Search

Rodent studies have shown that alternative splicing in neurons plays important roles in development and maturity, and is regulatable by signals such as electrical activity. However, rodent-human similarities are less well explored. We compared basal and activity-dependent exon splicing in cortical-patterned human ESC-derived neurons with that in cortical mouse ESC-derived neurons, primary mouse cortical neurons at two developmental stages, and mouse hippocampal neurons, focussing on conserved orthologous exons. Both basal exon inclusion levels and activity-dependent changes in splicing showed human-mouse correlation. Conserved activity regulated exons are enriched in RBFOX, SAM68, NOVA and PTBP targets, and centered on cytoskeletal organization, mRNA processing, and synaptic signaling genes. However, human-mouse correlations were weaker than inter-mouse comparisons of neurons from different brain regions, developmental stages and origin (ESC vs. primary), suggestive of some inter-species divergence. The set of genes where activity-dependent splicing was observed only in human neurons were dominated by those involved in lipid biosynthesis, signaling and trafficking. Study of human exon splicing in mouse Tc1 neurons carrying human chromosome-21 showed that neuronal basal exon inclusion was influenced by cis-acting sequences, although may not be sufficient to confer activity-responsiveness in an allospecific environment. Overall, these comparisons suggest that neuronal alternative splicing should be confirmed in a human-relevant system even when exon structure is evolutionarily conserved., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Dando, McQueen, Burr, Kind, Chandran, Hardingham and Qiu.)

Aims: 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, Positron Emission Tomography (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., Methods: 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 FTDtau10 + 16 and 13 controls)., Results: 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 FTDtau10 + 16 cortex as well as a loss of presynaptic protein staining and region-specific increased colocalization of phospho-tau with synapses in temporal cortex., Conclusions: Our data indicate that synaptic pathology likely contributes to pathogenesis in FTDtau10 + 16 caused by the MAPT 10 + 16 mutation., (© 2024 The Author(s). Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

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, Ca 2+ 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., (© 2024. The Author(s).)

Synapse loss correlates with cognitive decline in Alzheimer's disease, and soluble oligomeric amyloid beta (Aβ) is implicated in synaptic dysfunction and loss. An important knowledge gap is the lack of understanding of how Aβ leads to synapse degeneration. In particular, there has been difficulty in determining whether there is a synaptic receptor that binds Aβ 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 Aβ 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 Aβ generates a FRET signal with transmembrane protein 97. Further, Aβ 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 Aβ/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 Aβ. In human-induced pluripotent stem cell derived neurons, transmembrane protein 97 is present in synapses and colocalizes with Aβ when neurons are challenged with human Alzheimer's brain homogenate. Transcriptional changes are induced by Aβ 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 Aβ in human Alzheimer's disease brain where it may mediate synaptotoxicity., (© 2024. The Author(s).)

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., (© 2023 The Authors. GLIA published by Wiley Periodicals LLC.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jiwaji, Márkus, McQueen, Emelianova, He, Dando, Chandran and Hardingham.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Baxter, Dando and Hardingham.)

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., (© 2023. The Author(s).)

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., (© 2022 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

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., (© 2022. The Author(s).)

Dysregulated protein synthesis is a core pathogenic mechanism in Fragile X Syndrome (FX). The mGluR Theory of FX predicts that pathological synaptic changes arise from the excessive translation of mRNAs downstream of mGlu 1/5 activation. Here, we use a combination of CA1 pyramidal neuron-specific TRAP-seq and proteomics to identify the overtranslating mRNAs supporting exaggerated mGlu 1/5 -induced long-term synaptic depression (mGluR-LTD) in the FX mouse model (Fmr1 -/y ). Our results identify a significant increase in the translation of ribosomal proteins (RPs) upon mGlu 1/5 stimulation that coincides with a reduced translation of long mRNAs encoding synaptic proteins. These changes are mimicked and occluded in Fmr1 -/y neurons. Inhibiting RP translation significantly impairs mGluR-LTD and prevents the length-dependent shift in the translating population. Together, these results suggest that pathological changes in FX result from a length-dependent alteration in the translating population that is supported by excessive RP translation., (© 2022. The Author(s).)

Alzheimer's disease (AD) alters astrocytes, but the effect of Aß and Tau pathology is poorly understood. TRAP-seq translatome analysis of astrocytes in APP/PS1 ß-amyloidopathy and MAPT P301S tauopathy mice revealed that only Aß influenced expression of AD risk genes, but both pathologies precociously induced age-dependent changes, and had distinct but overlapping signatures found in human post-mortem AD astrocytes. Both Aß and Tau pathology induced an astrocyte signature involving repression of bioenergetic and translation machinery, and induction of inflammation pathways plus protein degradation/proteostasis genes, the latter enriched in targets of inflammatory mediator Spi1 and stress-activated cytoprotective Nrf2. Astrocyte-specific Nrf2 expression induced a reactive phenotype which recapitulated elements of this proteostasis signature, reduced Aß deposition and phospho-tau accumulation in their respective models, and rescued brain-wide transcriptional deregulation, cellular pathology, neurodegeneration and behavioural/cognitive deficits. Thus, Aß and Tau induce overlapping astrocyte profiles associated with both deleterious and adaptive-protective signals, the latter of which can slow patho-progression., (© 2022. The Author(s).)

The transcription factor Nrf2 is a stress-responsive master regulator of antioxidant, detoxification and proteostasis genes. In astrocytes, Nrf2-dependent gene expression drives cell-autonomous cytoprotection and also non-cell-autonomous protection of nearby neurons, and can ameliorate pathology in several acute and chronic neurological disorders associated with oxidative stress. However, the value of astrocytic Nrf2 as a therapeutic target depends in part on whether Nrf2 activation by disease-associated oxidative stress occludes the effect of any Nrf2-activating drug. Nrf2 activation classically involves the inhibition of interactions between Nrf2's Neh2 domain and Keap1, which directs Nrf2 degradation. Keap1 inhibition is mediated by the modification of cysteine residues on Keap1, and can be triggered by electrophilic small molecules such as tBHQ. Here we show that astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling. Keap1 deficiency elevates basal Nrf2 target gene expression in astrocytes and occludes the effects of tBHQ, oxidative stress still induced strong Nrf2-dependent gene expression in Keap1-deficient astrocytes. Moreover, while tBHQ prevented protein degradation mediated via Nrf2's Neh2 domain, oxidative stress did not, consistent with a Keap1-independent mechanism. Moreover the effects of oxidative stress and tBHQ on Nrf2 target gene expression are additive, not occlusive. Mechanistically, oxidative stress enhances the transactivation potential of Nrf2's Neh5 domain in a manner dependent on its Cys-191 residue. Thus, astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling, meaning that further Nrf2 activation by Keap1-inhibiting drugs may be a viable therapeutic strategy., (Copyright © 2021. Published by Elsevier B.V.)

Primary hippocampal cell cultures are routinely used as an experimentally accessible model platform for the hippocampus and brain tissue in general. Containing multiple cell types including neurons, astrocytes and microglia in a state that can be readily analysed optically, biochemically and electrophysiologically, such cultures have been used in many in vitro studies. To what extent the in vivo environment is recapitulated in primary cultures is an on-going question. Here, we compare the transcriptomic profiles of primary hippocampal cell cultures and intact hippocampal tissue. In addition, by comparing profiles from wild type and the PrP 101LL transgenic model of prion disease, we also demonstrate that gene conservation is predominantly conserved across genetically altered lines., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Microglia, brain-resident macrophages, require instruction from the CNS microenvironment to maintain their identity and morphology and regulate inflammatory responses, although what mediates this is unclear. Here, we show that neurons and astrocytes cooperate to promote microglial ramification, induce expression of microglial signature genes ordinarily lost in vitro and in age and disease in vivo, and repress infection- and injury-associated gene sets. The influence of neurons and astrocytes separately on microglia is weak, indicative of synergies between these cell types, which exert their effects via a mechanism involving transforming growth factor β2 (TGF-β2) signaling. Neurons and astrocytes also combine to provide immunomodulatory cues, repressing primed microglial responses to weak inflammatory stimuli (without affecting maximal responses) and consequently limiting the feedback effects of inflammation on the neurons and astrocytes themselves. These findings explain why microglia isolated ex vivo undergo de-differentiation and inflammatory deregulation and point to how disease- and age-associated changes may be counteracted., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Physiological disturbances in cortical network excitability and plasticity are established and widespread in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those harbouring the C9ORF72 repeat expansion (C9ORF72 RE ) mutation - the most common genetic impairment causal to ALS and FTD. Noting that perturbations in cortical function are evidenced pre-symptomatically, and that the cortex is associated with widespread pathology, cortical dysfunction is thought to be an early driver of neurodegenerative disease progression. However, our understanding of how altered network function manifests at the cellular and molecular level is not clear., Methods: To address this we have generated cortical neurons from patient-derived iPSCs harbouring C9ORF72 RE mutations, as well as from their isogenic expansion-corrected controls. We have established a model of network activity in these neurons using multi-electrode array electrophysiology. We have then mechanistically examined the physiological processes underpinning network dysfunction using a combination of patch-clamp electrophysiology, immunocytochemistry, pharmacology and transcriptomic profiling., Results: We find that C9ORF72 RE causes elevated network burst activity, associated with enhanced synaptic input, yet lower burst duration, attributable to impaired pre-synaptic vesicle dynamics. We also show that the C9ORF72 RE is associated with impaired synaptic plasticity. Moreover, RNA-seq analysis revealed dysregulated molecular pathways impacting on synaptic function. All molecular, cellular and network deficits are rescued by CRISPR/Cas9 correction of C9ORF72 RE . Our study provides a mechanistic view of the early dysregulated processes that underpin cortical network dysfunction in ALS-FTD., Conclusion: These findings suggest synaptic pathophysiology is widespread in ALS-FTD and has an early and fundamental role in driving altered network function that is thought to contribute to neurodegenerative processes in these patients. The overall importance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic plasticity, synaptic vesicle stores, and network propagation, which directly impact upon cortical function.

Many neurodegenerative diseases are associated with neuronal misfolded protein accumulation, indicating a need for proteostasis-promoting strategies. Here we show that de-repressing the transcription factor Nrf2, epigenetically shut-off in early neuronal development, can prevent protein aggregate accumulation. Using a paradigm of α-synuclein accumulation and clearance, we find that the classical electrophilic Nrf2 activator tBHQ promotes endogenous Nrf2-dependent α-synuclein clearance in astrocytes, but not cortical neurons, which mount no Nrf2-dependent transcriptional response. Moreover, due to neuronal Nrf2 shut-off and consequent weak antioxidant defences, electrophilic tBHQ actually induces oxidative neurotoxicity, via Nrf2-independent Jun induction. However, we find that epigenetic de-repression of neuronal Nrf2 enables them to respond to Nrf2 activators to drive α-synuclein clearance. Moreover, activation of neuronal Nrf2 expression using gRNA-targeted dCas9-based transcriptional activation complexes is sufficient to trigger Nrf2-dependent α-synuclein clearance. Thus, targeting reversal of the developmental shut-off of Nrf2 in forebrain neurons may alter neurodegenerative disease trajectory by boosting proteostasis.

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.

Microglia are resident tissue macrophages of the central nervous system (CNS) that arise from erythromyeloid progenitors during embryonic development. They play essential roles in CNS development, homeostasis and response to disease. Since microglia are difficult to procure from the human brain, several protocols have been developed to generate microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, some concerns remain over the purity and quality of in vitro generated microglia. Here, we describe a new protocol that does not require co-culture with neural cells and yields cultures of 100% P2Y 12 + 95% TMEM119 + ramified human microglia-like cells (hiPSC-MG). In the presence of neural precursor cell-conditioned media, hiPSC-MG expressed high levels of human microglia signature genes, including SALL1, CSF1R, P2RY12, TMEM119, TREM2, HEXB and SIGLEC11, as revealed by whole-transcriptome analysis. Stimulation of hiPSC-MG with lipopolysaccharide resulted in downregulation of P2Y 12 expression, induction of IL1B mRNA expression and increase in cell capacitance. HiPSC-MG were phagocytically active and maintained their cell identity after transplantation into murine brain slices and human brain spheroids. Together, our new protocol for the generation of microglia-like cells from human iPSCs will facilitate the study of human microglial function in health and disease., (Copyright © 2020. Published by Elsevier B.V.)

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., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© 2019 The Authors. Glia published by Wiley Periodicals, Inc.)

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 Ca 2+ 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 Ca 2+ signals in human stem cell-derived neurons. We propose that neurons interpret Ca 2+ signals in a similar way to other cell types sense redox imbalance, to broadly induce antioxidant and detoxification genes.

A key knowledge gap blocking development of effective therapeutics for Alzheimer's disease (AD) is the lack of understanding of how amyloid beta (Aβ) peptide and pathological forms of the tau protein cooperate in causing disease phenotypes. Within a mouse tau-deficient background, we probed the molecular, cellular, and behavioral disruption triggered by the influence of wild-type human tau on human Aβ-induced pathology. We find that Aβ and tau work cooperatively to cause a hyperactivity behavioral phenotype and to cause downregulation of transcription of genes involved in synaptic function. In both our mouse model and human postmortem tissue, we observe accumulation of pathological tau in synapses, supporting the potential importance of synaptic tau. Importantly, tau reduction in the mice initiated after behavioral deficits emerge corrects behavioral deficits, reduces synaptic tau levels, and substantially reverses transcriptional perturbations, suggesting that lowering synaptic tau levels may be beneficial in AD., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Fragile X Syndrome (FXS) is one of the most common monogenic forms of autism and intellectual disability. Preclinical studies in animal models have highlighted the potential of pharmaceutical intervention strategies for alleviating the symptoms of FXS. However, whether treatment strategies can be tailored to developmental time windows that define the emergence of particular phenotypes is unknown. Similarly, whether a brief, early intervention can have long-lasting beneficial effects, even after treatment cessation, is also unknown. To address these questions, we first examined the developmental profile for the acquisition of associative learning in a rat model of FXS. Associative memory was tested using a range of behavioral paradigms that rely on an animal's innate tendency to explore novelty. Fmr1 knockout (KO) rats showed a developmental delay in their acquisition of object-place recognition and did not demonstrate object-place-context recognition paradigm at any age tested (up to 23 weeks of age). Treatment of Fmr1 KO rats with lovastatin between 5 and 9 weeks of age, during the normal developmental period that this associative memory capability is established, prevents the emergence of deficits but has no effect in wild-type animals. Moreover, we observe no regression of cognitive performance in the FXS rats over several months after treatment. This restoration of the normal developmental trajectory of cognitive function is associated with the sustained rescue of both synaptic plasticity and altered protein synthesis. The findings provide proof of concept that the impaired emergence of the cognitive repertoire in neurodevelopmental disorders may be prevented by brief, early pharmacological intervention., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

Transcriptomic changes induced in one cell type by another mediate many biological processes in the brain and elsewhere; however, achieving artifact-free physical separation of cell types to study them is challenging and generally allows for analysis of only a single cell type. We describe an approach using a co-culture of distinct cell types from different species that enables physical cell sorting to be replaced by in silico RNA sequencing (RNA-seq) read sorting, which is possible because of evolutionary divergence of messenger RNA (mRNA) sequences. As an exemplary experiment, we describe the co-culture of purified neurons, astrocytes, and microglia from different species (12-14 d). We describe how to use our Python tool, Sargasso, to separate the reads from conventional RNA-seq according to species and to eliminate any artifacts borne of imperfect genome annotation (10 h). We show how this procedure, which requires no special skills beyond those that might normally be expected of wet lab and bioinformatics researchers, enables the simultaneous transcriptomic profiling of different cell types, revealing the distinct influence of microglia on astrocytic and neuronal transcriptomes under inflammatory conditions.

This corrects the article DOI: 10.1038/ncomms15132.

Mutations in C9ORF72 are the most common cause of familial amyotrophic lateral sclerosis (ALS). Here, through a combination of RNA-Seq and electrophysiological studies on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs), we show that increased expression of GluA1 AMPA receptor (AMPAR) subunit occurs in MNs with C9ORF72 mutations that leads to increased Ca 2+ -permeable AMPAR expression and results in enhanced selective MN vulnerability to excitotoxicity. These deficits are not found in iPSC-derived cortical neurons and are abolished by CRISPR/Cas9-mediated correction of the C9ORF72 repeat expansion in MNs. We also demonstrate that MN-specific dysregulation of AMPAR expression is also present in C9ORF72 patient post-mortem material. We therefore present multiple lines of evidence for the specific upregulation of GluA1 subunits in human mutant C9ORF72 MNs that could lead to a potential pathogenic excitotoxic mechanism in ALS.

Excessive mRNA translation downstream of group I metabotropic glutamate receptors (mGlu 1/5 ) is a core pathophysiology of fragile X syndrome (FX); however, the differentially translating mRNAs that contribute to altered neural function are not known. We used translating ribosome affinity purification (TRAP) and RNA-seq to identify mistranslating mRNAs in CA1 pyramidal neurons of the FX mouse model (Fmr1 -/y ) hippocampus, which exhibit exaggerated mGlu 1/5 -induced long-term synaptic depression (LTD). In these neurons, we find that the Chrm4 transcript encoding muscarinic acetylcholine receptor 4 (M 4 ) is excessively translated, and synthesis of M 4 downstream of mGlu 5 activation is mimicked and occluded. Surprisingly, enhancement rather than inhibition of M 4 activity normalizes core phenotypes in the Fmr1 -/y , including excessive protein synthesis, exaggerated mGluR-LTD, and audiogenic seizures. These results suggest that not all excessively translated mRNAs in the Fmr1 -/y brain are detrimental, and some may be candidates for enhancement to correct pathological changes in the FX brain., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The influence that neurons exert on astrocytic function is poorly understood. To investigate this, we first developed a system combining cortical neurons and astrocytes from closely related species, followed by RNA-seq and in silico species separation. This approach uncovers a wide programme of neuron-induced astrocytic gene expression, involving Notch signalling, which drives and maintains astrocytic maturity and neurotransmitter uptake function, is conserved in human development, and is disrupted by neurodegeneration. Separately, hundreds of astrocytic genes are acutely regulated by synaptic activity via mechanisms involving cAMP/PKA-dependent CREB activation. This includes the coordinated activity-dependent upregulation of major astrocytic components of the astrocyte-neuron lactate shuttle, leading to a CREB-dependent increase in astrocytic glucose metabolism and elevated lactate export. Moreover, the groups of astrocytic genes induced by neurons or neuronal activity both show age-dependent decline in humans. Thus, neurons and neuronal activity regulate the astrocytic transcriptome with the potential to shape astrocyte-neuron metabolic cooperation.

CDKL5 deficiency is a severe neurological disorder caused by mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5). The predominant human CDKL5 brain isoform is a 9.7kb transcript comprised of 18 exons with a large 6.6kb 3'-untranslated region (UTR). Mammalian models of CDKL5 disorder are currently limited to mouse, and little is known about Cdkl5 in other organisms used to model neurodevelopmental disorders, such as rat. In this study we characterise, both bioinformatically and experimentally, the rat Cdkl5 gene structure and its associated transcript isoforms. New exonic regions, splice sites and UTRs are described, confirming the presence of four distinct transcript isoforms. The predominant isoform in the brain, which we name rCdkl5_1, is orthologous to the human hCDKL5_1 and mouse mCdkl5_1 isoforms and is the most highly expressed isoform across all brain regions tested. This updated gene model of Cdkl5 in rat provides a framework for studies into its protein products and provides a reference for the development of molecular therapies for testing in rat models of CDKL5 disorder., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Evolutionary differences in gene regulation between humans and lower mammalian experimental systems are incompletely understood, a potential translational obstacle that is challenging to surmount in neurons, where primary tissue availability is poor. Rodent-based studies show that activity-dependent transcriptional programs mediate myriad functions in neuronal development, but the extent of their conservation in human neurons is unknown. We compared activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons with those induced in developing primary- or stem cell-derived mouse cortical neurons. While activity-dependent gene-responsiveness showed little dependence on developmental stage or origin (primary tissue vs. stem cell), notable species-dependent differences were observed. Moreover, differential species-specific gene ortholog regulation was recapitulated in aneuploid mouse neurons carrying human chromosome-21, implicating promoter/enhancer sequence divergence as a factor, including human-specific activity-responsive AP-1 sites. These findings support the use of human neuronal systems for probing transcriptional responses to physiological stimuli or indeed pharmaceutical agents., Competing Interests: The authors declare that no competing interests exist.

Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms.

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 β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABAARs. Taken together our data support the notion that hECN GABAARs have an α2/3β3γ2 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 β subunits., (© 2014 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)