Astrocytic Phenotype Research Articles

The gut microbiome regulates astrocyte reaction to Aβ amyloidosis through microglial dependent and independent mechanisms

BackgroundPrevious studies show that antibiotic-mediated (abx) alteration of the gut microbiome (GMB) results in a reduction of amyloid beta (Aβ) plaques and proinflammatory microglial phenotype in male APPPS1-21 mice. However, the effect of GMB perturbation on astrocyte phenotypes and microglial-astrocyte communication in the context of amyloidosis has not been examined.MethodsTo study whether the GMB modulates astrocyte phenotype in the context of amyloidosis, APPPS1-21 male and female mice were treated with broad-spectrum abx leading to GMB perturbation. GFAP + astrocytes, plaque-associated astrocytes (PAA), PAA morphological parameters, and astrocyte complement component C3 levels were quantified using a combination of immunohistochemistry, immunoblotting, widefield microscopy, and confocal microscopy. Furthermore, these same astrocyte phenotypes were assessed in abx-treated APPPS1-21 male mice that received either fecal matter transplant (FMT) from untreated APPPS1-21 male donors to restore their microbiome or vehicle control. To assess complete absence of the GMB on astrocyte phenotypes, the same astrocyte phenotypes were quantified in APPPS1-21 male mice raised in germ-free (GF) or specific-pathogen free conditions (SPF). Lastly, we assessed whether microglia are necessary for abx-induced astrocyte phenotypes by depleting microglia in APPPS1-21 male mice via treatment with a colony-stimulating factor 1 receptor (CSF1R) inhibitor (PLX5622) and vehicle control or PLX5622 and abx.ResultsHerein, we demonstrate that postnatal treatment of male APPPS1-21 mice with broad-spectrum abx leading to GMB perturbation reduces GFAP + reactive astrocytes and PAAs, suggesting that the GMB plays a role in regulating reactive astrocyte induction and recruitment to Aβ plaques. Additionally, we show that compared to controls, PAAs in abx-treated male APPPS1-21 mice exhibit an altered morphology with increased number and length of processes and reduced astrocytic complement C3, consistent with a homeostatic phenotype. GFAP + astrocyte reduction, PAA reduction, astrocyte morphological changes, and C3 levels are restored when abx-treated mice are subject to FMT from untreated APPPS1-21 male donor mice. Next, we found that APPPS1-21 male mice raised in GF conditions have similar astrocyte phenotypes as abx-treated male APPPS1-21 male mice. Correlational analysis revealed that pathogenic bacteria depleted by abx correlate with GFAP + astrocytosis, PAAs, and astrocyte morphological changes. Finally, we determined that abx-mediated reduction in GFAP + astrocytosis, PAAs, and astrocytic C3 expression is independent of microglia. However, abx-induced astrocyte morphological alterations are dependent on the presence of microglia, suggesting that there is both microglial independent and dependent GMB control of reactive astrocyte phenotypes.ConclusionsWe show for the first time, in the context of amyloidosis, that the GMB plays an important role in controlling reactive astrocyte induction, morphology, and astrocyte recruitment to Aβ plaques. GMB regulation of these astrocytic phenotypes is both independent and dependent on microglia.

Knockout of microglial Hv1 proton channel reduces neurotoxic A1 astrocytes and neuronal damage via the ROS/STAT3 pathway after spinal cord injury.

Spinal cord injury (SCI) causes severe functional deficits and neuronal damage, accompanied by intense glial activation. The voltage-gated proton channel Hv1, selectively expressed on microglia, is associated with SCI progression. However, the effect of Hv1 on the phenotypes and functions of reactive astrocytes after SCI remains unclear. Here, we combined Hv1 knockout (Hv1-/- ) mice and T10 spinal cord contusion to investigate the effects of microglial Hv1 on SCI pathophysiology and the phenotypes and functions of reactive astrocytes. After SCI, astrocytes proliferated and activated in the peri-injury area and exhibited an A1-dominant phenotype. Hv1 knockout reduced neurotoxic A1 astrocytes and shifted the dominant phenotype of reactive astrocytes from A1 to A2, enhancing synaptogenesis promotion, phagocytosis, and neurotrophy of astrocytes. Moreover, synaptic and axonal remodeling as well as motor recovery after SCI benefited from the improved astrocytic functions of Hv1 knockout. Furthermore, exogenous and endogenous reactive oxygen species (ROS) in astrocytes after SCI were reduced by Hv1 knockout. Our in vitro results showed that inhibition of ROS reduced the neurotoxic A1 phenotype in primary astrocytes via the STAT3 pathway. Similar to the effect of Hv1 knockout, the application of the ROS scavenger N-acetylcysteine reduced SCI-induced neurotoxic A1 astrocytes in vivo. Based on the in vivo and vitro results, we elucidated that microglial Hv1 knockout promotes synaptic and axonal remodeling in SCI mice by decreasing neurotoxic A1 astrocytes and increasing neuroprotective A2 astrocytes via the ROS/STAT3 pathway. Therefore, the Hv1 proton channel is a promising target for the treatment of SCI.

IPSC-derived reactive astrocytes from patients with multiple-sclerosis protect cocultured neurons in inflammatory conditions.

Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system (CNS). The individual course is highly variable with complete remission in some patients and relentless courses in others. We generated induced pluripotent stem cells (iPSCs) to investigate possible mechanisms in benign MS (BMS), compared to progressive MS (PMS). We differentiated neurons and astrocytes that were then stressed with inflammatory cytokines typically associated with MS. TNFα/IL-17A treatment increased neurite damage in MS neurons irrespective of clinical phenotypes. In contrast, TNFα/IL-17A-reactive BMS astrocytes cultured with healthy control (HC) neurons exhibited significantly decreased axonal damage, compared to PMS astrocytes. Accordingly, single cell transcriptomic analysis of BMS-astrocyte co-cultured neurons demonstrated upregulated pathways of neuronal resilience, namely these astrocytes revealed differential growth factor expression. Moreover, supernatants from BMS astrocyte-neuron co-cultures rescued TNFα/IL-17-induced neurite damage. This process was associated with the unique expression of the growth factors, LIF and TGF-β1, as induced by TNFα/IL-17 and JAK-STAT activation. Our findings highlight a potential therapeutic role of modulating astrocyte phenotypes that generate a neuroprotective milieu preventing permanent neuronal damage.

Effects of exercise intensity on the reactive astrocyte polarization in the medial prefrontal cortex.

Physical exercise contributes to neuroplasticity by promoting cognitive functions, such as learning and memory. The astrocytic phenotype is closely associated with synaptic plasticity. This study aimed to determine whether astrocyte polarization and synaptic alterations in the medial prefrontal cortex (mPFC) are affected differently by high- and moderate-intensity exercise. Mice were subjected to moderate-(MIE) and high-intensity treadmill running (HIE). Memory capacity was assessed using the novel object recognition and modified Y-maze tests. For immunohistochemistry, c-Fos-positive cells were counted in the mPFC. Using western blot analysis, astrocyte phenotype markers were quantified in whole-cell lysates, and synaptic molecules were determined in the synaptosomal fraction. Exercise lengthened the approach time to novel objects regardless of intensity in the NOR test, whereas MIE only improved spatial memory. Exercise induced c-Fos expression in the anterior cingulate cortex (ACC) and c-Fos-positive cells were higher in MIE than in HIE in the ACC area. In the prelimbic/infralimbic cortex region, the number of c-Fos-positive cells were enhanced in MIE and decreased in HIE mice. The A1 astrocyte marker (C3) was increased in HIE mice, while the A2 astrocyte markers were enhanced in exercised mice, regardless of the intensity. In the synaptosomal fraction, synaptic proteins were elevated by exercise regardless of intensity. These results suggest that exercise intensity affects neuronal plasticity by modulating the reactive state of astrocytes in the mPFC.

The link between SARS-CoV-2 related microglial reactivity and astrocyte pathology in the inferior olivary nucleus.

The pathological involvement of the central nervous system in SARS-CoV2 (COVID-19) patients is established. The burden of pathology is most pronounced in the brain stem including the medulla oblongata. Hypoxic/ischemic damage is the most frequent neuropathologic abnormality. Other neuropathologic features include neuronophagia, microglial nodules, and hallmarks of neurodegenerative diseases: astrogliosis and microglial reactivity. It is still unknown if these pathologies are secondary to hypoxia versus a combination of inflammatory response combined with hypoxia. It is also unknown how astrocytes react to neuroinflammation in COVID-19, especially considering evidence supporting the neurotoxicity of certain astrocytic phenotypes. This study aims to define the link between astrocytic and microglial pathology in COVID-19 victims in the inferior olivary nucleus, which is one of the most severely affected brain regions in COVID-19, and establish whether COVID-19 pathology is driven by hypoxic damage. Here, we conducted neuropathologic assessments and multiplex-immunofluorescence studies on the medulla oblongata of 18 COVID-19, 10 pre-pandemic patients who died of acute respiratory distress syndrome (ARDS), and 7-8 control patients with no ARDS or COVID-19. The comparison of ARDS and COVID-19 allows us to identify whether the pathology in COVID-19 can be explained by hypoxia alone, which is common to both conditions. Our results showed increased olivary astrogliosis in ARDS and COVID-19. However, microglial density and microglial reactivity were increased only in COVID-19, in a region-specific manner. Also, olivary hilar astrocytes increased YKL-40 (CHI3L1) in COVID-19, but to a lesser extent than ARDS astrocytes. COVID-19 astrocytes also showed lower levels of Aquaporin-4 (AQP4), and Metallothionein-3 in subsets of COVID-19 brain regions. Cluster analysis on immunohistochemical attributes of astrocytes and microglia identified ARDS and COVID-19 clusters with correlations to clinical history and disease course. Our results indicate that olivary glial pathology and neuroinflammation in the COVID-19 cannot be explained solely by hypoxia and suggest that failure of astrocytes to upregulate the anti-inflammatory YKL-40 may contribute to the neuroinflammation. Notwithstanding the limitations of retrospective studies in establishing causality, our experimental design cannot adequately control for factors external to our design. Perturbative studies are needed to confirm the role of the above-described astrocytic phenotypes in neuroinflammation.

Redistribution of the astrocyte phenotypes in the medial vestibular nuclei after unilateral labyrinthectomy.

Astrocytes are highly heterogeneous and involved in different aspects of fundamental functions in the central nervous system (CNS). However, whether and how this heterogeneous population of cells reacts to the pathophysiological challenge is not well understood. To investigate the response status of astrocytes in the medial vestibular nucleus (MVN) after vestibular loss, we examined the subtypes of astrocytes in MVN using single-cell sequencing technology in a unilateral labyrinthectomy mouse model. We discovered four subtypes of astrocytes in the MVN with each displaying unique gene expression profiles. After unilateral labyrinthectomy, the proportion of the astrocytic subtypes and their transcriptional features on the ipsilateral side of the MVN differ significantly from those on the contralateral side. With new markers to detect and classify the subtypes of astrocytes in the MVN, our findings implicate potential roles of the adaptive changes of astrocyte subtypes in the early vestibular compensation following peripheral vestibular damage to reverse behavioral deficits.

Transcriptomic Analyses of Neurotoxic Astrocytes Derived from Adult Triple Transgenic Alzheimer's Disease Mice.

Neurodegenerative diseases such as Alzheimer's disease have been classically studied from a purely neuronocentric point of view. More recent evidences support the notion that other cell populations are involved in disease progression. In this sense, the possible pathogenic role of glial cells like astrocytes is increasingly being recognized. Once faced with tissue damage signals and other stimuli present in disease environments, astrocytes suffer many morphological and functional changes, a process referred as reactive astrogliosis. Studies from murine models and humans suggest that these complex and heterogeneous responses could manifest as disease-specific astrocyte phenotypes. Clear understanding of disease-associated astrocytes is a necessary step to fully disclose neurodegenerative processes, aiding in the design of new therapeutic and diagnostic strategies. In this work, we present the transcriptomics characterization of neurotoxic astrocytic cultures isolated from adult symptomatic animals of the triple transgenic mouse model of Alzheimer's disease (3xTg-AD). According to the observed profile, 3xTg-AD neurotoxic astrocytes show various reactivity features including alteration of the extracellular matrix and release of pro-inflammatory and proliferative factors that could result in harmful effects to neurons. Moreover, these alterations could be a consequence of stress responses at the endoplasmic reticulum and mitochondria as well as of concomitant metabolic adaptations. Present results support the hypothesis that adaptive changes of astrocytic function induced by a stressed microenvironment could later promote harmful astrocyte phenotypes and further accelerate or induce neurodegenerative processes.

Regulatory T cells decrease A1‐like C3‐positive reactive astrocytes in Alzheimer‐like pathology

AbstractBackgroundIncreasing evidence support a key role of peripheral immune processes in the pathophysiology of Alzheimer’s disease (AD), highlighting an intricate interplay between brain resident glial cells and both innate and adaptive peripheral immune effectors. We previously showed that regulatory T cells (Tregs) have a beneficial impact on disease progression in AD‐like pathology, notably by modulating the microglial response associated with Ab deposits in a mouse model of amyloid pathology. Besides microglia, reactive astrocytes also play a critical role in neuroinflammatory processes associated with AD. Different phenotypes of reactive astrocytes have recently been characterized, including A1 neurotoxic and A2 neuroprotective subtypes. However, the precise impact of Tregs on astrocyte reactivity and phenotypes in AD still remains poorly defined.MethodsWe assessed the impact of Treg immunomodulation on astrocyte reactivity in a mouse model of AD‐like amyloid pathology. Using 3D imaging we carried out extensive morphological analyses of astrocytes following either depletion or amplification of Tregs. We further assessed the expression of several A1‐ and A2‐like markers by immunofluorescence and RT‐qPCR.ResultsModulation of Tregs did not significantly impact the magnitude of global astrocyte reactivity in the brain nor in the close vicinity of cortical amyloid deposits. We did not observe changes in the number, morphology, or branching complexity of astrocytes according to immunomodulation of Tregs. However, early transient depletion of Tregs modulated the balance of reactive astrocyte subtypes, resulting in increased C3‐positive A1‐like phenotypes associated with amyloid deposits. Conversely, early depletion of Tregs decreased A2‐like phenotypes of reactive astrocytes associated with larger amyloid deposits. Intriguingly, modulation of Tregs also impacted the cerebral expression of several markers of A1‐like subsets in healthy mice.ConclusionsOur study suggests that Tregs contribute to modulate and fine tune the balance of reactive astrocyte subtypes in AD‐like amyloid pathology, by dampening A1‐like astrocytes in favor of A2‐like phenotypes. This effect of Tregs may partly relate to their capacity at modulating steady state astrocyte reactivity and homeostasis. Our data further highlight the need for refined markers of astrocytes subsets and strategy of analysis for better deciphering the complexity of astrocyte reactivity in neurodegeneration.

Effects of liraglutide on astrocyte polarization and neuroinflammation in db/db mice: focus on iron overload and oxidative stress.

Neuroinflammation plays a crucial role in the occurrence and development of cognitive impairment in type 2 diabetes mellitus (T2DM), but the specific injury mechanism is not fully understood. Astrocyte polarization has attracted new attention and has been shown to be directly and indirectly involved in neuroinflammation. Liraglutide has been shown to have beneficial effects on neurons and astrocytes. However, the specific protection mechanism still needs to be clarified. In this study, we assessed the levels of neuroinflammation and A1/A2-responsive astrocytes in the hippocampus of db/db mice and examined their relationships with iron overload and oxidative stress. First, in db/db mice, liraglutide alleviated the disturbance of glucose and lipid metabolism, increased the postsynaptic density, regulated the expression of NeuN and BDNF, and partially restored impaired cognitive function. Second, liraglutide upregulated the expression of S100A10 and downregulated the expression of GFAP and C3, and decreased the secretion of IL-1β, IL-18, and TNF-α, which may confirm that it regulates the proliferation of reactive astrocytes and A1/A2 phenotypes polarize and attenuate neuroinflammation. In addition, liraglutide reduced iron deposition in the hippocampus by reducing the expression of TfR1 and DMT1 and increasing the expression of FPN1; at the same time, liraglutide by up-regulating the levels of SOD, GSH, and SOD2 expression, as well as downregulation of MDA levels and NOX2 and NOX4 expression to reduce oxidative stress and lipid peroxidation. The above may attenuate A1 astrocyte activation. This study preliminarily explored the effect of liraglutide on the activation of different astrocyte phenotypes and neuroinflammation in the hippocampus of a T2DM model and further revealed its intervention effect on cognitive impairment in diabetes. Focusing on the pathological consequences of astrocytes may have important implications for the treatment of diabetic cognitive impairment.

Integrative Transcriptomic Analyses of Hippocampal-Entorhinal System Subfields Identify Key Regulators in Alzheimer's Disease.

The hippocampal-entorhinal system supports cognitive function and is selectively vulnerable to Alzheimer's disease (AD). Little is known about global transcriptomic changes in the hippocampal-entorhinal subfields during AD. Herein, large-scale transcriptomic analysis is performed in five hippocampal-entorhinal subfields of postmortem brain tissues (262 unique samples). Differentially expressed genes are assessed across subfields and disease states, and integrated genotype data from an AD genome-wide association study. An integrative gene network analysis of bulk and single-nucleus RNA sequencing (snRNA-Seq) data identifies genes with causative roles in AD progression. Using a system-biology approach, pathology-specific expression patterns for cell types are demonstrated, notably upregulation of the A1-reactive astrocyte signature in the entorhinal cortex (EC) during AD. SnRNA-Seq data show that PSAP signaling is involved in alterations of cell- communications in the EC during AD. Further experiments validate the key role of PSAP in inducing astrogliosis and an A1-like reactive astrocyte phenotype. In summary, this study reveals subfield-, cell type-, and AD pathology-specific changes and demonstrates PSAP as a potential therapeutic target in AD.

Soluble epoxide hydrolase inhibitor blockage microglial cell activation in subnucleus caudalis in a persistent model of arthritis

Rheumatoid arthritis (RA) is a chronic condition characterized by pain and infiltration of immune cells into the joint. Immune cells can be activated, producing inflammatory cytokines, leading to continuously degenerative and inflammatory reactions and the temporomandibular joint (TMJ) can be affected by RA. In this scenario, novel targets are needed to increase treatment efficacy with minimized side effects. The epoxy-eicosatrienoic acids (EETs), are endogenous signaling molecules, playing important roles in diminishing inflammation and pain but are promptly metabolized by soluble epoxide hydrolase (sEH), generating less-bioactive acids.Therefore, sEH inhibitors is an interest therapeutic target to enhance the beneficial effect of natural EETs. TPPU is a potent sEH inhibitor that is capable of dampening EETs hydrolysis. Thus, we aimed to assess the impact of pharmacological sEH inhibition on a persistent model of albumin-induced arthritis in the TMJ, in two scenarios: first, as post-treatment, in an installed arthritic condition, and second, the protective role, in preventing the development of an arthritic condition. In addition, we investigate the influence of sEH inhibition on microglia cell activation in the trigeminal subnucleus caudalis (TSC) and in vitro experiments. Finally, we examined the astrocyte phenotype. Oral administration of TPPU, acts in multiple pathways, in a protective and reparative post-treatment, ameliorating the preservation of the TMJ morphology, reducing the hypernociception, with an immunosuppressive action reducing neutrophil and lymphocytes and pro-inflammatory cytokines in the TMJ of rats. In TSC, TPPU reduces the cytokine storm and attenuates the microglia activated P2X7/Cathepsin S/Fractalkine pathway and reduces the astrocyte activation and glutamate levels. Collectively, our findings revealed that sEH inhibition mitigates hypersensitive nociception through the regulation of microglia activation and astrocyte modulation, demonstrating the potential use of sEH inhibitors as immunoresolvents in the treatment of autoimmune disorders.

β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

Postoperative delirium (POD) is a frequent and debilitating complication, especially amongst high risk procedures, such as orthopedic surgery. This kind of neurocognitive disorder negatively affects cognitive domains, such as memory, awareness, attention, and concentration after surgery; however, its pathophysiology remains unknown. Multiple lines of evidence supporting the occurrence of inflammatory events have come forward from studies in human patients’ brain and bio-fluids (CSF and serum), as well as in animal models for POD. β-arrestins are downstream molecules of guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). As versatile proteins, they regulate numerous pathophysiological processes of inflammatory diseases by scaffolding with inflammation-linked partners. Here we report that β-arrestin1, one type of β-arrestins, decreases significantly in the reactive astrocytes of a mouse model for POD. Using β-arrestin1 knockout (KO) mice, we find aggravating effect of β-arrestin1 deficiency on the cognitive dysfunctions and inflammatory phenotype of astrocytes in POD model mice. We conduct the in vitro experiments to investigate the regulatory roles of β-arrestin1 and demonstrate that β-arrestin1 in astrocytes interacts with the dynamin-related protein 1 (Drp1) to regulate mitochondrial fusion/fission process. β-arrestin1 deletion cancels the combination of β-arrestin1 and cellular Drp1, thus promoting the translocation of Drp1 to mitochondrial membrane to provoke the mitochondrial fragments and the subsequent mitochondrial malfunctions. Using β-arrestin1-biased agonist, cognitive dysfunctions of POD mice and pathogenic activation of astrocytes in the POD-linked brain region are reduced. We, therefore, conclude that β-arrestin1 is a promising target for the understanding of POD pathology and development of POD therapeutics.

TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during Alzheimer’s diseases

Neuroinflammation and oxidative stress have been implicated in the pathogenesis of Alzheimer’s disease (AD). Neuroinflammation and oxidative stress are associated with neuronal death in AD. Astrocytes are linked to neuroinflammation during AD. Astrocytes are important contributors to AD progression. Although the role of thioredoxin-interacting protein (TXNIP) has been identified in inflammation and oxidative stress, the mechanism by which TXNIP regulates inflammation and oxidative stress in astrocytes during AD remains unclear. In the present study, we found that TXNIP gene levels were elevated in cerebral cortex of patients with AD. The protein levels of TXNIP were elevated in GFAP-positive astrocytes of cerebral cortex from patients with AD and APP/PS1 double-transgenic mouse model of AD. Our results showed that TXNIP increased expression of genes related to pro-inflammatory reactive astrocytes and pro-inflammatory cytokines and chemokines in human astrocytes. Moreover, TXNIP increased production of pro-inflammatory cytokines and chemokines in human astrocytes. TXNIP induced activation of NK-kB signaling and over-production of mitochondrial reactive oxygen species (mtROS) in human astrocytes. TXNIP also induced mitochondrial oxidative stress by reduction of mitochondrial respiration and ATP production in human astrocytes. Furthermore, elevated TXNIP levels are correlated with caspase-3 activation of GFAP-positive astrocytes in patients with AD and mouse AD. TXNIP induced mitochondria-dependent apoptosis via caspase-9 and caspase-3 activation in human astrocytes. These results suggest that TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during AD.

Differentiation of neurosphere after transplantation into the damaged spinal cord.

This study aimed to compare the differentiation and survival of human neural stem/progenitor cells of various origins in vitro and after transplantation into the injured spinal cord of laboratory animals. Rats with simulated spinal cord injury were transplanted with neurosphere cells obtained by directed differentiation of HUES6 cell lines. Fluorescence microscopy was used to visualize the obtained results. HUES6#1 and iPSC#1 neurospheres showed a wide range of markers associated with glial differentiation. The expression of the proliferation marker Ki67 did not exceed 25%, both in the lines of early and late neurospheres. Although neurospheres did not fully differentiate into astrocytes in vitro, they massively approached the GFAP+ astrocyte phenotype when exposed to the transplanted environment. PSC-derived neurospheres transplanted into the site of SM injury without additional growth factors showed only moderate survival, a significant degree of differentiation into astrocytes, and moderate differentiation into neurons. The difference in the survival and differentiation of HUES6#1 and iPSC#1 neurospheres, both in vitro and in vivo, can be explained by the difference in the regulatory behavior of signaling molecules corresponding to the source of origin of PSCs. Derivatives of human PSCs of various origins obtained according to the described differentiation protocol did not mature into astrocytic populations, nor did the glycogenic transition of PSC-derived NSCs occur in vitro. The study demonstrated the impact of the injured spinal cord microenvironment on the differentiation of transplanted HUES6#1 and iPSC#1 into astrocytes. The results showed that HUES6-derived neurospheres generated 90% of GFAP+ astrocytes and 5-10% of early neurons, while iPSC-derived neurospheres generated an average of 74% GFAP+ astrocytes and 5% of early neurons in vivo.

Strain-Dependent Morphology of Reactive Astrocytes in Human- and Animal-Vole-Adapted Prions.

Reactive astrogliosis is one of the pathological hallmarks of prion diseases. Recent studies highlighted the influence of several factors on the astrocyte phenotype in prion diseases, including the brain region involved, the genotype backgrounds of the host, and the prion strain. Elucidating the influence of prion strains on the astrocyte phenotype may provide crucial insights for developing therapeutic strategies. Here, we investigated the relationship between prion strains and astrocyte phenotype in six human- and animal-vole-adapted strains characterized by distinctive neuropathological features. In particular, we compared astrocyte morphology and astrocyte-associated PrPSc deposition among strains in the same brain region, the mediodorsal thalamic nucleus (MDTN). Astrogliosis was detected to some extent in the MDTN of all analyzed voles. However, we observed variability in the morphological appearance of astrocytes depending on the strain. Astrocytes displayed variability in thickness and length of cellular processes and cellular body size, suggesting strain-specific phenotypes of reactive astrocytes. Remarkably, four out of six strains displayed astrocyte-associated PrPSc deposition, which correlated with the size of astrocytes. Overall, these data show that the heterogeneous reactivity of astrocytes in prion diseases depends at least in part on the infecting prion strains and their specific interaction with astrocytes.

Celsr2-mediated morphological polarization and functional phenotype of reactive astrocytes in neural repair.

Neural repair is highly influenced by reactive astrocytes. Atypical cadherin Celsr2 regulates neuron development and axon regeneration, while its role in glial cells remains unexplored. In this study, we show that Celsr2 is highly expressed in spinal astrocytes of adult mice, and knockout of Celsr2 results in reactive astrocytes with longer protrusions preferentially orientated towards lesion borders in culture scratch assay and injured spinal cord, and elevation of total and active Cdc42 and Rac1 protein in western blots. Inactivation of Celsr2 enhances calcium influx in reactive astrocytes in time-lapse imaging. Morphological phenotypes of cultured Celsr2-/- astrocytes are rescued by Cdc42 or Rac1 inhibitors. Following spinal cord injury (SCI), Celsr2-/- mice exhibit smaller lesion cavity and glial scar, enhanced fiber regeneration, weaker microglial response, and improved functional recovery than control animals. Similar phenotypes are found in mice with conditional knockout of Celsr2 in astrocytes. In Celsr2-/- mice, astrocyte phenotype is changed and neuroinflammation is alleviated after injury. Inhibiting Cdc42/Rac1 activities compromises astrocyte polarization and the improvement of neural repair and functional recovery in Celsr2-/- mice with SCI. In conclusion, Celsr2 regulates morphological polarization and functional phenotype of reactive astrocytes and inactivating Celsr2 is a potential therapeutic strategy for neural repair.

It takes two to remyelinate: A bioengineered platform to study astrocyte-oligodendrocyte crosstalk and potential therapeutic targets in remyelination

The loss of the myelin sheath insulating axons is the hallmark of demyelinating diseases. These pathologies often lead to irreversible neurological impairment and patient disability. No effective therapies are currently available to promote remyelination.Several elements contribute to the inadequacy of remyelination, thus understanding the intricacies of the cellular and signaling microenvironment of the remyelination niche might help us to devise better strategies to enhance remyelination.Here, using a new in vitro rapid myelinating artificial axon system based on engineered microfibres, we investigated how reactive astrocytes influence oligodendrocyte (OL) differentiation and myelination ability. This artificial axon culture system enables the effective uncoupling of molecular cues from the biophysical properties of the axons, allowing the detailed study of the astrocyte-OL crosstalk. Oligodendrocyte precursor cells (OPCs) were cultured on poly(trimethylene carbonate-co-ε-caprolactone) copolymer electrospun microfibres that served as surrogate axons. This platform was then combined with a previously established tissue engineered glial scar model of astrocytes embedded in 1 % (w/v) alginate matrices, in which astrocyte reactive phenotype was acquired using meningeal fibroblast conditioned medium.OPCs were shown to adhere to uncoated engineered microfibres and differentiate into myelinating OL. Reactive astrocytes were found to significantly impair OL differentiation ability, after six and eight days in a co-culture system. Differentiation impairment was seen to be correlated with astrocytic miRNA release through exosomes. We found significantly reduction on the expression of pro-myelinating miRNAs (miR-219 and miR-338) and an increase in anti-myelinating miRNA (miR-125a-3p) content between reactive and quiescent astrocytes. Additionally, we show that OPC differentiation inhibition could be reverted by rescuing the activated astrocytic phenotype with ibuprofen, a chemical inhibitor of the small rhoGTPase RhoA.Overall, these findings show that modulating astrocytic function might be an interesting therapeutic avenue for demyelinating diseases. The use of these engineered microfibres as an artificial axon culture system will enable the screening for potential therapeutic agents that promote OL differentiation and myelination while providing valuable insight on the myelination/remyelination processes.

Cerebrospinal Fluid sTREM-2, GFAP, and β-S100 in Symptomatic Sporadic Alzheimer's Disease: Microglial, Astrocytic, and APOE Contributions Along the Alzheimer's Disease Continuum.

Many transversal mechanisms act synergistically at different time-points in the cascade of Alzheimer's disease (AD), since amyloid-β (Aβ) deposition, tau pathology, and neuroinflammation influence each other. We explored the contributions of microglia and astrocytes in patients with symptomatic sporadic AD stratified according to AT(N) system and APOE genotype. We compared the cerebrospinal fluid (CSF) levels of sTREM-2 and markers of astrocytic activation (GFAP; β-S100) from 71 patients with AD (23 A+T-,48 A+T+; 38 APOEɛ3, 33 APOEɛ4) and 30 healthy controls (HC). With multivariate analyses we investigated associations between glial biomarkers, Aβ42, and p-tau in all subgroups. CSF sTREM-2 was higher in A+T+ [1.437 (0.264)] and A+T- [1.355 (0.213)] than in HC [1.042 (0.198); both p < 0.001]; GFAP and β-S100 were comparable across groups. Considering all patients, sTREM-2 positively associated with Aβ 42 (p = 0.04) and p-tau (=0.016), with the first being present only in the A+T- subgroup (p = 0.023). GFAP positively associated with Aβ 42 in all patients (p = 0.020) and in the A+T+ subgroup (p = 0.04). Stratifying by APOE, a positive association of sTREM-2 and p-tau was confirmed selectively in carriers of ɛ4 (p = 0.018). Finally, sTREM-2 positively correlated with β-S100 in all subgroups, and with GFAP in A+T+ (p = 0.042). Our results confirm the increase of CSF sTREM-2 in AD, which associates with reduced amyloidopathy in A+T- patients. Moreover, microglial activation seems to increase CSF tau levels in carriers of APOEɛ4, is associated with astrocytic reactivity (GFAP) in A+T+, and likely leads the acquisition of a more neurotoxic astrocytic phenotype (β-S100).

131 Hedgehog Signaling Mediates Astrocytic Response and Blood-Brain Barrier Repair Following Traumatic Central Nervous System Injury

INTRODUCTION: Injury to the central nervous system (CNS) provokes a multicellular response to facilitate restoration of tissue homeostasis. Astrocytes consolidate at the lesion site to form a “glial scar” which restricts leukocyte infiltration and repairs the blood-brain barrier (BBB). However, this “scar” has been considered an impediment to regeneration and functional recovery. Therefore, uncovering molecular regulators that may be therapeutically targeted to promote pro-regenerative astrocyte phenotypes is of great interest. METHODS: Genetic reporter and conditional mutant mice were used to monitor, constitutively activate, or ablate Hh signaling following stereotactically-induced penetrating cortical or spinal cord injury. Outcomes including reactive astrocyte proliferation and polarization, leukocyte restriction, and blood-brain barrier repair were assessed via immunohistochemistry. RESULTS: Astrocytic expression of the Hh target gene Gli1 was downregulated acutely following injury but restored in the subacute phase. Constitutive activation of Hh signaling reduced astrocytic proliferation (%BrdU/EdU+ = 9.6% vs. 51.0%, p&lt;0.01) and polarization (L/W Ratio = 2.8 vs. 1.5, p &lt; 0.01). Additionally, in the spinal cord, constitutive activation impaired migration and integration of reactive astrocytes into the white matter lesion site. In animals with intact Hh signaling, the BBB recovered by 28 days after injury as evidenced by low levels of plasma protein and leukocyte infiltration. In contrast, conditional ablation of Hh signaling from 14-16 days after injury resulted in persistent BBB defects at 28 days. CONCLUSIONS: Our results suggest that acute downregulation of Hh signaling is required to specify reactive astrocyte properties including polarization and cell cycle re-entry. However, restoration of Hh signaling, which occurs subacutely from 10-28 days after injury and coincides with glial scar maturation, is required for leukocyte restriction and BBB repair. Understanding the temporospatial dynamics of postinjury Hh signaling is essential to modeling future therapeutic targets.

Astrocyte-associated fibronectin promotes the proinflammatory phenotype of astrocytes through β1 integrin activation

Astrocytes are key players in neuroinflammation. In response to central nervous system (CNS) injury or disease, astrocytes undergo reactive astrogliosis, which is characterized by increased proliferation, migration, and glial fibrillary acidic protein (GFAP) expression. Activation of the transcription factor nuclear factor-κB (NF-κB) and upregulation of downstream proinflammatory mediators in reactive astrocytes induce a proinflammatory phenotype in astrocytes, thereby exacerbating neuroinflammation by establishing an inflammatory loop. In this study, we hypothesized that excessive fibronectin (FN) derived from reactive astrocytes would induce this proinflammatory phenotype in astrocytes in an autocrine manner. We exogenously treated astrocytes with monomer FN, which can be incorporated into the extracellular matrix (ECM), to mimic plasma FN extravasated through a compromised blood–brain barrier in neuroinflammation. We also induced de novo synthesis and accumulation of astrocyte-derived FN through tumor necrosis factor-α (TNF-α) stimulation. The excessive FN deposition resulting from both treatments initiated reactive astrogliosis and triggered NF-κB signaling in the cultured astrocytes. In addition, inhibition of FN accumulation in the ECM by the FN inhibitor pUR4 strongly attenuated the FN- and TNF-α-induced GFAP expression, NF-κB activation, and proinflammatory mediator production of astrocytes by interrupting FN–β1 integrin coupling and thus the inflammatory loop. In an in vivo experiment, intrathecal injection of pUR4 considerably ameliorated FN deposition, GFAP expression, and NF-κB activation in inflamed spinal cord, suggesting the therapeutic potential of pUR4 for attenuating neuroinflammation and promoting neuronal function restoration.