Frontotemporal lobar degeneration (FTLD) is the second most common cause of presenile dementia. The predominant neuropathology is FTLD with TAR DNA-binding protein (TDP-43) inclusions (FTLD-TDP). FTLD-TDP is frequently familial, resulting from mutations in GRN (which encodes progranulin). We assembled an international collaboration to identify susceptibility loci for FTLD-TDP through a genome-wide association study of 515 individuals with FTLD-TDP. We found that FTLD-TDP associates with multiple SNPs mapping to a single linkage disequilibrium block on 7p21 that contains TMEM106B. Three SNPs retained genome-wide significance following Bonferroni correction (top SNP rs1990622, P = 1.08 x 10(-11); odds ratio, minor allele (C) 0.61, 95% CI 0.53-0.71). The association replicated in 89 FTLD-TDP cases (rs1990622; P = 2 x 10(-4)). TMEM106B variants may confer risk of FTLD-TDP by increasing TMEM106B expression. TMEM106B variants also contribute to genetic risk for FTLD-TDP in individuals with mutations in GRN. Our data implicate variants in TMEM106B as a strong risk factor for FTLD-TDP, suggesting an underlying pathogenic mechanism.

Early impairments to neurovascular coupling have been proposed to be a key pathogenic factor in the onset and progression of Alzheimer's disease (AD). Studies have shown impaired neurovascular function in several mouse models of AD, including the J20-hAPP mouse. In this study, we aimed to investigate early neurovascular changes using wild-type (WT) controls and J20-hAPP mice at 6 months of age, by measuring cerebral haemodynamics and neural activity to physiological sensory stimulations. A thinned cranial window was prepared to allow access to cortical vasculature and imaged using 2D-optical imaging spectroscopy (2D-OIS). After chronic imaging sessions where the skull was intact, a terminal acute imaging session was performed where an electrode was inserted into the brain to record simultaneous neural activity. We found that cerebral haemodynamic changes were significantly enhanced in J20-hAPP mice compared with controls in response to physiological stimulations, potentially due to the significantly higher neural activity (hyperexcitability) seen in the J20-hAPP mice. Thus, neurovascular coupling remained preserved under a chronic imaging preparation. Further, under hyperoxia, the baseline blood volume and saturation of all vascular compartments in the brains of J20-hAPP mice were substantially enhanced compared to WT controls, but this effect disappeared under normoxic conditions. This study highlights novel findings not previously seen in the J20-hAPP mouse model, and may point towards a potential therapeutic strategy.

Pathological interactions between β-amyloid (Aβ) and tau drive synapse loss and cognitive decline in Alzheimer's disease (AD). Reactive astrocytes, displaying altered functions, are also a prominent feature of AD brain. This large and heterogeneous population of cells are increasingly recognised as contributing to early phases of disease. However, the contribution of astrocytes to Aβ-induced synaptotoxicity in AD is not well understood.

Medulloblastoma is currently subclassified into distinct DNA methylation subgroups/subtypes with particular clinico-molecular features. Using RNA sequencing (RNA-seq) in large, well-annotated cohorts of medulloblastoma, we show that transcriptionally group 3 and group 4 medulloblastomas exist as intermediates on a bipolar continuum between archetypal group 3 and group 4 entities. Continuum position is prognostic, reflecting a propensity for specific DNA copy-number changes, and specific switches in isoform/enhancer usage and RNA editing. Examining single-cell RNA-seq (scRNA-seq) profiles, we show that intratumoral transcriptional heterogeneity along the continuum is limited in a subtype-dependent manner. By integrating with a human scRNA-seq reference atlas, we show that this continuum is mirrored by an equivalent continuum of transcriptional cell types in early fetal cerebellar development. We identify distinct developmental niches for all four major subgroups and link each to a common developmental antecedent. Our findings show a transcriptional continuum arising from oncogenic disruption of highly specific fetal cerebellar cell types, linked to almost every aspect of group 3/group 4 molecular biology and clinico-pathology.

Genetic risk factors for Alzheimer's disease imply that inflammation plays a causal role in development of the disease. Experimental studies suggest that microglia, as the brain macrophages, have diverse functions, with their main role in health being to survey the brain parenchyma through highly motile processes.

β-amyloid (Aβ) plaques are a key feature of Alzheimer's disease pathology but correlate poorly with dementia. They are associated with astrocytes which may modulate the effect of Aβ-deposition on the neuropil. This study characterised the astrocyte response to Aβ plaque subtypes, and investigated their association with cognitive impairment.

GGGGCC repeat expansions of C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We and others have proposed that RNA transcribed from the repeat sequence is toxic via sequestration of RNA-binding factors. Both GGGGCC-repeat (sense) and CCCCGG-repeat (antisense) molecules are detectable by fluorescence in situ hybridisation as RNA foci, but their relative expression pattern within the CNS and contribution to disease has not been determined. Blinded examination of CNS biosamples from ALS patients with a repeat expansion of C9ORF72 showed that antisense foci are present at a significantly higher frequency in cerebellar Purkinje neurons and motor neurons, whereas sense foci are present at a significantly higher frequency in cerebellar granule neurons. Consistent with this, inclusions containing sense or antisense derived dipeptide repeat proteins were present at significantly higher frequency in cerebellar granule neurons or motor neurons, respectively. Immunohistochemistry and UV-crosslinking studies showed that sense and antisense RNA molecules share similar interactions with SRSF2, hnRNP K, hnRNP A1, ALYREF, and hnRNP H/F. Together these data suggest that, although sense and antisense RNA molecules might be expected to be equally toxic via their shared protein binding partners, distinct patterns of expression in various CNS neuronal populations could lead to relative differences in their contribution to the pathogenesis of neuronal injury. Moreover in motor neurons, which are the primary target of pathology in ALS, the presence of antisense foci (χ (2), p < 0.00001) but not sense foci (χ (2), p = 0.75) correlated with mislocalisation of TDP-43, which is the hallmark of ALS neurodegeneration. This has implications for translational approaches to C9ORF72 disease, and furthermore interacting RNA-processing factors and transcriptional activators responsible for antisense versus sense transcription might represent novel therapeutic targets.

The insulin/IGF1 signalling (IIS) pathways are involved in longevity regulation and are dysregulated in neurons in Alzheimer's disease (AD). We previously showed downregulation in IIS gene expression in astrocytes with AD-neuropathology progression, but IIS in astrocytes remains poorly understood. We therefore examined the IIS pathway in human astrocytes and developed models to reduce IIS at the level of the insulin or the IGF1 receptor (IGF1R).

Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3-10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis.

Gene expression profiling has been used previously with spinal cord homogenates and laser capture microdissected motor neurons to determine the mechanisms involved in neurodegeneration in amyotrophic lateral sclerosis. However, while cellular and animal model work has focused on superoxide dismutase 1-related amyotrophic lateral sclerosis, the transcriptional profile of human mutant superoxide dismutase 1 motor neurons has remained undiscovered. The aim of this study was to apply gene expression profiling to laser captured motor neurons from human superoxide dismutase 1-related amyotrophic lateral sclerosis and neurologically normal control cases, in order to determine those pathways dysregulated in human superoxide dismutase 1-related neurodegeneration and to establish potential pathways suitable for therapeutic intervention. Identified targets were then validated in cultured cell models using lentiviral vectors to manipulate the expression of key genes. Microarray analysis identified 1170 differentially expressed genes in spinal cord motor neurons from superoxide dismutase 1-related amyotrophic lateral sclerosis, compared with controls. These genes encoded for proteins in multiple functional categories, including those involved in cell survival and cell death. Further analysis determined that multiple genes involved in the phosphatidylinositol-3 kinase signalling cascade were differentially expressed in motor neurons that survived the disease process. Functional experiments in cultured cells and primary motor neurons demonstrate that manipulating this pathway by reducing the expression of a single upstream target, the negative phosphatidylinositol-3 kinase regulator phosphatase and tensin homology, promotes a marked pro-survival effect. Therefore, these data indicate that proteins in the phosphatidylinositol-3 kinase pathway could represent a target for therapeutic manipulation in motor neuron degeneration.

Astrocytes contribute to a variety of functions in the brain, including homeostasis, synapse formation, plasticity, and metabolism. Astrocyte dysfunction may disrupt their normal role, including neuronal support, thereby contributing to neurodegenerative pathologies, including Alzheimer's disease (AD). To understand the role of astrocytes in the pathogenesis of age-related disorders, we isolated astrocytes by laser capture microdissection, using glial fibrillary acidic protein (GFAP) as a marker, and characterized the astrocyte transcriptome at different Braak neurofibrillary tangle stages in postmortem temporal cortex samples derived from the Medical Research Council Cognitive Function and Ageing Study (MRC CFAS) cohort, using microarray analysis. The largest number of significant, differentially expressed genes were identified when the expression profile of astrocytes from isocortical stages of neurofibrillary tangle pathology (Braak stages V-VI) were compared with entorhinal stages (Braak stages I-II). Dysregulation of genes associated with the actin cytoskeleton, proliferation, apoptosis, and ubiquitin-mediated proteolysis occurred at low Braak stages, while altered regulation of intracellular signaling pathways, including insulin, phosphatidylinositol 3-kinase (PI3K)/Akt, and mitogen-activated protein kinase (MAPK) pathways were primarily associated with high levels of Alzheimer-type pathology, and occurred at lower Braak stages in individuals with the APOEε4 allele. Our findings implicate astrocyte dysfunction in the pathogenesis of neurodegenerative pathology in the aging brain, and provide a basis for future candidate studies based on specific pathways.

The accumulation of reactive oxygen species leading to oxidative damage and cell death plays an important role in a number of neurodegenerative disorders. FOXO3a, the main isoform of FOXO transcription factors, mediates the cellular response to oxidative stress by regulating the expression of genes involved in DNA repair and glutamine metabolism, including glutamine synthetase (GS). Immunohistochemical investigation of the population-based neuropathology cohort of the Medical Research Council's Cognitive Function and Ageing Study (MRC CFAS) demonstrates that nuclear retention of FOXO3a significantly correlates with a DNA damage response and with GS expression by astrocytes. Furthermore, we show that GS expression correlates with increasing Alzheimer-type pathology in this ageing cohort. Our findings suggest that in response to oxidative stress, the nuclear retention of FOXO3a in astrocytes upregulates expression of GS as a neuroprotective mechanism. However, the activity of GS may be compromised by increasing levels of oxidative stress in the ageing brain resulting in dysfunctional enzyme activity, neuronal excitotoxic damage and cognitive impairment.

International consensus recognises four medulloblastoma molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGrp3), and group 4 (MBGrp4), each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions.

Hypoxia is a feature of neurodegenerative diseases, and can both directly and indirectly impact on neuronal function through modulation of glial function. Astrocytes play a key role in regulating homeostasis within the central nervous system, and mediate hypoxia-induced changes in response to reduced oxygen availability. The current study performed a detailed characterization of hypoxia-induced changes in the transcriptomic profile of astrocytes in vitro. Human astrocytes were cultured under normoxic (5% CO2, 95% air) or hypoxic conditions (1% O2, 5% CO2, 94% N2) for 24 h, and the gene expression profile assessed by microarray analysis. In response to hypoxia 4904 genes were significantly differentially expressed (1306 upregulated and 3598 downregulated, FC ≥ 2 and p ≤ 0.05). Analysis of the significant differentially expressed transcripts identified an increase in immune response pathways, and dysregulation of signalling pathways, including HIF-1 (p = 0.002), and metabolism, including glycolysis (p = 0.006). To assess whether the hypoxia-induced metabolic gene changes observed affected metabolism at a functional level, both the glycolytic and mitochondrial flux were measured using an XF bioanalyser. In support of the transcriptomic data, under physiological conditions hypoxia significantly reduced mitochondrial respiratory flux (p = 0.0001) but increased basal glycolytic flux (p = 0.0313). However, when metabolically stressed, hypoxia reduced mitochondrial spare respiratory capacity (p = 0.0485) and both glycolytic capacity (p = 0.0001) and glycolytic reserve (p < 0.0001). In summary, the current findings detail hypoxia-induced changes in the astrocyte transcriptome in vitro, identifying potential targets for modifying the astrocyte response to reduced oxygen availability in pathological conditions associated with ischaemia/hypoxia, including manipulation of mitochondrial function, metabolism, and the immune response.

Amyotrophic lateral sclerosis (ALS) is underpinned by an oligogenic rare variant architecture. Identified genetic variants of ALS include RNA-binding proteins containing prion-like domains (PrLDs). We hypothesized that screening genes encoding additional similar proteins will yield novel genetic causes of ALS. The most common genetic variant of ALS patients is a G4C2-repeat expansion within C9ORF72. We have shown that G4C2-repeat RNA sequesters RNA-binding proteins. A logical consequence of this is that loss-of-function mutations in G4C2-binding partners might contribute to ALS pathogenesis independently of and/or synergistically with C9ORF72 expansions. Targeted sequencing of genomic DNA encoding either RNA-binding proteins or known ALS genes (n = 274 genes) was performed in ALS patients to identify rare deleterious genetic variants and explore genotype-phenotype relationships. Genomic DNA was extracted from 103 ALS patients including 42 familial ALS patients and 61 young-onset (average age of onset 41 years) sporadic ALS patients; patients were chosen to maximize the probability of identifying genetic causes of ALS. Thirteen patients carried a G4C2-repeat expansion of C9ORF72. We identified 42 patients with rare deleterious variants; 6 patients carried more than one variant. Twelve mutations were discovered in known ALS genes which served as a validation of our strategy. Rare deleterious variants in RNA-binding proteins were significantly enriched in ALS patients compared to control frequencies (p = 5.31E-18). Nineteen patients featured at least one variant in a RNA-binding protein containing a PrLD. The number of variants per patient correlated with rate of disease progression (t-test, p = 0.033). We identified eighteen patients with a single variant in a G4C2-repeat binding protein. Patients with a G4C2-binding protein variant in combination with a C9ORF72 expansion had a significantly faster disease course (t-test, p = 0.025). Our data are consistent with an oligogenic model of ALS. We provide evidence for a number of entirely novel genetic variants of ALS caused by mutations in RNA-binding proteins. Moreover we show that these mutations act synergistically with each other and with C9ORF72 expansions to modify the clinical phenotype of ALS. A key finding is that this synergy is present only between functionally interacting variants. This work has significant implications for ALS therapy development.

We describe an autosomal dominant, multi-generational, amyotrophic lateral sclerosis (ALS) pedigree in which disease co-segregates with a heterozygous p.Y374X nonsense mutation within TDP-43. Mislocalization of TDP-43 and formation of insoluble TDP-43-positive neuronal cytoplasmic inclusions is the hallmark pathology in >95% of ALS patients. Neuropathological examination of the single case for which CNS tissue was available indicated typical TDP-43 pathology within lower motor neurons, but classical TDP-43-positive inclusions were absent from motor cortex. The mutated allele is transcribed and translated in patient fibroblasts and motor cortex tissue, but overall TDP-43 protein expression is reduced compared to wild-type controls. Despite absence of TDP-43-positive inclusions we confirmed deficient TDP-43 splicing function within motor cortex tissue. Furthermore, urea fractionation and mass spectrometry of motor cortex tissue carrying the mutation revealed atypical TDP-43 protein species but not typical C-terminal fragments. We conclude that the p.Y374X mutation underpins a monogenic, fully penetrant form of ALS. Reduced expression of TDP-43 combined with atypical TDP-43 protein species and absent C-terminal fragments extends the molecular phenotypes associated with TDP-43 mutations and with ALS more broadly. Future work will need to include the findings from this pedigree in dissecting the mechanisms of TDP-43-mediated toxicity.

Neurovascular coupling is a critical brain mechanism whereby changes to blood flow accompany localised neural activity. The breakdown of neurovascular coupling is linked to the development and progression of several neurological conditions including dementia. In this study, we examined cortical haemodynamics in mouse preparations that modelled Alzheimer's disease (J20-AD) and atherosclerosis (PCSK9-ATH) between 9 and 12 m of age. We report novel findings with atherosclerosis where neurovascular decline is characterised by significantly reduced blood volume, altered levels of oxyhaemoglobin and deoxyhaemoglobin, in addition to global neuroinflammation. In the comorbid mixed model (J20-PCSK9-MIX), we report a 3 x increase in hippocampal amyloid-beta plaques. A key finding was that cortical spreading depression (CSD) due to electrode insertion into the brain was worse in the diseased animals and led to a prolonged period of hypoxia. These findings suggest that systemic atherosclerosis can be detrimental to neurovascular health and that having cardiovascular comorbidities can exacerbate pre-existing Alzheimer's-related amyloid-plaques.

Although a variety of brain lesions may contribute to the pathological assessment of dementia, the relationship of these lesions to dementia, how they interact and how to quantify them remains uncertain. Systematically assessing neuropathological measures by their degree of association with dementia may lead to better diagnostic systems and treatment targets. This study aims to apply machine learning approaches to feature selection in order to identify critical features of Alzheimer-related pathologies associated with dementia. We applied machine learning techniques for feature ranking and classification to objectively compare neuropathological features and their relationship to dementia status during life using a cohort (n=186) from the Cognitive Function and Ageing Study (CFAS). We first tested Alzheimer's Disease and tau markers and then other neuropathologies associated with dementia. Seven feature ranking methods using different information criteria consistently ranked 22 out of the 34 neuropathology features for importance to dementia classification. Although highly correlated, Braak neurofibrillary tangle stage, beta-amyloid and cerebral amyloid angiopathy features were ranked the highest. The best-performing dementia classifier using the top eight neuropathological features achieved 79% sensitivity, 69% specificity and 75% precision. However, when assessing all seven classifiers and the 22 ranked features, a substantial proportion (40.4%) of dementia cases was consistently misclassified. These results highlight the benefits of using machine learning to identify critical indices of plaque, tangle and cerebral amyloid angiopathy burdens that may be useful for classifying dementia.

Poststroke dementia (PSD) is associated with pathology in frontal brain regions, in particular dorsolateral prefrontal cortex (DLPFC) neurons and white matter, remote from the infarct. We hypothesised that PSD results from progressive DLPFC neuronal damage, associated with frontal white matter gliovascular unit (GVU) alterations. We investigated the transcriptomic profile of the neurons and white matter GVU cells previously implicated in pathology. Laser-capture microdissected neurons, astrocytes and endothelial cells were obtained from the Cognitive Function After Stroke cohort of control, PSD and poststroke non-dementia (PSND) human subjects. Gene expression was assessed using microarrays and pathway analysis to compare changes in PSD with controls and PSND. Neuronal findings were validated using NanoString technology and compared with those in the bilateral common carotid artery stenosis (BCAS) mouse model. Comparing changes in PSD compared to controls with changes in PSND compared to controls identified transcriptomic changes associated specifically with dementia. DLPFC neurons showed defects in energy production (tricarboxylic acid (TCA) cycle, adenosine triphosphate (ATP) binding and mitochondria), signalling and communication (MAPK signalling, Toll-like receptor signalling, endocytosis). Similar changes were identified in neurons isolated from BCAS mice. Neuronal findings accompanied by altered astrocyte communication and endothelium immune changes in the frontal white matter, suggesting GVU dysfunction. We propose a pathogenic model in PSD whereby neuronal changes are associated with frontal white matter GVU dysfunction leading to astrocyte failure in supporting neuronal circuits resulting in delayed cognitive decline associated with PSD. Therefore, targeting these processes could potentially ameliorate the dementia seen in PSD.

A consistent clinical feature of amyotrophic lateral sclerosis (ALS) is the sparing of eye movements and the function of external sphincters, with corresponding preservation of motor neurons in the brainstem oculomotor nuclei, and of Onuf's nucleus in the sacral spinal cord. Studying the differences in properties of neurons that are vulnerable and resistant to the disease process in ALS may provide insights into the mechanisms of neuronal degeneration, and identify targets for therapeutic manipulation. We used microarray analysis to determine the differences in gene expression between oculomotor and spinal motor neurons, isolated by laser capture microdissection from the midbrain and spinal cord of neurologically normal human controls. We compared these to transcriptional profiles of oculomotor nuclei and spinal cord from rat and mouse, obtained from the GEO omnibus database. We show that oculomotor neurons have a distinct transcriptional profile, with significant differential expression of 1,757 named genes (q < 0.001). Differentially expressed genes are enriched for the functional categories of synaptic transmission, ubiquitin-dependent proteolysis, mitochondrial function, transcriptional regulation, immune system functions, and the extracellular matrix. Marked differences are seen, across the three species, in genes with a function in synaptic transmission, including several glutamate and GABA receptor subunits. Using patch clamp recording in acute spinal and brainstem slices, we show that resistant oculomotor neurons show a reduced AMPA-mediated inward calcium current, and a higher GABA-mediated chloride current, than vulnerable spinal motor neurons. The findings suggest that reduced susceptibility to excitotoxicity, mediated in part through enhanced GABAergic transmission, is an important determinant of the relative resistance of oculomotor neurons to degeneration in ALS.