Brain Astrocytes Research Articles

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro.

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes. Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

Construction of a novel blood brain barrier-glioma microfluidic chip model: Applications in the evaluation of permeability and anti-glioma activity of traditional Chinese medicine components

Since most anti-glioma drug candidates hardly permeate through the blood-brain barrier (BBB), preclinical models that can integrate the complexity of the tumor microenvironment and the structure and function of the BBB is urgently needed for the treatment of glioma. Herein, we constructed an in vitro BBB-glioma microfluidic chip model lined by primary human brain microvascular endothelial cells, pericytes, astrocytes and glioma cells, which could recapitulate the high level of barrier function of the in vivo human BBB and glioma microenvironment. The BBB unit in BBB-glioma microfluidic chip (BBB–U251 chip) displayed selective permeability to fluorescein isothiocyanate isomer-dextran (FITC-dextran) with different molecular weights and three model drugs with different permeability behavior across BBB, which indicated that this glioma model included a functional barrier. Six potential anti-glioma components in traditional Chinese medicine (TCM) were delivered into the blood channel and the permeated amount was quantified by high-performance liquid chromatography combined with ultraviolet (HPLC-UV). The permeated drugs then directly acted on 3D cultured glioma cells (U251) to evaluate the drug efficacy. The results of permeability coefficients of drugs showed that the data were closer to the in vivo data of traditional Transwell model. The effect of the drugs on U251 cells in the BBB-U251 chip was significantly lower due to the existence of BBB. Drug responses on glioma demonstrated the necessity to take BBB into account during the development of anti-glioma new drugs. Therefore, this 3D glioma microfluidic models integrating the BBB functionality can be a useful platform for screening the anticancer drug for brain tumors.

Sequential Isolation of Microglia and Astrocytes from Young and Aged Adult Mouse Brains for Downstream Transcriptomic Analysis.

In aging, the brain is more vulnerable to injury and neurodegenerative disease, but the mechanisms responsible are largely unknown. Evidence now suggests that neuroinflammation, mediated by resident brain astrocyte and microglia populations, are key players in the generation of inflammatory responses and may influence both age related processes and the initiation/progression of neurodegeneration. Consequently, targeting these cell types individually and collectively may aid in the development of novel disease-modifying therapies. We have optimized and characterized a protocol for the effective sequential isolation of both microglia and astrocytes from the adult mouse brain in young and aged mice. We demonstrate a technique for the sequential isolation of these immune cells by using magnetic beads technology, optimized to increase yield and limit potential artifacts in downstream transcriptomic applications, including RNA-sequencing pipelines. This technique is versatile, cost-effective, and reliable for the study of responses within the same biological context, simultaneously being advantageous in reducing mice numbers required to assess cellular responses in normal and age-related pathological conditions.

The Many Faces of Astrocytes in the Septic Brain.

Sepsis is a life-threatening organ dysfunction that is caused by a dysregulated host response to infection. Surviving patients have cognitive and memory damage that started during sepsis. These neurologic damages have been associated with increased BBB permeability and microglial activation. However, a few discrete studies have seen over the years pointing to the potential role of astrocytes in the pathophysiology of neurological damage after sepsis. The purpose of this article is to review information on the potential role of astrocytes during sepsis, as well as to provoke further studies in this area. These published articles show astrocytic activation after sepsis; they also evidence the release of inflammatory mediators by these cells. In this sense, the role of astrocytes should be better elucidated during sepsis progression.

The impaired distribution of adenosine deaminase isoenzymes in multiple sclerosis plasma and cerebrospinal fluid.

BackgroundAdenosine deaminase (ADA) via two isoenzymes, ADA1 and ADA2, regulates intra- and extracellular adenosine concentrations by converting it to inosine. In the central nervous system (CNS), adenosine modulates the processes of neuroinflammation and demyelination that together play a critical role in the pathophysiology of multiple sclerosis (MS). Except for their catalytic activities, ADA isoenzymes display extra-enzymatic properties acting as an adhesion molecule or a growth factor.AimsThis study aimed to explore the distribution and activity of ADA1 and ADA2 in the plasma and the CSF of MS patients as well as in the human brain microvascular endothelial cells (HBMEC), human brain vascular pericytes and human astrocytes.Methods and resultsThe enzyme assay following reverse phase-high performance liquid chromatography (HPLC) analysis was used to detect the ADA1 and ADA2 activities and revealed an increased ratio of ADA1 to ADA2 in both the plasma and the CSF of MS patients. Plasma ADA1 activity was significantly induced in MS, while ADA2 was decreased in the CSF, but significance was not reached. The brain astrocytes, pericytes and endothelial cells revealed on their surface the activity of ADA1, with its basal level being five times higher in the endothelial cells than in the astrocytes or the pericytes. In turn, ADA2 activity was only observed in pericytes and endothelial cells. Stimulation of the cells with pro-inflammatory cytokines TNFα/IL17 for 18 h decreased intracellular nucleotide levels measured by HPLC only in pericytes. The treatment with TNFα/IL17 did not modulate cell-surface ATP and AMP hydrolysis nor adenosine deamination in pericytes or astrocytes. Whereas in endothelial cells it downregulated AMP hydrolysis and ADA2 activity and upregulated the ADA1, which reflects the ADA isoenzyme pattern observed here in the CSF of MS patients.ConclusionIn this study, we determined the impaired distribution of both ADA isoenzymes in the plasma and the CSF of patients with MS. The increased ADA1 to ADA2 ratio in the CSF and plasma may translate to unfavorable phenotype that triggers ADA1-mediated pro-inflammatory mechanisms and decreases ADA2-dependent neuroprotective and growth-promoting effects in MS.

Perivascular Stromal Cells Instruct Glioblastoma Invasion, Proliferation, and Therapeutic Response within an Engineered Brain Perivascular Niche Model.

Glioblastoma (GBM) tumor cells are found in the perivascular niche microenvironment and are believed to associate closely with the brain microvasculature. However, it is largely unknown how the resident cells of the perivascular niche, such as endothelial cells, pericytes, and astrocytes, influence GBM tumor cell behavior and disease progression. A 3D in vitro model of the brain perivascular niche developed by encapsulating brain-derived endothelial cells, pericytes, and astrocytes in a gelatin hydrogel is described. It is shown that brain perivascular stromal cells, namely pericytes and astrocytes, contribute to vascular architecture and maturation. Cocultures of patient-derived GBM tumor cells with brain microvascular cells are used to identify a role for pericytes and astrocytes in establishing a perivascular niche environment that modulates GBM cell invasion, proliferation, and therapeutic response. Engineered models provide unique insight regarding the spatial patterning of GBM cell phenotypes in response to a multicellular model of the perivascular niche. Critically, it is shown that engineered perivascular models provide an important resource to evaluate mechanisms by which intercellular interactions modulate GBM tumor cell behavior, drug response, and provide a framework to consider patient-specific disease phenotypes.

Enterovirus-71 utilizes small extracellular vesicles to cross the blood-brain barrier for infecting the central nervous system via transcytosis.

Central nervous system (CNS) infections caused by Enterovirus 71 (EV71) pose a serious threat to children, causing severe neurogenic complications and even fatality in some patients. However, the pathogenesis of EV71 infections in the CNS remains unclear. An in vitro blood-brain barrier (BBB) model was constructed by coculturing brain microvascular endothelial cells (BMECs) and astrocytes in transwell inserts for simulating CNS infections. EV71 virions and small extracellular vesicles (sEVs) derived from EV71-infected cells (EV71-sEVs) were isolated from the cell culture supernatant by density gradient centrifugation. The BBB model was separately infected with EV71 virions and EV71-sEVs. The mechanism of crossing the BBB was determined by inhibiting the different endocytic modes. A murine model of EV71 infection was constructed for confirming the results of in vitro experiments. The EV71-sEVs containing viral components were endocytosed by BMECs and released on the abluminal side of the BBB model, where they infected the astrocytes without disrupting the BBB in the early stages of infection. The integrity of the tight junctions (TJs) between BMECs was breached via downregulation of PI3K/Akt signaling in the late stages of infection. EV71 utilized the circulating sEVs for infecting the CNS by crossing the BBB.

A potent and selective inhibitor for the modulation of MAGL activity in the neurovasculature.

Chronic inflammation and blood–brain barrier dysfunction are key pathological hallmarks of neurological disorders such as multiple sclerosis, Alzheimer’s disease and Parkinson’s disease. Major drivers of these pathologies include pro-inflammatory stimuli such as prostaglandins, which are produced in the central nervous system by the oxidation of arachidonic acid in a reaction catalyzed by the cyclooxygenases COX1 and COX2. Monoacylglycerol lipase hydrolyzes the endocannabinoid signaling lipid 2-arachidonyl glycerol, enhancing local pools of arachidonic acid in the brain and leading to cyclooxygenase-mediated prostaglandin production and neuroinflammation. Monoacylglycerol lipase inhibitors were recently shown to act as effective anti-inflammatory modulators, increasing 2-arachidonyl glycerol levels while reducing levels of arachidonic acid and prostaglandins, including PGE2 and PGD2. In this study, we characterized a novel, highly selective, potent and reversible monoacylglycerol lipase inhibitor (MAGLi 432) in a mouse model of lipopolysaccharide-induced blood–brain barrier permeability and in both human and mouse cells of the neurovascular unit: brain microvascular endothelial cells, pericytes and astrocytes. We confirmed the expression of monoacylglycerol lipase in specific neurovascular unit cells in vitro, with pericytes showing the highest expression level and activity. However, MAGLi 432 did not ameliorate lipopolysaccharide-induced blood–brain barrier permeability in vivo or reduce the production of pro-inflammatory cytokines in the brain. Our data confirm monoacylglycerol lipase expression in mouse and human cells of the neurovascular unit and provide the basis for further cell-specific analysis of MAGLi 432 in the context of blood–brain barrier dysfunction caused by inflammatory insults.

Acute Methylglyoxal-Induced Damage in Blood-Brain Barrier and Hippocampal Tissue.

Methylglyoxal (MG) is a reactive dicarbonyl compound formed mostly via the glycolytic pathway. Elevated blood glucose levels can cause MG accumulation in plasma and cerebrospinal fluid in patients with diabetes mellitus and Alzheimer's disease. Under these disease conditions, the high reactivity of MG leads to modification of proteins and other biomolecules, generating advanced glycation end products (AGEs), which are considered mediators in neurodegenerative diseases. We investigated the integrity of the blood-brain barrier (BBB) and astrocyte response in the hippocampus to acute insult induced by MG when it was intracerebroventricularly administered to rats. Seventy-two hours later, BBB integrity was lost, as assessed by the entry of Evans dye into the brain tissue and albumin in the cerebrospinal fluid, and a decrease in aquaporin-4 and connexin-43 in the hippocampal tissue. MG did not induce changes in the hippocampal contents of RAGE in this short interval, but decreased the expression of S100B, an astrocyte-secreted protein that binds RAGE. The expression of two important transcription factors of the antioxidant response, NF-κB and Nrf2, was unchanged. However, hemeoxigenase-1 was upregulated in the MG-treated group. These data corroborate the idea that hippocampal cells are targets of MG toxicity and that BBB dysfunction and specific glial alterations induced by this compound may contribute to the behavioral and cognitive alterations observed in these animals.

In vivo spatiotemporal dynamics of astrocyte reactivity following neural electrode implantation

Brain computer interfaces (BCIs), including penetrating microelectrode arrays, enable both recording and stimulation of neural cells. However, device implantation inevitably causes injury to brain tissue and induces a foreign body response, leading to reduced recording performance and stimulation efficacy. Astrocytes in the healthy brain play multiple roles including regulating energy metabolism, homeostatic balance, transmission of neural signals, and neurovascular coupling. Following an insult to the brain, they are activated and gather around the site of injury. These reactive astrocytes have been regarded as one of the main contributors to the formation of a glial scar which affects the performance of microelectrode arrays. This study investigates the dynamics of astrocytes within the first 2 weeks after implantation of an intracortical microelectrode into the mouse brain using two-photon microscopy. From our observation astrocytes are highly dynamic during this period, exhibiting patterns of process extension, soma migration, morphological activation, and device encapsulation that are spatiotemporally distinct from other glial cells, such as microglia or oligodendrocyte precursor cells. This detailed characterization of astrocyte reactivity will help to better understand the tissue response to intracortical devices and lead to the development of more effective intervention strategies to improve the functional performance of neural interfacing technology.

The CaMKII/MLC1 Axis Confers Ca2+-Dependence to Volume-Regulated Anion Channels (VRAC) in Astrocytes.

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1′s proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.

Natural Killer Cells Disrupt Nerve Fibers by Granzyme H in Atheriosclerotic Cerebral Small Vessel Disease.

Natural killer (NK) cells are enriched in the central nervous system in aging-related atheriosclerotic cerebral small vessel disease (aCSVD), but their roles and underlying mechanism remain to be elucidated. To identify potential cytotoxic molecules released by NK cells in aCSVD lesions, proteomic analysis of cerebrospinal fluid (CSF), plasma, and peripheral NK cells from patients with aCSVD were performed. We found that integrin β2 (ITGB2), cathepsin D (CTSD), and granzyme H (GZMH) were highly expressed in NK cells. ITGB2 interacted with intercellular adhesion molecule 1 in vascular endothelial cells. As assessed by immunofluorescence and scanning electron microscopy of the blood-brain barrier model, transwell membranes covered with primary human brain microvascular endothelial cells and astrocytes, we demonstrated that the CTSD-mediated degradation of collagen in the blood-brain barrier depended on the cytotoxicity of NK cells in aCSVD. With the immunostaining in vitro and in vivo, GZMH disruption of demyelinated nerve fibers was reversed by cotreatment with the inhibitor 3,4-DCIC during white matter hyperintensity (WMH) in aCSVD. Our results indicate that NK cells contribute to CTSD-induced damage to the blood-brain barrier and GZMH-induced disruption of nerve fibers during WMH in aCSVD.

Ascl1 phospho-site mutations enhance neuronal conversion of adult cortical astrocytes in vivo.

Direct neuronal reprogramming, the process whereby a terminally differentiated cell is converted into an induced neuron without traversing a pluripotent state, has tremendous therapeutic potential for a host of neurodegenerative diseases. While there is strong evidence for astrocyte-to-neuron conversion in vitro, in vivo studies in the adult brain are less supportive or controversial. Here, we set out to enhance the efficacy of neuronal conversion of adult astrocytes in vivo by optimizing the neurogenic capacity of a driver transcription factor encoded by the proneural gene Ascl1. Specifically, we mutated six serine phospho-acceptor sites in Ascl1 to alanines (Ascl1 SA 6) to prevent phosphorylation by proline-directed serine/threonine kinases. Native Ascl1 or Ascl1 SA 6 were expressed in adult, murine cortical astrocytes under the control of a glial fibrillary acidic protein (GFAP) promoter using adeno-associated viruses (AAVs). When targeted to the cerebral cortex in vivo, mCherry+ cells transduced with AAV8-GFAP-Ascl1 SA 6-mCherry or AAV8-GFAP-Ascl1-mCherry expressed neuronal markers within 14 days post-transduction, with Ascl1 SA 6 promoting the formation of more mature dendritic arbors compared to Ascl1. However, mCherry expression disappeared by 2-months post-transduction of the AAV8-GFAP-mCherry control-vector. To circumvent reporter issues, AAV-GFAP-iCre (control) and AAV-GFAP-Ascl1 (or Ascl1 SA 6)-iCre constructs were generated and injected into the cerebral cortex of Rosa reporter mice. In all comparisons of AAV capsids (AAV5 and AAV8), GFAP promoters (long and short), and reporter mice (Rosa-zsGreen and Rosa-tdtomato), Ascl1 SA 6 transduced cells more frequently expressed early- (Dcx) and late- (NeuN) neuronal markers. Furthermore, Ascl1 SA 6 repressed the expression of astrocytic markers Sox9 and GFAP more efficiently than Ascl1. Finally, we co-transduced an AAV expressing ChR2-(H134R)-YFP, an optogenetic actuator. After channelrhodopsin photostimulation, we found that Ascl1 SA 6 co-transduced astrocytes exhibited a significantly faster decay of evoked potentials to baseline, a neuronal feature, when compared to iCre control cells. Taken together, our findings support an enhanced neuronal conversion efficiency of Ascl1 SA 6 vs. Ascl1, and position Ascl1 SA 6 as a critical transcription factor for future studies aimed at converting adult brain astrocytes to mature neurons to treat disease.

Tanshinone IIA reduces AQP4 expression and astrocyte swelling after OGD/R by inhibiting the HMGB1/RAGE/NF-κB/IL-6 pro-inflammatory axis

This study aimed to investigate the role of tanshinone IIA (TSO IIA) in astrocytic swelling caused by ischemia–reperfusion-like injury in an in vitro model and the molecular mechanisms underlying this effect. Primary brain astrocytes were cultured under conditions of glucose and oxygen deprivation and reoxygenation (OGD/R). The study explored the effects of TSO IIA treatment on cell swelling and injury and the protein levels of aquaporin 4 (AQP4) in the plasma membrane. It then examined the involvement of the high-mobility group box protein 1 (HMGB1)/receptors for advanced-glycation end products (RAGE)/nuclear factor-kappa B (NF-κB)/interleukin-6 (IL-6) pro-inflammatory axis in TSO IIA-mediated protection. The treatment with TSO IIA alleviated OGD/R-induced astrocytic swelling and the overclustering of AQP4 protein in the plasma membrane. In addition, TSO IIA significantly reduced the overexpression of HMGB1 and the high levels of the NF-κB protein in the nucleus and of the IL-6 protein in the cytoplasm and extracellular media induced by OGD/R. The combination of TSO IIA and recombinant HMGB1 reversed these effects. The inhibition of the RAGE, the receptor of HMGB1, induced results similar to those of TSO IIA. In addition, exogenous IL-6 reversed TSO IIA-mediated effect on AQP4 overclustering and cell swelling. TSO IIA significantly reduced astrocyte swelling after OGD/R injury in vitro, via blocking the activation of the HMGB1/RAGE/NF-κB/IL-6 pro-inflammatory axis and thereby decreasing the expression of AQP4 in the plasma membrane.

Enhanced M-CSF/CSF1R Signaling Closely Associates with PrPSc Accumulation in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents.

Activation and proliferation of microglia are one of the hallmarks of prion disease and is usually accompanied by increased levels of various cytokines and chemokines. Our previous study demonstrated that the level of brain macrophage colony-stimulating factor (M-CSF) was abnormally elevated during prion infection, but its association with PrPSc is not completely clear. In this study, colocalization of the increased M-CSF with accumulated PrPSc was observed by IHC with serial brain sections. Reliable molecular interaction between total PrP and M-CSF was observed in the brain of 263K-infected hamsters and in cultured prion-infected cell line. Immunofluorescent assays showed that morphological colocalization of M-CSF with neurons and microglia, but not with astrocytes in brains of scrapie-infected animals. The transcriptional and expressing levels of CSF1R were also significantly increased in prion-infected cell line and mice, and colocalization of CSF1R with neurons and microglia was observed in the brains of prion-infected mouse models. Removal of PrPSc replication by resveratrol in SMB-S15 cells induced limited reductions of cellular levels of M-CSF and CSF1R. In addition, we found that the level of IL-34, another ligand of CSF1R, did not change significantly after prion infection, but its distribution on the cell types in the brains shifted from neurons in healthy mice to the proliferated astrocytes and microglia in scrapie-infected mice. Our data demonstrate activation of M-CSF/IL-34/CSF1R signaling in the microenvironment of prion infection, strongly indicating its vital role in the pathophysiology of prions. It provides solid scientific evidence for the therapeutic potential of inhibiting M-CSF/CSF1R signaling in prion diseases.

Astrocyte-specific loss of lactoferrin influences neuronal structure and function by interfering with cholesterol synthesis.

Growing evidence indicates that circulating lactoferrin (Lf) is implicated in peripheral cholesterol metabolism disorders. It has emerged that the distribution of Lf changes in astrocytes of aging brains and those exhibiting neurodegeneration; however, its physiological and/or pathological role remains unknown. Here, we demonstrate that astrocyte-specific knockout of Lf (designated cKO) led to decreased body weight and cognitive abnormalities during early life in mice. Accordingly, there was a reduction in neuronal outgrowth and synaptic structure in cKO mice. Importantly, Lf deficiency in the primary astrocytes led to decreased sterol regulatory element binding protein 2 (Srebp2) activation and cholesterol production, and cholesterol content in cKO mice and/or in astrocytes was restored by exogenous Lf or a Srebp2 agonist. Moreover, neuronal dendritic complexity and total dendritic length were decreased after culture with the culture medium of the primary astrocytes derived from cKO mice and that this decrease was reversed after cholesterol supplementation. Alternatively, these alterations were associated with an activation of AMP-activated protein kinase (AMPK) and inhibition of SREBP2 nuclear translocation. These data suggest that astrocytic Lf might directly or indirectly control in situ cholesterol synthesis, which may be implicated in neurodevelopment and several neurological diseases.

The presence of BBB hastens neuronal differentiation of cerebral organoids – The potential role of endothelial derived BDNF

Despite remaining the best in vitro model to resemble the human brain, a weakness of human cerebral organoids is the lack of the endothelial component that in vivo organizes in the blood brain barrier (BBB). Since the BBB is crucial to control the microenvironment of the nervous system, this study proposes a co-culture of BBB and cerebral organoids. We utilized a BBB model consisting of primary human brain microvascular endothelial cells and astrocytes in a transwell system. Starting from induced Pluripotent Stem Cells (iPSCs) we generated human cerebral organoids which were then cultured in the absence or presence of an in vitro model of BBB to evaluate potential effects on the maturation of cerebral organoids. By morphological analysis, it emerges that in the presence of the BBB the cerebral organoids are better organized than controls in the absence of the BBB. This effect might be due to Brain Derived Neurotrophic Factor (BDNF), a neurotrophic factor released by the endothelial component of the BBB, which is involved in neurodevelopment, neuroplasticity and neurosurvival.

Study of Calcium Signaling in Astrocytes with a Novel Endoplasmic Reticulum-Targeted GCaMP Sensor.

The endoplasmic reticulum (ER), the major organelle for the storage of Ca2+ , maintains a concentration of Ca2+ much higher than in the cytosol or other subcellular organelles, such as the mitochondria. A variety of tools have been developed for measuring Ca2+ activity in neuronal and glial cells, but most of these sensors target either the plasma membrane (PM) or the cytosol. Though these sensors are important for measuring Ca2+ transients, they lack the capability to measure activity in the periphery of the ER or to measure low-amplitude events resulting from Ca2+ exchange between the ER and other organelles, such as the mitochondria. We recently developed an ER-targeted GCaMP6f anchored to the cytosolic side of the ER that can measure minute calcium exchange occurring in this region. In this article, we discuss detailed methods to characterize the ER-GCaMP6f sensor, utilize it for calcium imaging in cultured astrocytes, and assess its expression and calcium imaging in astrocytes in rodent brains. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression and characterization of ER-GCaMP6f Support Protocol 1: ER-GCaMP6f-expressing stable cell line generation Basic Protocol 2: In vitro calcium imaging with ER-GCaMP6f Support Protocol 2: Imaging of drug-induced calcium activity Alternate Protocol 1: Transduction of astrocytes with ER-GCaMP6f AAV Alternate Protocol 2: Calcium imaging of astrocytes with Fluo-4 AM Basic Protocol 3: In vivo ER-GCaMP6f expression and slice calcium imaging Support Protocol 3: Pharmacological studies with 2-APB in brain slices.

Delay of endothelial cell senescence protects cerebral barrier against age-related dysfunction: role of senolytics and senomorphics

ABSTRACT Accumulation of senescent cells in cerebrovasculature is thought to play an important role in age-related disruption of blood–brain barrier (BBB). Using an in vitro model of human BBB, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, this study explored the so-called correlative link between BMEC senescence and the BBB dysfunction in the absence or presence of functionally distinct senotherapeutics. Replicative senescence was deemed present at passage ≥19 where BMECs displayed shortened telomere length, reduced proliferative and tubulogenic potentials and increased NADPH oxidase activity, superoxide anion production (markers of oxidative stress), S-β-galactosidase activity and γ-H2AX staining. Significant impairments observed in integrity and function of a model of BBB established with senescent BMECs, ascertained successively by decreases in transendothelial electrical resistance and increases in paracellular flux, revealed a close correlation between endothelial cell senescence and BBB dysfunction. Disruptions in the localization or expression of tight junction proteins, zonula occludens-1, occludin, and claudin-5 in senescent BMECs somewhat explained this dysfunction. Indeed, treatment of relatively old BMEC (passage 16) with a cocktail of senolytics (dasatinib and quercetin) or senomorphics targeting transcription factor NF-κB (QNZ), p38MAPK signaling pathway (BIRB-796) or pro-oxidant enzyme NADPH oxidase (VAS2870) until passage 20 rendered these cells more resistant to senescence and totally preserved BBB characteristics by restoring subcellular localization and expression of tight junction proteins. In conclusion, attempts that effectively mitigate accumulation of senescent endothelial cells in cerebrovasculature may prevent age-related BBB dysfunction and may be of prophylactic or therapeutic value to extend lifelong health and wellbeing.

Inhibition of astrocytic DRD2 suppresses CNS inflammation in an animal model of multiple sclerosis.

Astrocyte activation is associated with progressive inflammatory demyelination in multiple sclerosis (MS). The molecular mechanisms underlying astrocyte activation remain incompletely understood. Recent studies have suggested that classical neurotransmitter receptors are implicated in the modulation of brain innate immunity. We investigated the role of dopamine signaling in the process of astrocyte activation. Here, we show the upregulation of dopamine D2 receptor (DRD2) in reactive astrocytes in MS brain and noncanonical role of astrocytic DRD2 in MS pathogenesis. Mice deficient in astrocytic Drd2 exhibit a remarkable suppression of reactive astrocytes and amelioration of experimental autoimmune encephalomyelitis (EAE). Mechanistically, DRD2 regulates the expression of 6-pyruvoyl-tetrahydropterin synthase, which modulates NF-κB activity through protein kinase C-δ. Pharmacological blockade of astrocytic DRD2 with a DRD2 antagonist dehydrocorybulbine remarkably inhibits the inflammatory response in mice lacking neuronal Drd2. Together, our findings reveal previously an uncharted role for DRD2 in astrocyte activation during EAE-associated CNS inflammation. Its therapeutic inhibition may provide a potent lever to alleviate autoimmune diseases.