Role Of Tuberous Sclerosis Complex Research Articles

Pathogenic variants in TSC2 might cause premature ovarian insufficiency through activated mTOR induced hyperactivation of primordial follicles

To investigate the role of tuberous sclerosis complex (TSC) genes, including TSC1 and TSC2, in the pathogenesis of human premature ovarian insufficiency (POI). Genetic and functional study. University-based reproductive medical center. Six patients from a cohort of 1,030 cases with idiopathic POI. Variants in TSC1 and TSC2 were screened through the largest in-house database of whole exome sequencing performed in 1,030 patients with idiopathic POI. The pathogenic effects of the variants were further verified by functional studies. TSC1 or TSC2 variant and functional characteristics. Five pathogenic heterozygous variants in TSC2 were identified in 6 patients with POI. Functional studies showed these variants impaired the repressive effect of TSC2 on mammalian target of rapamycin (mTOR) pathway by disrupting the formation of TSC complex or its GTPase-activating protein activity. Furthermore, invitro ovarian culture assay showed that TSC2 p.R98Q led to hyperactivation of mTOR pathway thereby triggering primordial follicle activation. The present study identified pathogenic variants of TSC2 in patients with POI, firstly suggested defective TSC/mTOR pathway mediated hyperactivation of primordial follicle participating in the pathogenesis of POI, giving insights into new targets of genetic counseling and clinical prevention for POI. Considering the pivotal role of TSC2 variants in diagnosis of TSC syndrome, the present study also highlighted the importance of history collection and long-term follow-up for the TSC2 variants carriers.

TCS2 Increases Olaquindox-Induced Apoptosis by Upregulation of ROS Production and Downregulation of Autophagy in HEK293 Cells.

Olaquindox, a feed additive, has drawn public attention due to its potential mutagenicity, genotoxicity, hepatoxicity and nephrotoxicity. The purpose of this study was to investigate the role of tuberous sclerosis complex (TSC2) pathways in olaquindox-induced autophagy in human embryonic kidney 293 (HEK293) cells. The results revealed that olaquindox treatment reduced the cell viability of HEK293 cells and downregulated the expression of TSC2 in a dose- and time-dependent manner. Meanwhile, olaquindox treatment markedly induced the production of reactive oxygen species (ROS), cascaded to autophagy, oxidative stress, and apoptotic cell death, which was effectively eliminated by the antioxidant N-acetylcysteine (NAC). Furthermore, overexpression of TSC2 attenuated olaquindox-induced autophagy in contrast to inducing the production of ROS, oxidative stress and apoptosis. Consistently, knockdown of TSC2 upregulated autophagy, and decreased olaquindox-induced cell apoptosis. In conclusion, our findings indicate that TSC2 partly participates in olaquindox-induced autophagy, oxidative stress and apoptosis, and demonstrate that TSC2 has a negative regulation role in olaquindox-induced autophagy in HEK293 cells.

Tsc1 expression by dendritic cells is required to preserve T-cell homeostasis and response

Dendritic cells (DCs) are pivotal to the induction of adaptive T-cell immune responses. Recent evidence highlights a critical role of tuberous sclerosis complex 1 (Tsc1), a primarily upstream negative regulator of mammalian target of rapamycin (mTOR), in DC development, but whether and how Tsc1 directly regulate mature DC function in vivo remains elusive. Here we show that selective disruption of Tsc1 in DCs results in a lymphoproliferative disorder with the spontaneous activation of T cells. Tsc1 deficiency results in the activation of mTORC1-PPARγ pathway, which leads to the upregulation of neuropilin-1 (Nrp1) expression on DCs to stimulate naive T-cell proliferation. However, Tsc1-deficient DCs have defects in the ability to induce antigen-specific T-cell responses in vitro and in vivo owing to impaired survival during antigen transportation and presentation. Indeed, Tsc1 promotes DC survival through restraining independent mTORC1 and ROS-Bim pathways. Our study identifies Tsc1 as a crucial signaling checkpoint in DCs essential for preserving T-cell homeostasis and response.

Abstract 4783: Identification of new therapies for the treatment of BRAF/NRAS wild-type melanomas by functional screening approaches

Abstract Melanoma causes 9000 deaths each year in the USA alone, more than one every hour. It represents the common tumor whose incidence has increased the most in the last 30 years. Despite promising advances, including immunotherapies, the therapeutic regimens are curative in only a fraction of patients. Implementation and combination of different therapeutic modalities is therefore required to improve patient survival. Given the clinical efficacy of drug combinations for BRAF mutant melanomas, we performed a high-throughput drug screening to identify new drug combinations for the treatment of BRAF/NRAS wild type melanomas. Effective targeted therapies are not currently available for this sub-type of the disease, which represents up to 30% of melanomas. By viability assays we characterized the sensitivity to 240 combinations of clinically relevant drugs in a collection of 21 BRAF/NRAS wild type melanoma cell lines. We analysed 8360 survival curves and found that 16 (73%) and 5 (23%) cell lines are highly sensitive to temozolomide plus olaparib and to nilotinib plus trametinib combinations, respectively. Two independent experimental approaches validated these drug synergies. By -omics technologies we deeply characterized the cell line profiles of mutations, copy number changes, DNA methylation, and gene and microRNA expression. We generated gene expression signatures of synergistic and resistant cell lines for the 2 drug combinations and used them to classify 374 human melanomas. Tumors significantly associated to nilotinib plus trametinib synergism signature were significantly enriched for high immune response and proliferative Jonsson's expression classes, while tumors associated to the signature of drug resistance are enriched for pigmentation class. Melanomas associated to temozolomide plus olaparib resistance signature displayed enrichment for normal class. We are currently confirming the representation of these signatures in an independent cohort of melanomas and looking for other clinical correlates. We are also validating the predictivity of these gene expression signatures in independent cohorts of melanoma cell lines. Prospectively, this may represent an approach to identify patients that could have the maximal benefit from these drug combinations. Additionally, we have recently performed a genome-wide CRISPR/Cas9 screen to identify drug resistance genes for these 2 drug combinations. Preliminary results indicate a prominent role of tuberous sclerosis complex in the regulation of the sensitivity to nilotinib plus trametinib combination. These results may pave the way to the development of novel patient-tailored targeted therapies for the efficient eradication of BRAF/NRAS wild type melanomas. Citation Format: Marco Ranzani, Magali Michaut, Clara Alsinet, Vera Grinkevich, Kim Wong, Vivek Iyer, Nanne Aben, Martin Del Castillo Velasco-Herrera, Marcela Sjoberg, Mamunur Rashid, Graham Bignell, Ken Dutton-Regester, Antonia Pritchard, Daniel Vis, Gemma Turner, Ultan McDermott, Nicholas K. Hayward, Kosuke Yusa, Lodewyk Wessels, Mathew Garnett, David Adams. Identification of new therapies for the treatment of BRAF/NRAS wild-type melanomas by functional screening approaches. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4783.

Autism Spectrum Disorders

Autism spectrum disorders was chosen as the topic for the 14th Neurobiology of Disease in Children Symposium, held on October 22-23, 2014, in Columbus, Ohio. Program codirectors Drs Stewart Mostofsky, Manny DiCicco-Bloom, and Deborah Hirtz prepared an engaging agenda that included individual presentations as well as a panel discussion from leaders working to advance the autism field. The symposium covered a variety of topics, such as clinical features, recent advances in understanding the pathogenesis of autism spectrum disorder, and new investigations of characteristic symptoms and behaviors categorized under the broad ‘‘spectrum’’ of autism. This was followed by a session on controversial topics and unanswered questions. The symposium concluded with a panel discussion led by scientific leaders in the field of autism spectrum disorders. Dr Sarah Spence opened the symposium with an introduction to autism, including changes to and advances in the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Dr Spence explained how the DSM-5 criteria may provide clinicians with better diagnostic tools to properly diagnose certain symptoms, behaviors, and disabilities associated with autism spectrum disorder. Dr Craig Newschaffer then discussed the epidemiology of the disorder, addressing its rising prevalence in addition to genetic and environmental risk factors. Dr Lonnie Zwaigenbaum’s discussion stressed the importance of identifying early behavioral and neurological characteristics of autism spectrum disorder. In addition, he focused on the significance of early diagnosis and recent advances in early intervention. To conclude the first session, Dr Roberto Tuchman discussed the relationship between autism spectrum disorder and epilepsy, concentrating on the impact and shared mechanisms of both disorders. The next session of the symposium focused on recent progress in understanding the molecular mechanisms underlying different types of autism spectrum disorder. Dr Jonathan Sebat discussed the genetics of autism, summarizing studies of rare genetic variation, copy number variants, and exome sequencing, and new discoveries from comprehensive analysis of de novo mutations in autism. Dr Sebat described his work to identify copy number variation in the human genome as an important contributor to human disease. He also emphasized the genetic and hereditary factors contributing to various types of autism. Recently, Dr Sebat and his colleagues attempted to sequence the entire human genome in an effort to characterize patterns of mutation in both coding and noncoding regions of the genome. Dr Tim Roberts addressed the significance of biomarkers in autism, which can be used for diagnostic and prognostic purposes, in order to understand experimental models and to monitor response to therapy. Dr Roberts reviewed the application of magnetoencephalography for children with epilepsy or autism, discussing event-related potentials-type responses in auditory sensation and gamma-band activity. Dr Roberts also discussed his study of the motor cortex and concluded that g-aminobutyric (GABA) concentration predicted gamma band oscillation, in addition to GABA level variations observed in children with autism. To conclude the second session, Dr Cyndi Schumann focused on the current understanding of neuropathology and neurobiology of autism. Dr Schumann emphasized the importance of having brain tissue for autism research in order to make progress in the field. The third session of the symposium focused on translational and therapeutic targets in autism research. Dr Mustafa Sahin reviewed current translational work involving mice and human models with tuberous sclerosis complex. It is reported that about half of the patients who have tuberous sclerosis also might be affected by autism spectrum disorder. Therefore, his discussion was focused on the relationship between the mammalian target of rapamycin pathway and tuberous sclerosis complex, to test the role of tuberous sclerosis complex in autism. He also discussed studies in which the loss of 1 or both TSC1 and TSC2 genes resulted in disease. Dr Sahin and his colleagues conducted a study that focused on the non-tuber pathology of the brain in mice with tuberous sclerosis complex. His team demonstrated that miswiring the neural connectivity within the central nervous system had contributed to the pathogenesis of tuberous sclerosis. Last, Dr Sahin addressed the circuitry and localization of brain lesions and how these lesions might contribute to clinical abnormalities observed in patients.

Neurogenin 3–Directed Cre Deletion of Tsc1 Gene Causes Pancreatic Acinar Carcinoma

The role of tuberous sclerosis complex (TSC) in the pathogenesis of pancreatic cancers remains largely unknown. The present study shows that neurogenin 3 directed Cre deletion of Tsc1 gene induces the development of pancreatic acinar carcinoma. By cross-breeding the Neurog3-cre mice with Tsc1loxp/loxp mice, we generated the Neurog3-Tsc1−/− transgenic mice in which Tsc1 gene is deleted and mTOR signaling activated in the pancreatic progenitor cells. All Neurog3-Tsc1−/− mice developed notable adenocarcinoma-like lesions in pancreas starting from the age of 100days old. The tumor lesions are composed of cells with morphological and molecular resemblance to acinar cells. Metastasis of neoplasm to liver and lung was detected in 5% of animals. Inhibition of mTOR signaling by rapamycin significantly attenuated the growth of the neoplasm. Relapse of the neoplasm occurred within 14days upon cessation of rapamycin treatment. Our studies indicate that activation of mTOR signaling in the pancreatic progenitor cells may trigger the development of acinar carcinoma. Thus, mTOR may serve as a potential target for treatment of pancreatic acinar carcinoma.

Tuberous sclerosis complex 1-mechanistic target of rapamycin complex 1 signaling determines brown-to-white adipocyte phenotypic switch.

Interconversion of white and brown adipocytes occurs between anabolic and catabolic states. The molecular mechanism regulating this phenotypic switch remains largely unknown. This study explores the role of tuberous sclerosis complex 1 (TSC1)-mechanistic target of rapamycin (mTOR) signaling in the conversion of brown to white adipose tissue (WAT). A colony of Fabp4-Tsc1(-/-) mice, in which the Tsc1 gene was specifically deleted by the fatty acid binding protein 4 (FABP4)-Cre, was established. Western blotting and immunostaining demonstrated the absence of TSC1 and activation of ribosomal protein S6 kinase 1, the downstream target of mTOR complex 1 (mTORC1) signaling, in the brown adipose tissues (BATs) of Fabp4-Tsc1(-/-) mice. Accumulation of lipid droplets in BAT was significantly increased. Levels of brown adipocyte markers were markedly downregulated, while white adipocyte markers were upregulated. Rapamycin reversed the conversion from BAT to WAT in Fabp4-Tsc1(-/-) mice. Deletion of the Tsc1 gene in cultured brown preadipocytes significantly increased the conversion to white adipocytes. FoxC2 mRNA, the transcriptional factor for brown adipocyte determination, was significantly decreased, while mRNAs for retinoblastoma protein, p107 and RIP140, the transcriptional factors for white adipocyte determination, increased in the BAT of Fabp4-Tsc1(-/-) mice. Our study demonstrates that TSC1-mTORC1 signaling contributes to the brown-to-white adipocyte phenotypic switch.

Abstract 160: Tuberosis Sclerosis Complex 1 Mediated Neointima Formation and Arterial Thrombosis Following Vascular Injury

Objectives: Vascular injury and thrombosis are main leading causes of cardiovascular diseases. Tuberous sclerosis complex (TSC) is a genetic disorder caused by heterozygous mutations in either of two genes, TSC1 and TSC2. Although role of TSCs has been implicated in cardiovascular diseases, the tissue- and isoform-specific roles of TSCs in the vascular response to injury are not known. Methods and Results: To determine the role of TSC1 in arterial injury and thrombosis, we generated vascular smooth muscle cell-specific TSC1 conditional knockout mice (TSC1SM22-/-) by crossing vascular smooth muscle cell-specific Cre (SM22Cre) mice with TSC1flox/flox mice and performed carotid artery ligation in haploinsufficient TSC1 conditional knockout mice (TSC1SM22+/-) compared with that of WT or haploinsufficient TSC2 knockout mice (TSC2+/-). Acute carotid artery occlusion was investigated by 5% ferric chloride injury. Arterial thrombosis and neointima formation were measured at 14 days after arterial ligation. Expression of proteins was observed by immunoblot analysis. The neointima formation was significantly increased in TSC1SM22+/- mice (intimal thickness/medial thickness ratio; 1.14 ± 0.14, p<0.001) compared with that of WT mice (0.13 ± 0.03). Two weeks after arterial injury, arterial thrombus area was increased in TSC1SM22+/- mice (thrombus area/luminal area ratio; 72.1 ± 4.4, p<0.001) compared with that of WT mice (0.1 ± 0.0) but there are no significant effect in acute arterial thrombosis induced by ferric chloride. Loss of TSC1 and hyper-activation of mammalian target of rapamycin complex 1 including mTOR and S6 proteins were observed by immunoblot analysis of carotid artery tissue lysates of TSC1SM22+/- mice compared with that of WT. Conclusion: These findings suggest that regulation of TSC1 and mTOR might be useful for therapeutic intervention in vascular injury and thrombosis.

The Role of Tuberous Sclerosis Complex 1 in Regulating Innate Immunity

The mechanisms that control TLR-induced responses, including endotoxin tolerance, have been not well understood. The tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that inhibits the mammalian target of rapamycin (mTOR). We show in this study that deficiency of TSC1 results in enhanced activation of not only mTOR complex 1 (mTORC1), but also JNK1/2, following LPS stimulation in macrophages. TSC1-deficient macrophages produce elevated proinflammatory cytokines and NO in response to multiple TLR ligands. Such enhanced TLR-induced responses can be inhibited by reducing mTORC1 and JNK1/2 activities with chemical inhibitors or small hairpin RNA, suggesting that TSC1 negatively controls TLR responses through both mTORC1 and JNK1/2. The impact of TSC1 deficiency appeared not limited to TLRs, as NOD- and RIG-I/MDA-5-induced innate responses were also altered in TSC1-deficient macrophages. Furthermore, TSC1 deficiency appears to cause impaired induction of endotoxin tolerance in vitro and in vivo, which is correlated with increased JNK1/2 activation and can be reversed by JNK1/2 inhibition. Our results reveal a critical role of TSC1 in regulating innate immunity by negative control of mTORC1 and JNK1/2 activation.

TSC1/TSC2 Signaling in Pancreatic β-Cells

The Tuberous Sclerosis complex (TSC) integrates metabolic and growth signals. Recent data demonstrate that this pathway is a major player in regulation of metabolism and energy balance. In this review, we will focus on the role of TSC in modulation of -cell mass and function.

TACC3-TSC2 maintains nuclear envelope structure and controls cell division

Studies of the role of tuberous sclerosis complex (TSC) proteins (TSC1/TSC2) in pathology have focused mainly on their capacity to regulate translation and cell growth, but their relationship with alterations of cellular structures and the cell cycle is not yet fully understood. The transforming acidic coiled-coil (TACC) domain-containing proteins are central players in structures and processes connected to the centrosome. Here, TACC3 interactome mapping identified TSC2 and 15 other physical interactors, including the evolutionary conserved interactions with ch-TOG/CKAP5 and FAM161B. TACC3 and TSC2 co-localize and co-purify with components of the nuclear envelope, and their deficiency causes morphological alterations of this structure. During cell division, TACC3 is necessary for the proper localization of phospho-Ser939 TSC2 at spindle poles and cytokinetic bridges. Accordingly, abscission alterations and increased frequency of binucleated cells were observed in Tacc3- and Tsc2-deficient cells relative to controls. In regulating cell division, TSC2 acts epistatically to TACC3 and, in addition to canonical TSC/mTOR signaling and cytokinetic associations, converges to the early mitotic checkpoint mediated by CHFR, consistently with nuclear envelope associations. Our findings link TACC3 to novel structural and cell division functions of TSC2, which may provide additional explanations for the clinical and pathological manifestations of lymphangioleiomyomatosis (LAM) disease and TSC syndrome, including the greater clinical severity of TSC2 mutations compared to TSC1 mutations.

Biphasic Response of Pancreatic β-Cell Mass to Ablation of Tuberous Sclerosis Complex 2 in Mice

Recent studies have demonstrated the importance of insulin or insulin-like growth factor 1 (IGF-1) for regulation of pancreatic beta-cell mass. Given the role of tuberous sclerosis complex 2 (TSC2) as an upstream molecule of mTOR (mammalian target of rapamycin), we examined the effect of TSC2 deficiency on beta-cell function. Here, we show that mice deficient in TSC2, specifically in pancreatic beta cells (betaTSC2(-/-) mice), manifest increased IGF-1-dependent phosphorylation of p70 S6 kinase and 4E-BP1 in islets as well as an initial increased islet mass attributable in large part to increases in the sizes of individual beta cells. These mice also exhibit hypoglycemia and hyperinsulinemia at young ages (4 to 28 weeks). After 40 weeks of age, however, the betaTSC2(-/-) mice develop progressive hyperglycemia and hypoinsulinemia accompanied by a reduction in islet mass due predominantly to a decrease in the number of beta cells. These results thus indicate that TSC2 regulates pancreatic beta-cell mass in a biphasic manner.