• Publikační typ
• tisková chyba MeSH

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.

• MeSH
• dítě MeSH
• dospělí MeSH
• down regulace MeSH
• kojenec MeSH
• kontaktiny * genetika   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mozek metabolismus   MeSH
• novorozenec MeSH
• poruchy autistického spektra komplikace   metabolismus   MeSH
• předškolní dítě MeSH
• tuberózní skleróza * komplikace   metabolismus   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• astrocyty metabolismus   MeSH
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mikro RNA genetika   metabolismus   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mozek růst a vývoj   patologie   MeSH
• mozková kůra patologie   MeSH
• myši inbrední C57BL MeSH
• neurony patologie   MeSH
• předškolní dítě MeSH
• signální transdukce genetika   MeSH
• tuberózní skleróza komplikace   genetika   patologie   MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• bílá hmota patologie   MeSH
• lidé MeSH
• mozková kůra patologie   MeSH
• myelinová pochva patologie   MeSH
• oligodendroglie patologie   MeSH
• šedá hmota patologie   MeSH
• tuberózní skleróza patologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• astrocytom etiologie   genetika   metabolismus   MeSH
• astrocyty účinky léků   metabolismus   MeSH
• butadieny farmakologie   MeSH
• dítě MeSH
• dospělí MeSH
• extracelulárním signálem regulované MAP kinasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• hamartin genetika   MeSH
• inhibitory enzymů farmakologie   MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• kojenec MeSH
• lidé MeSH
• MAP kinasový signální systém genetika   MeSH
• mechanistické cílové místo rapamycinového komplexu 1 MeSH
• messenger RNA metabolismus   MeSH
• mikro RNA metabolismus   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nádorové buňky kultivované MeSH
• nádory mozku komplikace   genetika   metabolismus   MeSH
• nitrily farmakologie   MeSH
• předškolní dítě MeSH
• sekvenční analýza RNA MeSH
• sekvenování transkriptomu MeSH
• stanovení celkové genové exprese MeSH
• tuberin genetika   MeSH
• tuberózní skleróza komplikace   genetika   MeSH
• výměnné faktory guaninnukleotidů genetika   metabolismus   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• dítě MeSH
• dospělí MeSH
• epilepsie genetika   MeSH
• genetická transkripce genetika   MeSH
• hamartin genetika   MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mechanistické cílové místo rapamycinového komplexu 1 genetika   MeSH
• mikro RNA genetika   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mozková kůra fyziologie   MeSH
• mutace genetika   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• neurony fyziologie   MeSH
• předškolní dítě MeSH
• signální transdukce genetika   MeSH
• tuberin genetika   MeSH
• tuberózní skleróza genetika   MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• myši MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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, β3, 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.

• MeSH
• dítě MeSH
• epilepsie etiologie   MeSH
• kohortové studie MeSH
• lidé MeSH
• mozek růst a vývoj   metabolismus   patologie   MeSH
• nemoci mozku patologie   MeSH
• oocyty MeSH
• receptory GABA-A metabolismus   MeSH
• symportéry metabolismus   MeSH
• tuberózní skleróza genetika   patologie   patofyziologie   MeSH
• Xenopus MeSH
• záchvaty patofyziologie   MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• dítě MeSH
• dospělí MeSH
• epilepsie komplikace   metabolismus   patologie   chirurgie   MeSH
• glióza komplikace   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mozková kůra patologie   chirurgie   MeSH
• multiproteinové komplexy analýza   metabolismus   MeSH
• myelinová pochva metabolismus   patologie   MeSH
• neurony patologie   MeSH
• předškolní dítě MeSH
• TOR serin-threoninkinasy analýza   metabolismus   MeSH
• tuberózní skleróza komplikace   metabolismus   patologie   chirurgie   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

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 RORβ) 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.

• Publikační typ
• časopisecké články MeSH