Neural Crest-Specific TSC1 Deletion in Mice Leads to Sclerotic Craniofacial Bone Lesion.
ABSTRACT: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in either TSC1 or TSC2. TSC has high frequency of osseous manifestations such as sclerotic lesions in the craniofacial region. However, an animal model that replicates TSC craniofacial bone lesions has not yet been described. The roles of Tsc1 and the sequelae of Tsc1 dysfunction in bone are unknown. In this study, we generated a mouse model of TSC with a deletion of Tsc1 in neural crest-derived (NCD) cells that recapitulated the sclerotic craniofacial bone lesions in TSC. Analysis of this mouse model demonstrated that TSC1 deletion led to enhanced mTORC1 signaling in NCD bones and the increase in bone formation is responsible for the aberrantly increased bone mass. Lineage mapping revealed that TSC1 deficient NCD cells overpopulated the NCD bones. Mechanistically, hyperproliferation of osteoprogenitors at an early postnatal stage accounts for the increased osteoblast pool. Intriguingly, early postnatal treatment with rapamycin, an mTORC1 inhibitor, can completely rescue the aberrant bone mass, but late treatment cannot. Our data suggest that enhanced mTOR signaling in NCD cells can increase bone mass through enlargement of the osteoprogenitor pool, which likely explains the sclerotic bone lesion observed in TSC patients.
Project description:Increased mTORC1 signaling from TSC1/TSC2 inactivation is found in cancer and causes tuberous sclerosis complex (TSC). The role of mesenchymal-derived cells in TSC tumorigenesis was investigated through disruption of Tsc2 in craniofacial and limb bud mesenchymal progenitors. Tsc2cKOPrrx1-cre mice had shortened lifespans and extensive hamartomas containing abnormal tortuous, dilated vessels prominent in the forelimbs. Abnormalities were blocked by the mTORC1 inhibitor sirolimus. A Tsc2/mTORC1 expression signature identified in Tsc2-deficient fibroblasts was also increased in bladder cancers with TSC1/TSC2 mutations in the TCGA database. Signature component Lgals3 encoding galectin-3 was increased in Tsc2-deficient cells and serum of Tsc2cKOPrrx1-cre mice. Galectin-3 was increased in TSC-related skin tumors, angiomyolipomas, and lymphangioleiomyomatosis with serum levels in patients with lymphangioleiomyomatosis correlating with impaired lung function and angiomyolipoma presence. Our results demonstrate Tsc2-deficient mesenchymal progenitors cause aberrant morphogenic signals, and identify an expression signature including Lgals3 relevant for human disease of TSC1/TSC2 inactivation and mTORC1 hyperactivity.
Project description:PURPOSE: To determine if sclerotic bone lesions evident at body computed tomography (CT) are of value as a diagnostic criterion of tuberous sclerosis complex (TSC) and in the differentiation of TSC with lymphangioleiomyomatosis (LAM) from sporadic LAM. MATERIALS AND METHODS: Informed consent was signed by all patients in this HIPAA-compliant study approved by the institutional review board. Retrospective analysis was performed of the body CT studies of 472 patients: 365 with sporadic LAM, 82 with TSC/LAM, and 25 with TSC. The images were reviewed by using a picture archiving and communication system workstation with bone settings (window width, 1500 HU; window level, 300 HU) and fit-to-screen option. CT image characteristics assessed included shape, size, and distribution of sclerotic bone lesions with subsequent calculation of differences in the frequency of these lesions. RESULTS: Most commonly the sclerotic bone lesions were round, measured 0.3 cm (range, 0.2-3.2), and were distributed throughout the spine. The frequencies differed among the three patient groups Four or more sclerotic bone lesions were detected in all 25 (100%) of those with TSC, with a sensitivity of .89 (72 of 82) and specificity of .97 (355 of 367) in the differentiation of sporadic LAM from TSC/LAM (P < .01). CONCLUSION: The number of sclerotic bone lesions at body CT is of potential value in the diagnosis of TSC and in the differentiation of patients with sporadic LAM from those with TSC/LAM. (c) RSNA, 2010.
Project description:Tuberous Sclerosis Complex (TSC) is a disease caused by autosomal dominant mutations in the TSC1 or TSC2 genes, and is characterized by tumor susceptibility, brain lesions, seizures and behavioral impairments. The TSC1 and TSC2 genes encode proteins forming a complex (TSC), which is a major regulator and suppressor of mammalian target of rapamycin complex 1 (mTORC1), a signaling complex that promotes cell growth and proliferation. TSC1/2 loss of heterozygosity (LOH) and the subsequent complete loss of TSC regulatory activity in null cells causes mTORC1 dysregulation and TSC-associated brain lesions or other tissue tumors. However, it is not clear whether TSC1/2 heterozygous brain cells are abnormal and contribute to TSC neuropathology. To investigate this issue, we generated induced pluripotent stem cells (iPSCs) from TSC patients and unaffected controls, and utilized these to obtain neural progenitor cells (NPCs) and differentiated neurons in vitro. These patient-derived TSC2 heterozygous NPCs were delayed in their ability to differentiate into neurons. Patient-derived progenitor cells also exhibited a modest activation of mTORC1 signaling downstream of TSC, and a marked attenuation of upstream PI3K/AKT signaling. We further show that pharmacologic PI3K or AKT inhibition, but not mTORC1 inhibition, causes a neuronal differentiation delay, mimicking the patient phenotype. Together these data suggest that heterozygous TSC2 mutations disrupt neuronal development, potentially contributing to the disease neuropathology, and that this defect may result from dysregulated PI3K/AKT signaling in neural progenitor cells.
Project description:Tuberous Sclerosis Complex (TSC) is a disease caused by autosomal dominant mutations in the TSC1 or TSC2 genes, and is characterized by tumor susceptibility, brain lesions, seizures and behavioral impairments. The TSC1 and TSC2 genes encode proteins forming a complex (TSC), which is a major regulator and suppressor of mammalian target of rapamycin (mTOR) in complex 1 (mTORC1), a signaling complex that promotes cell growth and proliferation. TSC1/2 loss of heterozygosity (LOH) and the subsequent complete loss of TSC regulatory activity in null cells causes mTORC1 dysregulation and TSC-associated brain lesions or other tissue tumors. However, it is not clear whether TSC1/2 heterozygous brain cells are abnormal and contribute to TSC neuropathology. To investigate this issue, we generated induced pluripotent stem cells (iPSCs) from TSC patients and unaffected controls, and utilized these to obtain neural progenitor cells (NPCs) and differentiated neurons in vitro. These patient-derived TSC2 heterozygous NPCs were delayed in their ability to differentiate into neurons. Patient-derived progenitor cells also exhibited a modest activation of mTORC1 signaling downstream of TSC, and a marked attenuation of upstream PI3K/AKT signaling. We further show that pharmacologic AKT inhibition, but not mTORC1 inhibition, causes a neuronal differentiation delay, mimicking the patient phenotype. Together these data suggest that heterozygous TSC2 mutations disrupt neuronal development, potentially contributing to the disease neuropathology, and that this defect may result from dysregulated AKT signaling in neural progenitor cells. Overall design: Two replicates each of TSC#1 and CON#1 NPC cell RNA were prepared for sequencing library preparation and seqeuencing.
Project description:BACKGROUND:Tuberous sclerosis complex (TSC) is a genetic disorder that cause tumors to form in many organs. These lesions may lead to epilepsy, autism, developmental delay, renal, and pulmonary failure. Loss of function mutations in TSC1 and TSC2 genes by aberrant activation of the mechanistic target of rapamycin (mTORC1) signaling pathway are the known causes of TSC. Therefore, targeting mTORC1 becomes a most available therapeutic strategy for TSC. Although mTORC1 inhibitor rapamycin and Rapalogs have demonstrated exciting results in the recent clinical trials, however, tumors rebound and upon the discontinuation of the mTORC1 inhibition. Thus, understanding the underlying molecular mechanisms responsible for rapamycin-induced cell survival becomes an urgent need. Identification of additional molecular targets and development more effective remission-inducing therapeutic strategies are necessary for TSC patients. RESULTS:We have discovered an Mitogen-activated protein kinase (MAPK)-evoked positive feedback loop that dampens the efficacy of mTORC1 inhibition. Mechanistically, mTORC1 inhibition increased MEK1-dependent activation of MAPK in TSC-deficient cells. Pharmacological inhibition of MAPK abrogated this feedback loop activation. Importantly, the combinatorial inhibition of mTORC1 and MAPK induces the death of TSC2-deficient cells. CONCLUSIONS:Our results provide a rationale for dual targeting of mTORC1 and MAPK pathways in TSC and other mTORC1 hyperactive neoplasm.
Project description:Tuberous sclerosis complex (TSC) is a neurogenetic disorder that leads to elevated mechanistic targeting of rapamycin complex 1 (mTORC1) activity. Cilia can be affected by mTORC1 signaling, and ciliary deficits are associated with neurodevelopmental disorders. Here, we examine whether neuronal cilia are affected in TSC. We show that cortical tubers from TSC patients and mutant mouse brains have fewer cilia. Using high-content image-based assays, we demonstrate that mTORC1 activity inversely correlates with ciliation in TSC1/2-deficient neurons. To investigate the mechanistic relationship between mTORC1 and cilia, we perform a phenotypic screen for mTORC1 inhibitors with TSC1/2-deficient neurons. We identify inhibitors of the heat shock protein 90 (Hsp90) that suppress mTORC1 through regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Pharmacological inhibition of Hsp90 rescues ciliation through downregulation of Hsp27. Our study uncovers the heat-shock machinery as a druggable signaling node to restore mTORC1 activity and cilia due to loss of TSC1/2, and it provides broadly applicable platforms for studying TSC-related neuronal dysfunction.
Project description:Although mTORC1 negatively regulates autophagy in cultured cells, how autophagy impacts mTORC1 signaling, in particular in vivo, is less clear. Here we show that autophagy supports mTORC1 hyperactivation in NSCs lacking Tsc1, thereby promoting defects in NSC maintenance, differentiation, tumourigenesis, and the formation of the neurodevelopmental lesion of Tuberous Sclerosis Complex (TSC). Analysing mice that lack Tsc1 and the essential autophagy gene Fip200 in NSCs we find that TSC-deficient cells require autophagy to maintain mTORC1 hyperactivation under energy stress conditions, likely to provide lipids via lipophagy to serve as an alternative energy source for OXPHOS. In vivo, inhibition of lipophagy or its downstream catabolic pathway reverses defective phenotypes caused by Tsc1-null NSCs and reduces tumorigenesis in mouse models. These results reveal a cooperative function of selective autophagy in coupling energy availability with TSC pathogenesis and suggest a potential new therapeutic strategy to treat TSC patients.
Project description:Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in TSC1 or TSC2, which encode proteins that negatively regulate mTOR complex 1 (mTORC1). TSC is associated with significant cognitive, psychiatric, and behavioral problems, collectively termed TSC-Associated Neuropsychiatric Disorders (TAND), and the cell types responsible for these manifestations are largely unknown. Here we use cell type-specific Tsc1 deletion to test whether dopamine neurons, which modulate cognitive, motivational, and affective behaviors, are involved in TAND. We show that loss of Tsc1 and constitutive activation of mTORC1 in dopamine neurons causes somatodendritic hypertrophy, reduces intrinsic excitability, alters axon terminal structure, and impairs striatal dopamine release. These perturbations lead to a selective deficit in cognitive flexibility, preventable by genetic reduction of the mTOR-binding protein Raptor. Our results establish a critical role for Tsc1-mTORC1 signaling in setting the functional properties of dopamine neurons, and indicate that dopaminergic dysfunction may contribute to cognitive inflexibility in TSC.
Project description:Tuberous sclerosis complex (TSC) is characterized by tumor development in the brain, heart, kidney, and lungs. In TSC tumors, loss of the TSC1/TSC2 protein complex leads to activation of mTORC1 with downstream effects on anabolism and cell growth. Because mTORC1 activation enhances mRNA transcription, we hypothesized that aberrant mTORC1 activation might confer TSC-null cell dependence on transcriptional regulation. We demonstrate that TSC1- or TSC2-null cells, in contrast to their wild-type counterparts, are sensitive to pharmacological inhibition of CDK7. Mechanistic studies revealed that CDK7 inhibition markedly reduces glutathione levels and increases reactive oxygen species due to reduced expression of NRF2 and glutathione biosynthesis genes. Treatment of both Tsc2+/ - mice and a TSC1-null bladder cancer xenograft model with a CDK7 inhibitor showed marked reduction in tumor volume and absence of regrowth in the xenograft model. These results suggest that CDK7 inhibition is a promising therapeutic approach for treatment of TSC-associated tumors and cancers with mutations in either TSC1 or TSC2.