Astrocytes In Cortex Research Articles

NDRG2 Expression Correlates with Neurofibrillary Tangles and Microglial Pathology in the Ageing Brain.

Astrocytes play a major role in the pathogenesis of a range of neurodegenerative diseases, including Alzheimer’s disease (AD), undergoing dramatic morphological and molecular changes that can cause potentially both beneficial and detrimental effects. They comprise a heterogeneous population, requiring a panel of specific phenotype markers to identify astrocyte subtypes, changes in function and their relation to pathology. This study aimed to characterise expression of the astrocyte marker N-myc downstream regulated gene 2 (NDRG2) in the ageing brain, investigate the relationship between NDRG2 and a panel of astrocyte markers, and relate NDRG2 expression to pathology. NDRG2 specifically immunolabelled the cell body and radiating processes of astrocytes in the temporal cortex of the Cognitive Function and Ageing Study (CFAS) neuropathology cohort. Expression of NDRG2 did not correlate with other astrocyte markers, including glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 2 (EAAT2) and glutamine synthetase (GS). NDRG2 showed a relationship to AT8+ neurofibrillary tangles (p = 0.001) and CD68+ microglia (p = 0.047), but not β-amyloid plaques or astrocyte nuclear γH2AX immunoreactivity, a marker of DNA damage response. These findings provide new insight into the astrocyte response to pathology in the ageing brain, and suggest NDRG2 may be a potential target to modulate this response.

Chimera RNA interference knockdown of γ-synuclein in human cortical astrocytes results in mitotic catastrophe.

Elevated levels of γ-synuclein (γ-syn) expression have been noted in the progression of glioblastomas, and also in the cerebrospinal fluid of patients diagnosed with neurodegenerative diseases. γ-Syn can be either internalized from the extracellular milieu or expressed endogenously by human cortical astrocytes. Internalized γ-syn results in increased cellular proliferation, brain derived neurotrophic factor release and astroprotection. However, the function of endogenous γ-syn in primary astrocytes, and the relationship to these two opposing disease states are unknown. γ-Syn is expressed by astrocytes in the human cortex, and to gain a better understanding of the role of endogenous γ-syn, primary human cortical astrocytes were treated with chimera RNA interference (RNAi) targeting γ-syn after release from cell synchronization. Quantitative polymerase chain reaction analysis demonstrated an increase in endogenous γ-syn expression 48 hours after release from cell synchronization, while RNAi reduced γ-syn expression to control levels. Immunocytochemistry of Ki67 and 5-bromodeoxyuridine showed chimera RNAi γ-syn knockdown reduced cellular proliferation at 24 and 48 hours after release from cell synchronization. To further investigate the consequence of γ-syn knockdown on the astrocytic cell cycle, phosphorylated histone H3 pSer10 (pHH3) and phosphorylated cyclin dependent kinase-2 pTyr15 (pCDK2) levels were observed via western blot analysis. The results revealed an elevated expression of pHH3, but not pCDK2, indicating γ-syn knockdown leads to disruption of the cell cycle and chromosomal compaction after 48 hours. Subsequently, flow cytometry with propidium iodide determined that increases in apoptosis coincided with γ-syn knockdown. Therefore, γ-syn exerts its effect to allow normal astrocytic progression through the cell cycle, as evidenced by decreased proliferation marker expression, increased pHH3, and mitotic catastrophe after knockdown. In this study, we demonstrated that the knockdown of γ-syn within primary human cortical astrocytes using chimera RNAi leads to cell cycle disruption and apoptosis, indicating an essential role for γ-syn in regulating normal cell division in astrocytes. Therefore, disruption to γ-syn function would influence astrocytic proliferation, and could be an important contributor to neurological diseases.

Morphology of GFAP-Positive Cells in Male and Female Rats’ Cerebral Cortex during the Cerebral Hypoxia Development according to the Stress Tolerance Level

Correlation between individual typological features like sex-related characteristics and low initial level of stress resistance and unfavorable prognosis of ischemic brain damage is shown. At the same time, the astrocytes’ participation in neuroplasticity in this disease represents the purpose of the study. The goal was to study the cerebral cortex astrocytes’ morphometric characteristics during the cerebral hypoxia in rats of different sexes with different stress tolerance levels. We performed the study with 72 Wistar rats. According to the Open Field test results all the animals were divided into two subgroups: with high and low level of stress tolerance. Both carotid arteries of the experimental group animals (48 animals) were bandaged. Animals were removed from the experiment at 21, 60 and 90 days after surgery. Glial fibrillary acidic protein (GFAP), a marker of mature astrocytes, was detected on histological sections of the central brain gyrus using primary polyclonal rabbit antibodies. Progressive decrease of the astrocytes’ numerical density and the number of first order processes were obtained during the study. It was less pronounced in animals with high stress tolerance and females. Increase of the processes’ distribution area was reliably detected. Area decreased after 90th day of the experiment. It is concluded that the astrocytes’ alteration develops earlier in animals with high stress tolerance and males, later in females (60 days) and animals with low stress tolerance (90 days).

Effects of a High-Fat Diet on Neuroinflammation and Apoptosis in Acute Stage After Moderate Traumatic Brain Injury in Rats.

A high-fat diet (HFD) is correlated with a higher risk of metabolic syndrome. The effect of HFD on neuroinflammation and apoptosis in acute stage after traumatic brain injury (TBI) in rats is not well known. Five-week-old male Sprague-Dawley (SD) rats were fed a HFD or normal diet (ND) for 8weeks. Anesthetized male SD rats were divided into two subgroups: sham-operated and TBI. Motor function was measured using an inclined plane. The numbers of apoptotic neurons (markers Neu-N, TUNEL, caspase-3), activated astrocytes (marker GFAP) and microglia (marker OX42), and TNF-α expression in microglia and astrocytes in the ischemic cortex were investigated using an immunofluorescence assay. All of the parameters were measured on the 3rd day after TBI. Rats fed a HFD for 8weeks had higher body weight, serum cholesterol, and triglycerides. TBI-induced motor deficits, neuronal decrease, neuronal apoptosis, astrocyte and microglia activation, and TNF-α expression in microglia and astrocytes in the ischemia cortex were significantly increased in ND and HFD rats compared to sham rats. However, these parameters were not significantly different between the ND and HFD TBI groups, except motor deficit. HFD has no significant effects on neuronal apoptosis or neuroinflammation in acute stage compared with ND for 8weeks after moderate TBI in experimental rats.

EV71 infection induces neurodegeneration via activating TLR7 signaling and IL-6 production.

As a neurotropic virus, human Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease (HFMD) and may develop severe neurological disorders in infants. Toll-like receptor 7 (TLR7) acts as an innate immune receptor and is also a death receptor in the central nervous system (CNS). However, the mechanisms underlying the regulation of TLR7-mediated brain pathogenesis upon EV71 infection remain largely elusive. Here we reveal a novel mechanism by which EV71 infects astrocytes in the brain and induces neural pathogenesis via TLR7 and interleukin-6 (IL-6) in C57BL/6 mice and in human astroglioma U251 cells. Upon EV71 infection, wild-type (WT) mice displayed more significant body weight loss, higher clinical scores, and lower survival rates as compared with TLR7-/- mice. In the cerebral cortex of EV71-infected mice, neurofilament integrity was disrupted, and inflammatory cell infiltration and neurodegeneration were induced in WT mice, whereas these were largely absent in TLR7-/- mice. Similarly, IL-6 production, Caspase-3 cleavage, and cell apoptosis were significantly higher in EV71-infected WT mice as compared with TLR7-/- mice. Moreover, EV71 preferentially infected and induced IL-6 in astrocytes of mice brain. In U251 cells, EV71-induced IL-6 production and cell apoptosis were suppressed by shRNA-mediated knockdown of TLR7 (shTLR7). Moreover, in the cerebral cortex of EV71-infected mice, the blockade of IL-6 with anti-IL-6 antibody (IL-6-Ab) restored the body weight loss, attenuated clinical scores, improved survival rates, reduced the disruption of neurofilament integrity, decreased cell apoptotic induction, and lowered levels of Caspase-3 cleavage. Similarly, in EV71-infected U251 cells, IL-6-Ab blocked EV71-induced IL-6 production and cell apoptosis in response to viral infection. Collectively, it’s exhibited TLR7 upregulation, IL-6 induction and astrocytic cell apoptosis in EV71-infected human brain. Taken together, we propose that EV71 infects astrocytes of the cerebral cortex in mice and human and triggers TLR7 signaling and IL-6 release, subsequently inducing neural pathogenesis in the brain.

The Neuroprotective Effects of Simvastatin on High Cholesterol Following Traumatic Brain Injury in Rats

High cholesterol has been correlated with a greater risk of cerebrovascular diseases. Whether pre-existing high cholesterol exacerbates traumatic brain injury (TBI), and whether treatment with the cholesterol-lowering agent simvastatin has neuroprotective effects, especially anti-neuroinflammatory effects, after TBI are not well investigated. Five-week-old male Sprague-Dawley rats were fed a high-fat diet for 8 weeks to induce hypercholesterolemia. Anesthetized male Sprague-Dawley rats were divided into 5 groups, including the sham-operated control, TBI control, and TBI with simvastatin treatment (4 mg/kg, 10 mg/kg, or 20 mg/kg) groups. Simvastatin was intraperitoneally injected at 0, 24, and 48 hours after TBI. Motor function was measured using an inclined plane. Neuronal apoptosis (maker Neu-N, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling), tumor necrosis factor-α expression in microglia (marker OX42) and astrocytes (marker glial fibrillary acidic protein), and Tumor necrosis factor-alpha receptor (TNFR) 1 and TNFR2 expression in neurons in the ischemic cortex were investigated using an immunofluorescence assay. All of the parameters were measured on the third day after TBI. TBI significantly increased the serum levels of cholesterol. The TBI-induced motor deficit was significantly attenuated by 4, 10, and 20 mg/kg simvastatin therapy on the third day after TBI. TBI-induced neuronal TNFR1 activation and apoptosis, as well as tumor necrosis factor-α expression in astrocytes in the ischemic cortex, were significantly attenuated by simvastatin, particularly when 20 mg/kg was administered. Simultaneously, the serum cholesterol remained high despite simvastatin treatment. The neuroprotection effects of simvastatin on the pre-existing hypercholesterolemia during TBI in rats may be related to its anti-neuroinflammatory effects but not to its cholesterol-lowing effects.

Rehabilitative training-induced functional vicariation is associated with coordinated plasticity of synapses and astrocytes in peri-infarct motor cortex

Rehabilitative training-induced functional vicariation is associated with coordinated plasticity of synapses and astrocytes in peri-infarct motor cortex

Isorhynchophylline ameliorates cognitive impairment via modulating amyloid pathology, tau hyperphosphorylation and neuroinflammation: Studies in a transgenic mouse model of Alzheimer’s disease

Isorhynchophylline (IRN) has been demonstrated to have distinct anti-Alzheimer’s disease (AD) activity in several animal models of AD. In this study, we aimed at evaluating the preventive effect of IRN on the cognitive deficits and amyloid pathology in TgCRND8 mice. Male TgCRND8 mice were administered with IRN (20 or 40 mg/kg) by oral gavage daily for 4 months, followed by assessing the spatial learning and memory functions with the Radial Arm Maze (RAM) test. Brain tissues were determined immunohistochemically or biochemically for changes in amyloid pathology, tau hyperphosphorylation and neuroinflammation. Our results revealed that IRN (40 mg/kg) significantly ameliorated cognitive deficits in TgCRND8 mice. In addition, IRN (40 mg/kg) markedly reduced the levels of Aβ40, Aβ42 and tumor necrosis factor (TNF-α), interleukin 6 (IL-6) and IL-1β, and modulated the amyloid precursor protein (APP) processing and phosphorylation by altering the protein expressions of β-site APP cleaving enzyme-1 (BACE-1), phosphorylated APP (Thr668), presenilin-1 (PS-1) and anterior pharynx-defective-1 (APH-1), as well as insulin degrading enzyme (IDE), a major Aβ-degrading enzyme. IRN was also found to inhibit the phosphorylation of tau at the sites of Thr205 and Ser396. Immunofluorescence showed that IRN reduced the Aβ deposition, and suppressed the activation of microglia (Iba-1) and astrocytes (GFAP) in the cerebral cortex and hippocampus of TgCRND8 mice. Furthermore, IRN was able to attenuate the ratios of p-c-Jun/c-Jun and p-JNK/JNK in the brains of TgCRND8 mice. IRN also showed marked inhibitory effect on JNK signaling pathway in the Aβ-treated rat primary hippocampus neurons. We conclude that IRN improves cognitive impairment in TgCRND8 transgenic mice via reducing Aβ generation and deposition, tau hyperphosphorylation and neuroinflammation through inhibiting the activation of JNK signaling pathway, and has good potential for further development into pharmacological treatment for AD.

Cortical Plasticity Induced by Anodal Transcranial Pulsed Current Stimulation Investigated by Combining Two-Photon Imaging and Electrophysiological Recording.

Anodal-transcranial pulsed current stimulation (a-tPCS) has been used in human studies to modulate cortical excitability or improve behavioral performance in recent years. Multiple studies show crucial roles of astrocytes in cortical plasticity. The calcium activity in astrocytes could regulate synaptic transmission and synaptic plasticity. Whether the astrocytic activity is involved in a-tPCS-induced cortical plasticity is presently unknown. The purpose of this study is to investigate the calcium responses in neurons and astrocytes evoked by a-tPCS with different current intensities, and thereby provides some indication of the mechanisms underlying a-tPCS-induced cortical plasticity. Two-photon calcium imaging was used to record the calcium responses of neurons and astrocytes in mouse somatosensory cortex. Local field potential (LFP) evoked by sensory stimulation was used to assess the effects of a-tPCS on plasticity. We found that long-duration a-tPCS with high-intensity current could evoke large-amplitude calcium responses in both neurons and astrocytes, whereas long-duration a-tPCS with low-intensity current evoked large-amplitude calcium responses only in astrocytes. The astrocytic Ca2+ elevations are driven by noradrenergic-dependent activation of the alpha-1 adrenergic receptors (A1ARs), while the intense Ca2+ responses of neurons are driven by action potentials. LFP recordings demonstrated that low-intensity a-tPCS led to enhancement of cortical excitability while high-intensity a-tPCS resulted in diminution of cortical excitability. The results provide some evidence that the enhancement of a-tPCS-induced cortical excitability might be partly associated with calcium elevation in astrocytes, whereas the diminution of a-tPCS-induced cortical excitability might be caused by excessive calcium activity in neurons. These findings indicate that the appropriate current intensity should be used in the application of a-tPCS.

Shape-dependent toxicity of alumina nanoparticles in rat astrocytes

Nanosized alumina (Al2O3-NPs), a widely used nanoparticle in numerous commercial applications, is released into environment posing a threat to the health of wildlife and humans. Recent research has revealed essential roles of physicochemical properties of nanoparticles in determining their toxicity potencies. However, influence of shape on neurotoxicity induced by heterogeneous Al2O3-NPs remains unknown. We herein compared the neurotoxicity of two shapes of γ-Al2O3-NPs (flake versus rod) and their effects on metabolic profiles of astrocytes in rat cerebral cortex. While exposed to both shapes caused significant cytotoxicity and apoptosis in a dose-dependent manner after 72 h exposure, a significantly stronger response was observed for nanorods than for nanoflakes. These effects were associated with significantly greater ROS accumulation and inflammation induction, as indicated by increased concentrations of IL-1β, IL-2 and IL-6. Using untargeted metabolomics, significant alternations in metabolism of amino acids, lipids and purines, and pyrimidines were observed after exposure to both types. Moreover, changes in the metabolome caused by nanorods were significantly greater than those by nanoflakes as also indicated by physiological stress responses to ROS, inflammation, and apoptosis. Taken together, these findings demonstrated the critical role of morphology in determining toxic potencies of nano-alumina and its underlying mechanisms of toxic actions.

Neuroinflammation in Gulf War Illness is linked with HMGB1 and complement activation, which can be discerned from brain-derived extracellular vesicles in the blood

Cognitive dysfunction and neuroinflammation are conspicuously observed in Gulf War Illness (GWI). We investigated whether brain inflammation in GWI is associated with activation of high mobility group box-1 (HMGB1) and complement-related proteins in neurons and astrocytes, and brain inflammation can be tracked through neuron-derived extracellular vesicles (NDEVs) and astrocyte-derived EVs (ADEVs) found in the circulating blood. We exposed animals to GWI-related chemicals pyridostigmine bromide, DEET and permethrin, and moderate stress for 28 days. We performed behavioral tests 10 months post-exposure and quantified activated microglia and reactive astrocytes in the cerebral cortex. Then, we measured the concentration of HMGB1, proinflammatory cytokines, and complement activation-related proteins in the cerebral cortex, and NDEVs and ADEVs in the circulating blood. Cognitive impairments persisted in GWI rats at 10 months post-exposure, which were associated with increased density of activated microglia and reactive astrocytes in the cerebral cortex. Moreover, the level of HMGB1 was elevated in the cerebral cortex with altered expression in the cytoplasm of neuronal soma and dendrites as well as the extracellular space. Also, higher levels of proinflammatory cytokines (TNFa, IL-1b, and IL-6), and complement activation-related proteins (C3 and TccC5b-9) were seen in the cerebral cortex. Remarkably, increased levels of HMGB1 and proinflammatory cytokines observed in the cerebral cortex of GWI rats could also be found in NDEVs isolated from the blood. Similarly, elevated levels of complement proteins seen in the cerebral cortex could be found in ADEVs. The results provide new evidence that persistent cognitive dysfunction and chronic neuroinflammation in a model of GWI are linked with elevated HMGB1 concentration and complement activation. Furthermore, the results demonstrated that multiple biomarkers of neuroinflammation could be tracked reliably via analyses of NDEVs and ADEVs in the circulating blood. Execution of such a liquid biopsy approach is especially useful in clinical trials for monitoring the remission, persistence or progression of brain inflammation in GWI patients with drug treatment.

FGF Signaling Directs the Cell Fate Switch from Neurons to Astrocytes in the Developing Mouse Cerebral Cortex.

During mammalian neocortical development, neural precursor cells generate neurons first and astrocytes later. The cell fate switch from neurons to astrocytes is a key process generating proper numbers of neurons and astrocytes. Although the intracellular mechanisms regulating this cell fate switch have been well characterized, extracellular regulators are still largely unknown. Here, we uncovered that fibroblast growth factor (FGF) regulates the cell fate switch from neurons to astrocytes in the developing cerebral cortex using mice of both sexes. We found that the FGF signaling pathway is activated in radial glial cells of the ventricular zone at time points corresponding to the switch in cell fate. Our loss- and gain-of-function studies using in utero electroporation indicate that activation of FGF signaling is necessary and sufficient to change cell fates from neurons to astrocytes. We further found that the FGF-induced neuron-astrocyte cell fate switch is mediated by the MAPK pathway. These results indicate that FGF is a critical extracellular regulator of the cell fate switch from neurons to astrocytes in the mammalian cerebral cortex.SIGNIFICANCE STATEMENT Although the intracellular mechanisms regulating the neuron-astrocyte cell fate switch in the mammalian cerebral cortex during development have been well studied, their upstream extracellular regulators remain unknown. By using in utero electroporation, our study provides in vivo data showing that activation of FGF signaling is necessary and sufficient for changing cell fates from neurons to astrocytes. Manipulation of FGF signaling activity led to drastic changes in the numbers of neurons and astrocytes. These results indicate that FGF is a key extracellular regulator determining the numbers of neurons and astrocytes in the mammalian cerebral cortex, and is indispensable for the establishment of appropriate neural circuitry.

Ibuprofen Treatment Reduces the Neuroinflammatory Response and Associated Neuronal and White Matter Impairment in the Growth Restricted Newborn.

Intrauterine growth restriction (IUGR) is a condition where the fetus does not achieve optimal growth, commonly caused by placental insufficiency. The chronic decrease in blood flow restricts oxygen and nutrient supply to the fetus, which can damage numerous organ systems, with the fetal brain being particularly vulnerable. Although white matter and neuronal injury are evident in IUGR infants, the specific mechanisms underlying these changes are poorly understood. Inflammation is considered to be a main driver in exacerbating brain injury. Using a spontaneous piglet model of IUGR, we aim to determine whether administration of the anti-inflammatory drug ibuprofen will decrease inflammation at postnatal day 4 (P4). The treatment group received ibuprofen (20 mg/kg/day on day 1 and 10 mg/kg/day on days 2 and 3) in piglet formula during the morning feed each day and brains examined on P4. Markers of inflammation, apoptosis, cell proliferation, neuronal injury, and white matter injury were examined. Ibuprofen treatment ameliorated the increase in numbers of microglia and astrocytes in the parietal cortex and white matter tracts of the IUGR piglet brain on P4 as well as decreasing proinflammatory cytokines. Ibuprofen treatment prevented the reduction in apoptosis, neuronal cell counts, and myelin index in the IUGR piglets. Our findings demonstrate ibuprofen reduces the inflammatory response in the IUGR neonatal brain and concurrently reduces neuronal and white matter impairment.

Differences in glutamate uptake between cortical regions impact neuronal NMDA receptor activation

Removal of synaptically-released glutamate by astrocytes is necessary to spatially and temporally limit neuronal activation. Recent evidence suggests that astrocytes may have specialized functions in specific circuits, but the extent and significance of such specialization are unclear. By performing direct patch-clamp recordings and two-photon glutamate imaging, we report that in the somatosensory cortex, glutamate uptake by astrocytes is slower during sustained synaptic stimulation when compared to lower stimulation frequencies. Conversely, glutamate uptake capacity is increased in the frontal cortex during higher frequency synaptic stimulation, thereby limiting extracellular buildup of glutamate and NMDA receptor activation in layer 5 pyramidal neurons. This efficient glutamate clearance relies on Na+/K+-ATPase function and both GLT-1 and non-GLT-1 transporters. Thus, by enhancing their glutamate uptake capacity, astrocytes in the frontal cortex may prevent excessive neuronal excitation during intense synaptic activity. These results may explain why diseases associated with network hyperexcitability differentially affect individual brain areas.

Evidence of Aquaporin 4 Regulation by Thyroid Hormone During Mouse Brain Development and in Cultured Human Glioblastoma Multiforme Cells.

Accumulating evidence indicates that thyroid function and the thyroid hormones L-thyroxine (T4) and L-triiodothyronine (T3) are important factors contributing to the improvement of various pathologies of the central nervous system, including stroke, and various types of cancer, including glioblastoma multiforme (GBM). Low levels of T3 are correlated with the poorest outcome of post-stroke brain function, as well as an increased migration and proliferation of GBM tumor cells. Thyroid hormones are known to stimulate maturation and brain development. Aquaporin 4 (AQP4) is a key factor mediating the cell swelling and edema that occurs during ischemic stroke, and plays a potential role in the migration and proliferation of GBM tumor cells. In this study, as a possible therapeutic target for GBM, we investigated the potential role of T3 in the expression of AQP4 during different stages of mouse brain development. Pregnant mice at gestational day 18, or young animals at postnatal days 27 and 57, received injection of T3 (1 μg/g) or NaOH (0.02 N vehicle). The brains of mice sacrificed on postnatal days 0, 30, and 60 were perfused with 4% paraformaldehyde and sections were prepared for immunohistochemistry of AQP4. AQP4 immunofluorescence was measured in the mouse brains and human GBM cell lines. We found that distribution of AQP4 was localized in astrocytes of the periventricular, subpial, and cerebral parenchyma. Newborn mice treated with T3 showed a significant decrease in AQP4 immunoreactivity followed by an increased expression at P30 and a subsequent stabilization of aquaporin levels in adulthood. All GBM cell lines examined exhibited significantly lower AQP4 expression than cultured astrocytes. T3 treatment significantly downregulated AQP4 in GBM-95 cells but did not influence the rate of GBM cell migration measured 24 h after treatment initiation. Collectively, our results showed that AQP4 expression is developmentally regulated by T3 in astrocytes of the cerebral cortex of newborn and young mice, and is discretely downregulated in GBM cells. These findings indicate that higher concentrations of T3 thyroid hormone would be more suitable for reducing AQP4 in GBM tumorigenic cells, thereby resulting in better outcomes regarding the reduction of brain tumor cell migration and proliferation.

Propagation and Coordination of Intercellular Astrocyte Ca2+ Waves by Glutamatergic Signaling

Brain functions are highly dependent on a fine regulation of cerebral blood flow by a mechanism known as neurovascular coupling. This mechanism is mediated by astrocytes, which are located between neurons and parenchymal arterioles. Neurotransmitters released during an increase in synaptic activity activate receptors located on astrocytes, which initiates Ca2+ waves that are propagated to the astrocytic endfeet and neighboring astrocytes through gap junctions and activation of a purinergic signaling by ATP release via connexin (Cx)‐formed hemichannels. However, Cx hemichannels may also participate in the astrocyte signaling by providing the pathway for the release of D‐serine and glutamate. However, the relevance of this process in the astrocytic Ca2+ signaling has not been determined. We evaluated if activation of metabotropic glutamate receptors (mGluR) and NMDA receptors (NMDAR) contribute to the propagation of astrocytic Ca2+ signals in primary cultures of rat cortex astrocytes loaded with the Ca2+ indicator Fluo‐4. Ca2+ waves were initiated in a single astrocyte by applying a controlled mechanical stimulation (MS) using a micropipette. Stimulation with glutamate or t‐ACPD, a mGluR agonist, induced a rapid increase in astrocyte [Ca2+]i, which was abolished by MPEP, a mGluR5 antagonist, but, in addition, this response was attenuated by DL‐AP5, a NMDAR antagonist; phenazine methosulfate (Phen‐Meth), a serine racemase inhibitor; or D‐amino acid oxidase (DAOX), an enzyme that deaminates D‐serine. As expected, stimulation with NMDA plus D‐serine evoked an increase in astrocyte [Ca2+]i, but, interestingly, this NMDAR‐mediated Ca2+ signal was abolished by MPEP. On the other hand, blockade of connexin‐based hemichannels with the Cx blocking peptide 37,43Gap27 also attenuated the glutamate‐elicited increase in [Ca2+]i. Consistent with the involvement of hemichannels in the astrocyte communication, the intercellular propagation of the Ca2+ signaling initiated by MS was inhibited by MPEP, DL‐AP5 or Phen‐Meth. These results suggest that glutamate and D‐serine release through hemichannels coordinates the Ca2+ signal initiated by NMDAR and mGluR, which contributes to enhance the propagation of astrocytic Ca2+ waves.Support or Funding InformationGrants: FONDECYT 1150530 and CONICYT 21160646.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuro-protective effect of monomethyl fumarate on ischemia reperfusion injury in rats: Role of Nrf2/HO1 pathway in peri-infarct region

Post stroke recanalization has been associated with increased risk of oxidative stress. Stimulating endogenous antioxidant pathway by activation of nuclear factor erythroid-2-related factor-2 (Nrf2) plays a key role in neuronal defense against inflammation and oxidative stress in penumbra. Here, we explored whether monomethyl fumarate (MMF) could produce neuro-protection after ischemia/reperfusion (I/R) injury via Nrf2/HO1 activation. In male SD rats, middle cerebral artery was occluded for 90 min and confirmed using Laser Doppler flowmeter. MMF (10, 20 and 40 mg/kg) was administered in two divided doses at 30 min post ischemia and 5–10 min after reperfusion. After 24 h, effect on neurobehavioral parameters, infarct damage by TTC staining and MRI, oxidative stress and inflammatory cytokines were assessed. Expression studies of nuclear Nrf2 and cytoplasmic HO1 were performed in peri-infarct cortex and striatum; followed by dual immunofluorescence study to check the specific cell type. I/R induced neurobehavioral deficits and infarct damage were significantly (p < 0.05) attenuated by MMF (20 and 40 mg/kg). MMF, 20 mg/kg, significantly normalized I/R induced altered redox status and increased levels of TNF-α, IL-1β in the ipsilateral cortex. MRI data showed significantly reduced infarct in cortex but not in striatum after MMF treatment. Expression of nuclear Nrf2 and cytoplasmic HO1 were significantly (p < 0.05) increased in peri-infarct cortex after treatment with MMF. Additionally, dual immunofluorescence showed increased Nrf2 expression in neurons and HO1 expression in neurons as well as astrocytes in peri-infarct cortex after MMF treatment. Our results show the neuro-protective potential of MMF probably by restricting the progression of damage from striatum to cortex through activation of Nrf2/HO1 pathway in peri-infarct cortex.

Cortical interlaminar astrocytes across the therian mammal radiation.

Interlaminar astrocytes (ILA) in the cerebral cortex possess a soma in layer I and extend an interlaminar process that runs perpendicular to the pia into deeper cortical layers. We examined cerebral cortex from 46 species that encompassed most orders of therian mammalians, including 22 primate species. We described two distinct cell types with interlaminar processes that have been referred to as ILA, that we termed pial ILA and supial ILA. ILA subtypes differ in somatic morphology, position in layer I, and presence across species. We further described rudimentary ILA that have short GFAP+ processes that do not exit layer I, and "typical" ILA with longer GFAP+ processes that exit layer I. Pial ILA were present in all mammalian species analyzed, with typical ILA observed in Primates, Scandentia, Chiroptera, Carnivora, Artiodactyla, Hyracoidea, and Proboscidea. Subpial ILA were absent in Marsupialia, and typical subpial ILA were only found in Primate. We focused on the properties of pial ILA by investigating the molecular properties of pial ILA and confirming their astrocytic nature. We found that while the density of pial ILA somata only varied slightly, the complexity of ILA processes varied greatly across species. Primates, specifically bonobo, chimpanzee, orangutan, and human, exhibited pial ILA with the highest complexity. We showed that interlaminar processes contact neurons, pia, and capillaries, suggesting a potential role for ILA in the blood-brain barrier and facilitating communication among cortical neurons, astrocytes, capillaries, meninges, and cerebrospinal fluid.

Astrocytic changes with aging and Alzheimer's disease-type pathology in chimpanzees.

Astrocytes are the main homeostatic cell of the central nervous system. In addition, astrocytes mediate an inflammatory response when reactive to injury or disease known as astrogliosis. Astrogliosis is marked by an increased expression of glial fibrillary acidic protein (GFAP) and cellular hypertrophy. Some degree of astrogliosis is associated with normal aging and degenerative conditions such as Alzheimer's disease (AD) and other dementing illnesses in humans. The recent observation of pathological markers of AD (amyloid plaques and neurofibrillary tangles) in aged chimpanzee brains provided an opportunity to examine the relationships among aging, AD-type pathology, and astrocyte activation in our closest living relatives. Stereologic methods were used to quantify GFAP-immunoreactive astrocyte density and soma volume in layers I, III, and V of the prefrontal and middle temporal cortex, as well as in hippocampal fields CA1 and CA3. We found that the patterns of astrocyte activation in the aged chimpanzee brain are distinct from humans. GFAP expression does not increase with age in chimpanzees, possibly indicative of lower oxidative stress loads. Similar to humans, chimpanzee layer I astrocytes in the prefrontal cortex are susceptible to AD-like changes. Both prefrontal cortex layer I and hippocampal astrocytes exhibit a high degree of astrogliosis that is positively correlated with accumulation of amyloid beta and tau proteins. However, unlike humans, chimpanzees do not display astrogliosis in other cortical layers. These results demonstrate a unique pattern of cortical aging in chimpanzees and suggest that inflammatory processes may differ between humans and chimpanzees in response to pathology.

Astrocyte Specificity and Coverage of hGFAP-CreERT2 [Tg(GFAP-Cre/ERT2)13Kdmc] Mouse Line in Various Brain Regions.

Astrocyte is the most abundant cell type in the central nervous system and its importance has been increasingly recognized in the brain pathophysiology. To study in vivo function of astrocyte, astrocyte-specific gene-targeting is regarded as a powerful approach. Especially, hGFAP-CreERT2, which expresses tamoxifen-inducible Cre recombinase under the human GFAP promoter, has been developed and characterized from several research groups. However, one of these mouse lines, [Tg(GFAP-Cre/ERT2)13Kdmc] from Ken McCarthy group has not been quantitatively analyzed, despite its frequent use. Here, we performed comprehensive characterization of this mouse line with quantitative analysis. By crossing this mouse line with Ai14 (RCL-tdTomato), a very sensitive Cre reporter mouse line, we visualized the Cre-expressing cells in various brain regions. For quantitative analysis, we immunostained S100β as an astrocytic marker and NeuN, tyrosine hydroxylase or calbindin as a neuronal marker in different brain regions. We calculated ‘astrocyte specificity’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of tdTomato positive cells and the ‘astrocyte coverage’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of S100β positive cells. Interestingly, we found varying degree of astrocyte specificity and coverage in each brain region. In cortex, hypothalamus, substantia nigra pars compacta and cerebellar Purkinje layer, we observed high astrocyte specificity (over 89%) and relatively high astrocyte coverage (over 70%). In striatum, hippocampal CA1 layer, dentate gyrus and cerebellar granule layer, we observed high astrocyte specificity (over 80%), but relative low astrocyte coverage (50–60%). However, thalamus and amygdala showed low astrocyte specificity (about 65%) and significant neuron specificity (over 30%). This hGFAP-CreERT2 mouse line can be useful for genetic modulations of target gene either in gain-of-function or loss-of-function studies in the brain regions with high astrocyte specificity and coverage. However, the use of this mouse line should be restricted to gain-of-function studies in the brain regions with high astrocyte specificity but low coverage. In conclusion, hGFAP-CreERT2 mouse line could be a powerful tool for gene-targeting of astrocytes in cortex, striatum, hippocampus, hypothalamus, substantia nigra pars compacta and cerebellum, but not in thalamus and amygdala.