SorLA restricts TNFα release from microglia to shape a glioma-supportive brain microenvironment.

SorLA, encoded by the gene SORL1, is an intracellular sorting receptor of the VPS10P domain receptor gene family. Although SorLA is best recognized for its ability to shuttle target proteins between intracellular compartments in neurons, recent data suggest that also its microglial expression can be of high relevance for the pathogenesis of brain diseases, including glioblastoma (GBM). Here, we interrogated the impact of SorLA on the functional properties of glioma-associated microglia and macrophages (GAMs). In the GBM microenvironment, GAMs are re-programmed and lose the ability to elicit anti-tumor responses. Instead, they acquire a glioma-supporting phenotype, which is a key mechanism promoting glioma progression. Our re-analysis of published scRNA-seq data from GBM patients revealed that functional phenotypes of GAMs are linked to the level of SORL1 expression, which was further confirmed using in vitro models. Moreover, we demonstrate that SorLA restrains secretion of TNFα from microglia to restrict the inflammatory potential of these cells. Finally, we show that loss of SorLA exacerbates the pro-inflammatory response of microglia in the murine model of glioma and suppresses tumor growth.

miR-193b-3p/ PGC-1α pathway regulates an insulin dependent anti-inflammatory response in Parkinson's disease.

It has been shown that many miRNAs, including miR-193b-3p, are differentially expressed in Parkinson's disease (PD). Dysregulation of miR-193b-3p/PGC-1α pathway may alter homeostasis in cells and can induce an inflammatory response commonly accompanied by metabolic disturbances. The aim of the present study is to investigate if dysregulation of the miR-193-3p/PGC-1α axis may contribute to the pathological changes observed in the PD brain. Brain tissue were obtained from middle frontal gyrus of non-demented controls and individuals with a PD diagnosis. RT-qPCR was used to determine the expression of miR-193b-3p and in situ hybridization (ISH) and immunological analysis were employed to establish the cellular distribution of miR-193b-3p. Functional assays were performed using SH-SY5Y cells, including transfection and knock-down of miR-193b-3p. We found significantly lower expression of miR-193b-3p in the early stages of PD (PD4) which increased throughout disease progression. Furthermore, altered expression of PGC-1α suggested a direct inhibitory effect of miR-193b-3p in the brain of individuals with PD. Moreover, we observed changes in expression of insulin after transfection of SH-SY5Y cells with miR-193b-3p, which led to dysregulation in the expression of several pro- or anti - inflammatory genes. Our findings indicate that the miR-193b-3p/PGC-1α axis is involved in the regulation of insulin signaling. This regulation is crucial, since insulin induced inflammatory response may serve as a protective mechanism during acute situations but potentially evolve into a pathological process in chronic conditions. This novel regulatory mechanism may represent an interesting therapeutic target with potential benefits for various neurodegenerative diseases.

Activated Wake Systems in Narcolepsy Type 1.

OBJECTIVE: Narcolepsy type 1 (NT1) is assumed to be caused solely by a lack of hypocretin (orexin) neurotransmission. Recently, however, we found an 88% reduction in corticotropin-releasing hormone (CRH)-positive neurons in the paraventricular nucleus (PVN). We assessed the remaining CRH neurons in NT1 to determine whether they co-express vasopressin (AVP) to reflect upregulation. We also systematically assessed other wake-systems, since current NT1 treatments target histamine, dopamine, and norepinephrine pathways. METHODS: In postmortem tissue of people with NT1 and matched controls, we immunohistochemically stained and quantified neuronal populations expressing: CRH and AVP in the PVN, and CRH in the Barrington nucleus; the key neuronal histamine-synthesizing enzyme, histidine decarboxylase (HDC) in the hypothalamic tuberomammillary nucleus (TMN); the rate-limited-synthesizing enzyme, tyrosine hydroxylase (TH), for dopamine in the mid-brain and for norepinephrine in the locus coeruleus (LC). RESULTS: In NT1, there was: a 234% increase in the percentage of CRH cells co-expressing AVP, while there was an unchanged integrated optical density of CRH staining in the Barrington nucleus; a 36% increased number of histamine neurons expressing HDC, while the number of typical human TMN neuronal profiles was unchanged; a tendency toward an increased density of TH-positive neurons in the substantia nigra compacta; while the density of TH-positive LC neurons was unchanged. INTERPRETATION: Our findings suggest an upregulation of activity by histamine neurons and remaining CRH neurons in NT1. This may explain earlier reports of normal basal plasma cortisol levels but lower levels after dexamethasone suppression. Alternatively, CRH neurons co-expressing AVP neurons are less vulnerable. ANN NEUROL 2023;94:762-771.

Astroglial calcium signaling and homeostasis in tuberous sclerosis complex.

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by the development of benign tumors in various organs, including the brain, and is often accompanied by epilepsy, neurodevelopmental comorbidities including intellectual disability and autism. A key hallmark of TSC is the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway, which induces alterations in cortical development and metabolic processes in astrocytes, among other cellular functions. These changes could modulate seizure susceptibility, contributing to the progression of epilepsy and its associated comorbidities. Epilepsy is characterized by dysregulation of calcium (Ca2+) channels and intracellular Ca2+ dynamics. These factors contribute to hyperexcitability, disrupted synaptogenesis, and altered synchronization of neuronal networks, all of which contribute to seizure activity. This study investigates the intricate interplay between altered Ca2+ dynamics, mTOR pathway dysregulation, and cellular metabolism in astrocytes. The transcriptional profile of TSC patients revealed significant alterations in pathways associated with cellular respiration, ER and mitochondria, and Ca2+ regulation. TSC astrocytes exhibited lack of responsiveness to various stimuli, compromised oxygen consumption rate and reserve respiratory capacity underscoring their reduced capacity to react to environmental changes or cellular stress. Furthermore, our study revealed significant reduction of store operated calcium entry (SOCE) along with strong decrease of basal mitochondrial Ca2+ concentration and Ca2+ influx in TSC astrocytes. In addition, we observed alteration in mitochondrial membrane potential, characterized by increased depolarization in TSC astrocytes. Lastly, we provide initial evidence of structural abnormalities in mitochondria within TSC patient-derived astrocytes, suggesting a potential link between disrupted Ca2+ signaling and mitochondrial dysfunction. Our findings underscore the complexity of the relationship between Ca2+ signaling, mitochondria dynamics, apoptosis, and mTOR hyperactivation. Further exploration is required to shed light on the pathophysiology of TSC and on TSC associated neuropsychiatric disorders offering further potential avenues for therapeutic development.

Impaired GABAergic regulation and developmental immaturity in interneurons derived from the medial ganglionic eminence in the tuberous sclerosis complex.

GABAergic interneurons play a critical role in maintaining neural circuit balance, excitation-inhibition regulation, and cognitive function modulation. In tuberous sclerosis complex (TSC), GABAergic neuron dysfunction contributes to disrupted network activity and associated neurological symptoms, assumingly in a cell type-specific manner. This GABAergic centric study focuses on identifying specific interneuron subpopulations within TSC, emphasizing the unique characteristics of medial ganglionic eminence (MGE)- and caudal ganglionic eminence (CGE)-derived interneurons. Using single-nuclei RNA sequencing in TSC patient material, we identify somatostatin-expressing (SST+) interneurons as a unique and immature subpopulation in TSC. The disrupted maturation of SST+ interneurons may undergo an incomplete switch from excitatory to inhibitory GABAergic signaling during development, resulting in reduced inhibitory properties. Notably, this study reveals markers of immaturity specifically in SST+ interneurons, including an abnormal NKCC1/KCC2 ratio, indicating an imbalance in chloride homeostasis crucial for the postsynaptic consequences of GABAergic signaling as well as the downregulation of GABAA receptor subunits, GABRA1, and upregulation of GABRA2. Further exploration of SST+ interneurons revealed altered localization patterns of SST+ interneurons in TSC brain tissue, concentrated in deeper cortical layers, possibly linked to cortical dyslamination. In the epilepsy context, our research underscores the diverse cell type-specific roles of GABAergic interneurons in shaping seizures, advocating for precise therapeutic considerations. Moreover, this study illuminates the potential contribution of SST+ interneurons to TSC pathophysiology, offering insights for targeted therapeutic interventions.

Dysregulation of miR-543 in Parkinson's disease: Impact on the neuroprotective gene SIRT1.

AIMS: Parkinson's disease (PD) is a progressive and age-dependent neurodegenerative disease characterised clinically by a variety of motor symptoms and cognitive impairment. PD was initially considered to be a grey matter disease; however, recently, evidence has emerged that white matter changes in PD precede the neuronal loss seen in the grey matter. The cause of these initial white matter changes is yet to be elucidated. Here, we explored whether dysregulated miRNAs and their target mRNA could provide insight into the underlying mechanisms of early white matter changes in PD. METHODS: We analysed the expression of miRNAs in three different stages of PD through RNA-sequencing and validated the differential expression of miRNAs through quantitative reverse transcription polymerase chain reaction. With bioinformatic analyses, we predicted target genes of dysregulated miRNAs and investigated their biomarker potential. Finally, in vitro, we confirmed the targetting of the gene SIRT1 by miR-543. RESULTS: We identified 12 dysregulated miRNAs in PD and found that miR-543 holds potential as a biomarker for late-stage PD with dementia. We report upregulation of miR-543 in early PD white matter tissue and downregulation of SIRT1. In vitro experiments showed that the upregulation of miR-543 results in the downregulation of SIRT1 in the white matter, but not in the grey matter. CONCLUSIONS: We validated SIRT1 as a target of miR-543 in the brain and showed its function as a potential biomarker. Our results highlight the idea that dysregulation of miR-543 in early PD white matter, resulting in the dysregulation of SIRT1, potentially influencing the early white matter changes observed in PD.

Brain molecular mechanisms in Rasmussen encephalitis.

OBJECTIVE: This study was undertaken to identify molecular mechanisms in brain tissue of Rasmussen encephalitis (RE) when compared to people with non-RE epilepsy (PWE) and control cases using whole exome sequencing (WES), RNAseq, and proteomics. METHODS: Frozen brain tissue (ages = 2-19 years) was obtained from control autopsy (n = 14), surgical PWE (n = 10), and surgical RE cases (n = 27). We evaluated WES variants in RE associated with epilepsy, seizures, RE, and human leukocyte antigens (HLAs). Differential expression was evaluated by RNAseq (adjusted p < .05) and label-free quantitative mass spectrometry (false discovery rate < 5%) in the three groups. RESULTS: WES revealed no common pathogenic variants in RE, but several rare and likely deleterious variants of unknown significance (VUS; ANGPTL7/MTOR, SCN1A, FCGR3B, MTOR) and more common HLA VUS in >25% of RE cases (HLA-DRB1, HLA-DQA2), all with allele frequency < 5% in the general population. RNAseq in RE versus PWE (1516 altered transcripts) revealed significant activation of crosstalk between dendritic and natural killer cells (p = 7.94 × 10-6 , z = 2.65), in RE versus control (7466 transcripts) neuroinflammation signaling activation (p = 6.31 × 10-13 , z = 5.07), and in PWE versus control (945 transcripts) phagosome formation activation (p = 2.00 × 10-13 , z = 5.61). Proteomics detected fewer altered targets. SIGNIFICANCE: In RE, we identified activated immune signaling pathways and immune cell type annotation enrichment that suggest roles of the innate and adaptive immune responses, as well as HLA variants that may increase vulnerability to RE. Follow-up studies could evaluate cell type density and subregional localization associated with top targets, clinical history (neuropathology, disease duration), and whether modulating crosstalk between dendritic and natural killer cells may limit disease progression.

Histopathological characterization of cerebral small vessel disease in epilepsy patients with temporal lobe epilepsy submitted to surgery: A case-control study.

BACKGROUND: Cerebrovascular disease (CVD) is a major contributor to epilepsy; however, patients with epilepsy also have a significantly increased risk of stroke. The way in which epilepsy contributes to the increased risk of stroke is still uncertain and is ill-characterized in neuropathological studies. A neuropathological characterization of cerebral small vessel disease (cSVD) in patients with chronic epilepsy was performed. METHODS: Thirty-three patients with refractory epilepsy and hippocampal sclerosis (HS) submitted to epilepsy surgery from a reference center were selected between 2010 and 2020 and compared with 19 autopsy controls. Five randomly selected arterioles from each patient were analyzed using a previously validated scale for cSVD. The presence of CVD disease imaging markers in pre-surgical brain magnetic resonance imaging (MRI) was studied. RESULTS: There were no differences in age (43.8 vs. 41.6 years; p = 0.547) or gender distribution (female gender 60.6% vs. male gender 52.6%; p = 0.575) between groups. Most CVD findings in brain MRI were mild. Patients had a mean time between the epilepsy onset and surgery of 26 ± 14.7 years and were medicated with a median number of three antiseizure medication (ASMs) [IQR 2-3]. Patients had higher median scores in arteriolosclerosis (3 vs. 1; p < 0.0001), microhemorrhages (4 vs. 1; p < 0.0001) and total score value (12 vs. 8.9; p = 0.031) in comparison with controls. No correlation was found between age, number of years until surgery, number of ASMs or cumulative defined daily dosage of ASM. CONCLUSION: The present study provides evidence supporting the increased burden of cSVD in the neuropathological samples of patients with chronic epilepsy.

Distinct DNA Methylation Patterns of Subependymal Giant Cell Astrocytomas in Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is a monogenic disorder caused by mutations in either the TSC1 or TSC2 gene, two key regulators of the mechanistic target of the rapamycin complex pathway. Phenotypically, this leads to growth and formation of hamartomas in several organs, including the brain. Subependymal giant cell astrocytomas (SEGAs) are low-grade brain tumors commonly associated with TSC. Recently, gene expression studies provided evidence that the immune system, the MAPK pathway and extracellular matrix organization play an important role in SEGA development. However, the precise mechanisms behind the gene expression changes in SEGA are still largely unknown, providing a potential role for DNA methylation. We investigated the methylation profile of SEGAs using the Illumina Infinium HumanMethylation450 BeadChip (SEGAs n = 42, periventricular control n = 8). The SEGA methylation profile was enriched for the adaptive immune system, T cell activation, leukocyte mediated immunity, extracellular structure organization and the ERK1 & ERK2 cascade. More interestingly, we identified two subgroups in the SEGA methylation data and show that the differentially expressed genes between the two subgroups are related to the MAPK cascade and adaptive immune response. Overall, this study shows that the immune system, the MAPK pathway and extracellular matrix organization are also affected on DNA methylation level, suggesting that therapeutic intervention on DNA level could be useful for these specific pathways in SEGA. Moreover, we identified two subgroups in SEGA that seem to be driven by changes in the adaptive immune response and MAPK pathway and could potentially hold predictive information on target treatment response.

Increased expression of complement components in tuberous sclerosis complex and focal cortical dysplasia type 2B brain lesions.

OBJECTIVE: Increasing evidence supports the contribution of inflammatory mechanisms to the neurological manifestations of epileptogenic developmental pathologies linked to mammalian target of rapamycin (mTOR) pathway dysregulation (mTORopathies), such as tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD). In this study, we aimed to investigate the expression pattern and cellular distribution of the complement factors C1q and C3 in resected cortical tissue of clinically well-characterized patients with TSC and FCD2B. METHODS: We applied immunohistochemistry in TSC (n = 29) and FCD2B (n = 32) samples and compared them to autopsy and biopsy controls (n = 27). Furthermore, protein expression was observed via Western blot, and for descriptive colocalization studies immunofluorescence double labeling was performed. RESULTS: Protein expression for C3 was significantly upregulated in TSC and FCD2B white and gray matter lesions compared to controls. Staining of the synaptic vesicle protein synaptophysin showed a remarkable increase in the white matter of both TSC and FCD2B. Furthermore, confocal imaging revealed colocalization of complement factors with astroglial, microglial, neuronal, and abnormal cells in various patterns. SIGNIFICANCE: Our results demonstrate that the prominent activation of the complement pathway represents a common pathological hallmark of TSC and FCD2B, suggesting that complement overactivation may play a role in these mTORopathies.

Serotonin receptor expression in hippocampus and temporal cortex of temporal lobe epilepsy patients by postictal generalized electroencephalographic suppression duration.

OBJECTIVE: Prolonged postictal generalized electroencephalographic suppression (PGES) is a potential biomarker for sudden unexpected death in epilepsy (SUDEP), which may be associated with dysfunctional autonomic responses and serotonin signaling. To better understand molecular mechanisms, PGES duration was correlated to 5HT1A and 5HT2A receptor protein expression and RNAseq from resected hippocampus and temporal cortex of temporal lobe epilepsy patients with seizures recorded in preoperative evaluation. METHODS: Analyses included 36 cases (age = 14-64 years, age at epilepsy onset = 0-51 years, epilepsy duration = 2-53 years, PGES duration = 0-93 s), with 13 cases in all hippocampal analyses. 5HT1A and 5HT2A protein was evaluated by Western blot and histologically in hippocampus (n = 16) and temporal cortex (n = 9). We correlated PGES duration to our previous RNAseq dataset for serotonin receptor expression and signaling pathways, as well as weighted gene correlation network analysis (WGCNA) to identify correlated gene clusters. RESULTS: In hippocampus, 5HT2A protein by Western blot positively correlated with PGES duration (p = .0024, R2  = .52), but 5HT1A did not (p = .87, R2  = .0020). In temporal cortex, 5HT1A and 5HT2A had lower expression and did not correlate with PGES duration. Histologically, PGES duration did not correlate with 5HT1A or 5HT2A expression in hippocampal CA4, dentate gyrus, or temporal cortex. RNAseq identified two serotonin receptors with expression that correlated with PGES duration in an exploratory analysis: HTR3B negatively correlated (p = .043, R2  = .26) and HTR4 positively correlated (p = .049, R2  = .25). WGCNA identified four modules correlated with PGES duration, including positive correlation with synaptic transcripts (p = .040, Pearson correlation r = .52), particularly potassium channels (KCNA4, KCNC4, KCNH1, KCNIP4, KCNJ3, KCNJ6, KCNK1). No modules were associated with serotonin receptor signaling. SIGNIFICANCE: Higher hippocampal 5HT2A receptor protein and potassium channel transcripts may reflect underlying mechanisms contributing to or resulting from prolonged PGES. Future studies with larger cohorts should assess functional analyses and additional brain regions to elucidate mechanisms underlying PGES and SUDEP risk.

Impaired myelin production due to an intrinsic failure of oligodendrocytes in mTORpathies.

AIMS: We aim to evaluate if the myelin pathology observed in epilepsy-associated focal cortical dysplasia type 2B (FCD2B) and-histologically indistinguishable-cortical tubers of tuberous sclerosis complex (TSC) is primarily related to the underlying malformation or constitutes a secondary phenomenon due to the toxic microenvironment created by epileptic seizures. We also aim to investigate the possible beneficial effect of the mTOR pathway regulator everolimus on white matter pathology. METHODS: Primary mixed glial cell cultures derived from epilepsy surgery specimens of one TSC and seven FCD2B patients were grown on polycaprolactone fibre matrices and analysed using immunofluorescence and electron microscopy. Unaffected white matter from three age-matched epilepsy patients with mild malformations of cortical development (mMCD) and one with FCD3D served as controls. Additionally, TSC2 knock-out was performed using an oligodendroglial cell line. Myelination capacities of nanofibre grown cells in an inflammatory environment after mTOR-inhibitor treatment with everolimus were further investigated. RESULTS: Reduced oligodendroglial turnover, directly related to a lower myelin content was found in the patients' primary cells. In our culture model of myelination dynamics, primary cells grown under 'inflammatory condition' showed decreased myelination, that was repaired by treatment with everolimus. CONCLUSIONS: Results obtained in patient-derived primary oligodendroglial and TSC2 knock-out cells suggest that maturation of oligodendroglia and production of a proper myelin sheath seem to be impaired as a result of mTOR pathway disturbance. Hence, oligodendroglial pathology may reflect a more direct effect of the abnormal genetic programme rather than to be an inactive bystander of chronic epilepsy.

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.

The coding and non-coding transcriptional landscape of subependymal giant cell astrocytomas.

Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited neurocutaneous disorder caused by inactivating mutations in TSC1 or TSC2, key regulators of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. In the CNS, TSC is characterized by cortical tubers, subependymal nodules and subependymal giant cell astrocytomas (SEGAs). SEGAs may lead to impaired circulation of CSF resulting in hydrocephalus and raised intracranial pressure in patients with TSC. Currently, surgical resection and mTORC1 inhibitors are the recommended treatment options for patients with SEGA. In the present study, high-throughput RNA-sequencing (SEGAs n = 19, periventricular control n = 8) was used in combination with computational approaches to unravel the complexity of SEGA development. We identified 9400 mRNAs and 94 microRNAs differentially expressed in SEGAs compared to control tissue. The SEGA transcriptome profile was enriched for the mitogen-activated protein kinase (MAPK) pathway, a major regulator of cell proliferation and survival. Analysis at the protein level confirmed that extracellular signal-regulated kinase (ERK) is activated in SEGAs. Subsequently, the inhibition of ERK independently of mTORC1 blockade decreased efficiently the proliferation of primary patient-derived SEGA cultures. Furthermore, we found that LAMTOR1, LAMTOR2, LAMTOR3, LAMTOR4 and LAMTOR5 were overexpressed at both gene and protein levels in SEGA compared to control tissue. Taken together LAMTOR1-5 can form a complex, known as the 'Ragulator' complex, which is known to activate both mTORC1 and MAPK/ERK pathways. Overall, this study shows that the MAPK/ERK pathway could be used as a target for treatment independent of, or in combination with mTORC1 inhibitors for TSC patients. Moreover, our study provides initial evidence of a possible link between the constitutive activated mTORC1 pathway and a secondary driver pathway of tumour growth.

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.

Coding and non-coding transcriptome of mesial temporal lobe epilepsy: Critical role of small non-coding RNAs.

Our understanding of mesial temporal lobe epilepsy (MTLE), one of the most common form of drug-resistant epilepsy in humans, is derived mainly from clinical, imaging, and physiological data from humans and animal models. High-throughput gene expression studies of human MTLE have the potential to uncover molecular changes underlying disease pathogenesis along with novel therapeutic targets. Using RNA- and small RNA-sequencing in parrallel, we explored differentially expressed genes in the hippocampus and cortex of MTLE patients who had undergone surgical resection and non-epileptic controls. We identified differentially expressed genes in the hippocampus of MTLE patients and differentially expressed small RNAs across both the cortex and hippocampus. We found significant enrichment for astrocytic and microglial genes among up-regulated genes, and down regulation of neuron specific genes in the hippocampus of MTLE patients. The transcriptome profile of the small RNAs reflected disease state more robustly than mRNAs, even across brain regions which show very little pathology. While mRNAs segregated predominately by brain region for MTLE and controls, small RNAs segregated by disease state. In particular, our data suggest that specific miRNAs (e.g., let-7b-3p and let-7c-3p) may be key regulators of multiple pathways related to MTLE pathology. Further, we report a strong association of other small RNA species with MTLE pathology. As such we have uncovered novel elements that may contribute to the establishment and progression of MTLE pathogenesis and that could be leveraged as therapeutic targets.

Reduced expression of the glucocorticoid receptor in the hippocampus of patients with drug-resistant temporal lobe epilepsy and comorbid depression.

OBJECTIVE: Depressive disorders are common among about 50% of the patients with drug-resistant temporal lobe epilepsy (TLE). The underlying etiology remains elusive, but hypothalamus-pituitary-adrenal (HPA) axis activation due to changes in glucocorticoid receptor (GR) protein expression could play an important role. Therefore, we set out to investigate expression of the GR in the hippocampus, an important brain region for HPA axis feedback, of patients with drug-resistant TLE, with and without comorbid depression. METHODS: GR expression was studied using immunohistochemistry on hippocampal sections from well-characterized TLE patients with depression (TLE + D, n = 14) and without depression (TLE - D, n = 12) who underwent surgery for drug-resistant epilepsy, as well as on hippocampal sections from autopsy control cases (n = 9). Video-electroencephalography (EEG), magnetic resonance imaging (MRI), and psychiatric and memory assessments were performed prior to surgery. RESULTS: Abundant GR immunoreactivity was present in dentate gyrus granule cells and CA1 pyramidal cells of controls. In contrast, neuronal GR expression was lower in patients with TLE, particularly in the TLE + D group. Quantitative analysis showed a smaller GR+ area in TLE + D as compared to TLE - D patients and controls. Furthermore, the ratio between the number of GR+/NeuN+ cells was lower in patients with TLE + D as compared to TLE - D and correlated negatively with the depression severity based on psychiatric history. The expression of the GR was also lower in glial cells of TLE + D compared to TLE - D patients and correlated negatively to the severity of depression. SIGNIFICANCE: Reduced hippocampal GR expression may be involved in the etiology of depression in patients with TLE and could constitute a biological marker of depression in these patients.

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.