin biology. This translocation disrupts among others the DISC1 gene which plays a crucial role in brain development. We show that translocation-carrying patients display significant disruption of  white matter integrity compared with familial controls. At a cellular level, we observe dysregulation of key pathways controlling oligodendrocyte development and morphogenesis in induced pluripotent stem cell (iPSC) derived case oligodendrocytes. This is associated with reduced proliferation and a stunted morphology in vitro. Further, myelin internodes in a humanized mouse model that recapitulates the human translocation as well as after transplantation of t(1;11) oligodendrocyte progenitors were significantly reduced when  compared with controls. Thus we provide evidence that the t(1;11) translocation has biological effects at both the systems and cellular level that together suggest oligodendrocyte-myelin dysfunction.

Neuronal Activity and Its Role in Controlling Antioxidant Genes.

Forebrain neurons have relatively weak intrinsic antioxidant defenses compared to astrocytes, in part due to hypo-expression of Nrf2, an oxidative stress-induced master regulator of antioxidant and detoxification genes. Nevertheless, neurons do possess the capacity to auto-regulate their antioxidant defenses in response to electrical activity. Activity-dependent Ca2+ signals control the expression of several antioxidant genes, boosting redox buffering capacity, thus meeting the elevated antioxidant requirements associated with metabolically expensive electrical activity. These genes include examples which are reported Nrf2 target genes and yet are induced in a Nrf2-independent manner. Here we discuss the implications for Nrf2 hypofunction in neurons and the mechanisms underlying the Nrf2-independent induction of antioxidant genes by electrical activity. A significant proportion of Nrf2 target genes, defined as those genes controlled by Nrf2 in astrocytes, are regulated by activity-dependent Ca2+ signals in human stem cell-derived neurons. We propose that neurons interpret Ca2+ signals in a similar way to other cell types sense redox imbalance, to broadly induce antioxidant and detoxification genes.

Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones.

We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABAA receptors (GABAARs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC50 values of 278 µm and 182 µm, respectively, and the presence of a GABAAR population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABAAR-mediated currents, evoked by EC50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC50 values of 2.7 and 5.1 µm, respectively. hECN GABAARs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn(2+), together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the ß2 or ß3 subunit, with RNA-seq analysis indicating strong expression of ß3 in hECN GABAARs. Taken together our data support the notion that hECN GABAARs have an α2/3ß3γ2 subunit composition - a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 µm. Glycine-evoked (500 µm) currents were blocked by strychnine (IC50 = 630 nm) and picrotoxin (IC50 = 197 µm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and ß subunits.

Laboratory Automated Interrogation of Data: an interactive web application for visualization of multilevel data from biological experiments.

A key step in understanding the results of biological experiments is visualization of the data. Many laboratory experiments contain a range of measurements that exist within a hierarchy of interdependence. An automated and facile way to visualize and interrogate such multilevel data, across many experimental variables, would (i) lead to improved understanding of the results, (ii) help to avoid misleading interpretation of statistics and (iii) easily identify outliers and sources of batch and confounding effects. While many excellent graphing solutions already exist, they are often geared towards the production of publication-ready plots and the analysis of a single variable at a time, require programming expertise or are unnecessarily complex for the task at hand. Here, we present Laboratory Automated Interrogation of Data (LAB-AID), an interactive tool specifically designed to automatically visualize and query hierarchical data resulting from biological experiments.

Synaptic gene expression changes in frontotemporal dementia due to the MAPT 10+16 mutation.

Mutations in the MAPT gene encoding tau protein can cause autosomal dominant neurodegenerative tauopathies including frontotemporal dementia (often with Parkinsonism). In Alzheimer's disease, the most common tauopathy, synapse loss is the strongest pathological correlate of cognitive decline. Recently, PET imaging with synaptic tracers revealed clinically relevant loss of synapses in primary tauopathies; however, the molecular mechanisms leading to synapse degeneration in primary tauopathies remain largely unknown. In this study, we examined post-mortem brain tissue from people who died with frontotemporal dementia with tau pathology (FTDtau) caused by the MAPT intronic exon 10+16 mutation, which increases splice variants containing exon 10 resulting in higher levels of tau with four microtubule binding domains. We used RNA sequencing and histopathology to examine temporal cortex and visual cortex, to look for molecular phenotypes compared to age, sex, and RNA integrity matched participants who died without neurological disease (n=12 per group). Bulk tissue RNA sequencing reveals substantial downregulation of gene expression associated with synaptic function. Upregulated biological pathways in human MAPT 10+16 brain included those involved in transcriptional regulation, DNA damage response, and neuroinflammation. Histopathology confirmed increased pathological tau accumulation in FTDtau cortex as well as a loss of presynaptic protein staining, and region-specific increased colocalization of phospho-tau with synapses in temporal cortex. Our data indicate that synaptic pathology likely contributes to pathogenesis in FTDtau caused by the MAPT 10+16 mutation.

Enhanced hippocampal LTP but normal NMDA receptor and AMPA receptor function in a rat model of CDKL5 deficiency disorder.

BACKGROUND: Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and intellectual disability (ID). Impaired hippocampal function has been implicated in other models of monogenic forms of autism spectrum disorders and ID and is often linked to epilepsy and behavioural abnormalities. Many individuals with CDKL5 deficiency disorder (CDD) have null mutations and complete loss of CDKL5 protein, therefore in the current study we used a Cdkl5-/y rat model to elucidate the impact of CDKL5 loss on cellular excitability and synaptic function of CA1 pyramidal cells (PCs). We hypothesised abnormal pre and/or post synaptic function and plasticity would be observed in the hippocampus of Cdkl5-/y rats. METHODS: To allow cross-species comparisons of phenotypes associated with the loss of CDKL5, we generated a loss of function mutation in exon 8 of the rat Cdkl5 gene and assessed the impact of the loss of CDLK5 using a combination of extracellular and whole-cell electrophysiological recordings, biochemistry, and histology. RESULTS: Our results indicate that CA1 hippocampal long-term potentiation (LTP) is enhanced in slices prepared from juvenile, but not adult, Cdkl5-/y rats. Enhanced LTP does not result from changes in NMDA receptor function or subunit expression as these remain unaltered throughout development. Furthermore, Ca2+ permeable AMPA receptor mediated currents are unchanged in Cdkl5-/y rats. We observe reduced mEPSC frequency accompanied by increased spine density in basal dendrites of CA1 PCs, however we find no evidence supporting an increase in silent synapses when assessed using a minimal stimulation protocol in slices. Additionally, we found no change in paired-pulse ratio, consistent with normal release probability at Schaffer collateral to CA1 PC synapses. CONCLUSIONS: Our data indicate a role for CDKL5 in hippocampal synaptic function and raise the possibility that altered intracellular signalling rather than synaptic deficits contribute to the altered plasticity. LIMITATIONS: This study has focussed on the electrophysiological and anatomical properties of hippocampal CA1 PCs across early postnatal development. Studies involving other brain regions, older animals and behavioural phenotypes associated with the loss of CDKL5 are needed to understand the pathophysiology of CDD.

Differential splicing choices made by neurons and astrocytes and their importance when investigating signal-dependent alternative splicing in neural cells.

A variety of proteins can be encoded by a single gene via the differential splicing of exons. In neurons this form of alternative splicing can be controlled by activity-dependent calcium signaling, leading to the properties of proteins being altered, including ion channels, neurotransmitter receptors and synaptic cell adhesion molecules. The pre-synaptic cell adhesion molecule Neurexin 1 (Nrxn1) is alternatively spliced at splice-site 4 (SS4) which governs exon 22 inclusion (SS4+) and consequently postsynaptic NMDA receptor responses. Nrxn1 was reported to be subject to a delayed-onset shift in Nrxn1 SS4 splicing resulting in increased exon 22 inclusion, involving epigenetic mechanisms which, if disrupted, reduce memory stability. Exon inclusion at SS4 represented one of hundreds of exons reported to be subject to a genome-wide shift in fractional exon inclusion following membrane depolarization with high extracellular K+ that was delayed in onset. We report that high K+ does not increase the SS4+/SS4- ratio in cortical neurons, but does induce a delayed-onset NMDA receptor-dependent neuronal death. In mixed neuronal/astrocyte cultures this neuronal death results in an increase in the astrocyte: neuron ratio, and a misleading increase in SS4+/SS4- ratio attributable to astrocytes having a far higher SS4+/SS4- ratio than neurons, rather than any change in the neurons themselves. We reassessed the previously reported genome-wide delayed-onset shift in fractional exon inclusion after high K+ exposure. This revealed that the reported changes correlated strongly with differences in exon inclusion level between astrocytes and neurons, and was accompanied by a strong decrease in the ratio of neuron-specific: astrocyte-specific gene expression. As such, these changes can be explained by the neurotoxic nature of the stimulation paradigm, underlining the importance of NMDA receptor blockade when using high K+ depolarizing stimuli.

General anesthesia alters CNS and astrocyte expression of activity-dependent and activity-independent genes.

General anesthesia represents a common clinical intervention and yet can result in long-term adverse CNS effects particularly in the elderly or dementia patients. Suppression of cortical activity is a key feature of the anesthetic-induced unconscious state, with activity being a well-described regulator of pathways important for brain health. However, the extent to which the effects of anesthesia go beyond simple suppression of neuronal activity is incompletely understood. We found that general anesthesia lowered cortical expression of genes induced by physiological activity in vivo, and recapitulated additional patterns of gene regulation induced by total blockade of firing activity in vitro, including repression of neuroprotective genes and induction of pro-apoptotic genes. However, the influence of anesthesia extended beyond that which could be accounted for by activity modulation, including the induction of non activity-regulated genes associated with inflammation and cell death. We next focused on astrocytes, important integrators of both neuronal activity and inflammatory signaling. General anesthesia triggered gene expression changes consistent with astrocytes being in a low-activity environment, but additionally caused induction of a reactive profile, with transcriptional changes enriched in those triggered by stroke, neuroinflammation, and Aß/tau pathology. Thus, while the effects of general anesthesia on cortical gene expression are consistent with the strong repression of brain activity, further deleterious effects are apparent including a reactive astrocyte profile.

Cystatin F (Cst7) drives sex-dependent changes in microglia in an amyloid-driven model of Alzheimer's disease.

Microglial endolysosomal (dys)function is strongly implicated in neurodegenerative disease. Transcriptomic studies show that a microglial state characterised by a set of genes involved in endolysosomal function is induced in both mouse Alzheimer's disease (AD) models and human AD brain, and that the emergence of this state is emphasised in females. Cst7 (encoding cystatin F) is among the most highly upregulated genes in these microglia. However, despite such striking and robust upregulation, the function of Cst7 in neurodegenerative disease is not understood. Here, we crossed Cst7-/- mice with the AppNL-G-F mouse to test the role of Cst7 in a model of amyloid-driven AD. Surprisingly, we found that Cst7 plays a sexually dimorphic role regulating microglia in this model. In females, Cst7-/-AppNL-G-F microglia had greater endolysosomal gene expression, lysosomal burden, and amyloid beta (Aß) burden in vivo and were more phagocytic in vitro. However, in males, Cst7-/-AppNL-G-F microglia were less inflammatory and had a reduction in lysosomal burden but had no change in Aß burden. Overall, our study reveals functional roles for one of the most commonly upregulated genes in microglia across disease models, and the sex-specific profiles of Cst7-/--altered microglial disease phenotypes. More broadly, the findings raise important implications for AD including crucial questions on sexual dimorphism in neurodegenerative disease and the interplay between endolysosomal and inflammatory pathways in AD pathology.

Astrocyte-oligodendrocyte interaction regulates central nervous system regeneration.

Failed regeneration of myelin around neuronal axons following central nervous system damage contributes to nerve dysfunction and clinical decline in various neurological conditions, for which there is an unmet therapeutic demand. Here, we show that interaction between glial cells - astrocytes and mature myelin-forming oligodendrocytes - is a determinant of remyelination. Using in vivo/ ex vivo/ in vitro rodent models, unbiased RNA sequencing, functional manipulation, and human brain lesion analyses, we discover that astrocytes support the survival of regenerating oligodendrocytes, via downregulation of the Nrf2 pathway associated with increased astrocytic cholesterol biosynthesis pathway activation. Remyelination fails following sustained astrocytic Nrf2 activation in focally-lesioned male mice yet is restored by either cholesterol biosynthesis/efflux stimulation, or Nrf2 inhibition using the existing therapeutic Luteolin. We identify that astrocyte-oligodendrocyte interaction regulates remyelination, and reveal a drug strategy for central nervous system regeneration centred on targeting this interaction.