Astrocytic Phenotype Research Articles

Transcriptional and Chromatin Accessibility Profiling of Neural Stem Cells Differentiating into Astrocytes Reveal Dynamic Signatures Affected under Inflammatory Conditions

Astrocytes arise from multipotent neural stem cells (NSCs) and represent the most abundant cell type of the central nervous system (CNS), playing key roles in the developing and adult brain. Since the differentiation of NSCs towards a gliogenic fate is a precisely timed and regulated process, its perturbation gives rise to dysfunctional astrocytic phenotypes. Inflammation, which often underlies neurological disorders, including neurodevelopmental disorders and brain tumors, disrupts the accurate developmental process of NSCs. However, the specific consequences of an inflammatory environment on the epigenetic and transcriptional programs underlying NSCs' differentiation into astrocytes is unexplored. Here, we address this gap by profiling in mice glial precursors from neural tissue derived from early embryonic stages along their astrocytic differentiation trajectory in the presence or absence of tumor necrosis factor (TNF), a master pro-inflammatory cytokine. By using a combination of RNA- and ATAC-sequencing approaches, together with footprint and integrated gene regulatory network analyses, we here identify key differences during the differentiation of NSCs into astrocytes under physiological and inflammatory settings. In agreement with its role to turn cells resistant to inflammatory challenges, we detect Nrf2 as a master transcription factor supporting the astrocytic differentiation under TNF exposure. Further, under these conditions, we unravel additional transcriptional regulatory hubs, including Stat3, Smad3, Cebpb, and Nfkb2, highlighting the interplay among pathways underlying physiological astrocytic developmental processes and those involved in inflammatory responses, resulting in discrete astrocytic phenotypes. Overall, our study reports key transcriptional and epigenetic changes leading to the identification of molecular regulators of astrocytic differentiation. Furthermore, our analyses provide a valuable resource for understanding inflammation-induced astrocytic phenotypes that might contribute to the development and progression of CNS disorders with an inflammatory component.

Ethanol-induced cerebellar transcriptomic changes in a postnatal model of fetal alcohol spectrum disorders: Focus on disease onset.

Fetal alcohol spectrum disorders (FASD) are a group of neurodevelopmental disorders caused by ethanol exposure in utero, which can result in neurocognitive and behavioral impairments, growth defects, and craniofacial anomalies. FASD affects up to 1-5% of school-aged children in the United States, and there is currently no cure. The underlying mechanisms involved in ethanol teratogenesis remain elusive and need greater understanding to develop and implement effective therapies. Using a third trimester human equivalent postnatal mouse model of FASD, we evaluate the transcriptomic changes induced by ethanol exposure in the cerebellum on P5 and P6, after only 1 or 2 days of ethanol exposure, with the goal of shedding light on the transcriptomic changes induced early during the onset and development of FASD. We have highlighted key pathways and cellular functions altered by ethanol exposure, which include pathways related to immune function and cytokine signaling as well as the cell cycle. Additionally, we found that ethanol exposure resulted in an increase in transcripts associated with a neurodegenerative microglia phenotype, and acute- and pan-injury reactive astrocyte phenotypes. Mixed effects on oligodendrocyte lineage cell associated transcripts and cell cycle associated transcripts were observed. These studies help to elucidate the underlying mechanisms that may be involved with the onset of FASD and provide further insights that may aid in identifying novel targets for interventions and therapeutics.

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

BackgroundIncreasing evidence supports a key role for 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 Aβ 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 previously been characterized, including A1-like neurotoxic and A2-like 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 markers of 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 C3-positive 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.

GABAB receptor activation alters astrocyte phenotype changes induced by trimethyltin via ERK signaling in the dentate gyrus of mice

AimsWe examined the effect of γ-aminobutyric acid (GABA)B receptor activation on astrocyte phenotype changes induced by trimethyltin (TMT) in the dentate gyrus of mice. Main methodsMale C57BL/6N mice received TMT (2.6 mg/kg, i.p.), and the expression of GABAB receptors was evaluated in the hippocampus. The GABAB receptor agonist baclofen (2.5, 5, or 10 mg/kg, i.p. × 5 at 12-h intervals) was administered 3–5 days after TMT treatment, and the expression of Iba-1, GFAP, and astrocyte phenotype markers was evaluated 6 days after TMT. SL327 (30 mg/kg, i.p.), an extracellular signal-related kinase (ERK) inhibitor, was administered 1 h after each baclofen treatment. Key findingsTMT insult significantly induced the astroglial expression of GABAB receptors in the dentate molecular layer. Baclofen significantly promoted the expression of S100A10, EMP1, and CD109, but not that of C3, GGTA1, and MX1 induced by TMT. In addition, baclofen significantly increased the TMT-induced expression of p-ERK in the dentate molecular layer. Interestingly, p-ERK was more colocalized with S100A10 than with C3 after TMT insult, and a significant positive correlation was found between the expression of p-ERK and S100A10. Consistently, SL327 reversed the effect of baclofen on astrocyte phenotype changes. Baclofen also enhanced the TMT-induced astroglial expression of glial cell-derived neurotrophic factor (GDNF), an anti-inflammatory astrocytes-to-microglia mediator, and consequently attenuated Iba-1 expression and delayed apoptotic neuronal death. SignificanceOur results suggest that GABAB receptor activation increases S100A10-positive anti-inflammatory astrocytes and astroglial GDNF expression via ERK signaling after TMT excitotoxicity in the dentate molecular layer of mice.

Low-intensity pulsed ultrasound triggers a beneficial neuromodulation in dementia mice with chronic cerebral hypoperfusion via activation of hippocampal Fndc5/irisin signaling

BackgroundExercise-related signaling Fndc5/irisin expresses in brain and acts as a crucial regulator of cognitive function, but its detailed roles in vascular dementia (VaD) are still unclear. Low intensity pulsed ultrasound (LIPUS), a novel brain stimulation approach, has been suggested as a promising treatment for dementia. Here, we investigated the activity and efficacy of Fndc5/irisin in experimental VaD, further explored whether the potential effects of LIPUS on VaD is related to Fndc5/irisin.MethodsMouse model of VaD was established with chronic cerebral hypoperfusion (CCH) using bilateral common carotid arteries stenosis (BCAS). Transcranial LIPUS was applied 24 h after BCAS and subsequently daily with a stimulation time of 5 min at an ultrasound pressure of 0.51 MPa for a period of 28 days. The levels of Fndc5/irisin in different brain regions, the hippocampal long-term potentiation and anti-inflammatory cytokines were investigated at day 28 after cognitive evaluation. Global Fndc5 knock-out (F5KO), forced expression or knockdown of Fndc5, and recombinant irisin application were respectively employed for mechanism exploration. The neuron dendritic spine density and astrocyte phenotype were detected in vitro.ResultsFndc5/irisin was reduced in hippocampus of BCAS mice, forced expression hippocampal Fndc5 or bilateral intrahippocampal injection of recombinant irisin respectively improved hippocampal synaptic plasticity or inflammatory microenvironment, and then alleviated the cognitive impairments. LIPUS existed a positive efficacy in enhancing hippocampal Fndc5/irisin in BCAS mice, thus triggering a beneficial neuromodulation for VaD protection. Importantly, the neurorestorative effects of LIPUS on CCH-induced damages were totally reversed by knockdown the expression of hippocampal Fndc5 in WT mice, or in F5KO mice. Moreover, Fndc5 mediated the upregulated effects of LIPUS on spine density as well as irisin secretion of hippocampal neurons. The neuron-secreted irisin further drove reactive astrocytes to a neuroprotective phenotype.ConclusionLIPUS induced a neurorestorative stimulation against VaD may be through upregulation of the hippocampal Fndc5/irisin levels. Hippocampal Fndc5/irisin signaling might be a promising strategic target for VaD.

Roles of prokineticin 2 in electroconvulsive shock-induced memory impairment via regulation of phenotype polarization in astrocytes

Electroconvulsive shock (ECT) is the most effective treatment for depression but can impair learning and memory. ECT is increasingly being shown to activate astrocytes and induce neuroinflammation, resulting in cognitive decline. Activated astrocytes can differentiate into two subtypes, A1-type astrocytes and A2-type astrocytes. Regarding cognitive function, neurotoxic A1 astrocytes and neuroprotective A2 astrocytes may exhibit opposite effects. Specifically, prokineticin 2 (PK2) functions as an essential mediator of inflammation and induces a selective A2-protective phenotype in astrocytes. This study aimed to clarify how PK2 promotes improved learning memory following electroconvulsive shock (ECS). As part of the study, rats were modeled using chronic unpredictable mild stress. Behavioral experiments were conducted to assess their cognitive abilities and depression-like behaviors. Western blot was used to determine the expression of PK2. Immunohistochemical and electron microscopy analyses of the hippocampal CA1 region were conducted to study the activation of astrocyte subtypes and synaptic ultrastructure, respectively. In this study, rats' spatial learning and memory impairment began to improve as activated A1-subtype astrocytes gradually decreased, and PK2 and A2 phenotype activation peaked on the third day after ECS. PKRA7 (PK2 antagonist) inhibits A2-type astrocyte activation partially and suppresses spatial learning and memory improvement. Collectively, our findings support that PK2 may induce a selective modulation of astrocytic polarization to a protective phenotype to promote learning and memory improvement after ECS

Targeting Astrocyte Signaling Alleviates Cerebrovascular and Synaptic Function Deficits in a Diet-Based Mouse Model of Small Cerebral Vessel Disease.

Despite the indispensable role that astrocytes play in the neurovascular unit, few studies have investigated the functional impact of astrocyte signaling in cognitive decline and dementia related to vascular pathology. Diet-mediated induction of hyperhomocysteinemia (HHcy) recapitulates numerous features of vascular contributions to cognitive impairment and dementia (VCID). Here, we used astrocyte targeting approaches to evaluate astrocyte Ca2+ dysregulation and the impact of aberrant astrocyte signaling on cerebrovascular dysfunction and synapse impairment in male and female HHcy diet mice. Two-photon imaging conducted in fully awake mice revealed activity-dependent Ca2+ dysregulation in barrel cortex astrocytes under HHcy. Stimulation of contralateral whiskers elicited larger Ca2+ transients in individual astrocytes of HHcy diet mice compared with control diet mice. However, evoked Ca2+ signaling across astrocyte networks was impaired in HHcy mice. HHcy also was associated with increased activation of the Ca2+/calcineurin-dependent transcription factor NFAT4, which has been linked previously to the reactive astrocyte phenotype and synapse dysfunction in amyloid and brain injury models. Targeting the NFAT inhibitor VIVIT to astrocytes, using adeno-associated virus vectors, led to reduced GFAP promoter activity in HHcy diet mice and improved functional hyperemia in arterioles and capillaries. VIVIT expression in astrocytes also preserved CA1 synaptic function and improved spontaneous alternation performance on the Y maze. Together, the results demonstrate that aberrant astrocyte signaling can impair the major functional properties of the neurovascular unit (i.e., cerebral vessel regulation and synaptic regulation) and may therefore represent a promising drug target for treating VCID and possibly Alzheimer's disease and other related dementias.SIGNIFICANCE STATEMENT The impact of reactive astrocytes in Alzheimer's disease and related dementias is poorly understood. Here, we evaluated Ca2+ responses and signaling in barrel cortex astrocytes of mice fed with a B-vitamin deficient diet that induces hyperhomocysteinemia (HHcy), cerebral vessel disease, and cognitive decline. Multiphoton imaging in awake mice with HHcy revealed augmented Ca2+ responses in individual astrocytes, but impaired signaling across astrocyte networks. Stimulation-evoked arteriole dilation and elevated red blood cell velocity in capillaries were also impaired in cortex of awake HHcy mice. Astrocyte-specific inhibition of the Ca2+-dependent transcription factor, NFAT, normalized cerebrovascular function in HHcy mice, improved synaptic properties in brain slices, and stabilized cognition. Results suggest that astrocytes are a mechanism and possible therapeutic target for vascular-related dementia.

DSS-induced colitis activates the kynurenine pathway in serum and brain by affecting IDO-1 and gut microbiota.

Accumulative studies suggest that inflammatory bowel disease (IBD) may cause multiple central nervous system (CNS) pathologies. Studies have found that indoleamine-2,3-dioxygenase (IDO, rate-limiting enzyme of the kynurenine (Kyn) pathway) deficient mice were protected from endotoxin induced cognitive impairment, and Kyn administration induced cognitive memory deficits in both control and IDO-deficient mice. However, there is no investigation of the brain Kyn pathway in IBD, thus we investigated whether dextran sulfate sodium (DSS)-induced colitis could cause dysregulation of Kyn pathway in brain, and also in serum. C57BL/6J mice were given drinking water with 2% DSS for 10 consecutive days to induce colitis. In serum, we found significant increase in Kyn and kynurenic acid (Kyna) level, which was regulated by IDO-1 and KAT2 (rate-limiting enzymes of Trp-Kyn-Kyna pathway). Similarly, by analyzing GEO datasets, higher IDO-1 levels in peripheral blood monocytes and colon of UC patients was found. Furthermore, the Kyn pathway was significantly upregulated in the cerebral cortex under the action of IDO-1 after DSS treatment, which ultimately induced the neurotoxic phenotype of astrocytes. To investigate whether gut microbiota is involved in IBD-induced Kyn pathway dysregulation, we performed intestinal flora 16S rRNA sequencing and found that DSS-induced colitis significantly altered the composition and diversity of the gut microbiota. Metabolic function analysis also showed that Tryptophan metabolism, NOD-like receptor signaling pathway and MAPK signaling pathway were significantly up-regulated in the 2% DSS group. A significant association between intestinal flora and Trp metabolism (both in serum and brain) was found by correlation analysis. Overall, this study revealed that DSS-induced colitis causes dysregulation of the Kyn pathway in serum and brain by affecting rate-limiting enzymes and intestinal flora.

The Imbalance of Astrocytic Mitochondrial Dynamics Following Blast-Induced Traumatic Brain Injury.

Mild blast-induced traumatic brain injury (bTBI) is a modality of injury that has been of major concern considering a large number of military personnel exposed to explosive blast waves. bTBI results from the propagation of high-pressure static blast forces and their subsequent energy transmission within brain tissue. Exposure to this overpressure energy causes a diffuse injury that leads to acute cell damage and, if chronic, leads to detrimental long-term cognitive deficits. The literature presents a neuro-centric approach to the role of mitochondria dynamics dysfunction in bTBI, and changes in astrocyte-specific mitochondrial dynamics have not been characterized. The balance between fission and fusion events is known as mitochondrial dynamics. As a result of fission and fusion, the mitochondrial structure is constantly altering its shape to respond to physiological stimuli or stress, which in turn affects mitochondrial function. Astrocytic mitochondria are recognized to play an essential role in overall brain metabolism, synaptic transmission, and neuron protection. Mitochondria are vulnerable to injury insults, leading to the increase in mitochondrial fission, a mechanism controlled by the GTPase dynamin-related protein (Drp1) and the phosphorylation of Drp1 at serine 616 (p-Drp1s616). This site is critical to mediate the Drp1 translocation to mitochondria to promote fission events and consequently leads to fragmentation. An increase in mitochondrial fragmentation could have negative consequences, such as promoting an excessive generation of reactive oxygen species or triggering cytochrome c release. The aim of the present study was to characterize the unique pattern of astrocytic mitochondrial dynamics by exploring the role of DRP1 with a combination of in vitro and in vivo bTBI models. Differential remodeling of the astrocytic mitochondrial network was observed, corresponding with increases in p-Drp1S616 four hours and seven days post-injury. Further, results showed a time-dependent reactive astrocyte phenotype transition in the rat hippocampus. This discovery can lead to innovative therapeutics targets to help prevent the secondary injury cascade after blast injury that involves mitochondria dysfunction.

Astrocytes Transplanted during Early Postnatal Development Integrate, Mature, and Survive Long Term in Mouse Cortex.

Astrocytes have complex structural, molecular, and physiological properties and form specialized microenvironments that support circuit-specific functions in the CNS. To better understand how astrocytes acquire their unique features, we transplanted immature mouse cortical astrocytes into the developing cortex of male and female mice and assessed their integration, maturation, and survival. Within days, transplanted astrocytes developed morphologies and acquired territories and tiling behavior typical of cortical astrocytes. At 35-47 d post-transplantation, astrocytes appeared morphologically mature and expressed levels of EAAT2/GLT1 similar to nontransplanted astrocytes. Transplanted astrocytes also supported excitatory/inhibitory (E/I) presynaptic terminals within their territories, and displayed normal Ca2+ events. Transplanted astrocytes showed initially reduced expression of aquaporin 4 (AQP4) at endfeet and elevated expression of EAAT1/GLAST, with both proteins showing normalized expression by 110 d and one year post-transplantation, respectively. To understand how specific brain regions support astrocytic integration and maturation, we transplanted cortical astrocytes into the developing cerebellum. Cortical astrocytes interlaced with Bergmann glia (BG) in the cerebellar molecular layer to establish discrete territories. However, transplanted astrocytes retained many cortical astrocytic features including higher levels of EAAT2/GLT1, lower levels of EAAT1/GLAST, and the absence of expression of the AMPAR subunit GluA1. Collectively, our findings demonstrate that immature cortical astrocytes integrate, mature, and survive (more than one year) following transplantation and retain cortical astrocytic properties. Astrocytic transplantation can be useful for investigating cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms contributing to astrocytic development/diversity, and for determining the optimal timing for transplanting astrocytes for cellular delivery or replacement in regenerative medicine.SIGNIFICANCE STATEMENT The mechanisms that enable astrocytes to acquire diverse molecular and structural properties remain to be better understood. In this study, we systematically analyzed the properties of cortical astrocytes following their transplantation to the early postnatal brain. We found that immature cortical astrocytes transplanted into cerebral cortex during early postnatal mouse development integrate and establish normal astrocytic properties, and show long-term survival in vivo (more than one year). In contrast, transplanted cortical astrocytes display reduced or altered ability to integrate into the more mature cerebral cortex or developing cerebellum, respectively. This study demonstrates the developmental potential of transplanted cortical astrocytes and provides an approach to tease apart cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms that determine the structural, molecular, and physiological phenotype of astrocytes.

Diverse but unique astrocytic phenotypes during embryonic stem cell differentiation, culturing and development

Astrocytes are resident glial cells of the central nervous system (CNS) that play complex and heterogeneous roles in brain development, homeostasis and disease. Since their vast involvement in health and disease is becoming increasingly recognized, suitable and reliable tools for studying these cells in vivo and in vitro are of utmost importance. One of the key challenges hereby is to adequately mimic their context-dependent in vivo phenotypes and functions in vitro. To better understand the spectrum of astrocytic variations in defined settings we performed a side-by-side-comparison of murine embryonic stem cell (ESC)-derived astrocytes as well as primary neonatal and adult astrocytes, revealing major differences on a functional and transcriptomic level, specifically on proliferation, migration, calcium signaling and cilium activity. Our results highlight the need to carefully consider the choice of astrocyte origin and phenotype with respect to age, isolation and culture protocols based on the respective biological question.

Total flavones of Rhododendron induce the transformation of A1/A2 astrocytes via promoting the release of CBS-produced H2S

BackgroundWe previously found that total flavones of Rhododendron (TFR) protected against the cerebral ischemia/reperfusion (I/R) injury. But the detailed mechanism is not clear. Recent research revealed that reactive astrocytes were divided into A1 and A2 phenotypes for their morphological and functional remodeling and neurotoxic- vs-neuroprotective effect on the injury of the central nervous system (CNS). PurposeThe present study was undertaken to explore the role and mechanism of TFR on the phenotypic change of astrocytes following cerebral I/R in vivo and oxygen glucose deprivation/re-oxygenation (OGD/R) in vitro. Study design and methodsWe tested the expression of astrocytes marker glial fibrillary acidic protein (GFAP), A1 astrocytes marker C3 protein and A2 astrocytes marker S100a10, as well as the BrdU/GFAP-positive cells, GFAP/S100a10-positive cells and GFAP/C3-positive cells in mice hippocampal tissues to evaluate the phenotypic change of astrocytes. Besides, we assessed the change of astrocyte phenotypes following OGD/R in vitro. ResultsWe found that mice cerebral I/R promoted the astrocytes proliferation of both A1 and A2 phenotypes in hippocampal tissues. While treatment with TFR could promote the proliferation of A2 astrocytes but inhibit the A1 astrocytes proliferation in mice hippocampal tissues, suggesting that TFR could accelerate the astrocytes transformation into A2 subtype following cerebral I/R. Whereas, in OGD/R model of astrocytes, we found that TFR inhibited the proliferation of both A1 and A2 astrocytes. Besides, we found that TFR could up-regulate the release of cystathionine β-synthase (CBS)-produced hydrogen sulfide (H2S) and inhibit RhoA/Rho kinase pathway, and revealed that the inhibitory effect of TFR on astrocytes proliferation could be blocked by aminooxyacetic acid (AOAA), an CBS inhibitor. Furthermore, TFR could ameliorate the mice cerebral I/R injury and the OGD/R-induced astrocytic damage. ConclusionThese findings suggested that TFR could affect the transformation of astrocytes subtypes following cerebral I/R, which may be related to up-regulation of CBS-produced H2S and subsequent inhibition of RhoA/ROCK pathway.

Activation of innate immune cGAS-STING pathway contributes to Alzheimer's pathogenesis in 5×FAD mice.

cGAS senses microbial and host-derived double-stranded DNA in cytoplasm to trigger cellular innate immune response in a STING-dependent manner; however, it remains unknown whether the cGAS-STING pathway in innate immunity contributes to Alzheimer's disease (AD). Here we demonstrated the detectable binding of the cGAS double-stranded DNA in cytoplasm and the activation of the microglial cGAS-STING pathway in brains of human AD and aged mice. Cgas-/-;5×FAD mice were largely protected from cognitive impairment, amyloid-β pathology, neuroinflammation and other sequelae associated with AD. Furthermore, Cgas deficiency in microglia inhibited a neurotoxic A1 astrocytic phenotype and thus alleviated oligomeric amyloid-β peptide-induced neurotoxicity. Finally, administration of STING inhibitor H-151 potently suppressed the activation of the cGAS-STING pathway and ameliorated AD pathogenesis in 5×FAD mice. In conclusion, our present study has identified a critical molecular link between innate immunity and AD and suggests that therapeutic targeting of the cGAS-STING pathway activity might effectively interfere with the progression of AD.

Metabolic Heterogeneity of Cerebral Cortical and Cerebellar Astrocytes.

Astrocytes play critical roles in regulating neuronal synaptogenesis, maintaining blood-brain barrier integrity, and recycling neurotransmitters. Increasing numbers of studies have suggested astrocyte heterogeneity in morphology, gene profile, and function. However, metabolic phenotype of astrocytes in different brain regions have not been explored. In this paper, we investigated the metabolic signature of cortical and cerebellar astrocytes using primary astrocyte cultures. We observed that cortical astrocytes were larger than cerebellar astrocytes, whereas cerebellar astrocytes had more and longer processes than cortical astrocytes. Using a Seahorse extracellular flux analyzer, we demonstrated that cortical astrocytes had higher mitochondrial respiration and glycolysis than cerebellar astrocytes. Cerebellar astrocytes have lower spare capacity of mitochondrial respiration and glycolysis as compared with cortical astrocytes. Consistently, cortical astrocytes have higher mitochondrial oxidation and glycolysis-derived ATP content than cerebellar astrocytes. In addition, cerebellar astrocytes have a fuel preference for glutamine and fatty acid, whereas cortical astrocytes were more dependent on glucose to meet energy demands. Our study indicated that cortical and cerebellar astrocytes display distinct metabolic phenotypes. Future studies on astrocyte metabolic heterogeneity and brain function in aging and neurodegeneration may lead to better understanding of the role of astrocyte in brain aging and neurodegenerative disorders.

High-intensity interval training ameliorates Alzheimer's disease-like pathology by regulating astrocyte phenotype-associated AQP4 polarization.

Background: Alzheimer's disease (AD), one of the most common forms of dementia, is a widely studied neurodegenerative disease characterized by Aβ accumulation and tau hyperphosphorylation. Currently, there is no effective cure available for AD. The astrocyte AQP4 polarized distribution-mediated glymphatic system is essential for Aβ and abnormal tau clearance and is a potential therapeutic target for AD. However, the role of exercise on the AQP4 polarized distribution and the association between the AQP4 polarized distribution and astrocyte phenotype polarization are poorly understood. Methods: Using a streptozotocin (STZ)-induced sporadic AD rat model, we investigated the effects of high-intensity interval training on AD pathologies. The Branes maze task was conducted to measure spatial learning and memory. Immunofluorescence staining of NeuN with TUNEL, Fluoro-Jade C, and relative neuronal damage markers was applied to measure neuronal apoptosis, neurodegeneration, and damage. Sholl analysis was carried out to analyze the morphology of microglia. Line-scan analysis, 3D rendering, and the orthogonal view were applied to analyze the colocalization. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) analysis were conducted to examine AQP4 and Aβ, respectively. An APP/PS1 transgenic AD mice model was used to confirm the key findings. Results: High-intensity interval training (HIIT) alleviates cognitive dysfunction in STZ-induced AD-like rat models and provides neuroprotection against neurodegeneration, neuronal damage, and neuronal loss. Additionally, HIIT improved the drainage of abnormal tau and Aβ from the cortex and hippocampus via the glymphatic system to the kidney. Further mechanistic studies support that the beneficial effects of HIIT on AD might be due, in part, to the polarization of glial cells from a neurotoxic phenotype towards a neuroprotective phenotype. Furthermore, an intriguing finding of our study is that the polarized distribution of AQP4 was strongly correlated with astrocyte phenotype. We found A2 phenotype exhibited more evident AQP4 polarization than the A1 phenotype. Conclusion: Our findings indicate that HIIT ameliorates Alzheimer's disease-like pathology by regulating astrocyte phenotype and astrocyte phenotype-associated AQP4 polarization. These changes promote Aβ and p-tau clearance from the brain tissue through the glymphatic system and the kidney.

Significant Systemic Insulin Resistance is Associated With Unique Glioblastoma Multiforme Phenotype.

Some glioblastoma multiforme (GBM) are characterized by the presence of gemistocytes (GCs), a unique phenotype of reactive astrocytes. Certain GCs can be identified as neoplastic cells but these cells were also found to be associated with diabetes in non-neoplastic lesions of the central nervous system. Our aim was to find a correlation between insulin - resistance metabolic features and the presence of GCs in patients with newly diagnosed GBM. Medical records from histologically confirmed GBM patients were retrospectively extracted for different systemic metabolic variables. A statistic-based comparison was made between GBM, diabetic patients with and without GC. Patients with poorly controlled diabetes (ie, hemoglobin A1C ⩾ 8.0) were also compared between the 2 groups. A total of 220 newly diagnosed GBM patients were included in our study. 58 (26.3%) patients had a history of diabetes mellitus type 2 (DM2) at the time of admission. The rate of poorly-controlled DM2 was nearly as twice in the GC-GBM group than in the non-GC GBM group (18.75% vs 9.5%; P = .130). In the DM2 cohort, the subgroup of GC-GBM was significantly associated with demographic and metabolic features related to insulin resistance such as male gender predominance (89% vs 50%, P = .073) and morbid obesity (weight ⩾85 kg: OR 6.16; P = .0019 and mean BMI: 34.1 ± 11.42 vs 28.7 ± 5.44; P = .034 for group with and without GCs, respectively). In the poorly-controlled DM2 group, none of the GC-GBM patients were using insulin prior to diagnosis, compared to 61.1% in the non-GC GBM patients (OR = 0.04, P = .045). Systemic metabolic factors related to marked insulin resistance (DM2, morbid obesity, male gender) are associated with a unique histologic phenotype of GBM, characterized by the presence of GCs. This feature is prominent in poorly-controlled DM2 GBM patients who are not using synthetic insulin. This novel finding may add to the growing data on the relevance of glucose metabolism in astrocytes and in astrocytes associated with high-grade gliomas. In GBM patients, a correlation between patients' metabolic status, tumor's histologic phenotype, tumor's molecular changes, use of anti-diabetic drugs and the respective impact of these factor on survival warrants further investigation.

Micro-RNAs Shuttled by Extracellular Vesicles Secreted from Mesenchymal Stem Cells Dampen Astrocyte Pathological Activation and Support Neuroprotection in In-Vitro Models of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with no effective cure. Astrocytes display a toxic phenotype in ALS and contribute to motoneuron (MN) degeneration. Modulating astrocytes' neurotoxicity can reduce MN death. Our previous studies showed the beneficial effect of mesenchymal stem cell (MSC) administration in SOD1G93A ALS mice, but the mechanisms are still unclear. We postulated that the effects could be mediated by extracellular vesicles (EVs) secreted by MSCs. We investigated, by immunohistochemical, molecular, and in vitro functional analyses, the activity of MSC-derived EVs on the pathological phenotype and neurotoxicity of astrocytes isolated from the spinal cord of symptomatic SOD1G93A mice and human astrocytes (iAstrocytes) differentiated from inducible neural progenitor cells (iNPCs) of ALS patients. In vitro EV exposure rescued mouse and human ALS astrocytes' neurotoxicity towards MNs. EVs significantly dampened the pathological phenotype and neuroinflammation in SOD1G93A astrocytes. In iAstrocytes, exposure to EVs increased the antioxidant factor Nrf2 and reduced reactive oxygen species. We previously found nine miRNAs upregulated in MSC-derived EVs. Here, the transfection of SOD1G93A astrocytes with single miRNA mimics reduced astrocytes' activation and the expression of neuroinflammatory factors. Moreover, miR-466q and miR-467f mimics downregulate Mapk11, while miR-466m-5p and miR-466i-3p mimics promote the nuclear translocation of Nrf2. In iAstrocytes, transfection with miR-29b-3p mimic upregulated NQO1 antioxidant activity and reduced neurotoxicity towards MNs. MSC-derived EVs modulate astrocytes' reactive phenotype and neurotoxicity through anti-inflammatory and antioxidant-shuttled miRNAs, thus representing a therapeutic strategy in ALS.

Amyloid and tau‐driven signatures in hippocampal GFAP‐positive astrocytes

AbstractBackgroundDespite the known heterogeneity of astrocyte responses to injuries, Alzheimer’s disease (AD) pre‐clinical and clinical research relies on the glial fibrillary acidic protein (GFAP) as a proxy of astrocyte reactivity. Recently, it was demonstrated that plasma GFAP levels differentially relate to AD hallmarks, associating with amyloid‐β (Aβ), but not with tau pathology. However, the molecular underpinnings behind this specificity are still unexplored. Here, we aim to uncover the molecular signatures of GFAP‐positive astrocytes driven by either by Aβ or tau pathology.MethodHippocampal GFAP‐positive astrocytes data from 6 month‐old TauP301S (n = 5), 11.5 months‐old APP/PS2 (n = 5) and their wild‐type littermates (WT, n = 5) were acquired from GEO datasets (GSE129770;GSE129797) and used to identify differentially expressed genes (DEGs) and enriched biological processes. DEGs were evaluated using the DESeq2 method. Further data analysis included Gene Ontology (GO) functional evaluation.ResultPrincipal component analysis clearly differentiated gene expression of GFAP‐positive astrocytes from APP/PS2, TauP301S and their WT controls (Fig.1A). Interestingly, only 30 DEGs overlapped among GFAP‐positive astrocytes from these two transgenic mouse models, revealing multiple DEGs specifically associated with Tau or Aβ (Fig.1B). The functional enrichment analysis revealed 36 GO terms enriched with DEGs altered in the PS2APP mice compared to WT. The most significantly enriched terms include processes such as “protein localization to cell periphery and plasma membrane”, “interleukin−6 production and regulation”, “exocytosis” and “neuron death” (Fig.1C). For the comparison of the TauP301S mice versus WT controls, the functional enrichment analysis using DEGs revealed 95 terms. Among the most significantly enriched terms, we found “learning”, “cognition”, and “small GTPase mediated signal transduction” (Fig.1C).ConclusionOverall, we observed a small overlap of DEGs between the Aβ and tau mouse models, indicating the unique nature of GFAP‐positive astrocytic responses to different aspects of AD pathology. The findings presented in this study allowed the identification of functional categories and biological processes particular to Aβ or tau burden. Characterizing how these upstream triggers influence GFAP‐positive astrocyte phenotypes may contribute to the biological interpretation of GFAP as a biomarker of AD pathology and pave the way to unravel novel mechanisms and context‐specific astrocyte biomarkers for AD.

Perturbed astrocyte Ca2+ signaling in a diet‐based mouse model of small cerebral vessel disease

AbstractBackgroundThe hyperhomocysteinemia (HHcy) diet model of small cerebral vessel disease exhibits vascular inflammation, cerebral hypoperfusion, impaired neurovascular coupling, synapse dysfunction, and cognitive decline. Pathological changes in astrocyte structural features have also been noted, but functional astrocyte phenotypes have not been characterized. Here, we assessed Ca2+ signaling in individual astrocytes and astrocyte networks in barrel cortex of mice, pretreated with control or HHcy diet. Investigations were performed in fully awake mice to avoid confounding interactions between anesthetics, neural circuit activity, and vasoactive factors.MethodAdult C57/BL6 mice were fed for 15 weeks with control chow, or chow deficient in vitamins B6/B12 and enriched in methionine (HHcy diet). At week 11‐12, mice were injected with AAV‐Gfa104‐lck‐GCaMP6f into barrel cortex, followed by craniotomies and installation of a glass cranial windows with headmounts. Three weeks later, fully awake mice were head‐mounted under a two photon microscope to assess GCaMP6/Ca2+ transient activity in barrel cortex astrocytes during a 10 minute baseline (spontaneous activity) and also before, during, and air puff stimulation of contralateral whiskers (10Hz, 10 sec) . Mean Ca2+ transient amplitudes, transient rise/decay times, and number of transients per region of interest (ROI) were measured and averaged across the field of view (FOV). Functional relationships between multiple ROIs within the FOV were assessed to evaluate astrocyte network activityResultSpontaneous Ca2+ transients were comparable in amplitude and rise/decay kinetics across diet conditions. However, the number of transients per ROI was reduced in HHcy diet mice. Whisker stimulation evoked a primary Ca2+ elevation per ROI followed 10‐15 seconds later by a smaller amplitude Ca2+‐“aftershock”. Evoked Ca2+ signals were highly coordinated across ROIs within the FOV. However, the number of active ROIs and the functional connectivity between ROIs were significantly reduced in HHcy vs. control diet mice. Despite these network deficits, evoked Ca2+ transient amplitudes and aftershocks in individual ROIs were significantly larger with faster activation/decay kinetics in HHcy mice.ConclusionThe results suggest that altered astrocyte Ca2+ signaling, as a result of vascular pathology, may play an important role in the dynamic interplay between cerebral blood flow and neuronal activity, leading to overall neural dysfunction and cognitive impairment.

Knockout of transient receptor potential ankyrin 1 (TRPA1) modulates the glial phenotype and alleviates perihematomal neuroinflammation after intracerebral hemorrhage in mice via MAPK/NF-κB signaling.

The objective is to explore the role of astrocytic transient receptor potential ankyrin 1 (TRPA1) in glial phenotype transformation in neuroinflammation after intracerebral hemorrhage (ICH). Wild-type astrocytes and TRPA1-/- astrocytes were subjected to 6-h hemin treatment, and the calcium ions and transcriptome sequencing were assessed. A mouse autologous blood injection ICH model was established to evaluate the proliferation and phenotypes of astrocytes and microglia around the hematoma. The neuroinflammation and behavioral performance of wild-type ICH mice and TRPA1-/- ICH mice were assessed. Knockout of astrocytic TRPA1 decreased calcium ions of astrocytes after hemin treatment in-vitro, and microglial and astrocytes around the hematoma proliferated after the ICH model. Furthermore, RNA-sequencing (RNA-seq), immunofluorescence, and Western blotting results showed that the activated astrocytes transformed into the A2 phenotype in TRPA1-/- ICH mice. The 'ameboid' microglia were observed around the hematoma in TRPA1-/- ICH mice. The proliferation of A2 astrocytes and 'ameboid' microglia ameliorated the neuroinflammation after ICH. The inflammatory response was reduced by inhibiting the mitogen-activated protein kinase/nuclear factor kappa-B signaling pathway, and neurologic deficits were improved in TRPA1-/- ICH mice compared with wild-type ICH mice. This research suggests that astrocytic TRPA1 is a new therapeutic target to rescue neuroinflammation by modulating the glial phenotype after ICH.