A cell-autonomous role for border-associated macrophages in ApoE4 neurovascular dysfunction and susceptibility to white matter injury.

Apolipoprotein E4 (ApoE4), the strongest genetic risk factor for sporadic Alzheimer's disease, is also a risk factor for microvascular pathologies leading to cognitive impairment, particularly subcortical white matter injury. These effects have been attributed to alterations in the regulation of the brain blood supply, but the cellular source of ApoE4 and the underlying mechanisms remain unclear. In mice expressing human ApoE3 or ApoE4, we report that border-associated macrophages (BAMs), myeloid cells closely apposed to neocortical microvessels, are both sources and effectors of ApoE4 mediating the neurovascular dysfunction through reactive oxygen species. ApoE4 in BAMs is solely responsible for the increased susceptibility to oligemic white matter damage in ApoE4 mice and is sufficient to enhance damage in ApoE3 mice. The data unveil a new aspect of BAM pathobiology and highlight a previously unrecognized cell-autonomous role of BAM in the neurovascular dysfunction of ApoE4 with potential therapeutic implications.

Molecular and Functional Alterations in the Cerebral Microvasculature in an Optimized Mouse Model of Sepsis-Associated Cognitive Dysfunction.

Systemic inflammation has been implicated in the development and progression of neurodegenerative conditions such as cognitive impairment and dementia. Recent clinical studies indicate an association between sepsis, endothelial dysfunction, and cognitive decline. However, the investigations of the role and therapeutic potential of the cerebral microvasculature in sepsis-induced cognitive dysfunction have been limited by the lack of standardized experimental models for evaluating the alterations in the cerebral microvasculature and cognition induced by the systemic inflammatory response. Herein, we validated a mouse model of endotoxemia that recapitulates key pathophysiology related to sepsis-induced cognitive dysfunction, including the induction of an acute systemic hyperinflammatory response, blood-brain barrier (BBB) leakage, neurovascular inflammation, and memory impairment after recovery from the systemic inflammation. In the acute phase, we identified novel molecular (e.g., upregulation of plasmalemma vesicle-associated protein, PLVAP, a driver of endothelial permeability, and the procoagulant plasminogen activator inhibitor-1, PAI-1) and functional perturbations (i.e., albumin and small-molecule BBB leakage) in the cerebral microvasculature along with neuroinflammation. Remarkably, small-molecule BBB permeability, elevated levels of PAI-1, intra-/perivascular fibrin/fibrinogen deposition, and microglial activation persisted 1 month after recovery from sepsis. We also highlight molecular neuronal alterations of potential clinical relevance following systemic inflammation including changes in neurofilament phosphorylation and decreases in postsynaptic density protein 95 and brain-derived neurotrophic factor, suggesting diffuse axonal injury, synapse degeneration, and impaired neurotrophism. Our study serves as a standardized mouse model to support future mechanistic studies of sepsis-associated cognitive dysfunction and to identify novel endothelial therapeutic targets for this devastating condition.

A minimally invasive thrombotic stroke model to study circadian rhythm in awake mice.

Experimental stroke models in rodents are essential for mechanistic studies and therapeutic development. However, these models have several limitations negatively impacting their translational relevance. Here we aimed to develop a minimally invasive thrombotic stroke model through magnetic particle delivery that does not require craniotomy, is amenable to reperfusion therapy, can be combined with in vivo imaging modalities, and can be performed in awake mice. We found that the model results in reproducible cortical infarcts within the middle cerebral artery (MCA) with cytologic and immune changes similar to that observed with more invasive distal MCA occlusion models. Importantly, the injury produced by the model was ameliorated by tissue plasminogen activator (tPA) administration. We also show that MCA occlusion in awake animals results in bigger ischemic lesions independent of day/night cycle. Magnetic particle delivery had no overt effects on physiologic parameters and systemic immune biomarkers. In conclusion, we developed a novel stroke model in mice that fulfills many requirements for modeling human stroke.

Molecular and functional alterations in the cerebral microvasculature in an optimized mouse model of sepsis-associated cognitive dysfunction.

Systemic inflammation has been implicated in the development and progression of neurodegenerative conditions such as cognitive impairment and dementia. Recent clinical studies indicate an association between sepsis, endothelial dysfunction, and cognitive decline. However, the investigations of the role and therapeutic potential of the cerebral microvasculature in systemic inflammation-induced cognitive dysfunction have been limited by the lack of standardized experimental models for evaluating the alterations in the cerebral microvasculature and cognition induced by the systemic inflammatory response. Herein, we validated a mouse model of endotoxemia that recapitulates key pathophysiology related to sepsis-induced cognitive dysfunction, including the induction of an acute systemic hyperinflammatory response, blood-brain barrier (BBB) leakage, neurovascular inflammation, and memory impairment after recovery from the systemic inflammatory response. In the acute phase, we identified novel molecular (e.g. upregulation of plasmalemma vesicle associated protein, a driver of endothelial permeability, and the pro-coagulant plasminogen activator inhibitor-1, PAI-1) and functional perturbations (i.e., albumin and small molecule BBB leakage) in the cerebral microvasculature along with neuroinflammation. Remarkably, small molecule BBB permeability, elevated levels of PAI-1, intra/perivascular fibrin/fibrinogen deposition and microglial activation persisted 1 month after recovery from sepsis. We also highlight molecular neuronal alterations of potential clinical relevance following systemic inflammation including changes in neurofilament phosphorylation and decreases in postsynaptic density protein 95 and brain-derived neurotrophic factor suggesting diffuse axonal injury, synapse degeneration and impaired neurotrophism. Our study serves as a standardized model to support future mechanistic studies of sepsis-associated cognitive dysfunction and to identify novel endothelial therapeutic targets for this devastating condition. SIGNIFICANCE: The limited knowledge of how systemic inflammation contributes to cognitive decline is a major obstacle to the development of novel therapies for dementia and other neurodegenerative diseases. Clinical evidence supports a role for the cerebral microvasculature in sepsis-induced neurocognitive dysfunction, but the investigation of the underlying mechanisms has been limited by the lack of standardized experimental models. Herein, we optimized a mouse model that recapitulates important pathophysiological aspects of systemic inflammation-induced cognitive decline and identified key alterations in the cerebral microvasculature associated with cognitive dysfunction. Our study provides a reliable experimental model for mechanistic studies and therapeutic discovery of the impact of systemic inflammation on cerebral microvascular function and the development and progression of cognitive impairment.

Analysis of brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke.

Cerebral ischemia triggers a powerful inflammatory reaction involving peripheral leukocytes and brain resident cells that contribute to both tissue injury and repair. However, their dynamics and diversity remain poorly understood. To address these limitations, we performed a single-cell transcriptomic study of brain and blood cells 2 or 14 days after ischemic stroke in mice. We observed a strong divergence of post-ischemic microglia, monocyte-derived macrophages and neutrophils over time, while endothelial cells and brain-associated macrophages showed altered transcriptomic signatures at 2 days poststroke. Trajectory inference predicted the in situ trans-differentiation of macrophages from blood monocytes into day 2 and day 14 phenotypes, while neutrophils were projected to be continuously de novo recruited from the blood. Brain single-cell transcriptomes from both female and male aged mice were similar to that of young male mice, but aged and young brains differed in their immune cell composition. Although blood leukocyte analysis also revealed altered transcriptomes after stroke, brain-infiltrating leukocytes displayed higher transcriptomic divergence than their circulating counterparts, indicating that phenotypic diversification occurs within the brain in the early and recovery phases of ischemic stroke. A portal ( https://anratherlab.shinyapps.io/strokevis/ ) is provided to allow user-friendly access to our data.

Meningeal interleukin-17-producing T cells mediate cognitive impairment in a mouse model of salt-sensitive hypertension.

Hypertension (HTN), a disease afflicting over one billion individuals worldwide, is a leading cause of cognitive impairment, the mechanisms of which remain poorly understood. In the present study, in a mouse model of HTN, we find that the neurovascular and cognitive dysfunction depends on interleukin (IL)-17, a cytokine elevated in individuals with HTN. However, neither circulating IL-17 nor brain angiotensin signaling can account for the dysfunction. Rather, IL-17 produced by T cells in the dura mater is the mediator released in the cerebrospinal fluid and activating IL-17 receptors on border-associated macrophages (BAMs). Accordingly, depleting BAMs, deleting IL-17 receptor A in brain macrophages or suppressing meningeal T cells rescues cognitive function without attenuating blood pressure elevation, circulating IL-17 or brain angiotensin signaling. Our data unveil a critical role of meningeal T cells and macrophage IL-17 signaling in the neurovascular and cognitive dysfunction in a mouse model of HTN.

Border-associated macrophages promote cerebral amyloid angiopathy and cognitive impairment through vascular oxidative stress.

BACKGROUND: Cerebral amyloid angiopathy (CAA) is a devastating condition common in patients with Alzheimer's disease but also observed in the general population. Vascular oxidative stress and neurovascular dysfunction have been implicated in CAA but the cellular source of reactive oxygen species (ROS) and related signaling mechanisms remain unclear. We tested the hypothesis that brain border-associated macrophages (BAM), yolk sac-derived myeloid cells closely apposed to parenchymal and leptomeningeal blood vessels, are the source of radicals through the Aß-binding innate immunity receptor CD36, leading to neurovascular dysfunction, CAA, and cognitive impairment. METHODS: Tg2576 mice and WT littermates were transplanted with CD36-/- or CD36+/+ bone marrow at 12-month of age and tested at 15 months. This approach enables the repopulation of perivascular and leptomeningeal compartments with CD36-/- BAM. Neurovascular function was tested in anesthetized mice equipped with a cranial window in which cerebral blood flow was monitored by laser-Doppler flowmetry. Amyloid pathology and cognitive function were also examined. RESULTS: The increase in blood flow evoked by whisker stimulation (functional hyperemia) or by endothelial and smooth muscle vasoactivity was markedly attenuated in WT → Tg2576 chimeras but was fully restored in CD36-/- → Tg2576 chimeras, in which BAM ROS production was suppressed. CAA-associated Aß1-40, but not Aß1-42, was reduced in CD36-/- → Tg2576 chimeras. Similarly, CAA, but not parenchymal plaques, was reduced in CD36-/- → Tg2576 chimeras. These beneficial vascular effects were associated with cognitive improvement. Finally, CD36-/- mice were able to more efficiently clear exogenous Aß1-40 injected into the neocortex or the striatum. CONCLUSIONS: CD36 deletion in BAM suppresses ROS production and rescues the neurovascular dysfunction and damage induced by Aß. CD36 deletion in BAM also reduced brain Aß1-40 and ameliorated CAA without affecting parenchyma plaques. Lack of CD36 enhanced the vascular clearance of exogenous Aß. Restoration of neurovascular function and attenuation of CAA resulted in a near complete rescue of cognitive function. Collectively, these data implicate brain BAM in the pathogenesis of CAA and raise the possibility that targeting BAM CD36 is beneficial in CAA and other conditions associated with vascular Aß deposition and damage.

Comparative multi-omic analyses of cardiac mitochondrial stress in three mouse models of frataxin deficiency.

Cardiomyopathy is often fatal in Friedreich ataxia (FA). However, FA hearts maintain adequate function until advanced disease stages, suggesting initial adaptation to the loss of frataxin (FXN). Conditional cardiac knockout mouse models of FXN show transcriptional and metabolic profiles of the mitochondrial integrated stress response (ISRmt), which could play an adaptive role. However, the ISRmt has not been investigated in models with disease-relevant, partial decrease in FXN. We characterized the heart transcriptomes and metabolomes of three mouse models with varying degrees of FXN depletion: YG8-800, KIKO-700 and FXNG127V. Few metabolites were changed in YG8-800 mice, which did not provide a signature of cardiomyopathy or ISRmt; several metabolites were altered in FXNG127V and KIKO-700 hearts. Transcriptional changes were found in all models, but differentially expressed genes consistent with cardiomyopathy and ISRmt were only identified in FXNG127V hearts. However, these changes were surprisingly mild even at advanced age (18 months), despite a severe decrease in FXN levels to 1% of those of wild type. These findings indicate that the mouse heart has low reliance on FXN, highlighting the difficulty in modeling genetically relevant FA cardiomyopathy.

Cell autonomous role of border associated macrophages in ApoE4 neurovascular dysfunction and susceptibility to white matter injury.

Apolipoprotein-E4 (ApoE4), the strongest genetic risk factor for sporadic Alzheimer's disease, is also a risk factor for microvascular pathologies leading to cognitive impairment, particularly subcortical white matter injury. These effects have been attributed to alterations in the regulation of the brain blood supply, but the cellular source of ApoE4 and the underlying mechanisms remain unclear. In mice expressing human ApoE3 or ApoE4 we report that border associated macrophages (BAM), myeloid cells closely apposed to neocortical microvessels, are both the source and the target of the ApoE4 mediating the neurovascular dysfunction through reactive oxygen species. ApoE4 in BAM is solely responsible for the increased susceptibility to oligemic white matter damage in ApoE4 mice and is sufficient to enhance damage in ApoE3 mice. The data unveil a new aspect of BAM pathobiology and highlight a previously unrecognized cell autonomous role of BAM in the neurovascular dysfunction of ApoE4 with potential therapeutic implications.

Calcium transients in nNOS neurons underlie distinct phases of the neurovascular response to barrel cortex activation in awake mice.

Neuronal nitric oxide (NO) synthase (nNOS), a Ca2+ dependent enzyme, is expressed by distinct populations of neocortical neurons. Although neuronal NO is well known to contribute to the blood flow increase evoked by neural activity, the relationships between nNOS neurons activity and vascular responses in the awake state remain unclear. We imaged the barrel cortex in awake, head-fixed mice through a chronically implanted cranial window. The Ca2+ indicator GCaMP7f was expressed selectively in nNOS neurons using adenoviral gene transfer in nNOScre mice. Air-puffs directed at the contralateral whiskers or spontaneous motion induced Ca2+ transients in 30.2 ± 2.2% or 51.6 ± 3.3% of nNOS neurons, respectively, and evoked local arteriolar dilation. The greatest dilatation (14.8 ± 1.1%) occurred when whisking and motion occurred simultaneously. Ca2+ transients in individual nNOS neurons and local arteriolar dilation showed various degrees of correlation, which was strongest when the activity of whole nNOS neuron ensemble was examined. We also found that some nNOS neurons became active immediately prior to arteriolar dilation, while others were activated gradually after arteriolar dilatation. Discrete nNOS neuron subsets may contribute either to the initiation or to the maintenance of the vascular response, suggesting a previously unappreciated temporal specificity to the role of NO in neurovascular coupling.

Immune compartments at the brain's borders in health and neurovascular diseases.

Recent evidence implicates cranial border immune compartments in the meninges, choroid plexus, circumventricular organs, and skull bone marrow in several neuroinflammatory and neoplastic diseases. Their pathogenic importance has also been described for cardiovascular diseases such as hypertension and stroke. In this review, we will examine the cellular composition of these cranial border immune niches, the potential pathways through which they might interact, and the evidence linking them to cardiovascular disease.

Border-associated macrophages promote cerebral amyloid angiopathy and cognitive impairment through vascular oxidative stress.

Background: Cerebral amyloid angiopathy (CAA) is a devastating condition common in patients with Alzheimer's disease but also observed in the general population. Vascular oxidative stress and neurovascular dysfunction have been implicated in CAA but the cellular source of reactive oxygen species (ROS) and related signaling mechanisms remain unclear. We tested the hypothesis that brain border-associated macrophages (BAM), yolk sac-derived myeloid cells closely apposed to parenchymal and leptomeningeal blood vessels, are the source of radicals through the Aß-binding innate immunity receptor CD36, leading to neurovascular dysfunction, CAA, and cognitive impairment. Methods: Tg2576 mice and WT littermates were transplanted with CD36 -/- or CD36 +/+ bone marrow at 12-month of age and tested at 15 months. This approach enables the repopulation of perivascular and leptomeningeal compartments with CD36 -/- BAM. Neurovascular function was tested in anesthetized mice equipped with a cranial window in which cerebral blood flow was monitored by laser-Doppler flowmetry. Amyloid pathology and cognitive function were also examined. Results: The increase in blood flow evoked by whisker stimulation (functional hyperemia) or by endothelial and smooth muscle vasoactivity was markedly attenuated in WT®Tg2576 chimeras but was fully restored in CD36 -/- ®Tg2576 chimeras, in which BAM ROS production was suppressed. CAA-associated Aß 1-40 , but not Aß 1-42 , was reduced in CD36 -/- ®Tg2576 chimeras. Similarly, CAA, but not parenchymal plaques, was reduced in CD36 -/- ®Tg2576 chimeras. These beneficial vascular effects were associated with cognitive improvement. Finally, CD36 -/- mice were able to more efficiently clear exogenous Aß 1-40 injected into the neocortex or the striatum. Conclusions: CD36 deletion in BAM suppresses ROS production and rescues the neurovascular dysfunction and damage induced by Aß. CD36 deletion in BAM also reduced brain Aß 1-40 and ameliorated CAA without affecting parenchyma plaques. Lack of CD36 enhanced the vascular clearance of exogenous Aß. Restoration of neurovascular function and attenuation of CAA resulted in a near complete rescue of cognitive function. Collectively, these data implicate CNS BAM in the pathogenesis of CAA and raise the possibility that targeting BAM CD36 is beneficial in CAA and other conditions associated with vascular Aß deposition and damage.

Brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke.

Cerebral ischemia triggers a powerful inflammatory reaction involving both peripheral leukocytes and brain resident cells. Recent evidence indicates that their differentiation into a variety of functional phenotypes contributes to both tissue injury and repair. However, the temporal dynamics and diversity of post-stroke immune cell subsets remain poorly understood. To address these limitations, we performed a longitudinal single-cell transcriptomic study of both brain and mouse blood to obtain a composite picture of brain-infiltrating leukocytes, circulating leukocytes, microglia and endothelium diversity over the ischemic/reperfusion time. Brain cells and blood leukocytes isolated from mice 2 or 14 days after transient middle cerebral artery occlusion or sham surgery were purified by FACS sorting and processed for droplet-based single-cell transcriptomics. The analysis revealed a strong divergence of post-ischemic microglia, macrophages, and neutrophils over time, while such diversity was less evident in dendritic cells, B, T and NK cells. Conversely, brain endothelial cells and brain associated-macrophages showed altered transcriptomic signatures at 2 days post-stroke, but low divergence from sham at day 14. Pseudotime trajectory inference predicted the in-situ longitudinal progression of monocyte-derived macrophages from their blood precursors into day 2 and day 14 phenotypes, while microglia phenotypes at these two time points were not connected. In contrast to monocyte-derived macrophages, neutrophils were predicted to be continuously de-novo recruited from the blood. Brain single-cell transcriptomics from both female and male aged mice did not show major changes in respect to young mice, but aged and young brains differed in their immune cell composition. Furthermore, blood leukocyte analysis also revealed altered transcriptomes after stroke. However, brain-infiltrating leukocytes displayed higher transcriptomic divergence than their circulating counterparts, indicating that phenotypic diversification into cellular subsets occurs within the brain in the early and the recovery phase of ischemic stroke. In addition, this resource report contains a searchable database https://anratherlab.shinyapps.io/strokevis/ to allow user-friendly access to our data. The StrokeVis tool constitutes a comprehensive gene expression atlas that can be interrogated at the gene and cell type level to explore the transcriptional changes of endothelial and immune cell subsets from mouse brain and blood after stroke.

A preclinical randomized controlled multi-centre trial of anti-interleukin-17A treatment for acute ischaemic stroke.

Multiple consensus statements have called for preclinical randomized controlled trials to improve translation in stroke research. We investigated the efficacy of an interleukin-17A neutralizing antibody in a multi-centre preclinical randomized controlled trial using a murine ischaemia reperfusion stroke model. Twelve-week-old male C57BL/6 mice were subjected to 45 min of transient middle cerebral artery occlusion in four centres. Mice were randomly assigned (1:1) to receive either an anti-interleukin-17A (500 µg) or isotype antibody (500 µg) intravenously 1 h after reperfusion. The primary endpoint was infarct volume measured by magnetic resonance imaging three days after transient middle cerebral artery occlusion. Secondary analysis included mortality, neurological score, neutrophil infiltration and the impact of the gut microbiome on treatment effects. Out of 136 mice, 109 mice were included in the analysis of the primary endpoint. Mixed model analysis revealed that interleukin-17A neutralization significantly reduced infarct sizes (anti-interleukin-17A: 61.77 ± 31.04 mm3; IgG control: 75.66 ± 34.79 mm3; P = 0.01). Secondary outcome measures showed a decrease in mortality (hazard ratio = 3.43, 95% confidence interval = 1.157-10.18; P = 0.04) and neutrophil invasion into ischaemic cortices (anti-interleukin-17A: 7222 ± 6108 cells; IgG control: 28 153 ± 23 206 cells; P < 0.01). There was no difference in Bederson score. The analysis of the gut microbiome showed significant heterogeneity between centres (R = 0.78, P < 0.001, n = 40). Taken together, neutralization of interleukin-17A in a therapeutic time window resulted in a significant reduction of infarct sizes and mortality compared with isotype control. It suggests interleukin-17A neutralization as a potential therapeutic target in stroke.

The Neurovasculome: Key Roles in Brain Health and Cognitive Impairment: A Scientific Statement From the American Heart Association/American Stroke Association.

BACKGROUND: Preservation of brain health has emerged as a leading public health priority for the aging world population. Advances in neurovascular biology have revealed an intricate relationship among brain cells, meninges, and the hematic and lymphatic vasculature (the neurovasculome) that is highly relevant to the maintenance of cognitive function. In this scientific statement, a multidisciplinary team of experts examines these advances, assesses their relevance to brain health and disease, identifies knowledge gaps, and provides future directions. METHODS: Authors with relevant expertise were selected in accordance with the American Heart Association conflict-of-interest management policy. They were assigned topics pertaining to their areas of expertise, reviewed the literature, and summarized the available data. RESULTS: The neurovasculome, composed of extracranial, intracranial, and meningeal vessels, as well as lymphatics and associated cells, subserves critical homeostatic functions vital for brain health. These include delivering O2 and nutrients through blood flow and regulating immune trafficking, as well as clearing pathogenic proteins through perivascular spaces and dural lymphatics. Single-cell omics technologies have unveiled an unprecedented molecular heterogeneity in the cellular components of the neurovasculome and have identified novel reciprocal interactions with brain cells. The evidence suggests a previously unappreciated diversity of the pathogenic mechanisms by which disruption of the neurovasculome contributes to cognitive dysfunction in neurovascular and neurodegenerative diseases, providing new opportunities for the prevention, recognition, and treatment of these conditions. CONCLUSIONS: These advances shed new light on the symbiotic relationship between the brain and its vessels and promise to provide new diagnostic and therapeutic approaches for brain disorders associated with cognitive dysfunction.

DNA damage independent inhibition of NF-κB transcription by anthracyclines.

Anthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of histones from select sites in the genome. Here, we show that the extensively used anthracyclines Doxorubicin, Daunorubicin, and Epirubicin decrease the transcription of nuclear factor kappa B (NF-κB)-dependent gene targets, but not interferon-responsive genes in primary mouse (Mus musculus) macrophages. Using an NMR-based structural approach, we demonstrate that anthracyclines disturb the complexes formed between the NF-κB subunit RelA and its DNA-binding sites. The anthracycline variants Aclarubicin, Doxorubicinone, and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other anthracyclines but do not induce DNA damage, also suppressed inflammation, thus uncoupling DNA damage from the effects on inflammation. These findings have implications for anticancer therapy and for the development of novel anti-inflammatory drugs with limited side effects for life-threatening conditions such as sepsis.