Alberto Javier Ramos

Astrocytes are among the first cellular responders to central nervous system injury, yet the mechanisms governing their earliest responses remain incompletely understood. Here, we investigated astrocyte dynamics during the first hours after focal cortical injury induced by cortical devascularization in rats. We observed a rapid and spatially restricted increase in glial fibrillary acidic protein (GFAP) and aquaporin‐4 (AQP4) immunoreactivity surrounding the ischemic core as early as 1.5–3.5 h post‐lesion, in association with blood–brain barrier disruption and edema‐related changes. Within the injury core, astrocytes displayed differential GFAP detection by monoclonal and polyclonal antibodies, together with the appearance of lower‐molecular‐weight GFAP fragments both in vivo and after oxygen–glucose deprivation in vitro, suggesting GFAP cleavage in severely damaged astrocytes. At the chromatin level, astrocytes proximal to the lesion exhibited reduced histone H3 acetylation, particularly histone 3 acetylation at lysine 9 (H3K9ac), a phenomenon recapitulated in cultured astrocytes exposed to hypo‐osmolar stress. This reduction was transient, reversible upon recovery, and prevented by histone deacetylase (HDAC) inhibition. Functionally, hypo‐osmolar stress conditioned astrocyte responses to subsequent stimuli, attenuating nuclear factor kappa B (NF‐κB) activation and complement 3 (C3) induction after lipopolysaccharide exposure while enhancing proliferative capacity during recovery. Together, these findings identify edema‐associated osmotic stress as an early regulator of astrocyte epigenetic state and functional plasticity, suggesting that astrocytes exposed to edema are primed to adopt distinct responses that may contribute to tissue repair and scar formation following brain injury. image

Epilepsy is a debilitating neurological disorder characterized by recurrent seizures, affecting millions of patients worldwide. Retrospective studies in temporal lobe epilepsy (TLE) patients have shown a high incidence of an initial precipitating event (IPE) in early childhood, followed by a silent period where epileptogenesis occurs to end up in chronic epilepsy. Epileptogenesis, the process through which a normal brain undergoes structural and functional changes leading to epilepsy, is not completely understood. We hypothesized that epigenetics may be involved in epileptogenesis, specifically affecting astrocytes through pathological remodeling. To study this process, we used three approaches: the lithium–pilocarpine model of TLE in rats, primary astroglial cultures exposed to epileptogenic DAMP named HMGB1, and brain tissue samples resected from TLE patients with drug-resistant epilepsy. We found that the IPE achieved by lithium–pilocarpine treatment (127/30 mg/kg IP) induced the DNA methylation of astrocytes at 7-, 21-, and 35-days post-IPE, indicating persistent epigenetic alterations in astrocytes during the epileptogenic period. In addition, we observed the down-regulation of homeostatic astroglial genes, including AQP4, glutamine synthase (GS), and Kir4.1, along with increased expression of proinflammatory genes (C3, MAFG) and DNA methyltransferases (DNMT). These alterations were mimicked in primary astrocyte cultures exposed to the epileptogenic HMGB1 (500 ng/ml; 18 h), which resulted in the hypermethylation of homeostatic astroglial genes and repression of homeostatic genes. HMGB1-induced repression of astroglial homeostatic genes was prevented by the treatment with DNMT inhibitor decitabine. Interestingly, astrocytes from TLE patients brains showed reactive astrogliosis, increased DNA methylation, and down-regulation of homeostatic genes Kir4.1 and GS. Taken together, these findings show that astrocytes are pathologically altered during the epileptogenic period by epigenetic modifications, combining the proinflammatory gain of function with the loss of homeostatic profile. This may contribute to the long-term alterations underlying epileptogenesis.

Epilepsy is a debilitating neurological disorder characterized by recurrent seizures, affecting millions of patients worldwide. Retrospective studies in Temporal lobe epilepsy (TLE) patients have shown a high incidence of an initial precipitating event (IPE) in early childhood followed by a silent period where epileptogenesis occurs to end up in chronic epilepsy. Epileptogenesis, the process through which a normal brain undergoes structural and functional changes leading to epilepsy, remains an enigmatic phenomenon. We hypothesized that epigenetics may be involved in epileptogenesis and specifically astrocytes could be affected by pathological remodeling. To study this process, we used three approaches: The lithium-pilocarpine model of TLE in rats, primary astroglial cultures exposed to epileptogenic DAMP named HMGB1, and brain tissue samples resected from TLE patients with drug-resistant epilepsy. We found that the IPE achieved by lithium- pilocarpine treatment (127/30 mg/kg IP) induced the hypermethylation of astrocytes at 7-, 21-, and 35 days post-IPE, indicating persistent epigenetic alterations in astrocytes during the epileptogenic period. In addition, we observed the downregulation of homeostatic astroglial genes AQP4; glutamine synthase (GS), and Kir4.1 with increased proinflammatory genes (C3, MAFG) and DNA methyl transferases (DNMT) expression. These alterations were mimicked in primary astrocyte cultures exposed to the epileptogenic HMGB1 (500 ng/ml; 18 hours) that also induced the hypermethylation of homeostatic astroglial genes. Astrocytes from TLE patients brains showed reactive astrogliosis, increased DNA methylation, and downregulation of homeostatic genes Kir4.1 and GS. These findings show that astrocytes are pathologically altered during the epileptogenic period, combining the proinflammatory gain of function with the loss of homeostatic profile. This may sustain the long-term alterations underlining epileptogenesis.

This work demonstrates the need for caspase-3 activity to eliminate weaker synapses in the well-known visual system where most of the pruning work has been done. It presents several experiments aimed at demonstrating an interesting mechanism of synap...

Astrocytes provide metabolic support to neurons, maintain ionic and water homeostasis, and uptake and recycle neurotransmitters. After exposure to the prototypical PAMP lipopolysaccharide (LPS), reactive astrocytes increase the expression of pro‐inflammatory genes, facilitating neurodegeneration. In this study, we analyzed the expression of homeostatic genes in astrocytes exposed to LPS and identified the epigenetic factors contributing to the suppression of homeostatic genes in reactive astrocytes. Primary astrocytic cultures were acutely exposed to LPS and allowed to recover for 24, 72 h, and 7 days. As expected, LPS exposure induced reactive astrogliosis and increased the expression of pro‐inflammatory IL‐1B and IL‐6. Interestingly, the acute exposure resulted in persistent hypermethylation of astroglial DNA. Similar hypermethylation was observed in highly reactive astrocytes from the traumatic brain injury (TBI) penumbra in vivo. Hypermethylation was accompanied by decreased expression of homeostatic genes including LDHA and Scl16a1 (MCT1) both involved in the lactate shuttle to neurons; glutamine synthase (GS) responsible for glutamate processing; Kcnj10 (Kir4.1) important for K⁺ homeostasis, and the water channel aquaporin‐4 (Aqp4). Furthermore, the master regulator of DNA methylation, MAFG‐1, as well as DNA methyl transferases DNMT1 and DNMT3a were overexpressed. The downregulation of homeostatic genes correlated with increased methylation of CpG islands in their promoters, as assessed by methylation‐sensitive PCR and increased DNMT3a binding to the GS promoter. Treatment with decitabine, a DNMT inhibitor, prevented the LPS‐ and the HMGB‐1‐induced downregulation of homeostatic genes. Decitabine treatment also prevented the neurotoxic effects of these astrocytes in primary cortical cultures. In summary, our findings reveal that the pathological remodeling of reactive astrocytes encompasses not only the pro‐inflammatory response but, significantly, also entails a long‐term suppression of homeostatic gene expression with methylation of crucial CpG islands within their promoters. image

Traumatic brain injury is a global health crisis, causing significant death and disability worldwide. Neuroinflammation that follows traumatic brain injury has serious consequences for neuronal survival and cognitive impairments, with astrocytes involved in this response. Following traumatic brain injury, astrocytes rapidly become reactive, and astrogliosis propagates from the injury core to distant brain regions. Homeostatic astroglial proteins are downregulated near the traumatic brain injury core, while pro-inflammatory astroglial genes are overexpressed. This altered gene expression is considered a pathological remodeling of astrocytes that produces serious consequences for neuronal survival and cognitive recovery. In addition, glial scar formed by reactive astrocytes is initially necessary to limit immune cell infiltration, but in the long term impedes axonal reconnection and functional recovery. Current therapeutic strategies for traumatic brain injury are focused on preventing acute complications. Statins, cannabinoids, progesterone, beta-blockers, and cerebrolysin demonstrate neuroprotective benefits but most of them have not been studied in the context of astrocytes. In this review, we discuss the cell signaling pathways activated in reactive astrocytes following traumatic brain injury and we discuss some of the potential new strategies aimed to modulate astroglial responses in traumatic brain injury, especially using cell-targeted strategies with miRNAs or lncRNA, viral vectors, and repurposed drugs.

Epilepsy, a chronic neurological disorder characterized by recurrent seizures, affects millions of individuals worldwide. Despite extensive research, the underlying mechanisms leading to epileptogenesis, the process by which a normal brain develops epilepsy, remain elusive. We, here, explored the immune system and spleen responses triggered by pilocarpine-induced status epilepticus (SE) focusing on their role in the epileptogenesis that follows SE. Initial examination of spleen histopathology revealed transient disorganization of white pulp, in animals subjected to SE. This disorganization, attributed to immune activation, peaked at 1-day post-SE (1DPSE) but returned to control levels at 3DPSE. Alterations in peripheral blood lymphocyte populations, demonstrated a decrease following SE, accompanied by a reduction in CD3+ T-lymphocytes. Further investigations uncovered an increased abundance of T-lymphocytes in the piriform cortex and choroid plexus at 3DPSE, suggesting a specific mobilization toward the Central Nervous System. Notably, splenectomy mitigated brain reactive astrogliosis, neuroinflammation, and macrophage infiltration post-SE, particularly in the hippocampus and piriform cortex. Additionally, splenectomized animals exhibited reduced lymphatic follicle size in the deep cervical lymph nodes. Most significantly, splenectomy correlated with improved neuronal survival, substantiated by decreased neuronal loss and reduced degenerating neurons in the piriform cortex and hippocampal CA2-3 post-SE. Overall, these findings underscore the pivotal role of the spleen in orchestrating immune responses and neuroinflammation following pilocarpine-induced SE, implicating the peripheral immune system as a potential therapeutic target for mitigating neuronal degeneration in epilepsy.

Astrocytes are the main homeostatic cells in the central nervous system (CNS) and they have an essential role in preserving neuronal physiology. After brain injury, astrocytes become reactive, and that involves a profound change in the astroglial gene expression program as well as intense cytoskeleton remodeling that has been classically shown by the up‐regulation of glial fibrillary acidic protein (GFAP), a pan‐reactive gene over‐expressed in reactive astrocytes, independently of the type of injury. Using the stab wound rodent model of penetrating traumatic injury in the cortex, we here studied the reactive astroglial morphology and reactive microgliosis in detail at 1, 3, 7, 14, and 28 days post‐injury (dpi). By combining immunohistochemistry, morphometrical parameters, and Sholl analysis, we segmented the astroglial cell population into clusters of reactive astrocytes that were localized in the core, penumbra, and distal regions of the stab wound. Specifically, highly reactive clusters with more complex morphology, increased C3, decreased aquaporin‐4 (AQP4), and glutamine synthetase (GS) expression, were enriched at 7 dpi when behavioral alterations, microgliosis, and neuronal alterations in injured mice were most significant. While pro‐inflammatory gain of function with peripheral lipopolysaccharide (LPS) administration immediately after a stab wound expanded these highly reactive astroglial clusters, the treatment with the NF‐κB inhibitor sulfasalazine reduced the abundance of this highly reactive cluster. Increased neuronal loss and exacerbated reactive microgliosis at 7 dpi were associated with the expansion of the highly reactive astroglial cluster. We conclude that highly reactive astrocytes found in stab wound injury, but expanded in pro‐inflammatory conditions, are a population of astrocytes that become engaged in pathological remodeling with a pro‐inflammatory gain of function and loss of homeostatic capacity. Controlling this astroglial population may be a tempting strategy to reduce neuronal loss and neuroinflammation in the injured brain. image