Browsing by Subject "Astrocytes"
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Item Activated endothelial cells induce a distinct type of astrocytic reactivity(Springer Nature, 2022-03-29) Taylor, Xavier; Cisternas, Pablo; Jury, Nur; Martinez, Pablo; Huang, Xiaoqing; You, Yanwen; Redding-Ochoa, Javier; Vidal, Ruben; Zhang, Jie; Troncoso, Juan; Lasagna-Reeves, Cristian A.; Anatomy, Cell Biology and Physiology, School of MedicineReactive astrogliosis is a universal response of astrocytes to abnormal events and injuries. Studies have shown that proinflammatory microglia can polarize astrocytes (designated A1 astrocytes) toward a neurotoxic phenotype characterized by increased Complement Component 3 (C3) expression. It is still unclear if inflammatory stimuli from other cell types may also be capable of inducing a subset of C3+ neurotoxic astrocytes. Here, we show that a subtype of C3+ neurotoxic astrocytes is induced by activated endothelial cells that is distinct from astrocytes activated by microglia. Furthermore, we show that endothelial-induced astrocytes have upregulated expression of A1 astrocytic genes and exhibit a distinctive extracellular matrix remodeling profile. Finally, we demonstrate that endothelial-induced astrocytes are Decorin-positive and are associated with vascular amyloid deposits but not parenchymal amyloid plaques in mouse models and AD/CAA patients. These findings demonstrate the existence of potentially extensive and subtle functional diversity of C3+-reactive astrocytes.Item Activation of Egr-1 expression in astrocytes by HIV-1 Tat: new insights into astrocyte-mediated Tat neurotoxicity(Springer Nature, 2011-03) Fan, Yan; Zou, Wei; Green, Linden A.; oh Kim, Byung; He, Johnny J.; Microbiology and Immunology, School of MedicineHuman immunodeficiency virus type 1 (HIV-1) Tat plays an important role in HIV-associated neuropathogenesis; the underlying mechanisms are still evolving. We have recently shown that HIV-1 Tat induces expression of glial fibrillary acidic protein (GFAP), a characteristic of HIV-1 infection of the central nervous system (CNS). We have also shown that the Tat-induced GFAP expression in astrocytes is regulated by p300, and that deletion of the early growth response 1 (Egr-1) cis-transacting element within the p300 promoter abolishes Tat-induced GFAP expression. In this study, we further examined the relationship between Tat and Egr-1 in astrocytes. We found increased Egr-1 protein expression in Tat-expressing human astrocytoma cells and mouse primary astrocytes. Using the Egr-1 promoter-driven firefly luciferase reporter gene assay and the site-directed mutagenesis, we demonstrated that Tat increased Egr-1 expression by transactivating the Egr-1 promoter and involving specific serum response elements (SRE) within the promoter. Consistent with these data, we showed that Tat transactivation of the Egr-1 promoter was abrogated when astrocytes were cultured in serum-reduced media. Taken together, these results reveal that Tat directly transactivates Egr-1 expression and suggest that Tat interaction with Egr-1 is probably one of the very upstream molecular events that initiate Tat-induced astrocyte dysfunction and subsequent Tat neurotoxicity.Item Analyses of the development and function of stem cell derived cells in neurodegenerative diseases(2022-12) Lavekar, Sailee Sham; Meyer, Jason; Canfield, Scott; Belecky-Adams, Teri; Mastracci, Teresa; Perrin, BenjaminHuman pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction. Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.Item Association between Change in the peripheral biomarkers of inflammation, astrocyte activation, and neuroprotection at one week of critical illness and hospital mortality in patients with delirium: A prospective cohort study(Public Library of Science, 2023-09-01) Khan, Sikandar H.; Perkins, Anthony J.; Eltarras, Ahmed M.; Chi, Rosalyn; Athar, Ammar A.; Wang, Sophia; Campbell, Noll L.; Gao, Sujuan; Boustani, Malaz A.; Khan, Babar A.; Medicine, School of MedicineObjective: In critically ill adults with delirium, biomarkers of systemic inflammation, astrocyte activation, neuroprotection, and systemic inflammation measured at one week of critical illness may be associated with mortality. Design: Prospective observational study. Setting: Intensive care unit (ICU). Patients: 178 ICU patients with delirium, alive and remaining in ICU at one week. Interventions: None. Measurements and main results: Blood samples collected for a pair of previously published, negative, clinical trials were utilized. Samples were collected at study enrollment/ICU admission (Day 1 sample) and one week later (Day 8 sample), and analyzed for interleukins (IL)-6, 8, 10, Insulin-like Growth Factor (IGF), S100 Binding Protein (S100B), Tumor Necrosis Factor Alpha (TNF-A) and C-Reactive Protein (CRP). Delirium, delirium severity, and coma were assessed twice daily using Confusion Assessment Method for the Intensive Care Unit (CAM-ICU), CAM-ICU-7, and Richmond Agitation-Sedation Scale (RASS), respectively. Mortality was assessed until discharge using the electronic medical record. Logistic regression models adjusting for age, sex, severity of illness, comorbidities, sepsis, and randomization status, were used to assess the relationship among biomarkers and mortality. Higher IL-10 quartiles at day 8 were associated with increased odds of hospital mortality (IL-10: OR 2.00 95%CI: 1.1-3.65, p = 0.023). There was a significant interaction between day 1 and day 8 biomarker quartiles only for IL-6. Patients with IL-6 values in the first three quartiles on admission to the ICU that transitioned to higher IL-6 quartiles at day 8 had increased probability of hospital mortality. Conclusion: In this hypothesis-generating study, higher IL-6 and IL-10 quartiles at one week, and increase in IL-6 from day 1 to day 8 were associated with increased hospital mortality. Studies with larger sample sizes are needed to confirm the mechanisms for these observations.Item Astrocytic GABA transporter 1 deficit in novel SLC6A1 variants mediated epilepsy: Connected from protein destabilization to seizures in mice and humans(Elsevier, 2022) Mermer, Felicia; Poliquin, Sarah; Zhou, Shuizhen; Wang, Xiaodong; Ding, Yifeng; Yin, Fei; Shen, Wangzhen; Wang, Juexin; Rigsby, Kathryn; Xu, Dong; Mack, Taralynn; Nwosu, Gerald; Flamm, Carson; Stein, Matthew; Kang, Jing-Qiong; Biomedical Engineering and Informatics, Luddy School of Informatics, Computing, and EngineeringObjective: Mutations in γ-aminobutyric acid (GABA) transporter 1 (GAT-1)-encoding SLC6A1 have been associated with myoclonic atonic epilepsy and other phenotypes. We determined the patho-mechanisms of the mutant GAT-1, in order to identify treatment targets. Methods: We conducted whole-exome sequencing of patients with myoclonic atonic epilepsy (MAE) and characterized the seizure phenotypes and EEG patterns. We studied the protein stability and structural changes with homology modeling and machine learning tools. We characterized the function and trafficking of the mutant GAT-1 with 3H radioactive GABA uptake assay and confocal microscopy. We utilized different models including a knockin mouse and human astrocytes derived from induced pluripotent stem cells (iPSCs). We focused on astrocytes because of their direct impact of astrocytic GAT-1 in seizures. Results: We identified four novel SLC6A1 variants associated with MAE and 2 to 4 Hz spike-wave discharges as a common EEG feature. Machine learning tools predicted that the variant proteins are destabilized. The variant protein had reduced expression and reduced GABA uptake due to endoplasmic reticular retention. The consistent observation was made in cortical and thalamic astrocytes from variant-knockin mice and human iPSC-derived astrocytes. The Slc6a+/A288V mouse, representative of MAE, had increased 5-7 Hz spike-wave discharges and absence seizures. Interpretation: SLC6A1 variants in various locations of the protein peptides can cause MAE with similar seizure phenotypes and EEG features. Reduced GABA uptake is due to decreased functional GAT-1, which, in thalamic astrocytes, could result in increased extracellular GABA accumulation and enhanced tonic inhibition, leading to seizures and abnormal EEGs.Item BMP Pathway and Reactive Retinal Gliosis(2013-03-06) Dharmarajan, Subramanian; Belecky-Adams, Teri; Skalnik, David Gordon; Zhang, Xin; Atkinson, SimonReactive gliosis is known to have a beneficial and a degenerative effect following injury to neurons. Although many factors have been implicated in reactive gliosis, their role in regulating this change is still unclear. We investigated the role of bone morphogenetic proteins in reactive gliosis in vivo and in vitro. In vivo, IHC analysis indicated reactive gliosis in the 6 week Ins2Akita mouse and WPK rat retinas. Expression of BMP7 was upregulated in these models, leading to an increase in the phosphorylation of downstream SMAD1. In vitro, treatment of murine retinal astrocyte cells with a strong oxidizing agent such as sodium peroxynitrite regulated RNA levels of various markers, including GFAP, CSPGs, MMPs and TIMPs. BMP7 treatment also regulated RNA levels to a similar extent, suggesting reactive gliosis. Treatment with high glucose DMEM and BMP4, however, did not elicit increase in levels to a similar degree. Increase in SMAD levels and downstream targets of SMAD signaling such as ID1, ID3 and MSX2 was also observed following treatment with sodium peroxynitrite in vitro and in the 6 week Ins2Akita mouse retinas in vivo. These data concur with previously established data which show an increase in BMP7 levels following injury. It also demonstrates a role for BMP7 in gliosis following disease. Further, it suggests SMAD signaling to play a role in initiating reactivity in astrocytes as well as in remodeling the extracellular matrix following injury and in a disease condition.Item BMP PATHWAY AND RETINAL ASTROGLIOSIS(Office of the Vice Chancellor for Research, 2012-04-13) Dharmarajan, Subramanian; Sheibani, Nadeer; Belecky-Adams, TeriAstrocytes are the star shaped glial (non-neural) cells present in the cen-tral nervous system. The astrocytes perform a wide range of functions in-cluding: serving as an energy source, aiding neuronal development, main-taining homeostasis and detoxification. Astrocytes are also present in the eye, constituting the majority of the glial cell population and are found in the retina. Any injury to nearby neurons makes the astrocytes reactive, altering their function and morphology. One of the features of reactive astrocytes is to form a physical barrier around the injury site, called the glial scar. One group of molecules thought to play a role is the bone morphogenic proteins (BMPs). Although, the BMPs have been found to increase in CNS following injury, their role in making the retinal astrocytes reactive is not yet known. Our purpose in performing these studies was to clarify the role of BMP7 (a type of the BMP) in retinal astrocytes reactive gliosis. Mouse retinal astrocyte cells were incubated with sodium peroxynitrite (a strong oxidizing agent, which has been previously shown to make astrocytes reactive) or different cocncentrations of BMP7 for different time periods. Cells were lysed and total protein or total RNA was isolated to analyze the protein and gene expression levels of different markers. Treatment with peroxynitrite led to statistically significant increase in levels of expression in including astrocyte specific markers and certain in-hibitory molecules. In the BMP7 treated samples, similar increases in the levels of expression for the astrocyte specific markers and the inhibitory molecules. A comparison between the expression profiles of peroxynitrite- and BMP7-treated cells showed largely (but not completely) overlapping pro-files of expression. The work done here helps ascertain the role of BMP7 in reactive gliosis in vitro. Further, the observation of distinct reactivity profiles under different conditions is indicative of involvement of additional pathways.Item Bone morphogenetic protein 7 regulates reactive gliosis in retinal astrocytes and Müller glia(Emory, 2014-07-31) Dharmarajan, Subramanian; Gurel, Zafer; Wang, Shoujian; Sorenson, Christine M.; Sheibani, Nader; Belecky-Adams, Teri L.; Biology, School of SciencePurpose: The focus of this study was to determine whether bone morphogenetic proteins (BMPs) trigger reactive gliosis in retinal astrocytes and/or Müller glial cells. Methods: Retinal astrocytes and the Müller glial cell line MIO-M1 were treated with vehicle, BMP7, or BMP4. Samples from the treated cells were analyzed for changes in gliosis markers using reverse transcriptase - quantitative PCR (RT-qPCR) and western blotting. To determine potential similarities and differences in gliosis states, control and BMP-treated cells were compared to cells treated with sodium peroxynitrite (a strong oxidizing agent that will bring about some aspects of gliosis). Last, mature mice were microinjected intravitreally with BMP7 and analyzed for changes in gliosis markers using RT-qPCR, western blotting, and immunohistochemistry. Results: Treatment of retinal astrocyte cells and Müller glial cells with BMP7 regulated various reactive gliosis markers. When compared to the response of cells treated with sodium peroxynitrite, the profiles of gliosis markers regulated due to exposure to BMP7 were similar. However, as expected, the profiles including the oxidative agent and growth factor were not identical. Treatment of cells with BMP4, however, showed an attenuated response in comparison to peroxynitrite and BMP7 treatment. Injection of BMP7 into the mouse retina also triggered a reactive gliosis response 7 days after injection. Conclusions: BMP7 induced changes in levels of mRNA and protein markers typically associated with reactive gliosis in retinal astrocytes and Müller glial cells, including glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), a subset of chondroitin sulfate proteoglycans (CSPGs), matrix metalloproteinases (MMPs), and other molecules.Item Brain astrocytes and microglia express functional MR1 molecules that present microbial antigens to mucosal-associated invariant T (MAIT) cells(Elsevier, 2020-12-15) Priya, Raj; Brutkiewicz, Randy R.; Microbiology and Immunology, School of MedicineIt is unknown whether brain astrocytes and microglia have the capacity to present microbial antigens via the innate immune MR1/MAIT cell axis. We have detected MAIT cells in the normal mouse brain and found that both astrocytes and microglia are MR1+. When we stimulated brain astrocytes and microglia with E. coli, and then co-cultured them with MAIT cells, MR1 surface expression was upregulated and MAIT cells were activated in an antigen-dependent manner. Considering the association of MAIT cells with inflammatory conditions, including those in the CNS, the MR1/MAIT cell axis could be a novel therapeutic target in neuroinflammatory disorders.Item Cell surface proteoglycans control astrocyte migration and retinal angiogenesis by regulating basement membrane assembly(2015-12-15) Tao, Chenqi; Zhang, XinElaborate vascularization of the retina is crucial for the development and functioning of the eye. The proper patterning of astrocytes is a key event preceding retinal angiogenesis by providing guidance cues for endothelial cells, yet how this is regulated still remains obscure. The dual function of proteoglycans in both extracellular matrix (ECM) composition and cell signal transduction suggests their potential in the regulation of astrocyte migration. The current study demonstrated that non-cell-autonomous regulation by neuroretina cell surface proteoglycan is crucial for PDGF-A regulated astrocyte migration. Ablation of glycosaminoglycan side chains of proteoglycans in neuroretina led to impaired astrocyte migration, incomplete retinal angiogenesis, and hyaloid vessel persistence. This is followed by severe photoreceptor degeneration as a result of reactive gliosis, which cannot be rescued by constitutively activated Kras signaling. Notably, inner limiting membrane (ILM), the basement membrane of the retina, was breached in proteoglycan-deficient retinae prior to the formation of astrocytic network. Herein we propose that cell surface proteoglycans are essential for the initial assembly of ILM, and this cannot be compensated by secreted ECM proteoglycans. In support of this, after removal of ILM in retinal explant by Collagenase digestion, establishment of a new ILM can be achieved by incubation with exogenous laminin-supplemented Matrigel. This basement membrane reconstitution failed, however, in proteoglycan-deficient retinae or in wild type samples digested with a combination of Heparinase and ChABC in addition to Collagenase. Taken together, our study reveals a novel function of neuroretinal cell surface proteoglycans in the initial assembly of basement membrane which subsequently serves as a permissive substratum necessary for astrocyte migration.