Molecular EPISTOP, a comprehensive multi-omic analysis of blood from Tuberous Sclerosis Complex infants age birth to two years.

We present a comprehensive multi-omic analysis of the EPISTOP prospective clinical trial of early intervention with vigabatrin for pre-symptomatic epilepsy treatment in Tuberous Sclerosis Complex (TSC), in which 93 infants with TSC were followed from birth to age 2 years, seeking biomarkers of epilepsy development. Vigabatrin had profound effects on many metabolites, increasing serum deoxycytidine monophosphate (dCMP) levels 52-fold. Most serum proteins and metabolites, and blood RNA species showed significant change with age. Thirty-nine proteins, metabolites, and genes showed significant differences between age-matched control and TSC infants. Six also showed a progressive difference in expression between control, TSC without epilepsy, and TSC with epilepsy groups. A multivariate approach using enrollment samples identified multiple 3-variable predictors of epilepsy, with the best having a positive predictive value of 0.987. This rich dataset will enable further discovery and analysis of developmental effects, and associations with seizure development in TSC.

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.

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.

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.

Myelin Pathology Beyond White Matter in Tuberous Sclerosis Complex (TSC) Cortical Tubers.

Tuberous sclerosis complex (TSC) is a monogenetic disease that arises due to mutations in either the TSC1 or TSC2 gene and affects multiple organ systems. One of the hallmark manifestations of TSC are cortical malformations referred to as cortical tubers. These tubers are frequently associated with treatment-resistant epilepsy. Some of these patients are candidates for epilepsy surgery. White matter abnormalities, such as loss of myelin and oligodendroglia, have been described in a small subset of resected tubers but mechanisms underlying this phenomenon are unclear. Herein, we analyzed a variety of neuropathologic and immunohistochemical features in gray and white matter areas of resected cortical tubers from 46 TSC patients using semi-automated quantitative image analysis. We observed divergent amounts of myelin basic protein as well as numbers of oligodendroglia in both gray and white matter when compared with matched controls. Analyses of clinical data indicated that reduced numbers of oligodendroglia were associated with lower numbers on the intelligence quotient scale and that lower amounts of myelin-associated oligodendrocyte basic protein were associated with the presence of autism-spectrum disorder. In conclusion, myelin pathology in cortical tubers extends beyond the white matter and may be linked to cognitive dysfunction in TSC patients.

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.

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.

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.

Changes in vascular density in resected tissue of 97 patients with mild malformation of cortical development, focal cortical dysplasia or TSC-related cortical tubers.

Recent studies suggested a possible association between malformations of cortical development and microvascular density. In this study we aimed to further elucidate the relation between microvascular density and cortical developmental abnormalities in a cohort of 97 patients with epilepsy and histologically proven mild malformation of cortical development (mMCD), focal cortical dysplasia (FCD) or tuberous sclerosis complex (TSC). Surgical tissue samples were analyzed with quantitative measures of vessel density, T-cell response, microglial activation and myelin content. Subsequently, the results were compared to an age- and localization matched control group. We observed an increase in microvasculature in white matter of TSC cortical tubers, which is linked to inflammatory response. No increase was seen in mMCD or FCD subtypes compared to controls. In mMCD/FCD and tubers, lesional cortex and white matter showed increased vascular density compared to perilesional tissues. Moreover, cortical vessel density increased with longer epilepsy duration and older age at surgery while in controls it decreased with age. Our findings suggest for that the increase in white matter vascular density might be pathology-specific rather than a consequence of ongoing epileptic activity. Increased cortical vessel density with age and with longer epilepsy duration in mMCD/FCD's and tubers, however, could be a consequence of seizures.

Oxidative stress and inflammation in a spectrum of epileptogenic cortical malformations: molecular insights into their interdependence.

Oxidative stress (OS) occurs in brains of patients with epilepsy and coincides with brain inflammation, and both phenomena contribute to seizure generation in animal models. We investigated whether expression of OS and brain inflammation markers co-occurred also in resected brain tissue of patients with epileptogenic cortical malformations: hemimegalencephaly (HME), focal cortical dysplasia (FCD) and cortical tubers in tuberous sclerosis complex (TSC). Moreover, we studied molecular mechanisms linking OS and inflammation in an in vitro model of neuronal function. Untangling interdependency and underlying molecular mechanisms might pose new therapeutic strategies for treating patients with drug-resistant epilepsy of different etiologies. Immunohistochemistry was performed for specific OS markers xCT and iNOS and brain inflammation markers TLR4, COX-2 and NF-κB in cortical tissue derived from patients with HME, FCD IIa, IIb and TSC. Additionally, we studied gene expression of these markers using the human neuronal cell line SH-SY5Y in which OS was induced using H2 O2 . OS markers were higher in dysmorphic neurons and balloon/giant cells in cortex of patients with FCD IIb or TSC. Expression of OS markers was positively correlated to expression of brain inflammation markers. In vitro, 100 µM, but not 50 µM, of H2 O2 increased expression of TLR4, IL-1ß and COX-2. We found that NF-κB signaling was activated only upon stimulation with 100 µM H2 O2 leading to upregulation of TLR4 signaling and IL-1ß. The NF-κB inhibitor TPCA-1 completely reversed this effect. Our results show that OS positively correlates with neuroinflammation and is particularly evident in brain tissue of patients with FCD IIb and TSC. In vitro, NF-κB is involved in the switch to an inflammatory state after OS. We propose that the extent of OS can predict the neuroinflammatory state of the brain. Additionally, antioxidant treatments may prevent the switch to inflammation in neurons thus targeting multiple epileptogenic processes at once.

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.

mTOR dysregulation and tuberous sclerosis-related epilepsy.

INTRODUCTION: The mammalian target of rapamycin (mTOR) pathway has emerged as a key player for proper neural network development, and it is involved in epileptogenesis triggered by both genetic or acquired factors. Areas covered. The robust mTOR signaling deregulation observed in a large spectrum of epileptogenic developmental pathologies, such as focal cortical dysplasias and tuberous sclerosis complex (TSC), has been linked to germline and somatic mutations in mTOR pathway regulatory genes, increasing the spectrum of 'mTORopathies'. The significant advances in the field of TSC allowed for the validation of emerging hypotheses on the mechanisms of epileptogenesis and the identification of potential new targets of therapy. Recently, a double-blind phase III randomized clinical trial on patients with TSC related epilepsy, demonstrated that adjunctive treatment with mTOR inhibition is effective and safe in reducing focal drug resistant seizures. Expert commentary. mTOR signaling dysregulation represents a common pathogenic mechanism in a subset of malformations of cortical development, sharing histopathological and clinical features, including epilepsy, autism, and intellectual disability. EXIST-3 trial provided the first evaluation of the optimal dosage, conferring a higher chance of reducing seizure frequency and severity, with adverse events being similar to what observed with lower dosages.

Coding and small non-coding transcriptional landscape of tuberous sclerosis complex cortical tubers: implications for pathophysiology and treatment.

Tuberous Sclerosis Complex (TSC) is a rare genetic disorder that results from a mutation in the TSC1 or TSC2 genes leading to constitutive activation of the mechanistic target of rapamycin complex 1 (mTORC1). TSC is associated with autism, intellectual disability and severe epilepsy. Cortical tubers are believed to represent the neuropathological substrates of these disabling manifestations in TSC. In the presented study we used high-throughput RNA sequencing in combination with systems-based computational approaches to investigate the complexity of the TSC molecular network. Overall we detected 438 differentially expressed genes and 991 differentially expressed small non-coding RNAs in cortical tubers compared to autopsy control brain tissue. We observed increased expression of genes associated with inflammatory, innate and adaptive immune responses. In contrast, we observed a down-regulation of genes associated with neurogenesis and glutamate receptor signaling. MicroRNAs represented the largest class of over-expressed small non-coding RNA species in tubers. In particular, our analysis revealed that the miR-34 family (including miR-34a, miR-34b and miR-34c) was significantly over-expressed. Functional studies demonstrated the ability of miR-34b to modulate neurite outgrowth in mouse primary hippocampal neuronal cultures. This study provides new insights into the TSC transcriptomic network along with the identification of potential new treatment targets.

Functional aspects of early brain development are preserved in tuberous sclerosis complex (TSC) epileptogenic lesions.

Tuberous sclerosis complex (TSC) is a rare multi-system genetic disease characterized by several neurological disorders, the most common of which is the refractory epilepsy caused by highly epileptogenic cortical lesions. Previous studies suggest an alteration of GABAergic and glutamatergic transmission in TSC brain indicating an unbalance of excitation/inhibition that can explain, at least in part, the high incidence of epilepsy in these patients. Here we investigate whether TSC cortical tissues could retain GABAA and AMPA receptors at early stages of human brain development thus contributing to the generation and recurrence of seizures. Given the limited availability of pediatric human brain specimens, we used the microtransplantation method of injecting Xenopus oocytes with membranes from TSC cortical tubers and control brain tissues. Moreover, qPCR was performed to investigate the expression of GABAA and AMPA receptor subunits (GABAA α1-5, ß3, γ2, δ; GluA1, GluA2) and cation chloride co-transporters NKCC1 and KCC2. The evaluation of nine human cortical brain samples, from 15 gestation weeks to 15years old, showed a progressive shift towards more hyperpolarized GABAA reversal potential (EGABA). This shift was associated with a differential expression of the chloride cotransporters NKCC1 and KCC2. Furthermore, the GluA1/GluA2 mRNA ratio of expression paralleled the development process. On the contrary, in oocytes micro-transplanted with epileptic TSC tuber tissue from seven patients, neither the GABAA reversal potential nor the GluA1/GluA2 expression showed similar developmental changes. Our data indicate for the first time, that in the same cohort of TSC patients, the pattern of both GABAAR and GluA1/GluA2 functions retains features that are typical of an immature brain. These observations support the potential contribution of altered receptor function to the epileptic disorder of TSC and may suggest novel therapeutic approaches. Furthermore, our findings strengthen the novel hypothesis that other developmental brain diseases can share the same hallmarks of immaturity leading to intractable seizures.

Novel Histopathological Patterns in Cortical Tubers of Epilepsy Surgery Patients with Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is a genetic hamartoma syndrome frequently associated with severe intractable epilepsy. In some TSC patients epilepsy surgery is a promising treatment option provided that the epileptogenic zone can be precisely delineated. TSC brain lesions (cortical tubers) contain dysmorphic neurons, brightly eosinophilic giant cells and white matter alterations in various proportions. However, a histological classification system has not been established for tubers. Therefore, the aim of this study was to define distinct histological patterns within tubers based on semi-automated histological quantification and to find clinically significant correlations. In total, we studied 28 cortical tubers and seven samples of perituberal cortex from 28 TSC patients who had undergone epilepsy surgery. We assessed mammalian target of rapamycin complex 1 (mTORC1) activation, the numbers of giant cells, dysmorphic neurons, neurons, and oligodendrocytes, and calcification, gliosis, angiogenesis, inflammation, and myelin content. Three distinct histological profiles emerged based on the proportion of calcifications, dysmorphic neurons and giant cells designated types A, B, and C. In the latter two types we were able to subsequently associate them with specific features on presurgical MRI. Therefore, these histopathological patterns provide consistent criteria for improved definition of the clinico-pathological features of cortical tubers identified by MRI and provide a basis for further exploration of the functional and molecular features of cortical tubers in TSC.

Specific pattern of maturation and differentiation in the formation of cortical tubers in tuberous sclerosis omplex (TSC): evidence from layer-specific marker expression.

BACKGROUND: Tuberous sclerosis complex (TSC) is a multisystem disorder that results from mutations in the TSC1 or TSC2 genes, leading to constitutive activation of the mammalian target of rapamycin (mTOR) signaling pathway. Cortical tubers represent typical lesions of the central nervous system (CNS) in TSC. The pattern of cortical layering disruption observed in brain tissue of TSC patients is not yet fully understood, and little is known about the origin and phenotype of individual abnormal cell types recognized in tubers. METHODS: In the present study, we aimed to characterize dysmorphic neurons (DNs) and giant cells (GCs) of cortical tubers using neocortical layer-specific markers (NeuN, SMI32, Tbr1, Satb2, Cux2, ER81, and RORß) and to compare the features with the histo-morphologically similar focal cortical dysplasia (FCD) type IIb. We studied a cohort of nine surgically resected cortical tubers, five FCD type IIb, and four control samples using immunohistochemistry and in situ hybridization. RESULTS: Cortical tuber displayed a prominent cell loss in all cortical layers. Moreover, we observed altered proportions of layer-specific markers within the dysplastic region. DNs, in both tubers and FCD type IIb, were found positive for different cortical layer markers, regardless of their laminar location, and their immunophenotype resembles that of cortical projection neurons. CONCLUSIONS: These findings demonstrate that, similar to FCD type IIb, cortical layering is markedly disturbed in cortical tubers of TSC patients. Distribution of these disturbances is comparable in all tubers and suggests a dysmaturation affecting early and late migratory patterns, with a more severe impairment of the late stage of maturation.