Neuroinflammation In Mouse Model Research Articles

Spatial dynamics of mammalian brain development and neuroinflammation by multimodal tri-omics mapping.

The ability to spatially map multiple layers of the omics information over different time points allows for exploring the mechanisms driving brain development, differentiation, arealization, and alterations in disease. Herein we developed and applied spatial tri-omic sequencing technologies, DBiT ARP-seq (spatial ATAC-RNA-Protein-seq) and DBiT CTRP-seq (spatial CUT&Tag- RNA-Protein-seq) together with multiplexed immunofluorescence imaging (CODEX) to map spatial dynamic remodeling in brain development and neuroinflammation. A spatiotemporal tri-omic atlas of the mouse brain was obtained at different stages from postnatal day P0 to P21, and compared to the regions of interest in the human developing brains. Specifically, in the cortical area, we discovered temporal persistence and spatial spreading of chromatin accessibility for the layer-defining transcription factors. In corpus callosum, we observed dynamic chromatin priming of myelin genes across the subregions. Together, it suggests a role for layer specific projection neurons to coordinate axonogenesis and myelination. We further mapped the brain of a lysolecithin (LPC) neuroinflammation mouse model and observed common molecular programs in development and neuroinflammation. Microglia, exhibiting both conserved and distinct programs for inflammation and resolution, are transiently activated not only at the core of the LPC lesion, but also at distal locations presumably through neuronal circuitry. Thus, this work unveiled common and differential mechanisms in brain development and neuroinflammation, resulting in a valuable data resource to investigate brain development, function and disease.

Baicalin and baicalein from Scutellaria baicalensis Georgi alleviate aberrant neuronal suppression mediated by GABA from reactive astrocytes.

γ-aminobutyric acid (GABA) from reactive astrocytes is critical for the dysregulation of neuronal activity in various neuroinflammatory conditions. While Scutellaria baicalensis Georgi (S. baicalensis) is known for its efficacy in addressing neurological symptoms, its potential to reduce GABA synthesis in reactive astrocytes and the associated neuronal suppression remains unclear. This study focuses on the inhibitory action of monoamine oxidase B (MAO-B), the key enzyme for astrocytic GABA synthesis. Using a lipopolysaccharide (LPS)-induced neuroinflammation mouse model, we conducted immunohistochemistry to assess the effect of S. baicalensis on astrocyte reactivity and its GABA synthesis. High-performance liquid chromatography was performed to reveal the major compounds of S. baicalensis, the effects of which on MAO-B inhibition, astrocyte reactivity, and tonic inhibition in hippocampal neurons were validated by MAO-B activity assay, qRT-PCR, and whole-cell patch-clamp. The ethanolic extract of S. baicalensis ameliorated astrocyte reactivity and reduced excessive astrocytic GABA content in the CA1 hippocampus. Baicalin and baicalein exhibited significant MAO-B inhibition potential. These two compounds downregulate the mRNA levels of genes associated with reactive astrogliosis or astrocytic GABA synthesis. Additionally, LPS-induced aberrant tonic inhibition was reversed by both S. baicalensis extract and its key compounds. In summary, baicalin and baicalein isolated from S. baicalensis reduce astrocyte reactivity and alleviate aberrant tonic inhibition of hippocampal neurons during neuroinflammation.

ω3-PUFA alleviates neuroinflammation by upregulating miR-107 targeting PIEZO1/NFκB p65

BackgroundMiR-107 is reduced in sepsis and associated with inflammation regulation. Dietary supplementation with polyunsaturated fatty acids (ω3-PUFA) can increase the expression of miR-107; this study investigated whether the ω3-PUFA can effectively inhibit neuroinflammation and improve cognitive function by regulating miR-107 in the brain. MethodsThe LPS-induced mouse model of neuroinflammation and the BV2 cell inflammatory model were used to evaluate the effects of ω3-PUFA on miR-107 expression and inflammation. Intraventricular injection of Agomir and Antagomir was used to modulate miR-107 expression. HE and Nissl staining for analyzing hippocampal neuronal damage, immunofluorescence analysis for glial activation, RT-qPCR, and Western blot were conducted to examine miR-107 expression and inflammation signalling. ResultsThe result shows that LPS successfully induced the mouse neuroinflammation model and BV2 cell inflammation model. Supplementation of ω3-PUFA effectively reduced the secretion of pro-inflammatory factors TNFα, IL1β, and IL6 induced by LPS, improved cognitive function impairment, and increased miR-107 expression in the brain. Overexpression of miR-107 in the brain inhibited the nuclear factor κB (NFκB) pro-inflammatory signalling pathway by targeting PIEZO1, thus suppressing microglial and astrocyte activation and reducing the release of inflammatory mediators, which alleviated neuroinflammatory damage and improved cognitive function in mice. miR-107, as an intron of PANK1, PANK1 is subject to PPAR α Adjust. ω3-PUFA can activate PPARα, but ω3-PUFA upregulates brain miR-107, and PPARα/PANK1-related pathways may not be synchronized, and further research is needed to confirm the specific mechanism by which ω3-PUFA upregulates miR-107. ConclusionThe miR-107/PIEZO1/NFκB p65 pathway represents a novel mechanism underlying the improvement of neuroinflammation by ω3-PUFA.

Second Near-Infrared Macrophage-Biomimetic Nanoprobes for Photoacoustic Imaging of Neuroinflammation.

Neuroinflammation is a significant pathological event involving the neurodegenerative process associated with many neurological disorders. Diagnosis and treatment of neuroinflammation in its early stage are essential for the prevention and management of neurological diseases. Herein, we designed macrophage membrane-coated photoacoustic (PA) probes (MSINPs), with targeting specificities based on naturally existing target-ligand interactions for the early diagnosis of neuroinflammation. The second near-infrared dye, IR1061, was doped into silica as the core and was encapsulated with a macrophage membrane. In vitro as well as in vivo, the MSINPs could target inflammatory cells via the inflammation chemotactic effect. PA imaging was used to trace the MSINPs in a neuroinflammation mouse model and showed a great targeted effect of MSINPs in the prefrontal cortex. Therefore, the biomimetic nanoprobe prepared in this study offers a new strategy for PA molecular imaging of neuroinflammation, which can enhance our understanding of the evolution of neuroinflammation in specific brain regions.

MSC-Derived Extracellular Vesicles Alleviate NLRP3/GSDMD-Mediated Neuroinflammation in Mouse Model of Sporadic Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease, with sporadic form being the predominant type. Neuroinflammation plays a critical role in accelerating pathogenic processes in AD. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEVs) regulate inflammatory responses and show great promise for treating AD. Induced pluripotent stem cell (iPSC)-derived MSCs are similar to MSCs and exhibit low immunogenicity and heterogeneity, making them promising cell sources for clinical applications. This study examined the anti-inflammatory effects of MSC-sEVs in a streptozotocin-induced sporadic mouse model of AD (sAD). The intracisternal administration of iPSC-MSC-sEVs alleviated NLRP3/GSDMD-mediated neuroinflammation, decreased amyloid deposition and neuronal apoptosis, and mitigated cognitive dysfunction. Furthermore, it explored the role of miR-223-3p in the iPSC-MSC-sEVs-mediated anti-inflammatory effects in vitro. miR-223-3p directly targeted NLRP3, whereas inhibiting miR-223-3p almost completely reversed the suppression of NLRP3 by MSC-sEVs, suggesting that miR-223-3p may, at least partially, account for MSC-sEVs-mediated anti-inflammation. Results obtained suggest that intracisternal administration of iPSC-MSC-sEVs can reduce cognitive impairment by inhibiting NLRP3/GSDMD neuroinflammation in a sAD mouse model. Therefore, the present study provides a proof-of-principle for applying iPSC-MSC-sEVs to target neuroinflammation in sAD.

MRI of Neuroinflammation Using a Folate Receptor β‐Targeted Nanoparticle Contrast Agent

AbstractBackgroundNeuroinflammation plays an important role in Alzheimer’s disease (AD) pathogenesis and disease progression leading to cognitive decline. Current efforts for non‐invasive imaging of neuroinflammation are based on using PET radiotracers. An MRI‐based contrast agent for imaging of neuroinflammation would be highly desirable due to widespread use of MRI in imaging of neurological disorders. Folate receptor β, a marker of pro‐inflammatory phenotype, is expressed in microglia. In this work, we investigated a folate‐targeted liposomal‐Gd contrast agent for molecular MRI of neuroinflammation in mouse models of AD.Method In vivo studies were performed in APP/PSEN1 mice, a model of amyloid pathology, and P301S mice, a model of tau pathology. Transgenic mice and wild‐type (WT) littermates (n = 6/genotype, 24 total) underwent pre‐contrast MRI using T1‐weighted spin echo sequences with 160 µm in‐plane resolution and 1.2 mm slice thickness. Delayed MRI was performed 4 days after systemic administration of a high T1 relaxivity liposomal‐Gd contrast agent bearing folic acid molecules. Animals were euthanized after the final imaging session for microscopic analysis of brain. Signal changes between pre‐contrast and post‐contrast MRI were determined in target regions of the brain.ResultTransgenic APP/PSEN1 and P301S mice demonstrated signal enhancement in post‐contrast T1w‐MRI compared to pre‐contrast MRI. In comparison, WT control mice did not show signal enhancement in post‐contrast MRI. APP/PSEN1 transgenic mice exhibited greater relative enhancement (+5.9%, p<0.005) and lower inter‐subject variance than P301S mice (3.9%, p<0.05). Immunofluorescence analysis demonstrated higher burden of activated microglia in brains of transgenic mice compared to WT control mice.ConclusionA folate receptor β‐targeted liposomal‐Gd contrast agent enables in vivo MRI of neuroinflammation in mouse models of Alzheimer’s disease. Such imaging agents could play an important role in monitoring of Alzheimer’s disease progression leading to cognitive decline and evaluation of therapies that target neuroinflammation.

Complement C1q drives microglia-dependent synaptic loss and cognitive impairments in a mouse model of lipopolysaccharide-induced neuroinflammation

Activated microglia and subsequent release of pro-inflammatory cytokines result in neuroinflammatory status which further damage neurological function including cognitive impairments in various neurological conditions. However, the underlying molecular mechanisms during these pathological processing remain unknown. In the current study, mice received intraperitoneal administrations of LPS (0.5 mg/kg, daily, Escherichia coli O55:B5) for seven consecutive days and their different cohorts were used for behavioral assessment with open field, Y maze, and novel object recognition test or for electrophysiology recordings of mEPSC, LFP or LTP in in vivo or ex vivo preparation. The hippocampus from some cohorts were harvested for immunostaining or Western blotting of c1q, Iba-1, CD68, PSD95 and dendritic spine density or for transcriptome and proteomics analysis. Repeated LPS injections induced an up-regulation of complement system protein c1q and distinct microglial phenotype with an enrichment of the complement–phagosome pathway. Microglial synaptic engulfment and profound synaptic loss were found. These pathological changes were accompanied with the significantly decreased excitatory synaptic transmission, disturbed theta oscillations, impaired hippocampal long-term potentiation, and cognitive impairments. Notably, neutralization of c1q signaling robustly prevented these changes. Collectively, our data provide evidence that activated microglia and complement cascade c1q signaling in the hippocampus may account for synaptic loss and cognitive impairments in a mouse model of neuroinflammation induced by repeated LPS injections. Our work implicates that complement system may be a therapeutic target for developing therapies to prevent or treat cognitive disorders related to neuroinflammation or other disease conditions including neurodegenerative disease per se.

IL-27-containing exosomes secreted by innate B-1a cells suppress and ameliorate uveitis.

IL-27 is a heterodimeric cytokine composed of Ebi3 and IL-27p28 and can exert proinflammatory or immune suppressive effects depending on the physiological context. Ebi3 does not contain membrane-anchoring motifs, suggesting that it is a secreted protein while IL-27p28 is poorly secreted. How IL-27p28 and Ebi3 dimerize in-vivo to form biologically active IL-27 is unknown. Major impediment to clinical use of IL-27 derives from difficulty of determining exact amount of bioavailable heterodimeric IL-27 needed for therapy. To understand how IL-27 mediates immune suppression, we characterized an innate IL-27-producing B-1a regulatory B cell population (i27-Breg) and mechanisms i27-Bregs utilize to suppress neuroinflammation in mouse model of uveitis. We also investigated biosynthesis of IL-27 and i27-Breg immunobiology by FACS, immunohistochemical and confocal microscopy. Contrary to prevailing view that IL-27 is a soluble cytokine, we show that i27-Bregs express membrane-bound IL-27. Immunohistochemical and confocal analyses co-localized expression of IL-27p28 at the plasma membrane in association with CD81 tetraspanin, a BCR-coreceptor protein and revealed that IL-27p28 is a transmembrane protein in B cells. Most surprising, we found that i27-Bregs secrete IL-27-containing exosomes (i27-exosomes) and adoptive transfer of i27-exosomes suppressed uveitis by antagonizing Th1/Th17 cells, up-regulating inhibitory-receptors associated with T-cell exhaustion while inducing Treg expansion. Use of i27-exosomes thus obviates the IL-27 dosing problem, making it possible to determine bioavailable heterodimeric IL-27 needed for therapy. Moreover, as exosomes readily cross the blood-retina-barrier and no adverse effects were observed in mice treated with i27-exosome, results of this study suggest that i27-exosomes might be a promising therapeutic approach for CNS autoimmune diseases.

Dietary Intervention with the Gut Microbial Metabolite Urolithin A Attenuates Lipopolysaccharide-Induced Neuroinflammation and Cognitive Deficits via the Sirt1/acetyl-NF-κB Signaling Pathway.

The gut microbial metabolite Urolithin A (UA) exhibits anti-inflammatory properties in vivo and in vitro. Lipopolysaccharide (LPS), which is present in abundance in the gut, induces chronic neuroinflammation and triggers behavioral abnormalities. However, the neuroprotective effects of UA and the underlying mechanisms implicate in an LPS-elicited neuroinflammation mouse model remain elusive. Daily administration of UA (200mg kg-1 d-1 ; i.g.) for 21 days significantly mitigates cognitive deficits following LPS exposure. UA prevents LPS-induced neural loss and synaptic injury in the hippocampus. UA administration substantially represses LPS-triggered glial cell activation and the production of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). Further study reveals that UA promotes Sirt1 expression and NF-κB p65 deacetylation. Importantly, all the beneficial effects of UA, including biochemical and neuropathological changes and cognitive function, are abrogated by 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527), a specific Sirt1 inhibitor. The findings indicate that UA ameliorates LPS-triggered neural/synaptic damage and cognitive deficits, which potentially contributes to the inhibition of neuroinflammation by promoting the Sirt1/acetyl-NF-κB signaling pathway.

Effect and mechanism of chlorogenic acid on cognitive dysfunction in mice by lipopolysaccharide-induced neuroinflammation.

Neuroinflammation is an important factor causing numerous neurodegenerative pathologies. Inflammation can lead to abnormal neuronal structure and function and even death, followed by cognitive dysfunction. There is growing evidence that chlorogenic acid has anti-inflammatory effects and immunomodulatory activity. The aim of this study was to elucidate the potential targets and molecular mechanisms of chlorogenic acid in the treatment of neuroinflammation. We used the lipopolysaccharide-induced neuroinflammation mouse model and the lipopolysaccharide-stimulated BV-2 cells in vitro model. Behavioral scores and experiments were used to assess cognitive dysfunction in mice. HE staining and immunohistochemistry were used to assess neuronal damage in the mouse brain. Immunofluorescence detected microglia polarization in mouse brain. Western blot and flow cytometry detected the polarization of BV-2 cells. The migration of BV-2 cells was detected by wound healing assay and transwell assay. Potential targets for chlorogenic acid to exert protective effects were predicted by network pharmacology. These targets were then validated using molecular docking and experiments. The results of in vivo experiments showed that chlorogenic acid had an obvious ameliorating effect on neuroinflammation-induced cognitive dysfunction. We found that chlorogenic acid was able to inhibit BV-2 cells M1 polarization and promote BV-2 cells M2 polarization in vitro while also inhibiting the abnormal migration of BV-2 cells. Based on the network pharmacology results, we identified the TNF signaling pathway as a key signaling pathway in which chlorogenic acid exerts anti-neuroinflammatory effects. Among them, Akt1, TNF, MMP9, PTGS2, MAPK1, MAPK14, and RELA are the core targets for chlorogenic acid to function. Chlorogenic acid can inhibit microglial polarization toward the M1 phenotype and improve neuroinflammation-induced cognitive dysfunction in mice by modulating these key targets in the TNF signaling pathway.

First trimester human umbilical cord perivascular cells (HUCPVC) modulate the kynurenine pathway and glutamate neurotransmission in an LPS-induced mouse model of neuroinflammation

BackgroundThe Kynurenine Pathway (KP) of tryptophan degradation and glutamate toxicity is implicated in several neurological disorders, including depression. The therapeutic potential of mesenchymal stromal cells (MSC), owing to their well documented phagocytosis-driven mechanism of immunomodulation and neuroprotection, has been tested in many neurological disorders. However, their potential to influence KP and the glutamatergic system has not yet been investigated. Hence, this study sought to investigate the effect of HUCPVC, a rich and potent source of MSC, on Lipopolysaccharide (LPS)-activated KP metabolites, KP enzymes, and key components of glutamate neurotransmission.MethodsThe immunomodulatory effect of peripherally administered HUCPVC on the expression profile of kynurenine pathway metabolites and enzymes was assessed in the plasma and brain of mice treated with LPS using LCMS and QPCR. An assessment of the glutamatergic system, including selected receptors, transporters and related proteins was also conducted by QPCR, immunohistochemistry and Western blot.ResultsHUCPVC were found to modulate LPS-induced activation of KP enzymes and metabolites in the brain associated with neurotoxicity. Moreover, the reduced expression of the glutamatergic components due to LPS was also found to be significantly improved by HUCPVC.ConclusionsThe immunomodulatory properties of HUCPVC appear to confer neuroprotection, at least in part, through their ability to modulate the KP in the brain. This KP modulation enhances neuroprotective regulators and downregulates neurotoxic consequences, including glutamate neurotoxicity, which is associated with neuroinflammation and depressive behavior.

A Lipopolysaccharide Mouse Model Mirrors Neuroinflammatory Transcriptional Signatures of Human AD, and the Glucagon-Like Peptide-1 Receptor Agonist Semaglutide Attenuates Neuroinflammation in this Model (P1-6.007)

<h3>Objective:</h3> We aimed to investigate the molecular effects of semaglutide on lipopolysaccharide (LPS) induced hippocampal neuroinflammation in mice and further to compare human gene expression signatures in Alzheimer’s disease (AD) brain tissue with those induced by LPS. <h3>Background:</h3> Neuroinflammation is part of the pathophysiology in AD. AD neuroimaging studies highlight increased inflammation, while genome wide association studies indicate that many AD-associated genes are expressed in glial cells. The glucagon-like peptide-1 receptor agonist semaglutide is currently being investigated in two phase 3a randomized, placebo-controlled trials in people with early Alzheimer’s disease (evoke/evoke+). <h3>Design/Methods:</h3> Mice were treated daily with subcutaneous semaglutide (30nmol/kg) or vehicle for 28 days, and LPS or vehicle was administered on days 15–17. RNA-sequencing and immunohistochemistry were performed to assess transcriptional and morphological changes in the hippocampus at Day 19 and Day 28 (Day 2 and 11 post-LPS, respectively). RNA-sequencing analyses included differentially expressed gene analysis, weighted gene co-regulated network analysis, pathway analysis and gene set enrichment analysis on published human AD datasets. <h3>Results:</h3> Semaglutide treatment significantly decreased the area of ionized calcium binding adaptor molecule 1 (Iba1) positive microglia (quantitative assessment) and attenuated co-expressed inflammatory genes vs vehicle in LPS-treated mice (p&lt;0.01) on Day 28. Pathway analysis was consistent with an overall dampening of inflammatory processes in mice treated with LPS and semaglutide. The mouse LPS model mirrored the transcriptional signatures identified in people with AD in data of human hippocampal subtype transcriptional signatures, suggesting that semaglutide could potentially alleviate homologous inflammatory molecular processes in humans. <h3>Conclusions:</h3> In an LPS-induced neuroinflammation mouse model, semaglutide reduced hippocampal neuroinflammation as measured by transcriptional changes and microglial area (Iba1). This could represent a novel mechanism whereby semaglutide may affect neuronal integrity and function, and thereby AD pathophysiology. <b>Disclosure:</b> Dr. Ludwig has nothing to disclose. Dr. Rausch has nothing to disclose. Dr. Secher has received personal compensation for serving as an employee of Novo Nordisk. An immediate family member of Dr. Secher has received personal compensation for serving as an employee of Novo Nordisk. Dr. Secher has stock in Novo Nordisk. Dr. Bentsen has received personal compensation for serving as an employee of Novo Nordisk. An immediate family member of Dr. Bentsen has stock in Novo Nordisk. Dr. Hansen has received personal compensation for serving as an employee of Neumirna Therapeutics. Dr. Hansen has received intellectual property interests from a discovery or technology relating to health care. Dr. Bjerregaard has received personal compensation for serving as an employee of Novo Nordisk A/S. Dr. Niss has nothing to disclose. Kristoffer L Egerod has nothing to disclose. Dr. Hansen has received personal compensation for serving as an employee of Novo Nordisk A/S. Dr. Hansen has stock in Novo Nordisk A/S. Dr. Dalgaard has received personal compensation for serving as an employee of Novo Nordisk. Dr. Merkestein has received personal compensation for serving as an employee of Novo Nordisk A/S. Dorte Holst has nothing to disclose. Charles Pyke has nothing to disclose. Mrs. Wichern has received personal compensation for serving as an employee of Gubra ApS. Dr. Polex-Wolf has received personal compensation for serving as an employee of Novo Nordisk A/S. Tune H Pers has nothing to disclose. Lotte B Knudsen has nothing to disclose.

Menstrual blood-derived endometrial stem cells inhibit neuroinflammation by regulating microglia through the TLR4/MyD88/NLRP3/Casp1 pathway.

Neuroinflammation is a common response in various neurological disorders. Mesenchymal stem cell-based treatment has become a promising therapy for neuroinflammation-associated diseases. However, the effects of mesenchymal stem cells are controversial, and the underlying mechanism is incompletely understood. In the present study, menstrual blood-derived endometrial stem cells were intravenously transplanted into a mouse model of neuroinflammation established by peripheral injection of lipopolysaccharide. Microglial cells challenged with lipopolysaccharide were cultured with conditioned medium from endometrial stem cells. The levels of cytokines were detected by enzyme-linked immunosorbent assay. Cell proliferation and death were detected by Cell Counting Kit 8 and flow cytometry, respectively. The expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MyD88), NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (Casp1) were evaluated by western blotting. The results showed that intravenous transplantation of endometrial stem cells downregulated proinflammatory factors and upregulated anti-inflammatory factors in the brain of mice with neuroinflammation. Conditioned medium suppressed the inflammatory reaction and hyperactivation of microglial cells and protected microglial cells from cell death induced by lipopolysaccharide in vitro. The expression of TLR4, MyD88, NLRP3 and Casp1 in the brain of mice with neuroinflammation and in lipopolysaccharide-stimulated microglial cells was downregulated by endometrial stem cells and conditioned medium, respectively. These data suggested that menstrual blood-derived endometrial stem cells may suppress neuroinflammatory reactions partially by regulating microglia through the TLR4/MyD88/NLRP3/Casp1 signalling pathway. Our findings may be very useful for the development of an alternative stem cell-based therapy for neuroinflammation-associated disorders.

Direct implantation of hair-follicle-associated pluripotent (HAP) stem cells repairs intracerebral hemorrhage and reduces neuroinflammation in mouse model.

Intracerebral hemorrhage (ICH) is a leading cause of mortality with ineffective treatment. Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into neurons, glial cells and many other types of cells. HAP stem cells have been shown to repair peripheral-nerve and spinal-cord injury in mouse models. In the present study, HAP stem cells from C57BL/6J mice were implanted into the injured brain of C57BL/6J or nude mice with induced ICH. After allo transplantation, HAP stem cells differentiated to neurons, astrocytes, oligodendrocytes, and microglia in the ICH site of nude mice. After autologous transplantation in C57BL/6J mice, HAP stem cells suppressed astrocyte and microglia infiltration in the injured brain. The mRNA expression levels of IL-10 and TGF-β1, measured by quantitative Real-Time RT-PCR, in the brain of C57BL/6J mice with ICH was increased by HAP-stem-cell implantation compared to the non-implanted mice. Quantitative sensorimotor function analysis, with modified limb-placing test and the cylinder test, demonstrated a significant functional improvement in the HAP-stem-cell-implanted C57BL/6J mice, compared to non-implanted mice. HAP stem cells have critical advantages over induced pluripotent stem cells, embryonic stem cells as they do not develop tumors, are autologous, and do not require genetic manipulation. The present study demonstrates future clinical potential of HAP-stem-cell repair of ICH, currently a recalcitrant disease.

A multiple sclerosis-protective coding variant reveals an essential role for HDAC7 in regulatory T cells.

Genome-wide association studies identifying hundreds of susceptibility loci for autoimmune diseases indicate that genes active in immune cells predominantly mediate risk. However, identification and functional characterization of causal variants remain challenging. Here, we focused on the immunomodulatory role of a protective variant of histone deacetylase 7 (HDAC7). This variant (rs148755202, HDAC7.p.R166H) was identified in a study of low-frequency coding variation in multiple sclerosis (MS). Through transcriptomic analyses, we demonstrate that wild-type HDAC7 regulates genes essential for the function of Foxp3+ regulatory T cells (Tregs), an immunosuppressive subset of CD4 T cells that is generally dysfunctional in patients with MS. Moreover, Treg-specific conditional hemizygous deletion of HDAC7 increased the severity of experimental autoimmune encephalitis (EAE), a mouse model of neuroinflammation. In contrast, Tregs transduced with the protective HDAC7 R166H variant exhibited higher suppressive capacity in an in vitro functional assay, mirroring phenotypes previously observed in patient samples. In vivo modeling of the human HDAC7 R166H variant by generation of a knock-in mouse model bearing an orthologous R150H substitution demonstrated decreased EAE severity linked to transcriptomic alterations of brain-infiltrating Tregs, as assessed by single-cell RNA sequencing. Our data suggest that dysregulation of epigenetic modifiers, a distinct molecular class associated with disease risk, may influence disease onset. Last, our approach provides a template for the translation of genetic susceptibility loci to detailed functional characterization, using in vitro and in vivo modeling.

A lipopolysaccharide mouse model mirrors neuroinflammatory transcriptional signatures of human Alzheimer’s disease, and the Glucagon‐Like Peptide‐1 receptor agonist semaglutide attenuates neuroinflammation in this model

AbstractBackgroundNeuroinflammation is part of the pathophysiology in Alzheimer’s disease (AD). AD neuroimaging studies highlight increased inflammation, while genome wide association studies indicate that many AD‐associated genes are expressed in glial cells. The hormone and neuropeptide Glucagon‐Like Peptide‐1 (GLP‐1) was originally developed for treatment of type 2 diabetes but has equally important effects in weight loss control and provides cardiovascular benefits. The potential for applying a licensed therapeutic option with a well‐established safety profile is currently being investigated in two phase 3a randomized, placebo‐controlled trials with semaglutide in people with early Alzheimer’s disease (evoke/evoke+). Here, we investigate the molecular effects of semaglutide on lipopolysaccharide (LPS) induced hippocampal neuroinflammation in mice and compare the identified pathways with human gene signatures in AD brain tissue.MethodMice were treated daily subcutaneously with semaglutide (30nmol/kg) or vehicle for 28 days, and LPS or vehicle was administered on days 15‐17. RNA‐sequencing and immunohistochemistry was performed to assess transcriptional and morphological changes in the hippocampus at Day 19 and Day 28 (Day 2 and 11 post‐LPS, respectively). RNA‐sequencing analyses included differentially expressed gene analysis, weighted gene co‐regulated network analysis, pathway analysis and gene set enrichment analysis on published human AD datasets.ResultSemaglutide treatment significantly decreased the area of ionized calcium binding adaptor molecule 1 (Iba1) positive microglia (quantitative assessment) and attenuated co‐expressed inflammatory genes in LPS/semaglutide‐treated mice (p&lt;0.01 vs LPS+vehicle treated mice) on Day 28. Pathway analysis was consistent with an overall dampening of inflammatory processes in mice treated with LPS and semaglutide. The mouse LPS model mirrored the transcriptional signatures identified in people with AD in data of human hippocampal subtype transcriptional signatures, suggesting that semaglutide could potentially alleviate homologous inflammatory molecular processes in humans. A single nucleus RNA‐sequencing study in mice is ongoing to further elucidate the molecular effects of semaglutide on neuroinflammation and will be presented at the conference.ConclusionIn an LPS‐induced neuroinflammation mouse model, subcutaneously semaglutide reduced hippocampal neuroinflammation as measured by transcriptional changes and microglial area (Iba1). This could be a novel mechanism through which semaglutide, may affect neuronal integrity and function relevant for AD pathophysiology.

Phenotype-based screening rediscovered benzopyran-embedded microtubule inhibitors as anti-neuroinflammatory agents by modulating the tubulin–p65 interaction

Neuroinflammation is one of the critical processes implicated in central nervous system (CNS) diseases. Therefore, alleviating neuroinflammation has been highlighted as a therapeutic strategy for treating CNS disorders. However, the complexity of neuroinflammatory processes and poor drug transport to the brain are considerable hurdles to the efficient control of neuroinflammation using small-molecule therapeutics. Thus, there is a significant demand for new chemical entities (NCEs) targeting neuroinflammation. Herein, we rediscovered benzopyran-embedded tubulin inhibitor 1 as an anti-neuroinflammatory agent via phenotype-based screening. A competitive photoaffinity labeling study revealed that compound 1 binds to tubulin at the colchicine-binding site. Structure–activity relationship analysis of 1’s analogs identified SB26019 as a lead compound with enhanced anti-neuroinflammatory efficacy. Mechanistic studies revealed that upregulation of the tubulin monomer was critical for the anti-neuroinflammatory activity of SB26019. We serendipitously found that the tubulin monomer recruits p65, inhibiting its translocation from the cytosol to the nucleus and blocking NF-κB-mediated inflammatory pathways. Further in vivo validation using a neuroinflammation mouse model demonstrated that SB26019 suppressed microglial activation by downregulating lba-1 and proinflammatory cytokines. Intraperitoneal administration of SB26019 showed its therapeutic potential as an NCE for successful anti-neuroinflammatory regulation. Along with the recent growing demands on tubulin modulators for treating various inflammatory diseases, our results suggest that colchicine-binding site-specific modulation of tubulins can be a potential strategy for preventing neuroinflammation and treating CNS diseases.

A Biodistribution Study of the Radiolabeled Kv1.3-Blocking Peptide DOTA-HsTX1[R14A] Demonstrates Brain Uptake in a Mouse Model of Neuroinflammation.

The voltage-gated potassium channel Kv1.3 regulates the pro-inflammatory function of microglia and is highly expressed in the post-mortem brains of individuals with Alzheimer's and Parkinson's diseases. HsTX1[R14A] is a selective and potent peptide inhibitor of the Kv1.3 channel (IC50 ∼ 45 pM) that has been shown to decrease cytokine levels in a lipopolysaccharide (LPS)-induced mouse model of inflammation. Central nervous system exposure to HsTX1[R14A] was previously detected in this mouse model using liquid chromatography with tandem mass spectrometry, but this technique does not report on the spatial distribution of the peptide in the different brain regions or peripheral organs. Herein, the in vivo distribution of a [64Cu]Cu-labeled DOTA conjugate of HsTX1[R14A] was observed for up to 48 h by positron emission tomography (PET) in mice. After subcutaneous administration to untreated C57BL/6J mice, considerable uptake of the radiolabeled peptide was observed in the kidney, but it was undetectable in the brain. Biodistribution of a [68Ga]Ga-DOTA conjugate of HsTX1[R14A] was then investigated in the LPS-induced mouse model of neuroinflammation to assess the effects of inflammation on uptake of the peptide in the brain. A control peptide with very weak Kv1.3 binding, [68Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC50 ∼ 6 μM), was also tested. Significantly increased uptake of [68Ga]Ga-DOTA-HsTX1[R14A] was observed in the brains of LPS-treated mice compared to mice treated with control peptide, implying that the enhanced uptake was due to increased Kv1.3 expression rather than simply increased blood-brain barrier disruption. PET imaging also showed accumulation of [68Ga]Ga-DOTA-HsTX1[R14A] in inflamed joints and decreased clearance from the kidneys in LPS-treated mice. These biodistribution data highlight the potential of HsTX1[R14A] as a therapeutic for the treatment of neuroinflammatory diseases mediated by overexpression of Kv1.3.

CRISPRi screens in human iPSC-derived astrocytes elucidate regulators of distinct inflammatory reactive states.

Astrocytes become reactive in response to insults to the central nervous system by adopting context-specific cellular signatures and outputs, but a systematic understanding of the underlying molecular mechanisms is lacking. In this study, we developed CRISPR interference screening in human induced pluripotent stem cell-derived astrocytes coupled to single-cell transcriptomics to systematically interrogate cytokine-induced inflammatory astrocyte reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-κB activation drove two distinct inflammatory reactive signatures, one promoted by STAT3 and the other inhibited by STAT3. These signatures overlapped with those observed in other experimental contexts, including mouse models, and their markers were upregulated in human brains in Alzheimer's disease and hypoxic-ischemic encephalopathy. Furthermore, we validated that markers of these signatures were regulated by STAT3 in vivo using a mouse model of neuroinflammation. These results and the platform that we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.

How Microbiota-Derived Metabolites Link the Gut to the Brain during Neuroinflammation.

Microbiota-derived metabolites are important molecules connecting the gut to the brain. Over the last decade, several studies have highlighted the importance of gut-derived metabolites in the development of multiple sclerosis (MS). Indeed, microbiota-derived metabolites modulate the immune system and affect demyelination. Here, we discuss the current knowledge about microbiota-derived metabolites implications in MS and in different mouse models of neuroinflammation. We focus on the main families of microbial metabolites that play a role during neuroinflammation. A better understanding of the role of those metabolites may lead to new therapeutical avenues to treat neuroinflammatory diseases targeting the gut–brain axis.