Balloon cells promote immune system activation in focal cortical dysplasia type 2b.

AIMS: Focal cortical dysplasia (FCD) type 2 is an epileptogenic malformation of the neocortex associated with somatic mutations in the mammalian target of rapamycin (mTOR) pathway. Histopathologically, FCD 2 is subdivided into FCD 2a and FCD 2b, the only discriminator being the presence of balloon cells (BCs) in FCD 2b. While pro-epileptogenic immune system activation and inflammatory responses are commonly detected in both subtypes, it is unknown what contextual role BCs play. METHODS: The present study employed RNA sequencing of surgically resected brain tissue from FCD 2a (n = 11) and FCD 2b (n = 20) patients compared to autopsy control (n = 9) focusing on three immune system processes: adaptive immunity, innate immunity and cytokine production. This analysis was followed by immunohistochemistry on a clinically well-characterised FCD 2 cohort. RESULTS: Differential expression analysis revealed stronger expression of components of innate immunity, adaptive immunity and cytokine production in FCD 2b than in FCD 2a, particularly complement activation and antigen presentation. Immunohistochemical analysis confirmed these findings, with strong expression of leukocyte antigen I and II in FCD 2b as compared to FCD 2a. Moreover, T-lymphocyte tissue infiltration was elevated in FCD 2b. Expression of markers of immune system activation in FCD 2b was concentrated in subcortical white matter. Lastly, antigen presentation was strongly correlated with BC load in FCD 2b lesions. CONCLUSION: We conclude that, next to mutation-driven mTOR activation and seizure activity, BCs are crucial drivers of inflammation in FCD 2b. Our findings indicate that therapies targeting inflammation may be beneficial in FCD 2b.

MicroRNA-34a activation in tuberous sclerosis complex during early brain development may lead to impaired corticogenesis.

AIMS: Tuberous sclerosis complex (TSC) is a genetic disorder associated with dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. Neurodevelopmental disorders, frequently present in TSC, are linked to cortical tubers in the brain. We previously reported microRNA-34a (miR-34a) among the most upregulated miRs in tubers. Here, we characterised miR-34a expression in tubers with the focus on the early brain development and assessed the regulation of mTORC1 pathway and corticogenesis by miR-34a. METHODS: We analysed the expression of miR-34a in resected cortical tubers (n = 37) compared with autopsy-derived control tissue (n = 27). The effect of miR-34a overexpression on corticogenesis was assessed in mice at E18. The regulation of the mTORC1 pathway and the expression of the bioinformatically predicted target genes were assessed in primary astrocyte cultures from three patients with TSC and in SH-SY5Y cells following miR-34a transfection. RESULTS: The peak of miR-34a overexpression in tubers was observed during infancy, concomitant with the presence of pathological markers, particularly in giant cells and dysmorphic neurons. miR-34a was also strongly expressed in foetal TSC cortex. Overexpression of miR-34a in mouse embryos decreased the percentage of cells migrated to the cortical plate. The transfection of miR-34a mimic in TSC astrocytes negatively regulated mTORC1 and decreased the expression of the target genes RAS related (RRAS) and NOTCH1. CONCLUSIONS: MicroRNA-34a is most highly overexpressed in tubers during foetal and early postnatal brain development. miR-34a can negatively regulate mTORC1; however, it may also contribute to abnormal corticogenesis in TSC.

Seizure-mediated iron accumulation and dysregulated iron metabolism after status epilepticus and in temporal lobe epilepsy.

Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes.

microRNA-132 is overexpressed in glia in temporal lobe epilepsy and reduces the expression of pro-epileptogenic factors in human cultured astrocytes.

Temporal lobe epilepsy (TLE) is a chronic neurological disease in humans, which is refractory to pharmacological treatment in about 30% of the patients. Reactive glial cells are thought to play a major role during the development of epilepsy (epileptogenesis) via regulation of brain inflammation and remodeling of the extracellular matrix (ECM). These processes can be regulated by microRNAs (miRs), a class of small non-coding RNAs, which can control entire gene networks at a post-transcriptional level. The expression of miRs is known to change dynamically during epileptogenesis. miR-132 is one of the most commonly upregulated miRs in animal TLE models with important roles shown in neurons. However, the possible role of miR-132 in glia remains largely unknown. The aim of this study was to characterize the cell-type specific expression of miR-132 in the hippocampus of patients with TLE and during epileptogenesis in a rat TLE model. Furthermore, the potential role of miR-132 was investigated by transfection of human primary cultured astrocytes that were stimulated with the cytokines IL-1ß or TGF-ß1. We showed an increased expression of miR-132 in the human and rat epileptogenic hippocampus, particularly in glial cells. Transfection of miR-132 in human primary astrocytes reduced the expression of pro-epileptogenic COX-2, IL-1ß, TGF-ß2, CCL2, and MMP3. This suggests that miR-132, particularly in astrocytes, represents a potential therapeutic target that warrants further in vivo investigation.

SorCS2 facilitates release of endostatin from astrocytes and controls post-stroke angiogenesis.

SorCS2 is an intracellular sorting receptor of the VPS10P domain receptor gene family recently implicated in oxidative stress response. Here, we interrogated the relevance of stress-related activities of SorCS2 in the brain by exploring its role in ischemic stroke in mouse models and in patients. Although primarily seen in neurons in the healthy brain, expression of SorCS2 was massively induced in astrocytes surrounding the ischemic core in mice following stroke. Post-stroke induction was likely a result of increased levels of transforming growth factor ß1 in damaged brain tissue, inducing Sorcs2 gene transcription in astrocytes but not neurons. Induced astrocytic expression of SorCS2 was also seen in stroke patients, substantiating the clinical relevance of this observation. In astrocytes in vitro and in the mouse brain in vivo, SorCS2 specifically controlled release of endostatin, a factor linked to post-stroke angiogenesis. The ability of astrocytes to release endostatin acutely after stroke was lost in mice deficient for SorCS2, resulting in a blunted endostatin response which coincided with impaired vascularization of the ischemic brain. Our findings identified activated astrocytes as a source for endostatin in modulation of post-stroke angiogenesis, and the importance of the sorting receptor SorCS2 in this brain stress response.

miR147b: A novel key regulator of interleukin 1 beta-mediated inflammation in human astrocytes.

Astrocytes are important mediators of inflammatory processes in the brain and seem to play an important role in several neurological disorders, including epilepsy. Recent studies show that astrocytes produce several microRNAs, which may function as crucial regulators of inflammatory pathways and could be used as therapeutic target. We aim to study which miRNAs are produced by astrocytes during IL-1ß mediated inflammatory conditions in vitro, as well as their functional role and to validate these findings in human epileptogenic brain tissue. Sequencing was used to assess miRNA and mRNA expression in IL-1ß-stimulated human fetal astrocyte cultures. miRNAs were overexpressed in cell cultures using miRNA mimics. Expression of miRNAs in resected brain tissue from patients with tuberous sclerosis complex or temporal lobe epilepsy with hippocampal sclerosis was examined using in situ hybridization. Two differentially expressed miRNAs were found: miR146a and miR147b, which were associated with increased expression of genes related to the immune/inflammatory response. As previously reported for miR146a, overexpression of miR147b reduced the expression of the pro-inflammatory mediators IL-6 and COX-2 after IL-1ß stimulation in both astrocyte and tuberous sclerosis complex cell cultures. miR146a and miR147b overexpression decreased proliferation of astrocytes and promoted neuronal differentiation of human neural stem cells. Similarly to previous evidence for miR146a, miR147b was increased expressed in astrocytes in epileptogenic brain. Due to their anti-inflammatory effects, ability to restore aberrant astrocytic proliferation and promote neuronal differentiation, miR146a and miR147b deserve further investigation as potential therapeutic targets in neurological disorders associated with inflammation, such as epilepsy.

Increased expression of matrix metalloproteinase 3 can be attenuated by inhibition of microRNA-155 in cultured human astrocytes.

BACKGROUND: Temporal lobe epilepsy (TLE) is a chronic neurological disease, in which about 30% of patients cannot be treated adequately with anti-epileptic drugs. Brain inflammation and remodeling of the extracellular matrix (ECM) seem to play a major role in TLE. Matrix metalloproteinases (MMPs) are proteolytic enzymes largely responsible for the remodeling of the ECM. The inhibition of MMPs has been suggested as a novel therapy for epilepsy; however, available MMP inhibitors lack specificity and cause serious side effects. We studied whether MMPs could be modulated via microRNAs (miRNAs). Several miRNAs mediate inflammatory responses in the brain, which are known to control MMP expression. The aim of this study was to investigate whether an increased expression of MMPs after interleukin-1ß (IL-1ß) stimulation can be attenuated by inhibition of the inflammation-associated miR-155. METHODS: We investigated the expression of MMP2, MMP3, MMP9, and MMP14 in cultured human fetal astrocytes after stimulation with the pro-inflammatory cytokine IL-1ß. The cells were transfected with miR-155 antagomiR, and the effect on MMP3 expression was investigated using real-time quantitative PCR and Western blotting. Furthermore, we characterized MMP3 and miR-155 expression in brain tissue of TLE patients with hippocampal sclerosis (TLE-HS) and during epileptogenesis in a rat TLE model. RESULTS: Inhibition of miR-155 by the antagomiR attenuated MMP3 overexpression after IL-1ß stimulation in astrocytes. Increased expression of MMP3 and miR-155 was also evident in the hippocampus of TLE-HS patients and throughout epileptogenesis in the rat TLE model. CONCLUSIONS: Our experiments showed that MMP3 is dynamically regulated by seizures as shown by increased expression in TLE tissue and during different phases of epileptogenesis in the rat TLE model. MMP3 can be induced by the pro-inflammatory cytokine IL-1ß and is regulated by miR-155, suggesting a possible strategy to prevent epilepsy via reduction of inflammation.

Activation of the innate immune system is evident throughout epileptogenesis and is associated with blood-brain barrier dysfunction and seizure progression.

OBJECTIVE: Because brain inflammation may contribute to the pathophysiology of temporal lobe epilepsy (TLE), we investigated the expression of various inflammatory markers of the innate and adaptive immune system in the epileptogenic human and rat hippocampus in relation to seizure activity and blood-brain barrier (BBB) dysfunction. METHODS: Immunohistochemistry was performed using various immune cell markers (for microglia, monocytes, macrophages, T lymphocytes, and dendritic cells) on hippocampal sections of drug-resistant TLE patients and patients who died after status epilepticus. The expression of these markers was also studied in the electrical post-status epilepticus rat model for TLE, during the acute, latent, and chronic epileptic phase. BBB dysfunction was assessed using albumin immunohistochemistry and the BBB tracer fluorescein. RESULTS: Monocyte infiltration, microglia, and perivascular macrophage activation were persistently increased in both epileptogenic human and rat hippocampus, whereas T lymphocytes and dendritic cells were not or were scarcely detected. In addition to this, increased expression of C-C motif ligand 2 (CCL2) and osteopontin was observed. In humans, the expression of CD68 and CCL2 was related to the duration of epilepsy and type of pathology. In rats, the expression of CD68, CCL2, and the perivascular macrophage marker CD163 was related to the duration of the initial insult and to the number of spontaneous seizures. Interestingly, the number of CD163-positive perivascular macrophages was also positively correlated to BBB dysfunction in chronic epileptic rats. SIGNIFICANCE: These data suggest a proepileptogenic role for monocytes/macrophages and other cells of the innate immune response, possibly via increased BBB leakage, and indicate that T cells and dendritic cells, which are closely associated with the adaptive immune response, are only sparsely infiltrated during epileptogenesis in the electrical post-status epilepticus rat model. Future studies should reveal the relative importance of these immune cells and whether specific manipulation can modify or prevent epileptogenesis.

Increased expression of (immuno)proteasome subunits during epileptogenesis is attenuated by inhibition of the mammalian target of rapamycin pathway.

OBJECTIVE: Inhibition of the mammalian target of rapamycin (mTOR) pathway reduces epileptogenesis in various epilepsy models, possibly by inhibition of inflammatory processes, which may include the proteasome system. To study the role of mTOR inhibition in the regulation of the proteasome system, we investigated (immuno)proteasome expression during epileptogenesis, as well as the effects of the mTOR inhibitor rapamycin. METHODS: The expression of constitutive (ß1, ß5) and immunoproteasome (ß1i, ß5i) subunits was investigated during epileptogenesis using immunohistochemistry in the electrical post-status epilepticus (SE) rat model for temporal lobe epilepsy (TLE). The effect of rapamycin was studied on (immuno)proteasome subunit expression in post-SE rats that were treated for 6 weeks. (Immuno)proteasome expression was validated in the brain tissue of patients who had SE or drug-resistant TLE and the effect of rapamycin was studied in primary human astrocyte cultures. RESULTS: In post-SE rats, increased (immuno)proteasome expression was detected throughout epileptogenesis in neurons and astrocytes within the hippocampus and piriform cortex and was most evident in rats that developed a progressive form of epilepsy. Rapamycin-treated post-SE rats had reduced (immuno)proteasome protein expression and a lower number of spontaneous seizures compared to vehicle-treated rats. (Immuno)proteasome expression was also increased in neurons and astrocytes within the human hippocampus after SE and in patients with drug-resistant TLE. In vitro studies using cultured human astrocytes showed that interleukin (IL)-1ß-induced (immuno)proteasome gene expression could be attenuated by rapamycin. SIGNIFICANCE: Because dysregulation of the (immuno)proteasome system is observed before the occurrence of spontaneous seizures in rats, is associated with progression of epilepsy, and can be modulated via the mTOR pathway, it may represent an interesting novel target for drug treatment in epilepsy.

Effective reduction in seizure severity and prevention of a fatty liver by a novel low ratio ketogenic diet composition in the rapid kindling rat model of epileptogenesis.

Drug-resistant epilepsy patients may benefit from non-pharmacological therapies, such as the ketogenic diet (KD). However, its high fat content poses compliance challenges and metabolic risks. To mitigate this, we developed a novel KD composition with less fat and additional nutrients (citrate, nicotinamide riboside, and omega-3 fatty acids) for ketone-independent neuroprotection. The efficacy, metabolic and neuropathological effects of the novel KD and a classic KD were compared to a control diet in the rapid kindling model of temporal lobe epilepsy. Both KD groups entered ketosis before kindling onset, with higher ketone levels in the classic KD group. Remarkably, rats on the novel KD had slower progression of behavioral seizures as compared to rats on a control diet, while this was not the case for rats on a classic KD. Both KDs reduced electrographic after-discharge duration, preserved neurons in the dorsal hippocampus, and normalized activity in open field tests. The novel KD, despite lower fat and ketone levels, demonstrated effective reduction of behavioral seizure severity while the classic KD did not, suggesting alternative mode(s) of action are involved. Additionally, the novel KD significantly mitigated liver triglyceride and plasma fatty acid levels compared to the classic KD, indicating a reduced risk of long-term liver steatosis. Our findings highlight the potential of the novel KD to enhance therapeutic efficacy and compliance in epilepsy patients.

Loss of maturity and homeostatic functions in Tuberous Sclerosis Complex-derived astrocytes.

Introduction: Constitutive activation of the mTOR pathway, as observed in Tuberous Sclerosis Complex (TSC), leads to glial dysfunction and subsequent epileptogenesis. Although astrocytes are considered important mediators for synaptic clearance and phagocytosis, little is known on how astrocytes contribute to the epileptogenic network. Methods: We employed singlenuclei RNA sequencing and a hybrid fetal calf serum (FCS)/FCS-free cell culture model to explore the capacity of TSC-derived astrocytes to maintain glutamate homeostasis and clear debris in their environment. Results: We found that TSC astrocytes show reduced maturity on RNA and protein level as well as the inability to clear excess glutamate through the loss of both enzymes and transporters complementary to a reduction of phagocytic capabilities. Discussion: Our study provides evidence of mechanistic alterations in TSC astrocytes, underscoring the significant impairment of their supportive functions. These insights enhance our understanding of TSC pathophysiology and hold potential implications for future therapeutic interventions.

Altered Extracellular Matrix as an Alternative Risk Factor for Epileptogenicity in Brain Tumors.

Seizures are one of the most common symptoms of brain tumors. The incidence of seizures differs among brain tumor type, grade, location and size, but paediatric-type diffuse low-grade gliomas/glioneuronal tumors are often highly epileptogenic. The extracellular matrix (ECM) is known to play a role in epileptogenesis and tumorigenesis because it is involved in the (re)modelling of neuronal connections and cell-cell signaling. In this review, we discuss the epileptogenicity of brain tumors with a focus on tumor type, location, genetics and the role of the extracellular matrix. In addition to functional problems, epileptogenic tumors can lead to increased morbidity and mortality, stigmatization and life-long care. The health advantages can be major if the epileptogenic properties of brain tumors are better understood. Surgical resection is the most common treatment of epilepsy-associated tumors, but post-surgery seizure-freedom is not always achieved. Therefore, we also discuss potential novel therapies aiming to restore ECM function.

The Gelatinase Inhibitor ACT-03 Reduces Gliosis in the Rapid Kindling Rat Model of Epilepsy, and Attenuates Inflammation and Loss of Barrier Integrity In Vitro.

Matrix metalloproteinases (MMPs) are endopeptidases responsible for the cleavage of intra- and extracellular proteins. Several brain MMPs have been implicated in neurological disorders including epilepsy. We recently showed that the novel gelatinase inhibitor ACT-03 has disease-modifying effects in models of epilepsy. Here, we studied its effects on neuroinflammation and blood-brain barrier (BBB) integrity. Using the rapid kindling rat model of epilepsy, we examined whether ACT-03 affected astro- and microgliosis in the brain using immunohistochemistry. Cellular and molecular alterations were further studied in vitro using human fetal astrocyte and brain endothelial cell (hCMEC/D3) cultures, with a focus on neuroinflammatory markers as well as on barrier permeability using an endothelial and astrocyte co-culture model. We observed less astro- and microgliosis in the brains of kindled animals treated with ACT-03 compared to control vehicle-treated animals. In vitro, ACT-03 treatment attenuated stimulation-induced mRNA expression of several pro-inflammatory factors in human fetal astrocytes and brain endothelial cells, as well as a loss of barrier integrity in endothelial and astrocyte co-cultures. Since ACT-03 has disease-modifying effects in epilepsy models, possibly via limiting gliosis, inflammation, and barrier integrity loss, it is of interest to further evaluate its effects in a clinical trial.

Enhancing GAT-3 in thalamic astrocytes promotes resilience to brain injury in rodents.

Inflammatory processes induced by brain injury are important for recovery; however, when uncontrolled, inflammation can be deleterious, likely explaining why most anti-inflammatory treatments have failed to improve neurological outcomes after brain injury in clinical trials. In the thalamus, chronic activation of glial cells, a proxy of inflammation, has been suggested as an indicator of increased seizure risk and cognitive deficits that develop after cortical injury. Furthermore, lesions in the thalamus, more than other brain regions, have been reported in patients with viral infections associated with neurological deficits, such as SARS-CoV-2. However, the extent to which thalamic inflammation is a driver or by-product of neurological deficits remains unknown. Here, we found that thalamic inflammation in mice was sufficient to phenocopy the cellular and circuit hyperexcitability, enhanced seizure risk, and disruptions in cortical rhythms that develop after cortical injury. In our model, down-regulation of the GABA transporter GAT-3 in thalamic astrocytes mediated this neurological dysfunction. In addition, GAT-3 was decreased in regions of thalamic reactive astrocytes in mouse models of cortical injury. Enhancing GAT-3 in thalamic astrocytes prevented seizure risk, restored cortical states, and was protective against severe chemoconvulsant-induced seizures and mortality in a mouse model of traumatic brain injury, emphasizing the potential of therapeutically targeting this pathway. Together, our results identified a potential therapeutic target for reducing negative outcomes after brain injury.

Down-regulation of the brain-specific cell-adhesion molecule contactin-3 in tuberous sclerosis complex during the early postnatal period.

BACKGROUND: The genetic disorder tuberous sclerosis complex (TSC) is frequently accompanied by the development of neuropsychiatric disorders, including autism spectrum disorder and intellectual disability, with varying degrees of impairment. These co-morbidities in TSC have been linked to the structural brain abnormalities, such as cortical tubers, and recurrent epileptic seizures (in 70-80% cases). Previous transcriptomic analysis of cortical tubers revealed dysregulation of genes involved in cell adhesion in the brain, which may be associated with the neurodevelopmental deficits in TSC. In this study we aimed to investigate the expression of one of these genes - cell-adhesion molecule contactin-3. METHODS: Reverse transcription quantitative polymerase chain reaction for the contactin-3 gene (CNTN3) was performed in resected cortical tubers from TSC patients with drug-resistant epilepsy (n = 35, age range: 1-48 years) and compared to autopsy-derived cortical control tissue (n = 27, age range: 0-44 years), as well as by western blot analysis of contactin-3 (n = 7 vs n = 7, age range: 0-3 years for both TSC and controls) and immunohistochemistry (n = 5 TSC vs n = 4 controls). The expression of contactin-3 was further analyzed in fetal and postnatal control tissue by western blotting and in-situ hybridization, as well as in the SH-SY5Y neuroblastoma cell line differentiation model in vitro. RESULTS: CNTN3 gene expression was lower in cortical tubers from patients across a wide range of ages (fold change = - 0.5, p < 0.001) as compared to controls. Contactin-3 protein expression was lower in the age range of 0-3 years old (fold change = - 3.8, p < 0.001) as compared to the age-matched controls. In control brain tissue, contactin-3 gene and protein expression could be detected during fetal development, peaked around birth and during infancy and declined in the adult brain. CNTN3 expression was induced in the differentiated SH-SY5Y neuroblastoma cells in vitro (fold change = 6.2, p < 0.01). CONCLUSIONS: Our data show a lower expression of contactin-3 in cortical tubers of TSC patients during early postnatal period as compared to controls, which may affect normal brain development and might contribute to neuropsychiatric co-morbidities observed in patients with TSC.

Tuberous Sclerosis Complex as Disease Model for Investigating mTOR-Related Gliopathy During Epileptogenesis.

Tuberous sclerosis complex (TSC) represents the prototypic monogenic disorder of the mammalian target of rapamycin (mTOR) pathway dysregulation. It provides the rational mechanistic basis of a direct link between gene mutation and brain pathology (structural and functional abnormalities) associated with a complex clinical phenotype including epilepsy, autism, and intellectual disability. So far, research conducted in TSC has been largely neuron-oriented. However, the neuropathological hallmarks of TSC and other malformations of cortical development also include major morphological and functional changes in glial cells involving astrocytes, oligodendrocytes, NG2 glia, and microglia. These cells and their interglial crosstalk may offer new insights into the common neurobiological mechanisms underlying epilepsy and the complex cognitive and behavioral comorbidities that are characteristic of the spectrum of mTOR-associated neurodevelopmental disorders. This review will focus on the role of glial dysfunction, the interaction between glia related to mTOR hyperactivity, and its contribution to epileptogenesis in TSC. Moreover, we will discuss how understanding glial abnormalities in TSC might give valuable insight into the pathophysiological mechanisms that could help to develop novel therapeutic approaches for TSC or other pathologies characterized by glial dysfunction and acquired mTOR hyperactivation.

Increased expression of miR142 and miR155 in glial and immune cells after traumatic brain injury may contribute to neuroinflammation via astrocyte activation.

Traumatic brain injury (TBI) is associated with the pathological activation of immune-competent cells in the brain, such as astrocytes, microglia and infiltrating immune blood cells, resulting in chronic inflammation and gliosis. This may contribute to the secondary injury after TBI, thus understanding of these processes is crucial for the development of effective treatments of post-traumatic pathologies. MicroRNAs (miRNAs, miRs) are small noncoding RNAs, functioning as posttranscriptional regulators of gene expression. The increased expression of inflammation-associated microRNAs miR155 and miR142 has been reported after TBI in rats. However, expression of these miRNAs in the human brain post-TBI is not studied and their functions are not well understood. Moreover, circulating miR155 and miR142 are candidate biomarkers. Therefore, we characterized miR142 and miR155 expression in the perilesional cortex and plasma of rats that underwent lateral fluid-percussion injury, a model for TBI and in the human perilesional cortex post-TBI. We demonstrated higher miR155 and miR142 expression in the perilesional cortex of rats 2 weeks post-TBI. In plasma, miR155 was associated with proteins and miR142 with extracellular vesicles, however their expression did not change. In the human perilesional cortex miR155 was most prominently expressed by activated astrocytes, whereas miR142 was expressed predominantly by microglia, macrophages and lymphocytes. Pro-inflammatory medium from macrophage-like cells stimulated miR155 expression in astrocytes and overexpression of miR142 in these cells further potentiated a pro-inflammatory state of activated astrocytes. We conclude that miR155 and miR142 promote brain inflammation via astrocyte activation and may be involved in the secondary brain injury after TBI.

Dysregulation of the MMP/TIMP Proteolytic System in Subependymal Giant Cell Astrocytomas in Patients With Tuberous Sclerosis Complex: Modulation of MMP by MicroRNA-320d In Vitro.

Tuberous sclerosis complex (TSC), a rare genetic disorder caused by a mutation in the TSC1 or TSC2 gene, is characterized by the growth of hamartomas in several organs. This includes the growth of low-grade brain tumors, known as subependymal giant cell astrocytomas (SEGA). Previous studies have shown differential expression of genes related to the extracellular matrix in SEGA. Matrix metalloproteinases (MMPs), and their tissue inhibitors (TIMPs) are responsible for remodeling the extracellular matrix and are associated with tumorigenesis. This study aimed to investigate the MMP/TIMP proteolytic system in SEGA and the regulation of MMPs by microRNAs, which are important post-transcriptional regulators of gene expression. We investigated the expression of MMPs and TIMPs using previously produced RNA-Sequencing data, real-time quantitative PCR and immunohistochemistry in TSC-SEGA samples and controls. We found altered expression of several MMPs and TIMPs in SEGA compared to controls. We identified the lowly expressed miR-320d in SEGA as a potential regulator of MMPs, which can decrease MMP2 expression in human fetal astrocyte cultures. This study provides evidence of a dysregulated MMP/TIMP proteolytic system in SEGA of which MMP2 could be rescued by microRNA-320d. Therefore, further elucidating microRNA-mediated MMP regulation may provide insights into SEGA pathogenesis and identify novel therapeutic targets.