Glial Response Research Articles

Lipoxins A4 and B4 inhibit glial cell activation via CXCR3 signaling in acute retinal neuroinflammation

Lipoxins are small lipids that are potent endogenous mediators of systemic inflammation resolution in a variety of diseases. We previously reported that Lipoxins A4 and B4 (LXA4 and LXB4) have protective activities against neurodegenerative injury. Yet, lipoxin activities and downstream signaling in neuroinflammatory processes are not well understood. Here, we utilized a model of posterior uveitis induced by lipopolysaccharide endotoxin (LPS), which results in rapid retinal neuroinflammation primarily characterized by activation of resident macroglia (astrocytes and Müller glia), and microglia. Using this model, we observed that each lipoxin reduces acute inner retinal inflammation by affecting endogenous glial responses in a cascading sequence beginning with astrocytes and then microglia, depending on the timing of exposure; prophylactic or therapeutic. Subsequent analyses of retinal cytokines and chemokines revealed inhibition of both CXCL9 (MIG) and CXCL10 (IP10) by each lipoxin, compared to controls, following LPS injection. CXCL9 and CXCL10 are common ligands for the CXCR3 chemokine receptor, which is prominently expressed in inner retinal astrocytes and ganglion cells. We found that CXCR3 inhibition reduces LPS-induced neuroinflammation, while CXCR3 agonism alone induces astrocyte reactivity. Together, these data uncover a novel lipoxin–CXCR3 pathway to promote distinct anti-inflammatory and proresolution cascades in endogenous retinal glia.

Insights into neuroinflammatory mechanisms of deep brain stimulation in Parkinson's disease

Parkinson's disease, a progressive neurodegenerative disorder, involves gradual degeneration of the nigrostriatal dopaminergic pathway, leading to neuronal loss within the substantia nigra pars compacta and dopamine depletion. Molecular factors, including neuroinflammation, impaired protein homeostasis, and mitochondrial dysfunction, contribute to the neuronal loss. Deep brain stimulation, a form of neuromodulation, applies electric current through stereotactically implanted electrodes, effectively managing motor symptoms in advanced Parkinson's disease patients. Deep brain stimulation exerts intricate effects on neuronal systems, encompassing alterations in neurotransmitter dynamics, microenvironment restoration, neurogenesis, synaptogenesis, and neuroprotection. Contrary to initial concerns, deep brain stimulation demonstrates antiinflammatory effects, influencing cytokine release, glial activation, and neuronal survival.This review investigates the intricacies of deep brain stimulation mechanisms, including insertional effects, histological changes, and glial responses, and sheds light on the complex interplay between electrodes, stimulation, and the brain. This exploration delves into understanding the role of neuroinflammatory pathways and the effects of deep brain stimulation in the context of Parkinson's disease, providing insights into its neuroprotective capabilities.

Cell type-specific roles of APOE4 in Alzheimer disease.

The ɛ4 allele of the apolipoprotein E gene (APOE), which translates to the APOE4 isoform, is the strongest genetic risk factor for late-onset Alzheimer disease (AD). Within the CNS, APOE is produced by a variety of cell types under different conditions, posing a challenge for studying its roles in AD pathogenesis. However, through powerful advances in research tools and the use of novel cell culture and animal models, researchers have recently begun to study the roles of APOE4 in AD in a cell type-specific manner and at a deeper and more mechanistic level than ever before. In particular, cutting-edge omics studies have enabled APOE4 to be studied at the single-cell level and have allowed the identification of critical APOE4 effects in AD-vulnerable cellular subtypes. Through these studies, it has become evident that APOE4 produced in various types of CNS cell - including astrocytes, neurons, microglia, oligodendrocytes and vascular cells - has diverse roles in AD pathogenesis. Here, we review these scientific advances and propose a cell type-specific APOE4 cascade model of AD. In this model, neuronal APOE4 emerges as a crucial pathological initiator and driver of AD pathogenesis, instigating glial responses and, ultimately, neurodegeneration. In addition, we provide perspectives on future directions for APOE4 research and related therapeutic developments in the context of AD.

Cholesterol and oxysterols in retinal neuron-glia interactions: relevance for glaucoma.

Cholesterol is an essential component of cellular membranes, crucial for maintaining their structural and functional integrity. It is especially important for nervous tissues, including the retina, which rely on high amounts of plasma membranes for the transmission of the nervous signal. While cholesterol is by far the most abundant sterol, the retina also contains cholesterol precursors and metabolites, especially oxysterols, which are bioactive molecules. Cholesterol lack or excess is deleterious and some oxysterols are known for their effect on neuron survival. Cholesterol homeostasis must therefore be maintained. Retinal glial cells, especially Müller cells, the principal glial cells of the vertebrate retina, provide mechanical, nutritional, and metabolic support for the neighboring neurons. Several pieces of evidence indicate that Müller cells are major actors of cholesterol homeostasis in the retina, as it is known for other glial cells in the brain. This process is based on a close cooperation with neurons, and sterols can be signaling molecules participating in glia-neuron interactions. While some implication of cholesterol in age-related macular degeneration is now recognized, based on epidemiological and laboratory data, evidence for its role in glaucoma is still scarce. The association between cholesterolemia and glaucoma is controversial, but experimental data suggest that sterols could take part in the pathological processes. It has been demonstrated that Müller glial cells are implicated in the development of glaucoma through an ambivalent reactive retinal gliosis process. The early steps contribute to maintaining retinal homeostasis and favor the survival of ganglion cells, which are targeted during glaucoma. If gliosis persists, dysregulation of the neuroprotective functions, cytotoxic effects of gliotic Müller cells and disruption of glia-neuron interactions lead to an acceleration of ganglion cell death. Sterols could play a role in the glial cell response to glaucomatous injury. This represents an understudied but attractive topic to better understand glaucoma and conceive novel preventive or curative strategies. The present review describes the current knowledge on i) sterol metabolism in retinal glial cells, ii) the potential role of cholesterol in glaucoma, and iii) the possible relationships between cholesterol and oxysterols, glial cells and glaucoma. Focus is put on glia-neuron interactions.

Innovative Cyanine-Based Fluorescent Dye for Targeted Mitochondrial Imaging and Its Utility in Whole-Brain Visualization.

Conducting in vivo brain imaging can be a challenging task due to the complexity of brain tissue and the strict requirements for safe and effective imaging agents. However, a new fluorescent dye called Cy5-PEG2 has been developed that selectively accumulates in mitochondria, enabling the visualization of these essential organelles in various cell lines. This dye is versatile and can be used for the real-time monitoring of mitochondrial dynamics in living cells. Moreover, it can cross the blood-brain barrier, making it a promising tool for noninvasive in vivo brain imaging. Based on the assessment of glial cell responses in the hippocampus and neocortex regions using GFAP and Iba1 biomarkers, Cy5-PEG2 seems to have minimal adverse effects on brain immune response or neuronal health. Therefore, this mitochondria-targeting fluorescent dye has the potential to advance our understanding of mitochondrial dynamics and function within the broader context of whole-brain physiology and disease progression. However, further research is needed to evaluate the safety and efficacy of Cy5-PEG2.

Inflammatory response of both retinas and superior colliculi to unilateral optic nerve axotomy

Objective: Unilateral optic nerve crush (ONC) induces a bilateral inflammatory reaction affecting both injured and contralateral un‐injured retinas. However, few works have investigated the spread of inflammation along visual pathway. Our aim was to study the glial response and the underlying molecular changes in both retinas and the superior colliculi (SCi), the main projection areas of retinal ganglion cells in mice. In this species, >90% of RGCs project to the contralateral SC (cSCi).Methods: The left optic nerve of adult pigmented C57Bl/6 male mice were crushed (ONC) at 0.5 mm from the optic disk or underwent surgery without crushing (Sham). One group of intact mice was used as controls. Two studies were carried out:1) At 1, 3, 9 and 30 days post lesion (dpl) both retinas and SCi were freshly dissected and their RNA extracted. Using qPCR we measured the expression levels of TGF‐β1, IL‐1β, TNF‐α, Iba1, GFAP, MHC II and TSPO in retinas and SCi. In SCi extracts we also studied Casp3, CXCR1, Lcn2, IL‐6, CD206, IL‐4, and AQ4. Hptr and Gapdh were used as houskeepings. Intact retinas and SCi were used as controls.2) Three, 9 and 30 days after ONC or sham‐surgery immunodetection of microglia and macroglial cells was carried out in sagittal brain sections. The area occupied by GFAP and MHC II signal as well as the density of Iba1+ and CD68+ cells was quantified in both SCi. Intact brains were used as controls.Results: Sham surgery alone modified gene expression in both retinas and SCi compared to intact animals. Therefore, to isolate the effect of ONC, we compared ONC vs. Sham. After ONC, TNF‐α upregulated early in both retinas and IL‐β remained overexpressed in injured retina at day 30. GFAP overexpression was exclusively observed in injured retina while Iba1 overexpression was presented in both. TSPO and MHCII and Casp3 overexpression was observed only in the cSC.Anatomical microglial activation (rounded somas and CD68 expression) was observed in the cSC from day 3 onwards after ONC. In the ipsilateral SC (iSC) microglial cells did not show signs of activation. Astrocytes hypertrophied in both SCi from day 9 after ONC, but this response was also observed in Sham‐animals.Conclusions: Sham surgery promotes an inflammatory and glial response that must be considered in experimental designs. Unilateral ONC triggers a bilateral response which extends anterogradely and retrogradely along the visual pathway.

Blocking Palmitoylation of Apelin Receptor Alleviates Morphine Tolerance in Neuropathic Cancer Pain.

Neuropathic cancer pain (NCP) is an important symptom in patients with cancer. However, significant analgesic tolerance and other side effects critically hamper the administration of morphine. Protein palmitoylation mediated by the DHHC family may be involved in the glial activation and inflammatory responses underlying organ failure. In this study, we investigated the key role of protein palmitoylation in cancer pain and sought to target palmitoylation to suppress morphine tolerance. We found that long-term use of morphine led to the accumulation of the morphine metabolite, morphine-3-glucuronide, in vivo and activated ERK1/2 and microglia to release inflammatory factors through the apelin receptor APLNR. Palmitoyltransferase ZDHHC9 was upregulated in NCP, and APLNR was palmitylated to protect it from lysosomal degradation and to maintain its stability. We also designed competitive inhibitors of APLNR palmitoylation to inhibit the development of NCP, release of inflammatory factors, and attenuation of morphine tolerance. Therefore, targeting APLNR palmitoylation in combination with morphine is a potent method for cancer pain treatment. Our data provide a basis for the future clinical use of related drugs combined with morphine for the treatment of cancer-related pain.

Highly potent and selective PPARδ agonist reverses memory deficits in mouse models of Alzheimer's disease.

Rationale: Alzheimer's disease (AD) is a progressive neurodegenerative disease accompanied by neurotoxicity, excessive inflammation, and cognitive impairment. The peroxisome proliferator-activated receptor (PPAR) δ is a potential target for AD. However, its regulatory mechanisms and therapeutic potential in AD remain unclear. We aimed to investigate if the activation of PPARδ using a highly selective and potent agonist could provide an effective therapeutic strategy against AD. Methods: We synthesized a novel PPARδ agonist, 5a, containing a selenazole group and determined the X-ray crystal structure of its complex with PPARδ. The drug-like properties of 5a were assessed by analyzing cytochrome P450 (CYP) inhibition, microsomal stability, pharmacokinetics, and mutagenicity. We investigated the anti-inflammatory effects of 5a using lipopolysaccharide (LPS)-stimulated BV-2 microglia and neuroinflammatory mouse model. The therapeutic efficacy of 5a was evaluated in AD mice with scopolamine-induced memory impairment and APP/PS1 by analyzing cognitive function, glial reactivity, and amyloid pathology. Results: Compound 5a, the most potent and selective PPARδ agonist, was confirmed to bind hPPARδ in a complex by X-ray crystallographic analysis. PPARδ activation using 5a showed potent anti-inflammatory effects in activated glial cells and mouse model of neuroinflammation. Administration of 5a inhibited amyloid plaque deposition by suppressing the expression of neuronal beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), and reduced abnormal glial hyperactivation and inflammatory responses, resulting in improved learning and memory in the APP/PS1 mouse model of AD. Conclusion: We identified that specific activation of PPARδ provides therapeutic effects on multiple pathogenic phenotypes of AD, including neuroinflammation and amyloid deposition. Our findings suggest the potential of PPARδ as a promising drug target for treating AD.

Retinal glial cell response in a neurodevelopmental disease such as the Dravet syndrome

Dravet syndrome (DS) is an epileptic encephalopathy caused by mutations in the Scn1a gene encoding the α1 subunit of the Nav1.1 sodium channel, which is associated with recurrent and generalized seizures, highly resistant to antiepileptic drugs, which is related to cognitive impairment, developmental delays, hyperactivity, dysautonomia, attention deficit, language impairment, autistic features, even leading to death. GABAergic interneurons are particularly affected, where the Nav1.1 channel is mainly located and the consequent imbalance between excitation and inhibition triggers brain hyperexcitability and seizures, altering the correct functioning of neural networks involved in cognitive processes.Glial reactivity and neuroinflammatory processes have been shown to be present in childhood epileptic processes and therefore cytokines may exert a neuromodulatory role directly impacting neuronal excitability. Indeed, astrocytes have been documented to be involved in the pathogenesis of epilepsy, regulating neurotransmitter and ion concentrations.In our experimental model of DS (Syn‐Cre/Scn1aWT/A1783V), inflammatory events in the prefrontal cortex and dentate gyrus of the hippocampus expressed as an increase in GFAP (astrocyte marker) and Iba‐1 (microglia marker) immunoreactivity have been observed, in addition to morphological changes compatible with activated astrocytes and microglia.The retina is a projection of the brain, and in several pathologies affecting the central nervous system, such as neurodegenerative diseases, correlation between changes occurring in the brain and those occurring in the retina has been shown. Therefore, the analyse of the changes that may occur in retinal glial cells (astrocytes and microglia) in an experimental model of DS, and the correlation with the alterations observed in some specific cerebral areas of these animals could be good predictive and prognostic biomarkers for future treatment.This study analyzes the retinal histological changes in glial cells (microglia and astrocytes), retinal ganglion cells (RGCs) and GABAergic amacrine cells in an experimental model of DS (Syn‐Cre/Scn1aWT/A1783V) compared to a control group at postnatal day (PND) 25. Retinal whole‐mounts were labelled with anti‐GFAP, anti‐Iba‐1, anti‐Brn3a and anti‐GAD65/67. Signs of microglial and astroglial activation and the number of Brn3a+ and GAD65+ 67+ cells were quantified. We found retinal activation of astroglial and microglial cells but not death of RGCs and GABAergic amacrine cells. These changes are similar to those found at the level of the hippocampus in the same experimental model in PND25, indicating a relationship between brain and retinal changes in DS. This suggests that the retina could serve as a possible biomarker in DS.

Glial Cell Activation and Immune Responses in Glaucoma: A Systematic Review of Human Postmortem Studies of the Retina and Optic Nerve.

Although researched extensively the understanding regarding mechanisms underlying glaucoma pathogenesis remains limited. Further, the exact mechanism behind neuronal death remains elusive. The role of neuroinflammation in retinal ganglion cell (RGC) death has been prominently theorised. This review provides a comprehensive summary of neuroinflammatory responses in glaucoma. A systematic search of Medline and Embase for articles published up to 8th March 2023 yielded 32 studies using post-mortem tissues from glaucoma patients. The raw data were extracted from tables and text to calculate the standardized mean differences (SMDs). These studies utilized post-mortem tissues from glaucoma patients, totalling 490 samples, compared with 380 control samples. Among the included studies, 27 reported glial cell activation based on changes to cellular morphology and molecular staining. Molecular changes were predominantly attributed to astrocytes (62.5%) and microglia (15.6%), with some involvement of Muller cells. These glial cell changes included amoeboid microglial cells with increased CD45 or HLA-DR intensity and hypertrophied astrocytes with increased glial fibrillary acidic protein labelling. Further, changes to extracellular matrix proteins like collagen, galectin, and tenascin-C suggested glial cells' influence on structural changes in the optic nerve head. The activation of DAMPs-driven immune response and the classical complement cascade was reported and found to be associated with activated glial cells in glaucomatous tissue. Increased pro-inflammatory markers such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were also linked to glial cells. Glial cell activation was also associated with mitochondrial, vascular, metabolic and antioxidant component disruptions. Association of the activated glial cells with pro-inflammatory responses, dysregulation of homeostatic components and antigen presentation indicates that glial cell responses influence glaucoma progression. However, the exact mechanism triggering these responses and underlying interactions remains unexplored. This necessitates further research using human samples for an increased understanding of the precise role of neuroinflammation in glaucoma progression.

Substance Use and Addiction.

Efforts to reveal the molecular, cellular, and circuit mechanisms of addiction have largely focused on neurons. Yet accumulating data regarding the ability of glial cells to impact synaptic function, circuit activity, and behavior demands that we explore how these nonneuronal cells contribute to substance use disorders and addiction. Important work has shown that glial cells, including microglia, exhibit changes in phenotype following exposure to drugs of abuse and that modification of glial responses can impact behaviors related to drug seeking and drug taking. While these are critical first steps to understanding how microglia can impact addiction, there are still substantial gaps in knowledge that need to be addressed. This chapter reviews some of the key studies that have shown how microglia are affected by and can contribute to addiction. It also discusses areas where more knowledge is urgently needed to reveal new therapeutic and preventative approaches.

Old Age Exacerbates White Matter Neuroinflammation and Cognitive Deficits Following Closed-Head Injury, Particularly in Female Mice.

The increasing incidence of traumatic brain injury (TBI) among older adults, particularly mild injuries from falls, underscores the need to investigate age-related outcomes and potential sex differences in response to TBI. Although previous research has defined an aging-TBI signature (heightened glial responses and cognitive impairment) in open-skull moderate-to-severe TBI models, it is unknown whether this signature is also present in mild closed-head injuries (CHIs). This study explores the influences of age and sex on recovery in a mouse CHI model induced by an electromagnetic impactor device in 4-month-old and 18-month-old C57BL/6 mice. We assessed the righting reflex, body weight, behavior (radial arm water maze and active avoidance), and inflammation (GFAP, IBA1, CD45) in the neocortex, corpus callosum, and hippocampus. We observed that aged female mice exhibited more severe TBI-induced cognitive deficits. In addition, a more pronounced reactive neuroinflammatory response with age was noted within white matter regions. Conversely, gray matter regions in aged animals either showed no enhanced pathological changes in response to injury or the aged mice displayed hyporesponsive glia and signs of dystrophic glial degeneration that were not evident in their younger counterparts following CHI. These findings suggest that aging influences CHI outcomes, partially reflecting the aging-TBI signature seen in more severe injuries in white matter, while a distinct aging and mild-TBI signature was identified in gray matter. The heightened vulnerability of females to the combined effects of age and mild CHI establishes a foundation for further investigation into the mechanisms underlying the sexually dimorphic response in aging females.

Memory dysfunction in mild cognitive impairment (MCI) and early Alzheimer disease (AD) dementia is closely associated with synapse loss, and shows significant correlation with pathological tau in synapses and inflammatory glial cell responses in postmortem human brain

AbstractBackgroundSynapse density loss is among the strongest histopathological correlates of dementia in AD. Prodromal stages of AD, comprising amnestic MCI and mild AD dementia, represent the most promising timepoints for interventions aimed at attaining efficacious neurobiological and clinical effects. Yet, the understanding of other contributing neuropathologic changes in these prodromal disease stages and their impact on early cognitive dysfunction remains limited.MethodsWe studied the visual cortex of 28 demented and non‐demented human brains who had died at Braak III‐IV stages of NFT pathology, and who showed a comparable β‐amyloid plaque burden and absence of NFT deposits in the studied brain region, and a group of age‐matched healthy controls. We used immunohistochemical analyses with artificial intelligence quantifications, expansion microscopy, synaptosome extractions, and Western blotting, to assess associations between antemortem MMSE scores and synapse densities, glial cell phenotypes, and pathological tau.ResultsWe found a significant loss of presynapses (Synapsin 1+), excitatory postsynapses (PSD95+), and colocalized synapses (Synapsin1‐PSD95+) in the brains of demented compared to non‐demented and controls. Moreover, we observed an association between excitatory synapse loss and memory dysfunction (MMSE score) as well as rate of disease progression (MMSE loss/time). MMSE scores were further associated with increase in tau oligomers (TOC1+) and phospho‐tau (AT270+) in synaptosomes, but not with 4G8+ β‐amyloid burden. Additionally, disease severity and rate of progression were significantly correlated with inflammatory glial changes.ConclusionsOur data suggest that excitatory synapse loss could serve as an early and robust neuropathologic correlate of disease severity and rate of progression from MCI stages of the disease, and be a closer and better determinant of cognitive decline than β‐amyloid and tau deposits. Moreover, early increases in soluble tau in synapses together with local inflammatory glial cell responses could drive development of incipient changes in memory function and serve as potential early biomarkers. Our results point to the importance of early detection and interventions aimed at synapse preservation from prodromal stages of AD, and suggest soluble tau and inflammatory glial cell changes as key targets in the early AD phases.

Establishing glial responses to anti‐Aβ immunotherapy through single‐cell RNAseq to gain insights into mechanisms of ARIA

AbstractBackgroundWhile anti‐Aβ immunotherapies effectively clear Aβ plaques, mouse and human data show consistent and significant cerebrovascular adverse events of this treatment approach. Anti‐Aβ immunotherapies have been plagued in clinical trials by the occurrence of amyloid‐related imaging abnormalities (ARIA), signifying cerebrovascular disruptions of the edema (ARIA‐E) or hemorrhagic (ARIA‐H) type in the brain. Despite these cerebrovascular adverse effects, the FDA granted accelerated approval to aducanumab and lecanemab. However, our mechanistic understanding underlying ARIA remains minimal. Elucidating the mechanism(s) by which anti‐Aβ antibodies cause ARIA is critical towards improving safety of these therapies for AD. MethodIn the current study, we aimed to determine the glial responses to anti‐Ab antibodies with the hypothesis that astrocytes and microglia have temporal cellular shifts that contribute to cerebrovascular dysfunction after acute anti‐Aβ antibody administration. To identify changes in glial gene expression in response to acute anti‐Aβ antibody we performed a single cell sequencing and digital spatial profiling (DSP) on 14mo Tg2576 APP mice (Fig. 1). We observed differences in size of clusters between anti‐Aβ (6E10) and IgG control (IgG1) intracranial injections after 24hr and 3days, suggesting some clusters of cells respond to anti‐Aβ in a time‐dependent or antibody manner (Fig. 2).ResultPreliminary data identified some of the clusters as microglia (Cx3cr1) and astrocytes (Aldoc), as well as oligodendrocytes (Plp). Apoe was increased in multiple cell types. DSP on these mice is ongoing, however we aim to further characterize the transcriptional profile of glia at the vasculature, and in the vicinity of Aβ plaques and cerebral amyloid angiopathy (CAA). Contralateral hemispheres are utilized for downstream validation studies.ConclusionOur work will elucidate the mechanisms of anti‐Aβ antibody‐induced glial response and establish the role of astrocytes and microglia in cerebrovascular dysfunction and ARIA. This work is critical to improve safety and increase generalizability of these therapies.

Glial and neuronal mechanisms contributing to differential risks in TREM2 R47H and R62H variants in Alzheimer’s Disease

AbstractBackgroundCoding variants in the microglial TREM2 ectodomain differentially (R47H> R62H) increase the risk of Alzheimer’s disease (AD). To define mechanisms responsible in glia and neurons, we aimed to characterise neuropathology and transcriptomic responses in heterozygotes for these TREM2 variant alleles and for common allele homozygotes (CV) in non‐diseased (n = 16) and AD (n = 42) brain cortical tissue from 58 donors.MethodUsing immunohistochemistry (IHC) and imaging mass cytometry (IMC), we characterised neocortical b‐amyloid and AT8/PHF1 pTau pathology with CV (n = 30) and the R47H (n = 11) or R62H (n = 17) TREM2 carriers. We performed single nuclear RNA sequencing to test for the differential gene expression (DGE) in TREM2 and CV cortical tissues with greater 4G8+ b‐amyloid‐ or PHF1+ pTau‐immunostaining.ResultThere was a two‐fold increase in 4G8+, a three‐fold reduction in AT8+ but no change in PHF1+ immunostaining with AD in both variants versus CV. We found a reduced expression of genes associated with TREM2 function in R47H carriers relative to CV however enhanced microglial activation in R62H carriers. This was associated with an up‐regulation of pathways such as ‘neuroinflammation signalling pathway’ in R62H not seen in R47H. CV astrocytes had little DGE with increasing pathology however both variants (R47H>R62H) had an increased proportion of differentially expressed genes suggestive of a heightened activation state. R47H and R62H astrocytes impacted pathways had opposing actions such as ‘synaptogenesis signalling pathway’ and ‘PDGF signalling’ pathways downregulated in R47H but upregulated in R62H. Neuronal gene expression changes were evident in all variants, however greatly enhanced in R47H in response to 4G8+ b‐amyloid. With increasing PHF1+ pTau gene expression changes were mainly seen in R47H. Excitatory and inhibitory neurons both exhibited DGE in R47H carriers and impacted pathways centred around those involved in ‘synaptogenesis signalling pathway’, ‘SNARE signalling pathway’ and ‘synaptic long term depression’.ConclusionThis analysis identified novel disease‐associated transcriptomic differences in the glial response to TREM2 and the secondary neuronal responses that can explain differences in the genetic AD risk conferred by these two TREM2 risk variants.

Retromer pathways can regulate AD‐associated neuronal and microglial phenotypes

AbstractThe relationship between retromer‐dependent pathways in neurons and neighboring glial remains unknown. To address this question, we turn to a genetic mouse model where the retromer core protein VPS35 is depleted in hippocampal neurons, and then we replete VPS35 using an optimized viral vector protocol. The VPS35 depletion‐repletion studies strengthen the causal link between the neuronal retromer and AD‐associated neuronal phenotypes, including the acceleration of amyloid precursor protein cleavage and the loss of synaptic glutamate receptors. More interestingly, the studies show that the neuronal retromer can regulate a distinct, dystrophic, microglia morphology, phenotypic of hippocampal microglia in AD. Finally, the neuronal and, in part, the glial responses to VPS35 depletion were found to occur independent of tau. Future studies are required to determine the mechanisms by which retromer‐dependent dysfunction in neurons provokes neighboring microglia.

β-adrenergic signaling triggers enteric glial reactivity and acute enteric gliosis during surgery

BackgroundEnteric glia contribute to the pathophysiology of various intestinal immune-driven diseases, such as postoperative ileus (POI), a motility disorder and common complication after abdominal surgery. Enteric gliosis of the intestinal muscularis externa (ME) has been identified as part of POI development. However, the glia-restricted responses and activation mechanisms are poorly understood. The sympathetic nervous system becomes rapidly activated by abdominal surgery. It modulates intestinal immunity, innervates all intestinal layers, and directly interfaces with enteric glia. We hypothesized that sympathetic innervation controls enteric glia reactivity in response to surgical trauma.MethodsSox10iCreERT2/Rpl22HA/+ mice were subjected to a mouse model of laparotomy or intestinal manipulation to induce POI. Histological, protein, and transcriptomic analyses were performed to analyze glia-specific responses. Interactions between the sympathetic nervous system and enteric glia were studied in mice chemically depleted of TH+ sympathetic neurons and glial-restricted Sox10iCreERT2/JellyOPfl/+/Rpl22HA/+ mice, allowing optogenetic stimulation of β-adrenergic downstream signaling and glial-specific transcriptome analyses. A laparotomy model was used to study the effect of sympathetic signaling on enteric glia in the absence of intestinal manipulation. Mechanistic studies included adrenergic receptor expression profiling in vivo and in vitro and adrenergic agonism treatments of primary enteric glial cell cultures to elucidate the role of sympathetic signaling in acute enteric gliosis and POI.ResultsWith ~ 4000 differentially expressed genes, the most substantial enteric glia response occurs early after intestinal manipulation. During POI, enteric glia switch into a reactive state and continuously shape their microenvironment by releasing inflammatory and migratory factors. Sympathetic denervation reduced the inflammatory response of enteric glia in the early postoperative phase. Optogenetic and pharmacological stimulation of β-adrenergic downstream signaling triggered enteric glial reactivity. Finally, distinct adrenergic agonists revealed β-1/2 adrenoceptors as the molecular targets of sympathetic–driven enteric glial reactivity.ConclusionsEnteric glia act as early responders during post-traumatic intestinal injury and inflammation. Intact sympathetic innervation and active β-adrenergic receptor signaling in enteric glia is a trigger of the immediate glial postoperative inflammatory response. With immune-activating cues originating from the sympathetic nervous system as early as the initial surgical incision, adrenergic signaling in enteric glia presents a promising target for preventing POI development.

Orosomucoid-1 Arises as a Shared Altered Protein in Two Models of Multiple Sclerosis

Multiple sclerosis (MS) is a complex autoimmune and neurodegenerative disorder that affects the central nervous system (CNS). It is characterized by a heterogeneous disease course involving demyelination and inflammation. In this study, we utilized two distinct animal models, cuprizone (CPZ)-induced demyelination and experimental autoimmune encephalomyelitis (EAE), to replicate various aspects of the disease. We aimed to investigate the differential CNS responses by examining the proteomic profiles of EAE mice during the peak disease (15 days post-induction) and cuprizone-fed mice during the acute phase (38 days). Specifically, we focused on two different regions of the CNS: the dorsal cortex (Cx) and the entire spinal cord (SC). Our findings revealed varied glial, synaptic, dendritic, mitochondrial, and inflammatory responses within these regions for each model. Notably, we identified a single protein, Orosomucoid-1 (Orm1), also known as Alpha-1-acid glycoprotein 1 (AGP1), that consistently exhibited alterations in both models and regions. This study provides insights into the similarities and differences in the responses of these regions in two distinct demyelinating models.

The Analgesia Effect of Aucubin on CFA-Induced Inflammatory Pain by Inhibiting Glial Cells Activation-Mediated Inflammatory Response via Activating Mitophagy.

Inflammatory pain, characterized by sustained nociceptive hypersensitivity, represents one of the most prevalent conditions in both daily life and clinical settings. Aucubin, a natural plant iridoid glycoside, possesses potent biological effects, encompassing anti-inflammatory, antioxidant, and neuroprotective properties. However, its impact on inflammatory pain remains unclear. The aim of this study is to investigate the therapeutic effects and underlying mechanism of aucubin in addressing inflammatory pain induced by complete Freund's adjuvant (CFA). The CFA-induced inflammatory pain model was employed to assess whether aucubin exerts analgesic effects and its potential mechanisms. Behavioral tests evaluated mechanical and thermal hyperalgesia as well as anxiety-like behaviors in mice. The activation of spinal glial cells and the expression of pro-inflammatory cytokines were examined to evaluate neuroinflammation. Additionally, RNA sequencing was utilized for the identification of differentially expressed genes (DEGs). Molecular biology experiments were conducted to determine the levels of the PINK1 gene and autophagy-related genes, along with PINK1 distribution in neural cells. Furthermore, mitophagy induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) was employed to examine the roles of PINK1 and mitophagy in pain processing. Aucubin significantly ameliorated pain and anxiety-like behaviors induced by CFA in mice and reduced spinal inflammation. RNA sequencing indicated PINK1 as a pivotal gene, and aucubin treatment led to a significant downregulation of PINK1 expression. Further GO and KEGG analyses suggested the involvement of mitochondrial function in the therapeutic regulation of aucubin. Western blotting revealed that aucubin markedly decreased PINK1, Parkin, and p62 levels while increasing LC3B expression. Immunofluorescence showed the predominant co-localization of PINK1 with neuronal cells. Moreover, CCCP-induced mitophagy alleviated mechanical and thermal hyperalgesia caused by CFA and reversed CFA-induced mitochondrial dysfunction. In summary, our data suggest that aucubin effectively alleviates CFA-induced inflammatory pain, potentially through triggering the PINK1 pathway, promoting mitophagy, and suppressing inflammation. These results provide a novel theoretical foundation for addressing the treatment of inflammatory pain.

Mapping of neuronal and glial primary cilia contactome and connectome in the human cerebral cortex

Primary cilia act as antenna receivers of environmental signals and enable effective neuronal or glial responses. Disruption of their function is associated with circuit disorders. To understand the signals these cilia receive, we comprehensively mapped cilia's contacts within the human cortical connectome using serial-section EM reconstruction of a 1mm3 cortical volume, spanning the entire cortical thickness. We mapped the "contactome" of cilia emerging from neurons and astrocytes in every cortical layer. Depending on the layer and cell type, cilia make distinct patterns of contact. Primary cilia display cell-type- and layer-specific variations in size, shape, and microtubule axoneme core, which may affect their signaling competencies. Neuronal cilia are intrinsic components of a subset of cortical synapses and thus a part of the connectome. This diversity in the structure, contactome, and connectome of primary cilia endows each neuron or glial cell with a unique barcode of access to the surrounding neural circuitry.