Primary Human Astrocytes Research Articles

Human Primary Astrocytes Differently Respond to Pro- and Anti-Inflammatory Stimuli.

For a long time, astrocytes were considered a passive brain cell population. However, recently, many studies have shown that their role in the central nervous system (CNS) is more active. Previously, it was stated that there are two main functional phenotypes of astrocytes. However, nowadays, it is clear that there is rather a broad spectrum of these phenotypes. The major goal of this study was to evaluate the production of some inflammatory chemokines and neurotrophic factors by primary human astrocytes after pro- or anti-inflammatory stimulation. We observed that only astrocytes induced by inflammatory mediators TNFα/IL-1a/C1q produced CXCL10, CCL1, and CXCL13 chemokines. Unstimulated astrocytes and those cultured with anti-inflammatory cytokines (IL-4, IL-10, or TGF-β1) did not produce these chemokines. Interestingly, astrocytes cultured in proinflammatory conditions significantly decreased the release of neurotrophic factor PDGF-A, as compared to unstimulated astrocytes. However, in response to anti-inflammatory cytokine TGF-β1, astrocytes significantly increased PDGF-A production compared to the medium alone. The production of another studied neurotrophic factor BDNF was not influenced by pro- or anti-inflammatory stimulation. The secretory response was accompanied by changes in HLA-DR, CD83, and GFAP expression. Our study confirms that astrocytes differentially respond to pro- and anti-inflammatory stimuli, especially to inflammatory cytokines TNF-α, IL-1a, and C1q, suggesting their role in leukocyte recruitment.

Increased Intrathecal Activity of Follicular Helper T Cells in Patients With Relapsing-Remitting Multiple Sclerosis.

Follicular helper T (Tfh) cells play a critical role in protective immunity helping B cells produce antibodies against foreign pathogens and are likely implicated in the pathogenesis of various autoimmune diseases. The purpose of this study was to investigate the role of Tfh cells in the pathogenesis of multiple sclerosis (MS). Using flow cytometry, we investigated phenotype, prevalence, and function of Tfh cells in blood and CSF from controls and patients with relapsing-remitting MS (RRMS) and primary progressive MS (PPMS). In addition, an in vitro blood-brain barrier coculture assay of primary human astrocytes and brain microvascular endothelial cells grown in a Boyden chamber was used to assess the migratory capacity of peripheral Tfh cells. This study identified 2 phenotypically and functionally distinct Tfh cell populations: CD25- Tfh cells (Tfh1-like) and CD25int Tfh cells (Tfh17-like). Whereas minor differences in Tfh cell populations were found in blood between patients with MS and controls, we observed an increased frequency of CD25- Tfh cells in CSF of patients with RRMS and PPMS and CD25int Tfh cells in patients with RRMS, compared with controls. Increasing frequencies of CSF CD25- Tfh cells and the CD25- Tfh/Tfr ratio scaled with increasing IgG index in patients with RRMS. Despite an increased prevalence of intrathecal Tfh cells in patients with MS, no difference in the migratory capacity of circulating Tfh cells was observed between controls and patients with MS. Instead, CSF concentrations of CXCL13 scaled with total counts of Tfh and Tfr cell subsets in the CSF. Our study indicates substantial changes in intrathecal Tfh dynamics, particularly in patients with RRMS, and suggests that the intrathecal inflammatory environment in patients with RRMS promotes recruitment of peripheral Tfh cells rather than the Tfh cells having an increased capacity to migrate to CNS.

HIV-1 Tat and cocaine impact astrocytic energy reservoirs and epigenetic regulation by influencing the LINC01133-hsa-miR-4726-5p-NDUFA9 axis

Clinical research has proven that HIV-positive (HIV+) individuals with cocaine abuse show behavioral and neurocognitive disorders. Noncoding RNAs (ncRNAs), such as long ncRNAs (lncRNAs) and microRNAs (miRNAs), are known to regulate gene expression in the contexts of HIV infection and drug abuse. However, there are no specific lncRNA or miRNA biomarkers associated with HIV-1 Transactivator of transcription protein (Tat) and cocaine coexposure. In the central nervous system (CNS), astrocytes are the primary regulators of energy metabolism, and impairment of the astrocytic energy supply can trigger neurodegeneration. The aim of this study was to uncover the roles of lncRNAs and miRNAs in the regulation of messenger RNA (mRNA) targets affected by HIV infection and cocaine abuse. Integrative bioinformatics analysis revealed altered expression of 10 lncRNAs, 10 miRNAs, and 4 mRNA/gene targets in human primary astrocytes treated with cocaine and HIV-1 Tat. We assessed the alterations in the expression of two miRNAs, hsa-miR-2355 and hsa-miR-4726-5p; four lncRNAs, LINC01133, H19, HHIP-AS1, and NOP14-AS1; and four genes, NDUFA9, KYNU, HKDC1, and LIPG. The results revealed interactions in the LINC01133-hsa-miR-4726-5p-NDUFA9 axis that may eventually help us understand cocaine- and HIV-1 Tat-induced astrocyte dysfunction that may ultimately result in neurodegeneration.

Transcription factor-based direct conversion of human fibroblasts to functional astrocytes.

SummaryAstrocytes are emerging key players in neurological disorders. However, their role in disease etiology is poorly understood owing to inaccessibility of primary human astrocytes. Pluripotent stem cell-derived cells fail to mimic age and due to their clonal origin do not mimic genetic heterogeneity of patients. In contrast, direct conversion constitutes an attractive approach to generate human astrocytes that capture age and genetic diversity. We describe efficient direct conversion of human fibroblasts to functional induced astrocytes (iAs). Expression of the minimal combination Sox9 and Nfib generates iAs with molecular, phenotypic, and functional properties resembling primary human astrocytes. iAs could be obtained by conversion of fibroblasts covering the entire human lifespan. Importantly, iAs supported function of induced neurons obtained through direct conversion from the same fibroblast population. Fibroblast-derived iAs will become a useful tool to elucidate the biology of astrocytes and complement current in vitro models for studies of late-onset neurological disorders.

A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders

Astrocytes are one of the most numerous glial cells in the central nervous system (CNS) and provide essential support to neurons to ensure CNS health and function. During a neuropathological challenge, such as during human immunodeficiency virus (HIV)-1 infection or (METH)amphetamine exposure, astrocytes shift their neuroprotective functions and can become neurotoxic. Identifying cellular and molecular mechanisms underlying astrocyte dysfunction are of heightened importance to optimize the coupling between astrocytes and neurons and ensure neuronal fitness against CNS pathology, including HIV-1-associated neurocognitive disorders (HAND) and METH use disorder. Mitochondria are essential organelles for regulating metabolic, antioxidant, and inflammatory profiles. Moreover, endoplasmic reticulum (ER)-associated signaling pathways, such as calcium and the unfolded protein response (UPR), are important messengers for cellular fate and function, including inflammation and mitochondrial homeostasis. Increasing evidence supports that the three arms of the UPR are involved in the direct contact and communication between ER and mitochondria through mitochondria-associated ER membranes (MAMs). The current study investigated the effects of HIV-1 infection and chronic METH exposure on astrocyte ER and mitochondrial homeostasis and then examined the three UPR messengers as potential regulators of astrocyte mitochondrial dysfunction. Using primary human astrocytes infected with pseudotyped HIV-1 or exposed to low doses of METH for 7 days, astrocytes had increased mitochondrial oxygen consumption rate (OCR), cytosolic calcium flux and protein expression of UPR mediators. Notably, inositol-requiring protein 1α (IRE1α) was most prominently upregulated following both HIV-1 infection and chronic METH exposure. Moreover, pharmacological inhibition of the three UPR arms highlighted IRE1α as a key regulator of astrocyte metabolic function. To further explore the regulatory role of astrocyte IRE1α, astrocytes were transfected with an IRE1α overexpression vector followed by activation with the proinflammatory cytokine interleukin 1β. Overall, our findings confirm IRE1α modulates astrocyte mitochondrial respiration, glycolytic function, morphological activation, inflammation, and glutamate uptake, highlighting a novel potential target for regulating astrocyte dysfunction. Finally, these findings suggest both canonical and non-canonical UPR mechanisms of astrocyte IRE1α. Thus, additional studies are needed to determine how to best balance astrocyte IRE1α functions to both promote astrocyte neuroprotective properties while preventing neurotoxic properties during CNS pathologies.

Δ133p53α Protects Human Astrocytes from Amyloid-beta Induced Senescence and Neurotoxicity

Cellular senescence is an important contributor to aging and age-related diseases such as Alzheimer’s disease (AD). Senescent cells are characterized by a durable cell proliferation arrest and the acquisition of a proinflammatory senescence-associated secretory phenotype (SASP), which participates in the progression of neurodegenerative disorders. Clearance of senescent glial cells in an AD mouse model prevented cognitive decline suggesting pharmacological agents targeting cellular senescence might provide novel therapeutic approaches for AD. Δ133p53α, a natural protein isoform of p53, was previously shown to be a negative regulator of cellular senescence in primary human astrocytes, with clinical implications from its diminished expression in brain tissues from AD patients. Here we show that treatment of proliferating human astrocytes in culture with amyloid-beta oligomers (Aβ), an endogenous pathogenic agent of AD, results in reduced expression of Δ133p53α, as well as induces the cells to become senescent and express proinflammatory SASP cytokines such as IL-6, IL-1β and TNFα. Our data suggest that Aβ-induced astrocyte cellular senescence is associated with accelerated DNA damage, and upregulation of full-length p53 and its senescence-inducing target gene p21WAF1. We also show that exogenously enhanced expression of Δ133p53α rescues human astrocytes from Aβ-induced cellular senescence and SASP through both protection from DNA damage and dominant-negative inhibition of full-length p53, leading to inhibition of Aβ-induced, astrocyte-mediated neurotoxicity. The results presented here demonstrate that Δ133p53α manipulation could modulate cellular senescence in the context of AD, possibly opening new therapeutic avenues.

Deletion of RE1-silencing transcription factor in striatal astrocytes exacerbates manganese-induced neurotoxicity in mice.

Chronic manganese (Mn) overexposure causes a neurological disorder, referred to as manganism, exhibiting symptoms similar to parkinsonism. Dysfunction of the repressor element-1 silencing transcription factor (REST) is associated with various neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Mn-induced neurotoxicity, but its cellular and molecular mechanisms have yet to be fully characterized. Although neuronal REST is known to be neuroprotective, the role of astrocytic REST in neuroprotection remains to be established. We investigated if astrocytic REST in the striatal region of the mouse brain where Mn preferentially accumulates plays a role in Mn-induced neurotoxicity. Striatal astrocytic REST was deleted by infusion of adeno-associated viral vectors containing sequences of the glial fibrillary acidic protein promoter-driven Cre recombinase into the striatum of RESTflox/flox mice for 3 weeks, followed by Mn exposure (30 mg/kg, daily, intranasally) for another 3 weeks. Striatal astrocytic REST deletion exacerbated Mn-induced impairment of locomotor activity and cognitive function with further decrease in Mn-reduced protein levels of tyrosine hydroxylase and glutamate transporter 1 (GLT-1) in the striatum. Astrocytic REST deletion also exacerbated the Mn-induced proinflammatory mediator COX-2, as well as cytokines such as TNF-α, IL-1β, and IL-6, in the striatum. Mn-induced detrimental astrocytic products such as proinflammatory cytokines on neuronal toxicity were attenuated by astrocytic REST overexpression, but exacerbated by REST inhibition in an in vitro model using primary human astrocytes and Lund human mesencephalic (LUHMES) neuronal culture. These findings indicate that astrocytic REST plays a critical role against Mn-induced neurotoxicity by modulating astrocytic proinflammatory factors and GLT-1.

ADAR Dysfunction in Brain Aging and Alzheimer's Disease

Neuroinflammation is a central mechanism of brain aging and neurodegenerative disease, but why neuroinflammation develops with aging is incompletely understood. Adenosine deaminase RNA specific (ADAR) proteins are potential suppressors of neuroinflammation through their adenosine‐to‐inosine (A‐to‐I) editing and subsequent inhibition of double‐stranded RNA (dsRNA, an innate immune activator). However, the role of ADARs in brain aging and/or neurodegenerative disease (e.g., Alzheimer’s disease, AD) is unclear. To address this, we looked for age‐related changes in ADAR expression and function (indicated by A‐to‐I editing) in multiple, brain‐specific transcriptomic datasets. We found evidence of age‐related declines in ADAR expression in: 1) astrocytes (glial cells linked with immune/inflammatory signaling in aging and AD) in old vs. young mice; 2) induced astrocytes from old vs. young human subjects; and 3) brain tissue of AD patients vs. healthy age‐matched controls. We also found declines in A‐to‐I editing related to reduced ADAR expression, suggesting that ADAR and ADAR activity decline with age and disease. We followed up on these observations via in vitro experiments in primary human astrocytes, in which we suppressed ADAR via siRNA knockdown or with an ADAR inhibitor (8‐Azaadenosine). These treatments increased levels of the proinflammatory cytokine interleukin 6 (IL‐6) and intercellular adhesion molecule 1 (ICAM‐1, a marker of pro‐inflammatory astrocytes), and this was associated with accumulation/changes in the cellular distribution of dsRNA (assessed via immunofluorescence). Preliminary data showed that ADAR knockdown tended to increased levels of protein kinase R (PKR, a key pro‐inflammatory dsRNA sensor), and that dsRNA colocalizes with PKR. To identify related mechanisms, we are currently performing RNA‐seq to profile whole‐transcriptome changes associated with ADAR inhibition in astrocytes. Taken together, our data suggest that ADAR may be an important modulator of neuroinflammation in aging, and ultimately neurodegenerative diseases.

New In Vitro Model to Study Multicellular and Flow Control of Blood‐Brain Barrier

Brain endothelial cells of the blood‐brain barrier (BBB) have been shown to be regulated by supportive cells, such as pericytes and astrocytes, and shear stress exposure. However, studies investigating the impact of pericytes and astrocytes on brain endothelial cell function have identified both beneficial and detrimental results. Additionally, most studies investigating the relationship between shear stress and brain endothelial cell function lack physiological relevance via the use of sub‐physiological shear stress magnitudes and/or via the absence of pericytes and astrocytes. In this study, we developed a millifluidic device compatible with standard transwell inserts to investigate BBB function. In contrast to standard polydimethylsiloxane (PDMS) microfluidic devices, this model allows for easy, reproducible shear stress exposure without common limitations of PDMS devices such as inadequate nutrient diffusion and air bubble formation. In no‐flow conditions, we first used the device to examine the impact of primary human pericytes and astrocytes on human brain microvascular endothelial cell (HBMEC) barrier integrity. We found that astrocytes, pericytes, and a 1:1 ratio of both cell types increased HBMEC barrier integrity via reduced permeability to 40 kDa fluorescent dextran, which was associated with increased expression of tight junction protein, claudin‐5. Interestingly, we also observed a significantly lower permeability to 3 kDa dextran in HBMEC‐pericyte co‐cultures compared to HBMEC‐astrocyte and HBMEC‐astrocyte‐pericyte co‐cultures. Based on these findings, we hypothesize pericytes may be providing increased barrier support to the BBB model compared to astrocytes although they both function as permeability reducers. After using the device to generate 24‐hour flow at 12 dynes/cm2, we observed that shear stress exposure significantly reduced dextran permeability in HBMEC monolayers, but not in tri‐culture models consisting of HBMECs, pericytes, and astrocytes. These results indicate that co‐cultures may demonstrate a more pronounced impact on overall BBB permeability than flow exposure. In both cases, flow exposure was interestingly associated with reduced expression of both claudin‐5 and occludin. However, ZO‐1 expression, and localization at cell‐cell junctions increased in the tri‐culture but exhibited no apparent change in the HBMEC monolayer. Under flow conditions, we also observed alignment of HBMECs in the tri‐culture while no such phenomenon was observed in HBMEC monolayers, indicating supportive cells and flow are both essential to observe brain endothelial cell alignment in vitro. Collectively, these results support the necessity of physiologically relevant, multicellular BBB models when investigating brain endothelial cell function in relation to the BBB. Additionally, our findings provide clues on the role of shear stress and supportive cells within the BBB, a critical step to elucidating the physiology of the neurovascular unit.

Inositol‐requiring enzyme 1 alpha (IRE1α) regulates astrocyte metabolic and inflammatory phenotypes: Implications in (METH)amphetamine use disorder and HIV‐associated neurocognitive disorders

Human immunodeficiency virus 1 (HIV‐1) invades the central nervous system (CNS) early during infection and can persist in the CNS for life despite effective antiretroviral treatment. Infection and activation of residential glial cells leads to low viral replication and chronic inflammation, which damage neurons contributing to a spectrum of HIV‐associated neurocognitive disorders (HAND). Astrocytes are the most numerous glial cells in the CNS and provide essential support to neurons. During a neuropathological challenge, such as HIV‐1 infection, astrocytes can shift their neurotrophic functions to become neurotoxic and even serve as latent reservoirs for HIV‐1 infection. Notably, substance use disorders, including methamphetamine (METH) are disproportionately elevated among people living with HIV‐1. METH use can induce neurotoxic and neurodegenerative consequences, which can increase one’s risk and severity of HAND. Thus, a better understanding of HIV‐1 infection and METH exposure both alone and in combination on astrocyte function could help identify key cellular or molecular targets that can regulate astrocyte neuroprotective versus neurotoxic phenotypes to optimize astrocyte and neuronal coupling and combat CNS pathology.Direct contact sites between the endoplasmic reticulum (ER) and the mitochondria, termed mitochondria‐associated ER membranes (MAMs), are central hubs for regulating several cellular processes, including inflammation and mitochondrial function and dynamics. In fact, the transfer of calcium from the ER to mitochondria is essential for mitochondrial bioenergetics. Interestingly, increasing evidence supports that the three arms of the unfolded protein response (UPR) are key cell signaling messengers within the ER‐mitochondrial interface, beyond their classical ER stress functions. Briefly, protein kinase RNA‐like endoplasmic reticulum kinase (PERK) has been determined as a regulator for MAM tethering and mitochondrial morphology. Inositol‐requiring enzyme 1 alpha (IRE1α) is implicated in regulating MAM‐mediated calcium transfer. Activating transcription factor 6 (ATF6) is suspected to participate in MAM formation as it is known to mediate ER elongation and lipid homeostasis. However, these regulatory mechanisms have not yet been fully elucidated.Our studies specifically highlight IRE1α as a key regulator of astrocyte metabolic and inflammatory phenotypes. Using primary human astrocytes infected with pseudotyped HIV and/or exposed to low doses of METH for seven days, astrocytes have increased protein expression of select UPR/MAM mediators. Under the same paradigms, we see increased cytosolic calcium flux and mitochondrial oxygen consumption rate, which were associated with increased mitochondria calcium uptake. Manipulation of IRE1α using both pharmacological inhibitors and an overexpression plasmid, confirms IRE1α modulates astrocyte calcium signaling, metabolic activity, glutamate clearance, and cytokine release. These findings identify a novel target for regulating astrocyte metabolic and inflammatory phenotypes, which could help combat astrocyte‐mediated neurotoxicity and potentially promote a neurotrophic phenotype during CNS pathologies.

Cellular Model of Malignant Transformation of Primary Human Astrocytes Induced by Deadhesion/Readhesion Cycles

Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG—a human glioblastoma-derived cell lineage—reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/β-catenin signaling pathways, in addition to upregulation of genes related to epithelial–mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.

SRPX Emerges as a Potential Tumor Marker in the Extracellular Vesicles of Glioblastoma.

Simple SummaryGlioblastoma is the most common malignant primary brain tumor and remains incurable. Additionally, there are only a few non-invasive early diagnostic and prognostic markers for this disease. The stability of extracellular vesicles (EVs) and their availability in patient serum make them ideal for discovery of early markers associated with diagnosis, prognosis, and treatment response for glioblastoma. In this study, we used proteomics analysis to discover a novel tumor biomarker in glioblastoma human primary cell-derived EVs and found that sushi-repeat containing protein X-linked (SRPX) was the only protein identified in the majority of glioblastoma EVs that was absent in the HPA-derived EVs. Moreover, we further analyzed the possible role of SRPX in glioblastoma tumorigenesis and found that SRPX is involved in glioblastoma cell growth, and SRPX depletion sensitizes glioblastoma to temozolomide (TMZ). Taken together, our results suggest that SRPX can be used as a novel tumor biomarker for diagnostic and prognostic purposes for glioblastomas.Extracellular vesicles (EVs) may be used as a non-invasive screening platform to discover markers associated with early diagnosis, prognosis, and treatment response. Such an approach is invaluable for diseases such as glioblastoma, for which only a few non-invasive diagnostic or prognostic markers are available. We used mass spectrometry to analyze proteomics profiles of EVs derived from four glioblastoma cell lines and human primary astrocytes (HPAs) and found that SRPX is the only protein enriched in the majority of glioblastoma EVs that was absent in the HPA-derived EVs. Then, we evaluated the relationship between SRPX protein expression and tumor grade using immunohistochemical staining (IHC) and performed colony formation and viability assays to analyze the possible function of SRPX in glioblastoma. SRPX mRNA and protein expression were associated with tumor grade. Moreover, temozolomide (TMZ)-resistant tumor tissues showed highly positive SRPX staining, compared to all other tumor grades. Additionally, glioblastoma cells displayed enhanced SRPX gene expression when exposed to TMZ. Knockdown of SRPX gene expression via siRNA inhibited cell viability. Taken together, the results of this study suggest that SRPX can be used as a novel tumor marker for diagnostic and prognostic purposes and can also be a therapeutic target for glioblastomas.

Intranasal delivery of a small-molecule ErbB inhibitor promotes recovery from acute and late-stage CNS inflammation.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the CNS that is characterized by demyelination and axonal degeneration. Although several established treatments reduce relapse burden, effective treatments to halt chronic progression are scarce. Single-cell transcriptomic studies in MS and its animal models have described astrocytes and their spatial and functional heterogeneity as important cellular determinants of chronic disease. We combined CNS single-cell transcriptome data and small-molecule screens in primary mouse and human astrocytes to identify glial interactions, which could be targeted by repurposing FDA-approved small-molecule modulators for the treatment of acute and late-stage CNS inflammation. Using hierarchical in vitro and in vivo validation studies, we demonstrate that among selected pathways, blockade of ErbB by the tyrosine kinase inhibitor afatinib efficiently mitigates proinflammatory astrocyte polarization and promotes tissue-regenerative functions. We found that i.n. delivery of afatinib during acute and late-stage CNS inflammation ameliorates disease severity by reducing monocyte infiltration and axonal degeneration while increasing oligodendrocyte proliferation. We used unbiased screening approaches of astrocyte interactions to identify ErbB signaling and its modulation by afatinib as a potential therapeutic strategy for acute and chronic stages of autoimmune CNS inflammation.

Extracellular Cardiolipin Modulates Select Immune Functions of Astrocytes in Toll-Like Receptor (TLR) 4-Dependent Manner.

Alzheimer's disease (AD) is characterized by chronic neuroinflammation, which is partially mediated by dysregulated functions of glial cells. Cardiolipin (CL) is a phospholipid normally confined to the inner mitochondrial membrane; however, it has been detected in human sera, indicating that it can exist in the extracellular space where it may interact with nearby cells. Although CL has been shown to modulate several functions of microglia in a toll-like receptor (TLR) 4-dependent manner, the effects of extracellular CL on astrocytes are unknown. In addition to their homeostatic functions, astrocytes participate in neuroimmune responses of the brain and express TLR 4. Therefore, we hypothesized that extracellular CL (1) modulates the secretion of cytokines and cytotoxins by astrocytes, as well as their phagocytic activity, and (2) acts by interacting with astrocyte TLR 4. We demonstrate that CL inhibits the lipopolysaccharide- (LPS-) induced secretion of cytotoxins and expression of glial fibrillary acidic protein (GFAP) by human U118 MG astrocytic cells. CL alone upregulates the phagocytic activity of human astrocytic cells and primary murine astrocytes. CL in combination with LPS upregulates secretion of interleukin (IL)-1β by astrocytic cells. Furthermore, CL alone increases the secretion of monocyte chemoattractant protein (MCP)-1 by astrocytic cells, which is blocked by the TLR 4-specific antagonist TAK-242. We demonstrate that CL upregulates MCP-1 secretion in the absence of its natural carrier protein, β2-glycoprotein 1, indicating that CL may be bioactive in the brain where this protein is not present. Lastly, we show that CL downregulates the expression of astrocytic TLR 4, implying that CL engages this receptor, as its activation has been shown to lead to its degradation. Overall, our study extends the list of cell type functions of which CL modulates and provides evidence that CL, or liposomes containing this phospholipid can be used to modulate specific neuroimmune functions of astrocytes.

Protective Effect of Low Dose of Methamphetamine on The Amount of Extracellular Glutamine in Primary Fetal Human Astrocytes Induced by Amyloid Beta.

Objective Change in astrocytes is one of the first pathological symptoms of Alzheimer’s disease (AD). Understanding the signaling pathways in astrocytes can be a great help in treating of AD. This study aimed to investigate signaling pathway relations between low dose of methamphetamine (METH), the apoptosis, cell cycle, and glutamine (Gln) pathways in the activated astrocyte. Materials and Methods In this experimental study, the activated astrocyte cells were exposed to a low dose of METH (12.5 µM) which was determined by Thiazolyl blue tetrazolium bromide (MTT) method. The groups were: group 1 cells with Aβ, group 2 cells with METH, group 3 cells with METH after 24 hours of adding Aβ (Aβ+METH, treated group), group 4 cells with Aβ after 24 hours of adding METH (METH+Aβ, prevention group), and group 5 as the control. The Gln was assayed by high-performance liquid chromatography (HPLC), and also the apoptosis, and cell cycle and BAX, BCL-X expression was evaluated. Results The amount of Gln was increased, and the value of late and early apoptosis was reduced in the treatment groups, and necrosis is decreased in the prevention group (group 4 compared to group 1). Moreover, it was revealed through cell cycle analysis that G2 in group 4 was reduced compared to group 1 and the expression of BAX, BAX/ BCL-X, and BCL-X in group 3 and group 4, was decreased and increased, respectively compared to group 1. ConclusionThese findings suggest that perhaps a non-toxic dosage of METH (low dose) can reduce the amount of apoptosis and BAX expression and increase the expression of BCL-X. Furthermore, the cells are arrested in the G2 phase and can raise the amount of extracellular glutamine, which has a protective role in neuron cells. These findings may provide a new perspective to design a new drug with less toxic results.

HIV-1 Tat and cocaine coexposure impacts piRNAs to affect astrocyte energy metabolism.

Aim: To understand the effect of HIV infection and cocaine exposure on piRNA expression in human primary astrocytes. Materials & methods: We used small RNA sequencing analysis to investigate the impacts of HIV-1 Tat and cocaine coexposure on the expression of piRNAs in human primary astrocytes. Results: We identified 27,700 piRNAs and analyzed them by small RNA next-generation sequencing. A total of 239 piRNAs were significantly altered by HIV-1 Tat and cocaine coexposure. We also identified PIWIL1, PIWIL2, PIWIL3 and PIWIL4 as interacting partners of piRNAs that were affected by cocaine and HIV-1 Tat coexposure. Epigenetic changes in the expression levels of these piRNA targets were associated with Kyoto Encyclopedia of Genes and Genomes pathways of energy metabolism and neurodegeneration. Conclusion: These findings provide evidence that cocaine exposure and HIV infection affect the expression levels of piRNA, PIWIL1, PIWIL2, PIWIL3 and PIWIL4.

HIV-1 Tat endocytosis and retention in endolysosomes affects HIV-1 Tat-induced LTR transactivation in astrocytes.

The presence of latent HIV-1 reservoirs in the periphery and brain represents a major obstacle to curing HIV-1 infection. As an essential protein for HIV-1 viral replication, HIV-1Tat, mostly intracellular, has been implicated in latent HIV-1 infection. From HIV-1 infected cells, HIV-1Tat is actively secreted and bystander cells uptake the released Tat whereupon it is endocytosed and internalized into endolysosomes. However, to activate the HIV-1 LTR promoter and increase HIV-1 replication, HIV-1Tat must first escape from the endolysosomes and then enter the nucleus. Here, we tested the hypothesis that HIV-1Tat can accumulate in endolysosomes and contribute to the activation of latent HIV-1 in astrocytes. Using U87MG astrocytoma cells expressing HIV-1 LTR-driven luciferase and primary human astrocytes we found that exogenous HIV-1Tat enters endolysosomes, resides in endolysosomes for extended periods of time, and induces endolysosome de-acidification as well as enlargement. The weak base chloroquine promoted the release of HIV-1Tat from endolysosomes and induced HIV-1 LTR transactivation. Similar results were observed by activating endolysosome Toll-like receptor 3 (TLR3) and TLR7/8. Conversely, pharmacological block of TLRs and knocking down expression levels of TLR3 and TLR7, but not TLR8, prevented endolysosome leakage and attenuated HIV-1Tat-mediated HIV-1 LTR transactivation. Our findings suggest that HIV-1Tat accumulation in endolysosomes may play an important role in controlling HIV-1 transactivation.

Coronin 1A facilitates calcium mobilization and promotes astrocyte reactivity in HIV-1 neuropathogenesis.

Astrocyte reactivity, a phenomenon observed in a variety of neurodegenerative disorders, can have both beneficial and detrimental manifestations which significantly affect neuronal physiology. In neuroAIDS, reactive astrocytes have been observed to severely affect the neuronal population present in their vicinity. Calcium signaling plays a central role in mediating astrocyte reactivity. Coronin 1A, an actin‐binding protein, majorly reported in hematopoietic cells, regulates cell activity in a calcium‐dependent manner, but its role in astrocyte physiology and reactivity is largely unknown. Using a well‐characterized primary culture of human astroglia and neurons, we explored the roles of coronin 1A in astrocyte physiology and its involvement in facilitating astrocyte reactivity. In this study, we report coronin 1A expression in human primary astrocytes and autopsy brain sections, and that it plays activity‐dependent roles by facilitating calcium mobilization from the intracellular stores. HIV‐1 Tat, a potent neurotoxicant that turns astrocytes reactive, augments coronin 1A expression, apart from affecting GFAP and pro‐inflammatory molecules. Also, the autopsy brain tissue of HIV‐1 infected individuals has a higher expression of coronin 1A. Downregulation of coronin 1A attenuated the HIV‐1 Tat‐induced deleterious effects of reactive astrocytes, measured as the upregulated expression of GFAP, pro‐inflammatory molecules, and enhanced release of IL‐6, and hence reduced astrocyte‐mediated neurodegeneration. Our findings also suggest that out of a pool of dysregulated miRNAs studied by us, hsa‐miR‐92b‐5p regulates coronin 1A expression under the effect of HIV‐1 Tat. These findings highlight the novel roles of coronin 1A in regulating astrocyte activity in stimulated conditions and astrocyte reactivity observed in HIV‐1 neuropathogenesis.

MicroRNA-210 regulates the metabolic and inflammatory status of primary human astrocytes

BackgroundAstrocytes are the most numerous glial cell type with important roles in maintaining homeostasis and responding to diseases in the brain. Astrocyte function is subject to modulation by microRNAs (miRs), which are short nucleotide strands that regulate protein expression in a post-transcriptional manner. Understanding the miR expression profile of astrocytes in disease settings provides insight into the cellular stresses present in the microenvironment and may uncover pathways of therapeutic interest.MethodsLaser-capture microdissection was used to isolate human astrocytes surrounding stroke lesions and those from neurological control tissue. Astrocytic miR expression profiles were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Primary human fetal astrocytes were cultured under in vitro stress conditions and transfection of a miR mimic was used to better understand how altered levels of miR-210 affect astrocyte function. The astrocytic response to stress was studied using qPCR, enzyme-linked immunosorbent assays (ELISAs), measurement of released lactate, and Seahorse.ResultsHere, we measured miR expression levels in astrocytes around human ischemic stroke lesions and observed differential expression of miR-210 in chronic stroke astrocytes compared to astrocytes from neurological control tissue. We also identified increased expression of miR-210 in mouse white matter tissue around middle cerebral artery occlusion (MCAO) brain lesions. We aimed to understand the role of miR-210 in primary human fetal astrocytes by developing an in vitro assay of hypoxic, metabolic, and inflammatory stresses. A combination of hypoxic and inflammatory stresses was observed to upregulate miR-210 expression. Transfection with miR-210-mimic (210M) increased glycolysis, enhanced lactate export, and promoted an anti-inflammatory transcriptional and translational signature in astrocytes. Additionally, 210M transfection resulted in decreased expression of complement 3 (C3) and semaphorin 5b (Sema5b).ConclusionsWe conclude that miR-210 expression in human astrocytes is modulated in response to ischemic stroke disease and under in vitro stress conditions, supporting a role for miR-210 in the astrocytic response to disease conditions. Further, the anti-inflammatory and pro-glycolytic impact of miR-210 on astrocytes makes it a potential candidate for further research as a neuroprotective agent.

Ponesimod inhibits astrocyte-mediated neuroinflammation and protects against cingulum demyelination via S1P1 -selective modulation.

Ponesimod is a sphingosine 1‐phosphate (S1P) receptor (S1PR) modulator that was recently approved for treating relapsing forms of multiple sclerosis (MS). Three other FDA‐approved S1PR modulators for MS—fingolimod, siponimod, and ozanimod—share peripheral immunological effects via common S1P1 interactions, yet ponesimod may access distinct central nervous system (CNS) mechanisms through its selectivity for the S1P1 receptor. Here, ponesimod was examined for S1PR internalization and binding, human astrocyte signaling and single‐cell RNA‐seq (scRNA‐seq) gene expression, and in vivo using murine cuprizone‐mediated demyelination. Studies confirmed ponesimod’s selectivity for S1P1 without comparable engagement to the other S1PR subtypes (S1P2,3,4,5). Ponesimod showed pharmacological properties of acute agonism followed by chronic functional antagonism of S1P1. A major locus of S1P1 expression in the CNS is on astrocytes, and scRNA‐seq of primary human astrocytes exposed to ponesimod identified a gene ontology relationship of reduced neuroinflammation and reduction in known astrocyte disease‐related genes including those of immediate early astrocytes that have been strongly associated with disease progression in MS animal models. Remarkably, ponesimod prevented cuprizone‐induced demyelination selectively in the cingulum, but not in the corpus callosum. These data support the CNS activities of ponesimod through S1P1, including protective, and likely selective, effects against demyelination in a major connection pathway of the brain, the limbic fibers of the cingulum, lesions of which have been associated with several neurologic impairments including MS fatigue.