Cellular senescence may affect the post-mitotic cells of the brain. We examined the expression of senescence markers, including p16, p21, γH2Ax and H3K9me3, in the frontal cortex of brain donations from the Cognitive Function and Ageing Study to assess their relationship to Alzheimer’s disease neuropathological change (ADNC) and dementia. p21, γH2Ax and H3K9me3 were expressed in pyramidal neurons and glia, whilst p16 was confined to glial cells. p21 and γH2Ax were correlated in neurons, and with p16 in glia. They did not increase with ADNC, tending to be higher at early Braak neurofibrillary tangle stages. Transcriptomic profiling of pyramidal neuron-enriched samples at low Braak stages showed that higher neuronal p21 expression was associated with altered pathways for neuronal function, neurodegeneration, protein homeostasis, mitochondrial dysfunction and synaptic signalling. In conclusion, the different expression profile of senescence markers in neurons and glia suggest possible differences in senescence-related mechanisms. Expression at lower ADNC stages suggests senescence may be important at earlier stages of Alzheimer’s pathogenesis, whilst transcriptomic changes suggest an impact on neuronal function. The lack of association of senescence markers with dementia status indicates that more work is needed to determine the value of senescence as a therapeutic target for dementia.

Animals recognize mechanosensory stimuli and generate targeted responses. Flies perform distinct leg rubbing movements depending on which of the legs is stimulated. While the leg that receives stimulus is always involved in cleaning, different adjacent legs are recruited. For front and hind legs, the contralateral homolog or ipsilateral middle leg is used, but for the middle leg, one or both hind legs are engaged. Here, we identify six segmentally repeated command-like interneurons, LegPNs, whose activation induces leg rubbing. Sensorimotor circuits, repeated in each neuromere, include mechanosensory inputs and reciprocal excitatory connections with pre-motor LegPLs. Activation of LegPLs causes leg flexion. There are segmental differences in the circuits downstream of LegPNs for the middle legs-T2 LegPCs lack commissural connections but include additional ipsilateral intersegmental projections. LegPC activation causes simultaneous front and back leg rubbing. These behavioral and anatomical results demonstrate how shared and modified serially homologous neural circuits coordinate segmentally distinct grooming movements.

Limbs execute diverse actions coordinated by the nervous system through multiple motor programs. The basic architecture of motor neurons that activate muscles which articulate joints for antagonistic flexion and extension movements is conserved from flies to vertebrates. While excitatory premotor circuits are expected to establish sets of leg motor neurons that work together, our study uncovered an instructive role for inhibitory circuits - including their ability to generate rhythmic leg movements. Using electron microscopy data in the Drosophila nerve cord, we categorized ~120 GABAergic inhibitory neurons from the 13A and 13B hemilineages into classes based on similarities in morphology and connectivity. By mapping their connections, we uncovered pathways for inhibiting specific groups of motor neurons, disinhibiting antagonistic counterparts, and inducing alternation between flexion and extension. We tested the function of specific inhibitory neurons through optogenetic activation and silencing, using high resolution quantitative analysis of leg movements during grooming. We combined findings from anatomical and behavioral analyses to construct a computational model that can reproduce major aspects of the observed behavior, demonstrating that these premotor inhibitory circuits can generate rhythmic leg movements.

A key role for inflammation in amyotrophic lateral sclerosis/motor neuron disease (ALS/MND) has been identified. It is vital to assess which central nervous system structures are most affected and which inflammatory processes are responsible in humans. The inflammatory transcriptome was characterized in the cervical spinal cord and motor cortex in post-mortem frozen and formalin-fixed paraffin-embedded specimens from human sporadic ALS/MND and control cases using the nCounter (R) Neuroinflammation Panel. Archival data were reanalyzed and compared with the nCounter data. Immunohistochemistry was used to examine the inflammatory response in the spinal cord and motor cortex and validate changes found during transcriptomic analyses. In the spinal cord, marked inflammation was observed, while less inflammation was detected in the motor cortex. Examination of differentially expressed genes in the spinal cord highlighted TREM2, TYROBP, APOE, and CD163, as well as phagocytic pathways. In sporadic ALS/MND spinal cord, significant microglial reactivity and involvement of TREM2, ApoE (encoded by APOE), and TYROBP were confirmed, suggesting the involvement of the disease-associated microglial (DAM) phenotype. The corticospinal tracts showed greater inflammation than the ventral horns. The precentral gyrus of ALS/MND again showed less immune reactivity to disease when compared to controls. Finally, in the largest cohort assessed to date, we demonstrate an association between the APOE variant and ALS/MND risk, age of onset, and survival. We find confirmed associations between APOE epsilon 3/epsilon 3 and disease and between epsilon 2/epsilon 2 and absence of disease. Further, epsilon 4/epsilon 4 appears to be associated with earlier disease onset and a more aggressive course. We conclude that while there is widespread inflammation in the CNS in sporadic ALS/MND, this is more marked in the spinal cord, especially the corticospinal tract. The specific markers stress the DAM phenotype as having a key role together with a possible influx of somatic macrophages. In addition, APOE function and genotype may be relevant in ALS/MND.

A key role for inflammation in amyotrophic lateral sclerosis/motor neuron disease (ALS/MND) has been identified. It is vital to assess which central nervous system structures are most affected and which inflammatory processes are responsible in humans. The inflammatory transcriptome was characterized in the cervical spinal cord and motor cortex in post mortem frozen and formalin-fixed paraffin embedded specimens from human sporadic ALS/MND and control cases using the nCounter(R) Neuroinflammation Panel. Archival data were re-analysed and compared with the nCounter data. Immunohistochemistry was used to examine the inflammatory response in the spinal cord, motor cortex, and regions across the brain and validate changes found at during transcriptomic analyses. In the spinal cord, marked inflammation was observed while less inflammation was detected in the motor cortex. Examination of differentially expressed genes in the spinal cord highlighted TREM2, TYROBP, APOE, and CD163 as well as phagocytic pathways. In sporadic ALS/MND spinal cord, significant microglial reactivity, and involvement of TREM2, ApoE (encoded by APOE) and TYROBP was confirmed, suggesting the involvement of the disease-associated microglial (DAM) phenotype. The corticospinal tracts showed greater inflammation than the ventral horns. The precentral gyrus of ALS/MND again showed less immune reactivity to disease when compared to controls. Finally, in the largest cohort assessed to date, we demonstrate an association between the APOE haplotype and ALS/MND risk, age of onset and survival. We confirm associations between APOE ε4 and a more aggressive disease; and between ε2 and a less severe disease phenotype. We conclude that while there is widespread inflammation in the CNS in sporadic ALS/MND, this is more marked in the spinal cord, especially the corticospinal tract. The specific markers stress the DAM phenotype as having a key role together with a possible influx of somatic macrophages. In addition, APOE function and genotype may be relevant in ALS/MND. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Funders: The Pathological Society of Great Britain, The British Neuropathological Society, Bruker Spatial Biology who provided two nCounter Neuroinflammation panels. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Sheffield Brain Tissue Bank (SBTB) which provided the tissue used here has ethical permission to function as a Research Tissue Bank. At the time the work was undertaken, this was covered by a favourable opinion from the Scotland A Research Ethics Committee (Reference 19/SS/0029). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

To determine the algorithmic rules and neural circuits controlling selection amongst competing behaviors, we established assays where adult Drosophila melanogaster choose between grooming and feeding, grooming and courting, or feeding and courting. We found that there is not an absolute hierarchy: while flies typically perform grooming first, they can choose to feed if sufficiently starved, or court if an appropriate female is available. Flies alternate between competing behaviors, performing short bouts of each action rather than completely satisfying one drive before transitioning to another. While we did not do an exhaustive screen, from the candidates we examined, we did not find evidence for a specific genetic or neuronal locus that affects all decisions. We did identify genetic background effects, suggesting that multiple genes may contribute to decision-making priorities. Our results add to a growing body of work on decision making in Drosophila and provide a foundation for future investigation of the exact neural circuits required to achieve appropriate choices.

Gluten ataxia (GA) is the primary neurological manifestation of gluten sensitivity, characterised by loss of Purkinje cells throughout the cerebellar cortex and rooted in autoimmunity to transglutaminase 6 (TG6). Previous studies have shown the contribution of serum anti-TG6 antibodies to disease progression; however, it remains unclear where these antibodies are produced and how they gain access into the brain parenchyma. This study aims to provide an immunological assessment of the CSF in patients with GA to better define the humoral response contributing to disease pathophysiology. In this observational study we assessed the presence of plasma cells in the CSF of 20 patients with GA and 6 controls. CSF from 16 of the GA patients and from all 6 controls was investigated for the presence of anti-TG6 IgA antibodies. Immunohistochemistry for CD138 was performed to assess the presence of plasma cells in the cerebellum and spinal cord of 4 cases with GA, 4 ataxia controls and 4 neurologically healthy controls. A significant increase in anti-TG6 IgA antibodies was detected in the CSF of patients with GA compared to controls, with no correlation between CSF and serum levels of anti-TG6 IgA antibodies for either experimental group. CD138+ cells were present in the CSF of 2 patients with GA and in the cerebellum and spinal cord of 3 post-mortem cases of GA. In a subpopulation of patients with GA intrathecal presence of plasma cells and TG6 antibodies is a feature of the disease, likely associated with prolonged disease duration and continuous exposure to gluten.

Flies groom in response to competing mechanosensory cues in an anterior-to-posterior order using specific legs. From behavior screens, we identified a pair of cholinergic command-like neurons, Mago-no-Te (MGT), whose optogenetic activation elicits thoracic grooming by the back legs. Thoracic grooming is typically composed of body sweeps and leg rubs in alternation, but clonal analysis coupled with amputation experiments revealed that MGT activation only commands the body sweeps: initiation of leg rubbing requires contact between the leg and thorax. With new electron microscopy (EM) connectome data for the ventral nerve cord (VNC), we uncovered a circuit-based explanation for why stimulation of posterior thoracic mechanosensory bristles initiates cleaning by the back legs. Our previous work showed that flies weigh mechanosensory inputs across the body to select which part to groom, but we did not know why the thorax was always cleaned last. Here, the connectome for the VNC enabled us to identify a pair of GABAergic inhibitory neurons, UMGT1, that receives diverse sensory inputs and synapses onto both MGT and components of its downstream circuits. Optogenetic activation of UMGT1 suppresses thoracic cleaning, representing a mechanism by which mechanosensory stimuli on other body parts could take precedence in the grooming hierarchy. We also anatomically mapped the pre-motor circuit downstream of MGT, including inhibitory feedback connections that may enable rhythmicity and coordination of limb movement during thoracic grooming. The combination of behavioral screens and connectome analysis allowed us to identify a neural circuit connecting sensory-to- motor neurons that contributes to thoracic grooming.

Altered cholesterol metabolism is implicated in brain ageing and Alzheimer’s disease. We examined whether key genes regulating cholesterol metabolism and levels of brain cholesterol are altered in dementia and Alzheimer’s disease neuropathological change (ADNC). Temporal cortex (n=99) was obtained from the Cognitive Function and Ageing Study. Expression of the cholesterol biosynthesis rate-limiting enzyme HMG-CoA reductase (HMGCR) and its regulator, SREBP2, were detected using immunohistochemistry. Expression of HMGCR, SREBP2, CYP46A1 and ABCA1 were quantified by qPCR in samples enriched for astrocyte and neuronal RNA following laser-capture microdissection. Total cortical cholesterol was measured using the Amplex Red assay. HMGCR and SREBP2 proteins were predominantly expressed in pyramidal neurones, and in glia. Neuronal HMGCR did not vary with ADNC, oxidative stress, neuroinflammation or dementia status. Expression of HMGCR neuronal mRNA decreased with ADNC (p=0.022) and increased with neuronal DNA damage (p=0.049), whilst SREBP2 increased with ADNC (p=0.005). High or moderate tertiles for cholesterol levels were associated with increased dementia risk (OR 1.44, 1.58). APOE ε4 allele was not associated with cortical cholesterol levels. ADNC is associated with gene expression changes that may impair cholesterol biosynthesis in neurones but not astrocytes, whilst levels of cortical cholesterol show a weak relationship to dementia status.

Age-associated deep-subcortical white matter lesions (DSCLs) are an independent risk factor for dementia, displaying high levels of CD68+ microglia. This study aimed to characterize the transcriptomic profile of microglia in DSCLs and surrounding radiologically normal-appearing white matter (NAWM) compared to non-lesional control white matter. CD68+ microglia were isolated from white matter groups (n = 4 cases per group) from the Cognitive Function and Ageing Study neuropathology cohort using immuno-laser capture microdissection. Microarray gene expression profiling, but not RNA-sequencing, was found to be compatible with immuno-LCM-ed post-mortem material in the CFAS cohort and identified significantly differentially expressed genes (DEGs). Functional grouping and pathway analysis were assessed using the Database for Annotation Visualization and Integrated Discovery (DAVID) software, and immunohistochemistry was performed to validate gene expression changes at the protein level. Transcriptomic profiling of microglia in DSCLs compared to non-lesional control white matter identified 181 significant DEGs (93 upregulated and 88 downregulated). Functional clustering analysis in DAVID revealed dysregulation of haptoglobin–haemoglobin binding (Enrichment score 2.5, p = 0.017), confirmed using CD163 immunostaining, suggesting a neuroprotective microglial response to blood–brain barrier dysfunction in DSCLs. In NAWM versus control white matter, microglia exhibited 347 DEGs (209 upregulated, 138 downregulated), with significant dysregulation of protein de-ubiquitination (Enrichment score 5.14, p < 0.001), implying an inability to maintain protein homeostasis in NAWM that may contribute to lesion spread. These findings enhance understanding of microglial transcriptomic changes in ageing white matter pathology, highlighting a neuroprotective adaptation in DSCLs microglia and a potentially lesion-promoting phenotype in NAWM microglia.

Gluten sensitivity has long been recognized exclusively for its gastrointestinal involvement; however, more recent research provides evidence for the existence of neurological manifestations that can appear in combination with or independent of the small bowel manifestations. Amongst all neurological manifestations of gluten sensitivity, gluten ataxia is the most commonly occurring one, accounting for up to 40% of cases of idiopathic sporadic ataxia. However, despite its prevalence, its neuropathological basis is still poorly defined. Here, we provide a neuropathological characterization of gluten ataxia and compare the presence of neuroinflammatory markers glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, major histocompatibility complex II and cluster of differentiation 68 in the central nervous system of four gluten ataxia cases to five ataxia controls and seven neurologically healthy controls. Our results demonstrate that severe cerebellar atrophy, cluster of differentiation 20+ and cluster of differentiation 8+ lymphocytic infiltration in the cerebellar grey and white matter and a significant upregulation of microglial immune activation in the cerebellar granular layer, molecular layer and cerebellar white matter are features of gluten ataxia, providing evidence for the involvement of both cellular and humoral immune-mediated processes in gluten ataxia pathogenesis.

The descending neurons connecting the fly's brain to its ventral nerve cord respond to sensory stimuli and evoke motor programs of varying complexity. Anatomical characterization of the descending neurons and their synaptic connections suggests how these circuits organize movements, while optogenetic manipulation of their activity reveals what behaviors they can induce. Monitoring their responses to sensory stimuli or during behavior performance indicates what information they may encode. Recent advances in all three approaches make the descending neurons an excellent place to better understand the sensorimotor integration and transformation required for nervous systems to govern the motor sequences that constitute animal behavior.

While animals swim, crawl, walk, and fly with apparent ease, building robots capable of robust locomotion remains a significant challenge. In this review, we draw attention to mechanosensation--the sensing of mechanical forces generated within and outside the body--as a key sense that enables robust locomotion in animals. We discuss differences between mechanosensation in animals and current robots with respect to 1) the encoding properties and distribution of mechanosensors and 2) the integration and regulation of mechanosensory feedback. We argue that robotics would benefit greatly from a detailed understanding of these aspects in animals. To that end, we highlight promising experimental and engineering approaches to study mechanosensation, emphasizing the mutual benefits for biologists and engineers that emerge from moving forward together.

The blood–brain barrier (BBB) comprises a highly specialised complex of cells within the neurovascular unit, and is responsible for tightly regulating homeostasis within the central nervous system, which is critical for maintaining neuronal function [...]

The epidemiological neuropathology perspective of population and community-based studies allows unbiased assessment of the prevalence of various pathologies and their relationships to late-life dementia. In addition, this approach provides complementary insights to conventional case-control studies, which tend to be more representative of a younger clinical cohort. The Cognitive Function and Ageing Study (CFAS) is a longitudinal study of cognitive impairment and frailty in the general United Kingdom population. In this review, we provide an overview of the major findings from CFAS, alongside other studies, which have demonstrated a high prevalence of pathology in the ageing brain, particularly Alzheimer's disease neuropathological change and vascular pathology. Increasing burdens of these pathologies are the major correlates of dementia, especially neurofibrillary tangles, but there is substantial overlap in pathology between those with and without dementia, particularly at intermediate burdens of pathology and also at the oldest ages. Furthermore, additional pathologies such as limbic-predominant age-related TDP-43 encephalopathy, ageing-related tau astrogliopathy and primary age-related tauopathies contribute to late-life dementia. Findings from ageing population-representative studies have implications for the understanding of dementia pathology in the community. The high prevalence of pathology and variable relationship to dementia status has implications for disease definition and indicate a role for modulating factors on cognitive outcome. The complexity of late-life dementia, with mixed pathologies, indicates a need for a better understanding of these processes across the life-course to direct the best research for reducing risk in later life of avoidable clinical dementia syndromes.

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.

The brain coordinates the movements that constitute behavior, but how descending neurons convey the myriad of commands required to activate the motor neurons of the limbs in the right order and combinations to produce those movements is not well understood. For anterior grooming behavior in the fly, we show that its component head sweeps and leg rubs can be initiated separately, or as a set, by different descending neurons. Head sweeps and leg rubs are mutually exclusive movements of the front legs that normally alternate, and we show that circuits in the ventral nerve cord as well as in the brain can resolve competing commands. Finally, the left and right legs must work together to remove debris. The coordination for leg rubs can be achieved by unilateral activation of a single descending neuron, while a similar manipulation of a different descending neuron decouples the legs to produce single-sided head sweeps. Taken together, these results demonstrate that distinct descending neurons orchestrate the complex alternation between the movements that make up anterior grooming.

With the hypothesis that perivascular microglia are involved as neuroinflammatory components of the gliovascular unit contributing to white matter hyperintensities on MRI and pathophysiology, we assessed their status in stroke survivors who develop dementia. Immunohistochemical and immunofluorescent methods were used to assess the distribution and quantification of total and perivascular microglial cell densities in 68 brains focusing on the frontal lobe WM and overlying neocortex in post‐stroke dementia (PSD), post‐stroke non‐dementia (PSND) and similar age control subjects. We primarily used CD68 as a marker of phagocytic microglia, as well as other markers of microglia including Iba‐1 and TMEM119, and the myeloid cell marker TREM2 to assess dementia‐specific changes. We first noted greater total densities of CD68 + and TREM2 + cells per mm 2 in the frontal WM compared to the overlying cortex across the stroke cases and controls ( p = 0.001). PSD subjects showed increased percentage of activated perivascular CD68 + cells distinct from ramified or primed microglia in the WM ( p < 0.05). However, there was no apparent change in perivascular TREM2 + cells. Total densities of TREM2 + cells were only ~10% of CD68 + cells but there was high degree of overlap (>70%) between them in both the WM and the cortex. CD68 and Iba‐1 or CD68 and TMEM119 markers were colocalised by ~55%. Within the deep WM, ~30% of CD68+ cells were co‐localised with fragments of degraded myelin basic protein. Among fragmented CD68+ cells in adjacent WM of PSD subjects, >80% of the cells expressed cleaved caspase‐3. Our observations suggest although the overall repertoire of perivascular microglial cells is not changed in the parenchyma, PSD subjects accrue more perivascular‐activated CD68+ microglia rather than TREM2+ cells. This implies there is a subset of CD68+ cells, which are responsible for the differential response in perivascular inflammation within the gliovascular unit of the deep WM.