Microglia-targeted inhibition of miR-17 via mannose-coated lipid nanoparticles improves pathology and behavior in a mouse model of Alzheimer's disease.

Neuroinflammation and accumulation of Amyloid Beta (Aß) accompanied by deterioration of special memory are hallmarks of Alzheimer's disease (AD). Effective preventative and treatment options for AD are still needed. Microglia in AD brains are characterized by elevated levels of microRNA-17 (miR-17), which is accompanied by defective autophagy, Aß accumulation, and increased inflammatory cytokine production. However, the effect of targeting miR-17 on AD pathology and memory loss is not clear. To specifically inhibit miR-17 in microglia, we generated mannose-coated lipid nanoparticles (MLNPs) enclosing miR-17 antagomir (Anti-17 MLNPs), which are targeted to mannose receptors readily expressed on microglia. We used a 5XFAD mouse model (AD) that recapitulates many AD-related phenotypes observed in humans. Our results show that Anti-17 MLNPs, delivered to 5XFAD mice by intra-cisterna magna injection, specifically deliver Anti-17 to microglia. Anti-17 MLNPs downregulated miR-17 expression in microglia but not in neurons, astrocytes, and oligodendrocytes. Anti-17 MLNPs attenuated inflammation, improved autophagy, and reduced Aß burdens in the brains. Additionally, Anti-17 MLNPs reduced the deterioration in spatial memory and decreased anxiety-like behavior in 5XFAD mice. Therefore, targeting miR-17 using MLNPs is a viable strategy to prevent several AD pathologies. This selective targeting strategy delivers specific agents to microglia without the adverse off-target effects on other cell types. Additionally, this approach can be used to deliver other molecules to microglia and other immune cells in other organs.

Chronic Cortical Inflammation, Cognitive Impairment, and Immune Reactivity Associated with Diffuse Brain Injury Are Ameliorated by Forced Turnover of Microglia.

Traumatic brain injury (TBI) is associated with an increased risk of cognitive, psychiatric, and neurodegenerative complications that may develop after injury. Increased microglial reactivity following TBI may underlie chronic neuroinflammation, neuropathology, and exaggerated responses to immune challenges. Therefore, the goal of this study was to force turnover of trauma-associated microglia that develop after diffuse TBI and determine whether this alleviated chronic inflammation, improved functional recovery and attenuated reduced immune reactivity to lipopolysaccharide (LPS) challenge. Male mice received a midline fluid percussion injury (mFPI) and 7 d later were subjected to a forced microglia turnover paradigm using CSF1R antagonism (PLX5622). At 30 d postinjury (dpi), cortical gene expression, dendritic complexity, myelin content, neuronal connectivity, cognition, and immune reactivity were assessed. Myriad neuropathology-related genes were increased 30 dpi in the cortex, and 90% of these gene changes were reversed by microglial turnover. Reduced neuronal connectivity was evident 30 dpi and these deficits were attenuated by microglial turnover. TBI-associated dendritic remodeling and myelin alterations, however, remained 30 dpi independent of microglial turnover. In assessments of functional recovery, increased depressive-like behavior, and cognitive impairment 30 dpi were ameliorated by microglia turnover. To investigate microglial priming and reactivity 30 dpi, mice were injected intraperitoneally with LPS. This immune challenge caused prolonged lethargy, sickness behavior, and microglial reactivity in the TBI mice. These extended complications with LPS in TBI mice were prevented by microglia turnover. Collectively, microglial turnover 7 dpi alleviated behavioral and cognitive impairments associated with microglial priming and immune reactivity 30 dpi.SIGNIFICANCE STATEMENT A striking feature of traumatic brain injury (TBI), even mild injuries, is that over 70% of individuals have long-term neuropsychiatric complications. Chronic inflammatory processes are implicated in the pathology of these complications and these issues can be exaggerated by immune challenge. Therefore, our goal was to force the turnover of microglia 7 d after TBI. This subacute 7 d postinjury (dpi) time point is a critical transitional period in the shift toward chronic inflammatory processes and microglia priming. This forced microglia turnover intervention in mice attenuated the deficits in behavior and cognition 30 dpi. Moreover, microglia priming and immune reactivity after TBI were also reduced with microglia turnover. Therefore, microglia represent therapeutic targets after TBI to reduce persistent neuroinflammation and improve recovery.

Traumatic Brain Injury Causes Chronic Cortical Inflammation and Neuronal Dysfunction Mediated by Microglia.

Traumatic brain injury (TBI) can lead to significant neuropsychiatric problems and neurodegenerative pathologies, which develop and persist years after injury. Neuroinflammatory processes evolve over this same period. Therefore, we aimed to determine the contribution of microglia to neuropathology at acute [1 d postinjury (dpi)], subacute (7 dpi), and chronic (30 dpi) time points. Microglia were depleted with PLX5622, a CSF1R antagonist, before midline fluid percussion injury (FPI) in male mice and cortical neuropathology/inflammation was assessed using a neuropathology mRNA panel. Gene expression associated with inflammation and neuropathology were robustly increased acutely after injury (1 dpi) and the majority of this expression was microglia independent. At 7 and 30 dpi, however, microglial depletion reversed TBI-related expression of genes associated with inflammation, interferon signaling, and neuropathology. Myriad suppressed genes at subacute and chronic endpoints were attributed to neurons. To understand the relationship between microglia, neurons, and other glia, single-cell RNA sequencing was completed 7 dpi, a critical time point in the evolution from acute to chronic pathogenesis. Cortical microglia exhibited distinct TBI-associated clustering with increased type-1 interferon and neurodegenerative/damage-related genes. In cortical neurons, genes associated with dopamine signaling, long-term potentiation, calcium signaling, and synaptogenesis were suppressed. Microglial depletion reversed the majority of these neuronal alterations. Furthermore, there was reduced cortical dendritic complexity 7 dpi, reduced neuronal connectively 30 dpi, and cognitive impairment 30 dpi. All of these TBI-associated functional and behavioral impairments were prevented by microglial depletion. Collectively, these studies indicate that microglia promote persistent neuropathology and long-term functional impairments in neuronal homeostasis after TBI.SIGNIFICANCE STATEMENT Millions of traumatic brain injuries (TBIs) occur in the United States alone each year. Survivors face elevated rates of cognitive and psychiatric complications long after the inciting injury. Recent studies of human brain injury link chronic neuroinflammation to adverse neurologic outcomes, suggesting that evolving inflammatory processes may be an opportunity for intervention. Here, we eliminate microglia to compare the effects of diffuse TBI on neurons in the presence and absence of microglia and microglia-mediated inflammation. In the absence of microglia, neurons do not undergo TBI-induced changes in gene transcription or structure. Microglial elimination prevented TBI-induced cognitive changes 30 d postinjury (dpi). Therefore, microglia have a critical role in disrupting neuronal homeostasis after TBI, particularly at subacute and chronic timepoints.

Wolframin is a novel regulator of tau pathology and neurodegeneration.

Selective neuronal vulnerability to protein aggregation is found in many neurodegenerative diseases including Alzheimer's disease (AD). Understanding the molecular origins of this selective vulnerability is, therefore, of fundamental importance. Tau protein aggregates have been found in Wolframin (WFS1)-expressing excitatory neurons in the entorhinal cortex, one of the earliest affected regions in AD. The role of WFS1 in Tauopathies and its levels in tau pathology-associated neurodegeneration, however, is largely unknown. Here we report that WFS1 deficiency is associated with increased tau pathology and neurodegeneration, whereas overexpression of WFS1 reduces those changes. We also find that WFS1 interacts with tau protein and controls the susceptibility to tau pathology. Furthermore, chronic ER stress and autophagy-lysosome pathway (ALP)-associated genes are enriched in WFS1-high excitatory neurons in human AD at early Braak stages. The protein levels of ER stress and autophagy-lysosome pathway (ALP)-associated proteins are changed in tau transgenic mice with WFS1 deficiency, while overexpression of WFS1 reverses those changes. This work demonstrates a possible role for WFS1 in the regulation of tau pathology and neurodegeneration via chronic ER stress and the downstream ALP. Our findings provide insights into mechanisms that underpin selective neuronal vulnerability, and for developing new therapeutics to protect vulnerable neurons in AD.

Neuron-Specific HuR-Deficient Mice Spontaneously Develop Motor Neuron Disease.

Human Ag R (HuR) is an RNA binding protein in the ELAVL protein family. To study the neuron-specific function of HuR, we generated inducible, neuron-specific HuR-deficient mice of both sexes. After tamoxifen-induced deletion of HuR, these mice developed a phenotype consisting of poor balance, decreased movement, and decreased strength. They performed significantly worse on the rotarod test compared with littermate control mice, indicating coordination deficiency. Using the grip-strength test, it was also determined that the forelimbs of neuron-specific HuR-deficient mice were much weaker than littermate control mice. Immunostaining of the brain and cervical spinal cord showed that HuR-deficient neurons had increased levels of cleaved caspase-3, a hallmark of cell apoptosis. Caspase-3 cleavage was especially strong in pyramidal neurons and α motor neurons of HuR-deficient mice. Genome-wide microarray and real-time PCR analysis further indicated that HuR deficiency in neurons resulted in altered expression of genes in the brain involved in cell growth, including trichoplein keratin filament-binding protein, Cdkn2c, G-protein signaling modulator 2, immediate early response 2, superoxide dismutase 1, and Bcl2. The additional enriched Gene Ontology terms in the brain tissues of neuron-specific HuR-deficient mice were largely related to inflammation, including IFN-induced genes and complement components. Importantly, some of these HuR-regulated genes were also significantly altered in the brain and spinal cord of patients with amyotrophic lateral sclerosis. Additionally, neuronal HuR deficiency resulted in the redistribution of TDP43 to cytosolic granules, which has been linked to motor neuron disease. Taken together, we propose that this neuron-specific HuR-deficient mouse strain can potentially be used as a motor neuron disease model.

Concussion, microvascular injury, and early tauopathy in young athletes after impact head injury and an impact concussion mouse model.

The mechanisms underpinning concussion, traumatic brain injury, and chronic traumatic encephalopathy, and the relationships between these disorders, are poorly understood. We examined post-mortem brains from teenage athletes in the acute-subacute period after mild closed-head impact injury and found astrocytosis, myelinated axonopathy, microvascular injury, perivascular neuroinflammation, and phosphorylated tau protein pathology. To investigate causal mechanisms, we developed a mouse model of lateral closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanaesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of a concussion-like syndrome characterized by altered arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioural deficits. Experimental impact injury was associated with axonopathy, blood-brain barrier disruption, astrocytosis, microgliosis (with activation of triggering receptor expressed on myeloid cells, TREM2), monocyte infiltration, and phosphorylated tauopathy in cerebral cortex ipsilateral and subjacent to impact. Phosphorylated tauopathy was detected in ipsilateral axons by 24 h, bilateral axons and soma by 2 weeks, and distant cortex bilaterally at 5.5 months post-injury. Impact pathologies co-localized with serum albumin extravasation in the brain that was diagnostically detectable in living mice by dynamic contrast-enhanced MRI. These pathologies were also accompanied by early, persistent, and bilateral impairment in axonal conduction velocity in the hippocampus and defective long-term potentiation of synaptic neurotransmission in the medial prefrontal cortex, brain regions distant from acute brain injury. Surprisingly, acute neurobehavioural deficits at the time of injury did not correlate with blood-brain barrier disruption, microgliosis, neuroinflammation, phosphorylated tauopathy, or electrophysiological dysfunction. Furthermore, concussion-like deficits were observed after impact injury, but not after blast exposure under experimental conditions matched for head kinematics. Computational modelling showed that impact injury generated focal point loading on the head and seven-fold greater peak shear stress in the brain compared to blast exposure. Moreover, intracerebral shear stress peaked before onset of gross head motion. By comparison, blast induced distributed force loading on the head and diffuse, lower magnitude shear stress in the brain. We conclude that force loading mechanics at the time of injury shape acute neurobehavioural responses, structural brain damage, and neuropathological sequelae triggered by neurotrauma. These results indicate that closed-head impact injuries, independent of concussive signs, can induce traumatic brain injury as well as early pathologies and functional sequelae associated with chronic traumatic encephalopathy. These results also shed light on the origins of concussion and relationship to traumatic brain injury and its aftermath.awx350media15713427811001.

Traumatic brain injury-induced neuronal damage in the somatosensory cortex causes formation of rod-shaped microglia that promote astrogliosis and persistent neuroinflammation.

Microglia undergo dynamic structural and transcriptional changes during the immune response to traumatic brain injury (TBI). For example, TBI causes microglia to form rod-shaped trains in the cerebral cortex, but their contribution to inflammation and pathophysiology is unclear. The purpose of this study was to determine the origin and alignment of rod microglia and to determine the role of microglia in propagating persistent cortical inflammation. Here, diffuse TBI in mice was modeled by midline fluid percussion injury (FPI). Bone marrow chimerism and BrdU pulse-chase experiments revealed that rod microglia derived from resident microglia with limited proliferation. Novel data also show that TBI-induced rod microglia were proximal to axotomized neurons, spatially overlapped with dense astrogliosis, and aligned with apical pyramidal dendrites. Furthermore, rod microglia formed adjacent to hypertrophied microglia, which clustered among layer V pyramidal neurons. To better understand the contribution of microglia to cortical inflammation and injury, microglia were eliminated prior to TBI by CSF1R antagonism (PLX5622). Microglial elimination did not affect cortical neuron axotomy induced by TBI, but attenuated rod microglial formation and astrogliosis. Analysis of 262 immune genes revealed that TBI caused profound cortical inflammation acutely (8 hr) that progressed in nature and complexity by 7 dpi. For instance, gene expression related to complement, phagocytosis, toll-like receptor signaling, and interferon response were increased 7 dpi. Critically, these acute and chronic inflammatory responses were prevented by microglial elimination. Taken together, TBI-induced neuronal injury causes microglia to structurally associate with neurons, augment astrogliosis, and propagate diverse and persistent inflammatory/immune signaling pathways.

Genetically enhancing the expression of chemokine domain of CX3CL1 fails to prevent tau pathology in mouse models of tauopathy.

BACKGROUND: Fractalkine (CX3CL1) and its receptor (CX3CR1) play an important role in regulating microglial function. We have previously shown that Cx3cr1 deficiency exacerbated tau pathology and led to cognitive impairment. However, it is still unclear if the chemokine domain of the ligand CX3CL1 is essential in regulating neuronal tau pathology. METHODS: We used transgenic mice lacking endogenous Cx3cl1 (Cx3cl1-/-) and expressing only obligatory soluble form (with only chemokine domain) and lacking the mucin stalk of CX3CL1 (referred to as Cx3cl1105Δ mice) to assess tau pathology and behavioral function in both lipopolysaccharide (LPS) and genetic (hTau) mouse models of tauopathy. RESULTS: First, increased basal tau levels accompanied microglial activation in Cx3cl1105Δ mice compared to control groups. Second, increased CD45+ and F4/80+ neuroinflammation and tau phosphorylation were observed in LPS, hTau/Cx3cl1-/-, and hTau/Cx3cl1105Δ mouse models of tau pathology, which correlated with impaired spatial learning. Finally, microglial cell surface expression of CX3CR1 was reduced in Cx3cl1105Δ mice, suggesting enhanced fractalkine receptor internalization (mimicking Cx3cr1 deletion), which likely contributes to the elevated tau pathology. CONCLUSIONS: Collectively, our data suggest that overexpression of only chemokine domain of CX3CL1 does not protect against tau pathology.

Cellular players that shape evolving pathology and neurodegeneration following traumatic brain injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide, and has emerged as a critical risk factor for multiple neurodegenerative diseases, particularly Alzheimer's disease (AD). How the inflammatory cascade resulting from mechanical stress, axonal shearing and the loss of neurons and glia following initial impact in TBI, contributes to the development of AD-like disease is unclear. Neuroinflammation, characterized by blood-brain barrier (BBB) dysfunction and activation of brain-resident microglia and astrocytes, resulting in secretion of inflammatory mediators and subsequent recruitment of peripheral immune cells has been the focus of extensive research in attempts to identify drug-targets towards improving functional outcomes post TBI. While knowledge of intricate cellular interactions that shape lesion pathophysiology is incomplete, a major limitation in the field is the lack of understanding of how distinct cell types differentially alter TBI pathology. The aim of this review is to highlight functional differences between populations of bone marrow derived, infiltrating monocytes/macrophages and brain-resident microglia based on differential expression of the chemokine receptors CCR2 and CX3CR1. This review will focus on how unique subsets of mononuclear phagocytes shape TBI pathophysiology, neurotoxicity and BBB function, in a disease-stage dependent manner. Additionally, this review summarizes the role of multiple microglia and macrophage receptors, namely CCR2, CX3CR1 and Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) in pathological neuroinflammation and neurodegeneration vs. recovery following TBI. TREM2 has been implicated in mediating AD-related pathology, and variants in TREM2 are particularly important due to their correlation with exacerbated neurodegeneration. Finally, this review highlights behavioral outcomes associated with microglial vs. macrophage variances, the need for novel treatment strategies that target unique subpopulations of peripheral macrophages, and the importance of development of therapeutics to modulate inflammatory functions of brain-resident microglia at specific stages of TBI.

Neutrophil depletion after subarachnoid hemorrhage improves memory via NMDA receptors.

Cognitive deficits after aneurysmal subarachnoid hemorrhage (SAH) are common and disabling. Patients who experience delayed deterioration associated with vasospasm are likely to have cognitive deficits, particularly problems with executive function, verbal and spatial memory. Here, we report neurophysiological and pathological mechanisms underlying behavioral deficits in a murine model of SAH. On tests of spatial memory, animals with SAH performed worse than sham animals in the first week and one month after SAH suggesting a prolonged injury. Between three and six days after experimental hemorrhage, mice demonstrated loss of late long-term potentiation (L-LTP) due to dysfunction of the NMDA receptor. Suppression of innate immune cell activation prevents delayed vasospasm after murine SAH. We therefore explored the role of neutrophil-mediated innate inflammation on memory deficits after SAH. Depletion of neutrophils three days after SAH mitigates tissue inflammation, reverses cerebral vasoconstriction in the middle cerebral artery, and rescues L-LTP dysfunction at day 6. Spatial memory deficits in both the short and long-term are improved and associated with a shift of NMDA receptor subunit composition toward a memory sparing phenotype. This work supports further investigating suppression of innate immunity after SAH as a target for preventative therapies in SAH.

Reactive microglia drive tau pathology and contribute to the spreading of pathological tau in the brain.

Pathological aggregation of tau is a hallmark of Alzheimer's disease and related tauopathies. We have previously shown that the deficiency of the microglial fractalkine receptor (CX3CR1) led to the acceleration of tau pathology and memory impairment in an hTau mouse model of tauopathy. Here, we show that microglia drive tau pathology in a cell-autonomous manner. First, tau hyperphosphorylation and aggregation occur as early as 2 months of age in hTauCx3cr1(-/-) mice. Second, CD45(+) microglial activation correlates with the spatial memory deficit and spread of tau pathology in the anatomically connected regions of the hippocampus. Third, adoptive transfer of purified microglia derived from hTauCx3cr1(-/-) mice induces tau hyperphosphorylation within the brains of non-transgenic recipient mice. Finally, inclusion of interleukin 1 receptor antagonist (Kineret®) in the adoptive transfer inoculum significantly reduces microglia-induced tau pathology. Together, our results suggest that reactive microglia are sufficient to drive tau pathology and correlate with the spread of pathological tau in the brain.

Microglial derived tumor necrosis factor-α drives Alzheimer's disease-related neuronal cell cycle events.

Massive neuronal loss is a key pathological hallmark of Alzheimer's disease (AD). However, the mechanisms are still unclear. Here we demonstrate that neuroinflammation, cell autonomous to microglia, is capable of inducing neuronal cell cycle events (CCEs), which are toxic for terminally differentiated neurons. First, oligomeric amyloid-beta peptide (AßO)-mediated microglial activation induced neuronal CCEs via the tumor-necrosis factor-α (TNFα) and the c-Jun Kinase (JNK) signaling pathway. Second, adoptive transfer of CD11b+ microglia from AD transgenic mice (R1.40) induced neuronal cyclin D1 expression via TNFα signaling pathway. Third, genetic deficiency of TNFα in R1.40 mice (R1.40-Tnfα(-/-)) failed to induce neuronal CCEs. Finally, the mitotically active neurons spatially co-exist with F4/80+ activated microglia in the human AD brain and that a portion of these neurons are apoptotic. Together our data suggest a cell-autonomous role of microglia, and identify TNFα as the responsible cytokine, in promoting neuronal CCEs in the pathogenesis of AD.

Deplete and repeat: microglial CSF1R inhibition and traumatic brain injury.

Traumatic brain injury (TBI) is a public health burden affecting millions of people. Sustained neuroinflammation after TBI is often associated with poor outcome. As a result, increased attention has been placed on the role of immune cells in post-injury recovery. Microglia are highly dynamic after TBI and play a key role in the post-injury neuroinflammatory response. Therefore, microglia represent a malleable post-injury target that could substantially influence long-term outcome after TBI. This review highlights the cell specific role of microglia in TBI pathophysiology. Microglia have been manipulated via genetic deletion, drug inhibition, and pharmacological depletion in various pre-clinical TBI models. Notably, colony stimulating factor 1 (CSF1) and its receptor (CSF1R) have gained much traction in recent years as a pharmacological target on microglia. CSF1R is a transmembrane tyrosine kinase receptor that is essential for microglia proliferation, differentiation, and survival. Small molecule inhibitors targeting CSF1R result in a swift and effective depletion of microglia in rodents. Moreover, discontinuation of the inhibitors is sufficient for microglia repopulation. Attention is placed on summarizing studies that incorporate CSF1R inhibition of microglia. Indeed, microglia depletion affects multiple aspects of TBI pathophysiology, including neuroinflammation, oxidative stress, and functional recovery with measurable influence on astrocytes, peripheral immune cells, and neurons. Taken together, the data highlight an important role for microglia in sustaining neuroinflammation and increasing risk of oxidative stress, which lends to neuronal damage and behavioral deficits chronically after TBI. Ultimately, the insights gained from CSF1R depletion of microglia are critical for understanding the temporospatial role that microglia develop in mediating TBI pathophysiology and recovery.

Context is key: glucocorticoid receptor and corticosteroid therapeutics in outcomes after traumatic brain injury.

Traumatic brain injury (TBI) is a global health burden, and survivors suffer functional and psychiatric consequences that can persist long after injury. TBI induces a physiological stress response by activating the hypothalamic-pituitary-adrenal (HPA) axis, but the effects of injury on the stress response become more complex in the long term. Clinical and experimental evidence suggests long lasting dysfunction of the stress response after TBI. Additionally, pre- and post-injury stress both have negative impacts on outcome following TBI. This bidirectional relationship between stress and injury impedes recovery and exacerbates TBI-induced psychiatric and cognitive dysfunction. Previous clinical and experimental studies have explored the use of synthetic glucocorticoids as a therapeutic for stress-related TBI outcomes, but these have yielded mixed results. Furthermore, long-term steroid treatment is associated with multiple negative side effects. There is a pressing need for alternative approaches that improve stress functionality after TBI. Glucocorticoid receptor (GR) has been identified as a fundamental link between stress and immune responses, and preclinical evidence suggests GR plays an important role in microglia-mediated outcomes after TBI and other neuroinflammatory conditions. In this review, we will summarize GR-mediated stress dysfunction after TBI, highlighting the role of microglia. We will discuss recent studies which target microglial GR in the context of stress and injury, and we suggest that cell-specific GR interventions may be a promising strategy for long-term TBI pathophysiology.

Arid1b haploinsufficiency in pyramidal neurons causes cellular and circuit changes in neocortex but is not sufficient to produce behavioral or seizure phenotypes.

Arid1b is a high confidence risk gene for autism spectrum disorder that encodes a subunit of a chromatin remodeling complex expressed in neuronal progenitors. Haploinsufficiency causes a broad range of social, behavioral, and intellectual disability phenotypes, including Coffin-Siris syndrome. Recent work using transgenic mouse models suggests pathology is due to deficits in proliferation, survival, and synaptic development of cortical neurons. However, there is conflicting evidence regarding the relative roles of excitatory projection neurons and inhibitory interneurons in generating abnormal cognitive and behavioral phenotypes. Here, we conditionally knocked out either one or both copies of Arid1b from excitatory projection neuron progenitors and systematically investigated the effects on intrinsic membrane properties, synaptic physiology, social behavior, and seizure susceptibility. We found that disrupting Arid1b expression in excitatory neurons alters their membrane properties, including hyperpolarizing action potential threshold; however, these changes depend on neuronal subtype. Using paired whole-cell recordings, we found increased synaptic connectivity rate between projection neurons. Furthermore, we found reduced strength of excitatory synapses to parvalbumin (PV)-expression inhibitory interneurons. These data suggest an increase in the ratio of excitation to inhibition. However, the strength of inhibitory synapses from PV interneurons to excitatory neurons was enhanced, which may rebalance this ratio. Indeed, Arid1b haploinsufficiency in projection neurons was insufficient to cause social deficits and seizure phenotypes observed in a preclinical germline haploinsufficient mouse model. Our data suggest that while excitatory projection neurons likely contribute to autistic phenotypes, pathology in these cells is not the primary cause.

Sleep fragmentation after traumatic brain injury impairs behavior and conveys long-lasting impacts on neuroinflammation.

Traumatic brain injury (TBI) causes a prolonged inflammatory response in the central nervous system (CNS) driven by microglia. Microglial reactivity is exacerbated by stress, which often provokes sleep disturbances. We have previously shown that sleep fragmentation (SF) stress after experimental TBI increases microglial reactivity and impairs hippocampal function 30 days post-injury (DPI). The neuroimmune response is highly dynamic the first few weeks after TBI, which is also when injury induced sleep-wake deficits are detected. Therefore, we hypothesized that even a few weeks of TBI SF stress would synergize with injury induced sleep-wake deficits to promote neuroinflammation and impair outcome. Here, we investigated the effects of environmental SF in a lateral fluid percussion model of mouse TBI. Half of the mice were undisturbed, and half were exposed to 5 h of SF around the onset of the light cycle, daily, for 14 days. All mice were then undisturbed 15-30 DPI, providing a period for SF stress recovery (SF-R). Mice exposed to SF stress slept more than those in control housing 7-14 DPI and engaged in more total daily sleep bouts during the dark period. However, SF stress did not exacerbate post-TBI sleep deficits. Testing in the Morris water maze revealed sex dependent differences in spatial reference memory 9-14 DPI with males performing worse than females. Post-TBI SF stress suppressed neurogenesis-related gene expression and increased inflammatory signaling in the cortex at 14 DPI. No differences in sleep behavior were detected between groups during the SF stress recovery period 15-30 DPI. Microscopy revealed cortical and hippocampal IBA1 and CD68 percent-area increased in TBI SF-R mice 30 DPI. Additionally, neuroinflammatory gene expression was increased, and synaptogenesis-related gene expression was suppressed in TBI-SF mice 30 DPI. Finally, IPA canonical pathway analysis showed post-TBI SF impaired and delayed activation of synapse-related pathways between 14 and 30 DPI. These data show that transient SF stress after TBI impairs recovery and conveys long-lasting impacts on neuroimmune function independent of continuous sleep deficits. Together, these finding support that even limited exposure to post-TBI SF stress can have lasting impacts on cognitive recovery and regulation of the immune response to trauma.

DNA hypomethylation promotes the expression of CASPASE-4 which exacerbates inflammation and amyloid-ß deposition in Alzheimer's disease.

Alzheimer's disease (AD) is the sixth leading cause of death in the USA. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release pro-inflammatory products such as IL-1ß which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed reduced representation bisulfite sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed promotes the generation of IL-1ß and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4 and CASP11, which is encoded by mouse Caspase-4 (5xFAD/Casp4-/-). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-ß (Aß) and increased microglial production of IL-1ß in 5xFAD mice. Utilizing RNA-sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1ß from macrophages in response to cytosolic Aß through cleavage of downstream effector Gasdermin D (GSDMD). Therefore, here we unravel the role for CASP11 and GSDMD in the generation of IL-1ß in response to Aß and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential DNA methylation in AD microglia contributes to the progression of AD pathobiology. Thus, we identify CASP4 as a potential target for immunotherapies for the treatment and prevention of AD.

Multiparity Differentially Affects Specific Aspects of the Acute Neuroinflammatory Response to Traumatic Brain Injury in Female Mice.

Pregnancy is associated with profound acute and long-term physiological changes, but the effects of such changes on brain injury outcomes are unclear. Here, we examined the effects of previous pregnancy and maternal experience (parity) on acute neuroinflammatory responses to lateral fluid percussion injury (FPI), a well-defined experimental traumatic brain injury (TBI) paradigm. Multiparous (2-3 pregnancies and motherhood experiences) and age-matched nulliparous (no previous pregnancy or motherhood experience) female mice received either FPI or sham injury and were euthanized 3 days post-injury (DPI). Increased cortical Iba1, GFAP, and CD68 immunolabeling was observed following TBI independent of parity and microglia morphology did not differ between TBI groups. However, multiparous females had fewer CD45+ cells near the site of injury compared to nulliparous females, which was associated with preserved aquaporin-4 polarization, suggesting that parity may influence leukocyte recruitment to the site of injury and maintenance of blood brain barrier permeability following TBI. Additionally, relative cortical Il6 gene expression following TBI was dependent on parity such that TBI increased Il6 expression in nulliparous, but not multiparous, mice. Together, this work suggests that reproductive history may influence acute neuroinflammatory outcomes following TBI in females.

Divergent Spatial Learning, Enhanced Neuronal Transcription, and Blood-Brain Barrier Disruption Develop During Recovery from Post-Injury Sleep Fragmentation.

Traumatic brain injury (TBI) causes pathophysiology that may significantly decrease quality of life over time. A major propagator of this response is chronic, maladaptive neuroinflammation, which can be exacerbated by stressors such as sleep fragmentation (SF). This study determined whether post-TBI SF had lasting behavioral and inflammatory effects even with a period of recovery. To test this, male and female mice received a moderate lateral fluid percussion TBI or sham surgery. Half the mice were left undisturbed, and half were exposed to daily SF for 30 days. All mice were then undisturbed between 30 and 60 days post-injury (DPI), allowing mice to recover from SF (SF-R). SF-R did not impair global Barnes maze performance. Nonetheless, TBI SF-R mice displayed retrogression in latency to reach the goal box within testing days. These nuanced behavioral changes in TBI SF-R mice were associated with enhanced expression of neuronal processing/signaling genes and indicators of blood-brain barrier (BBB) dysfunction. Aquaporin-4 (AQP4) expression, a marker of BBB integrity, was differentially altered by TBI and TBI SF-R. For example, TBI enhanced cortical AQP4 whereas TBI SF-R mice had the lowest cortical expression of perivascular AQP4, dysregulated AQP4 polarization, and the highest number of CD45+ cells in the ipsilateral cortex. Altogether, post-TBI SF caused lasting, divergent behavioral responses associated with enhanced expression of neuronal transcription and BBB disruption even after a period of recovery from SF. Understanding lasting impacts from post-TBI stressors can better inform both acute and chronic post-injury care to improve long-term outcome post-TBI.