Search

How astrocytes grow and integrate into neural circuits remains poorly defined. Zebrafish are well-suited for such investigations, but bona fide astrocytes have not been described in this system. Here, we characterize a zebrafish cell type that is remarkably similar to mammalian astrocytes that derive from radial glial cells and elaborate processes to establish their territories at early larval stages. Zebrafish astrocytes associate closely with synapses, tile with one another, and express markers including Glast and glutamine synthetase. Once integrated into circuits, they exhibit whole-cell and microdomain Ca2+ transients, which are sensitive to norepinephrine. Finally, using a cell-specific CRISPR/Cas9 approach we demonstrate that fgfr3/4 are required for vertebrate astrocyte morphogenesis. This work provides the first visualization of astrocyte morphogenesis from stem cell to post-mitotic astrocyte in vivo, identifies a role for Fgf receptors in vertebrate astrocytes, and establishes zebrafish as a valuable new model system to study astrocyte biology in vivo.

Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand., Here, the authors report the development of a versatile academic, SARSCoV-2 RT-qPCR molecular diagnostic test that uses 3D printed technology for sample collection, is implemented in rural setting in the US state of Virginia and validated in its population.

Focal traumatic brain injury (TBI) induces astrogliosis, a process essential to protecting uninjured brain areas from secondary damage. However, astrogliosis can cause loss of astrocyte homeostatic functions and possibly contributes to comorbidities such as posttraumatic epilepsy (PTE). Scar-forming astrocytes seal focal injuries off from healthy brain tissue. It is these glial scars that are associated with epilepsy originating in the cerebral cortex and hippocampus. However, the vast majority of human TBIs also present with diffuse brain injury caused by acceleration-deceleration forces leading to tissue shearing. The resulting diffuse tissue damage may be intrinsically different from focal lesions that would trigger glial scar formation. Here, we used mice of both sexes in a model of repetitive mild/concussive closed-head TBI, which only induced diffuse injury, to test the hypothesis that astrocytes respond uniquely to diffuse TBI and that diffuse TBI is sufficient to cause PTE. Astrocytes did not form scars and classic astrogliosis characterized by upregulation of glial fibrillary acidic protein was limited. Surprisingly, an unrelated population of atypical reactive astrocytes was characterized by the lack of glial fibrillary acidic protein expression, rapid and sustained downregulation of homeostatic proteins and impaired astrocyte coupling. After a latency period, a subset of mice developed spontaneous recurrent seizures reminiscent of PTE in human TBI patients. Seizing mice had larger areas of atypical astrocytes compared with nonseizing mice, suggesting that these atypical astrocytes might contribute to epileptogenesis after diffuse TBI.SIGNIFICANCE STATEMENTTraumatic brain injury (TBI) is a leading cause of acquired epilepsies. Reactive astrocytes have long been associated with seizures and epilepsy in patients, particularly after focal/lesional brain injury. However, most TBIs also include nonfocal, diffuse injuries. Here, we showed that repetitive diffuse TBI is sufficient for the development of spontaneous recurrent seizures in a subset of mice. We identified an atypical response of astrocytes induced by diffuse TBI characterized by the rapid loss of homeostatic proteins and lack of astrocyte coupling while reactive astrocyte markers or glial scar formation was absent. Areas with atypical astrocytes were larger in animals that later developed seizures suggesting that this response may be one root cause of epileptogenesis after diffuse TBI.

In the adult brain, multiple cell types are known to produce factors that regulate blood-brain barrier properties, including astrocytes. Yet several recent studies disputed a role for mature astrocytes at the blood-brain barrier. To determine if astrocytes contribute a non-redundant and necessary function in maintaining the adult blood-brain barrier, we used a mouse model of tamoxifen-inducible astrocyte ablation. In adult mice, tamoxifen induction caused sparse apoptotic astrocyte cell death within 2 hours. Indicative of BBB damage, leakage of the small molecule Cadaverine and the large plasma protein fibrinogen into the brain parenchyma indicative of BBB damage was detected as early as astrocyte ablation was present. Vessels within and close to regions of astrocyte loss had lower expression of the tight junction protein zonula occludens-1 while endothelial glucose transporter 1 expression was undisturbed. Cadaverine leakage persisted for several weeks suggesting a lack of barrier repair. This is consistent with the finding that ablated astrocytes were not replaced. Adjacent astrocytes responded with partial non-proliferative astrogliosis, characterized by morphological changes and delayed phosphorylation of STAT3, which restricted dye leakage to the brain and vessel surface areas lacking coverage by astrocytes one month after ablation. In conclusion, astrocytes are necessary to maintain blood-brain barrier integrity in the adult brain. Blood-brain barrier-regulating factors secreted by other cell types, such as pericytes, are not sufficient to compensate for astrocyte loss.

9 páginas, 5 figuras, 1 tabla, Lafora disease (LD) is a fatal rare neurodegenerative disorder characterized by epilepsy, neurodegeneration and insoluble polyglucosan accumulation in brain and other peripheral tissues. Although in the last two decades we have increased our knowledge on the molecular basis underlying the pathophysiology of LD, only a small part of the research in LD has paid attention to the mechanisms triggering one of the most lethal features of the disease: epilepsy. Recent studies in our laboratory suggested that a dysfunction in the activity of the mouse astrocytic glutamate transporter 1 (GLT-1) could contribute to epilepsy in LD. In this work, we present new in vivo evidence of a GLT-1 dysfunction, contributing to increased levels of extracellular glutamate in the hippocampus of a mouse model of Lafora disease (Epm2b-/-, lacking the E3-ubiquitin ligase malin). According to our results, Epm2b-/- mice showed an increased neuronal activity, as assessed by c-fos expression, in the hippocampus, an area directly correlated to epileptogenesis. This brain area presented lesser ability to remove synaptic glutamate after local GLT-1 blockade with dihydrokainate (DHK), in comparison to Epm2b+/+ animals, suggesting that these animals have a compromised glutamate clearance when a challenging condition was presented. These results correlate with a hippocampal upregulation of the minor isoform of the Glt-1 gene, named Glt-1b, which has been associated with compensatory mechanisms activated in response to neuronal stress. In conclusion, the hippocampus of Epm2b-/- mice presents an in vivo impairment in glutamate uptake which could contribute to epileptogenesis., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness SAF2014-54604-C3-1-R, SAF2017-83151-R (to P.S.) and SAF2015-68346-P (to F.A.), co-funded by the European Regional Development Fund “A way to build Europe” (to F.A.), a grant from Fundación Ramón Areces (CIVP18A3935) and a grant from the National Institute of Health (NIH-NINDS) P01NS097197, which established the Lafora Epilepsy Cure Initiative (LECI), to P.S. The Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM) and de Enfermedades Raras (CIBERER) are also acknowledged for financial support. We also thank Letizia Campa and Verónica Paz for their outstanding technical assistance. C.M.-B. holds a FPU fellowship from the Spanish Ministry of Education, Culture and Sports.

Unprovoked recurrent seizures are a serious comorbidity affecting most patients who suffer from glioma, a primary brain tumor composed of malignant glial cells. Cellular mechanisms contributing to the development of recurrent spontaneous seizures include the release of the excitatory neurotransmitter glutamate from glioma into extracellular space. Under physiological conditions, astrocytes express two high affinity glutamate transporters, Glt-1 and Glast, which are responsible for the removal of excess extracellular glutamate. In the context of neurological disease or brain injury, astrocytes become reactive which can negatively affect neuronal function, causing hyperexcitability and/or death. Using electrophysiology, immunohistochemistry, fluorescent in situ hybridization, and Western blot analysis in different orthotopic xenograft and allograft models of human and mouse gliomas, we find that peritumoral astrocytes exhibit astrocyte scar formation characterized by proliferation, cellular hypertrophy, process elongation, and increased GFAP and pSTAT3. Overall, peritumoral reactive astrocytes show a significant reduction in glutamate and potassium uptake, as well as decreased glutamine synthetase activity. A subset of peritumoral astrocytes displayed a depolarized resting membrane potential, further contributing to reduced potassium and glutamate homeostasis. These changes may contribute to the propagation of peritumoral neuronal hyperexcitability and excitotoxic death. Funding was provided by the National Institutes of Health (NIH) RO1 NS036692 (HS), RO1 NS082851 (HS), RO1 NS052634 (HS), RO1 NS105807 (SR). SR was also supported by the Epilepsy Foundation and the American Brain Tumor Association (ABTA). Accepted version