Carbonic Anhydrase 2 Deletion Delays the Growth of Kidney Cysts Whereas Foxi1 Deletion Completely Abrogates Cystogenesis in TSC.

Tuberous sclerosis complex (TSC) presents with renal cysts and benign tumors, which eventually lead to kidney failure. The factors promoting kidney cyst formation in TSC are poorly understood. Inactivation of carbonic anhydrase 2 (Car2) significantly reduced, whereas, deletion of Foxi1 completely abrogated the cyst burden in Tsc1 KO mice. In these studies, we contrasted the ontogeny of cyst burden in Tsc1/Car2 dKO mice vs. Tsc1/Foxi1 dKO mice. Compared to Tsc1 KO, the Tsc1/Car2 dKO mice showed few small cysts at 47 days of age. However, by 110 days, the kidneys showed frequent and large cysts with overwhelming numbers of A-intercalated cells in their linings. The magnitude of cyst burden in Tsc1/Car2 dKO mice correlated with the expression levels of Foxi1 and was proportional to mTORC1 activation. This is in stark contrast to Tsc1/Foxi1 dKO mice, which showed a remarkable absence of kidney cysts at both 47 and 110 days of age. RNA-seq data pointed to profound upregulation of Foxi1 and kidney-collecting duct-specific H+-ATPase subunits in 110-day-old Tsc1/Car2 dKO mice. We conclude that Car2 inactivation temporarily decreases the kidney cyst burden in Tsc1 KO mice but the cysts increase with advancing age, along with enhanced Foxi1 expression.

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.

Targeting the EGFR pathway: An alternative strategy for the treatment of tuberous sclerosis complex?

INTRODUCTION: Tuberous sclerosis complex (TSC) is caused by variants in TSC1/TSC2, leading to constitutive activation of the mammalian target of rapamycin (mTOR) complex 1. Therapy with everolimus has been approved for TSC, but variations in success are frequent. Recently, caudal late interneuron progenitor (CLIP) cells were identified as a common origin of the TSC brain pathologies such as subependymal giant cell astrocytomas (SEGA) and cortical tubers (CT). Further, targeting the epidermal growth factor receptor (EGFR) with afatinib, which is expressed in CLIP cells, reduces cell growth in cerebral TSC organoids. However, investigation of clinical patient-derived data is lacking. AIMS: Observation of EGFR expression in SEGA, CT and focal cortical dysplasia (FCD) 2B human brain specimen and investigation of whether its inhibition could be a potential therapeutic intervention for these patients. METHODS: Brain specimens of 23 SEGAs, 6 CTs, 20 FCD2Bs and 17 controls were analysed via immunohistochemistry to characterise EGFR expression, cell proliferation (via Mib1) and mTOR signalling. In a cell-based assay using primary patient-derived cells (CT n = 1, FCD2B n = 1 and SEGA n = 4), the effects of afatinib and everolimus on cell proliferation and cell viability were observed. RESULTS: EGFR overexpression was observed in histological sections of SEGA, CT and FCD2B patients. Both everolimus and afatinib decreased the proliferation and viability in primary SEGA, tuber and FCD2B cells. CONCLUSION: Our study demonstrates that EGFR suppression might be an effective alternative treatment option for SEGAs and tubers, as well as other mTOR-associated malformations of cortical development, including FCD2B.

Inhibition of p70 Ribosomal S6 Kinase (S6K1) Reduces Cortical Blood Flow in a Rat Model of Autism-Tuberous Sclerosis.

The manifestations of tuberous sclerosis complex (TSC) in humans include epilepsy, autism spectrum disorders (ASD) and intellectual disability. Previous studies suggested the linkage of TSC to altered cerebral blood flow and metabolic dysfunction. We previously reported a significant elevation in cerebral blood flow in an animal model of TSC and autism of young Eker rats. Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin could restore normal oxygen consumption and cerebral blood flow. In this study, we investigated whether inhibiting a component of the mTOR signaling pathway, p70 ribosomal S6 kinase (S6K1), would yield comparable effects. Control Long Evans and Eker rats were divided into vehicle and PF-4708671 (S6K1 inhibitor, 75 mg/kg for 1 h) treated groups. Cerebral regional blood flow (14C-iodoantipyrine) was determined in isoflurane anesthetized rats. We found significantly increased basal cortical (+ 32%) and hippocampal (+ 15%) blood flow in the Eker rats. PF-4708671 significantly lowered regional blood flow in the cortex and hippocampus of the Eker rats. PF-4708671 did not significantly lower blood flow in these regions in the control Long Evans rats. Phosphorylation of S6-Ser240/244 and Akt-Ser473 was moderately decreased in Eker rats but only the latter reached statistical significance upon PF-4708671 treatment. Our findings suggest that moderate inhibition of S6K1 with PF-4708671 helps to restore normal cortical blood flow in Eker rats and that this information might have therapeutic potential in tuberous sclerosis complex and autism.

Loss of macrophage TSC1 exacerbates sterile inflammatory liver injury through inhibiting the AKT/MST1/NRF2 signaling pathway.

Tuberous sclerosis complex 1 (TSC1) plays important roles in regulating innate immunity. However, the precise role of TSC1 in macrophages in the regulation of oxidative stress response and hepatic inflammation in liver ischemia/reperfusion injury (I/R) remains unknown. In a mouse model of liver I/R injury, deletion of myeloid-specific TSC1 inhibited AKT and MST1 phosphorylation, and decreased NRF2 accumulation, whereas activated TLR4/NF-κB pathway, leading to increased hepatic inflammation. Adoptive transfer of AKT- or MST1-overexpressing macrophages, or Keap1 disruption in myeloid-specific TSC1-knockout mice promoted NRF2 activation but reduced TLR4 activity and mitigated I/R-induced liver inflammation. Mechanistically, TSC1 in macrophages promoted AKT and MST1 phosphorylation, and protected NRF2 from Keap1-mediated ubiquitination. Furthermore, overexpression AKT or MST1 in TSC1-knockout macrophages upregulated NRF2 expression, downregulated TLR4/NF-κB, resulting in reduced inflammatory factors, ROS and inflammatory cytokine-mediated hepatocyte apoptosis. Strikingly, TSC1 induction in NRF2-deficient macrophages failed to reverse the TLR4/NF-κB activity and production of pro-inflammatory factors. Conclusions: Macrophage TSC1 promoted the activation of the AKT/MST1 signaling pathway, increased NRF2 levels via reducing Keap1-mediated ubiquitination, and modulated oxidative stress-driven inflammatory responses in liver I/R injury. Our findings underscore the critical role of macrophage TSC1 as a novel regulator of innate immunity and imply the therapeutic potential for the treatment of sterile liver inflammation in transplant recipients. Schematic illustration of macrophage TSC1-mediated AKT/MST1/NRF2 signaling pathway in I/R-triggered liver inflammation. Macrophage TSC1 can be activated in I/R-stressed livers. TSC1 activation promotes phosphorylation of AKT and MST1, which in turn increases NRF2 expression and inhibits ROS production and TLR4/NF-κB activation, resulting in reduced hepatocellular apoptosis in I/R-triggered liver injury.

Predictive model for epileptogenic tubers from all tubers in patients with tuberous sclerosis complex based on 18F-FDG PET: an 8-year single-centre study.

BACKGROUND: More than half of patients with tuberous sclerosis complex (TSC) suffer from drug-resistant epilepsy (DRE), and resection surgery is the most effective way to control intractable epilepsy. Precise preoperative localization of epileptogenic tubers among all cortical tubers determines the surgical outcomes and patient prognosis. Models for preoperatively predicting epileptogenic tubers using 18F-FDG PET images are still lacking, however. We developed noninvasive predictive models for clinicians to predict the epileptogenic tubers and the outcome (seizure freedom or no seizure freedom) of cortical tubers based on 18F-FDG PET images. METHODS: Forty-three consecutive TSC patients with DRE were enrolled, and 235 cortical tubers were selected as the training set. Quantitative indices of cortical tubers on 18F-FDG PET were extracted, and logistic regression analysis was performed to select those with the most important predictive capacity. Machine learning models, including logistic regression (LR), linear discriminant analysis (LDA), and artificial neural network (ANN) models, were established based on the selected predictive indices to identify epileptogenic tubers from multiple cortical tubers. A discriminating nomogram was constructed and found to be clinically practical according to decision curve analysis (DCA) and clinical impact curve (CIC). Furthermore, testing sets were created based on new PET images of 32 tubers from 7 patients, and follow-up outcome data from the cortical tubers were collected 1, 3, and 5 years after the operation to verify the reliability of the predictive model. The predictive performance was determined by using receiver operating characteristic (ROC) analysis. RESULTS: PET quantitative indices including SUVmean, SUVmax, volume, total lesion glycolysis (TLG), third quartile, upper adjacent and standard added metabolism activity (SAM) were associated with the epileptogenic tubers. The SUVmean, SUVmax, volume and TLG values were different between epileptogenic and non-epileptogenic tubers and were associated with the clinical characteristics of epileptogenic tubers. The LR model achieved the better performance in predicting epileptogenic tubers (AUC = 0.7706; 95% CI 0.70-0.83) than the LDA (AUC = 0.7506; 95% CI 0.68-0.82) and ANN models (AUC = 0.7425; 95% CI 0.67-0.82) and also demonstrated good calibration (Hosmer‒Lemeshow goodness-of-fit p value = 0.7). In addition, DCA and CIC confirmed the clinical utility of the nomogram constructed to predict epileptogenic tubers based on quantitative indices. Intriguingly, the LR model exhibited good performance in predicting epileptogenic tubers in the testing set (AUC = 0.8502; 95% CI 0.71-0.99) and the long-term outcomes of cortical tubers (1-year outcomes: AUC = 0.7805, 95% CI 0.71-0.85; 3-year outcomes: AUC = 0.8066, 95% CI 0.74-0.87; 5-year outcomes: AUC = 0.8172, 95% CI 0.75-0.87). CONCLUSIONS: The 18F-FDG PET image-based LR model can be used to noninvasively identify epileptogenic tubers and predict the long-term outcomes of cortical tubers in TSC patients.

Translatome analysis of tuberous sclerosis complex 1 patient-derived neural progenitor cells reveals rapamycin-dependent and independent alterations.

BACKGROUND: Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in the TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD) and intellectual disability. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using TSC1 patient-derived neural progenitor cells (NPCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in TSC1-null NPCs, which were unaffected by the mTORC1 inhibitor rapamycin. METHODS: Here, we used polysome profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level, to compare CRISPR-edited TSC1-null with CRISPR-corrected TSC1-WT NPCs generated from one TSC donor (one clone/genotype). To assess the relevance of identified gene expression alterations, we performed polysome profiling in postmortem brains from ASD donors and age-matched controls. We further compared effects on translation of a subset of transcripts and rescue of early ND phenotypes in NPCs following inhibition of mTORC1 using the allosteric inhibitor rapamycin versus a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272. RESULTS: Polysome profiling of NPCs revealed numerous TSC1-associated alterations in mRNA translation that were largely recapitulated in human ASD brains. Moreover, although rapamycin treatment partially reversed the TSC1-associated alterations in mRNA translation, most genes related to neural activity/synaptic regulation or ASD were rapamycin-insensitive. In contrast, treatment with RMC-6272 inhibited rapamycin-insensitive translation and reversed TSC1-associated early ND phenotypes including proliferation and neurite outgrowth that were unaffected by rapamycin. CONCLUSIONS: Our work reveals ample mRNA translation alterations in TSC1 patient-derived NPCs that recapitulate mRNA translation in ASD brain samples. Further, suppression of TSC1-associated but rapamycin-insensitive translation and ND phenotypes by RMC-6272 unveils potential implications for more efficient targeting of mTORC1 as a superior treatment strategy for TAND.

Paradigm shift in the treatment of tuberous sclerosis: Effectiveness of everolimus.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterised by abnormal cell proliferation and differentiation that affects multiple organs and can lead to the growth of hamartomas. Tuberous sclerosis complex is caused by the disinhibition of the protein mTOR (mammalian target of rapamycin). In the past, various therapeutic approaches, even if only symptomatic, have been attempted to improve the clinical effects of this disease. While all of these therapeutic strategies are useful and are still used and indicated, they are symptomatic therapies based on the individual symptoms of the disease and therefore not fully effective in modifying long-term outcomes. A new therapeutic approach is the introduction of allosteric inhibitors of mTORC1, which allow restoration of metabolic homeostasis in mutant cells, potentially eliminating most of the clinical manifestations associated with Tuberous sclerosis complex. Everolimus, a mammalian target of the rapamycin inhibitor, is able to reduce hamartomas, correcting the specific molecular defect that causes Tuberous sclerosis complex. In this review, we report the findings from the literature on the use of everolimus as an effective and safe drug in the treatment of TSC manifestations affecting various organs, from the central nervous system to the heart.

Prdx5 in the Regulation of Tuberous Sclerosis Complex Mutation-Induced Signaling Mechanisms.

(1) Background: Tuberous sclerosis complex (TSC) mutations directly affect mTORC activity and, as a result, protein synthesis. In several cancer types, TSC mutation is part of the driver mutation panel. TSC mutations have been associated with mitochondrial dysfunction, tolerance to reactive oxygen species due to increased thioredoxin reductase (TrxR) enzyme activity, tolerance to endoplasmic reticulum (ER) stress, and apoptosis. The FDA-approved drug rapamycin is frequently used in clinical applications to inhibit protein synthesis in cancers. Recently, TrxR inhibitor auranofin has also been involved in clinical trials to investigate the anticancer efficacy of the combination treatment with rapamycin. We aimed to investigate the molecular background of the efficacy of such drug combinations in treating neoplasia modulated by TSC mutations. (2) Methods: TSC2 mutant and TSC2 wild-type (WT) cell lines were exposed to rapamycin and auranofin in either mono- or combination treatment. Mitochondrial membrane potential, TrxR enzyme activity, stress protein array, mRNA and protein levels were investigated via cell proliferation assay, electron microscopy, etc. (3) Results: Auranofin and rapamycin normalized mitochondrial membrane potential and reduced proliferation capacity of TSC2 mutant cells. Database analysis identified peroxiredoxin 5 (Prdx5) as the joint target of auranofin and rapamycin. The auranofin and the combination of the two drugs reduced Prdx5 levels. The combination treatment increased the expression of heat shock protein 70, a cellular ER stress marker. (4) Conclusions: After extensive analyses, Prdx5 was identified as a shared target of the two drugs. The decreased Prdx5 protein level and the inhibition of both TrxR and mTOR by rapamycin and auranofin in the combination treatment made ER stress-induced cell death possible in TSC2 mutant cells.

Increased degradation of FMRP contributes to neuronal hyperexcitability in tuberous sclerosis complex.

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder, but new therapies have been impeded by a lack of understanding of the pathological mechanisms. Tuberous sclerosis complex (TSC) and fragile X syndrome are associated with alterations in the mechanistic target of rapamycin (mTOR) and fragile X messenger ribonucleoprotein 1 (FMRP), which have been implicated in the development of ASD. Previously, we observed that transcripts associated with FMRP were down-regulated in TSC2-deficient neurons. In this study, we find that FMRP turnover is dysregulated in TSC2-deficient rodent primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons and is dependent on the E3 ubiquitin ligase anaphase-promoting complex. We also demonstrate that overexpression of FMRP can partially rescue hyperexcitability in TSC2-deficient iPSC-derived neurons. These data indicate that FMRP dysregulation represents an important pathological mechanism in the development of abnormal neuronal activity in TSC and illustrate a molecular convergence between these two neurogenetic disorders.

Design of negative-regulating proteins of Rheb/mTORC1 with much-reduced sizes of the tuberous sclerosis protein complex.

The mTORC1 signaling pathway regulates cell growth and metabolism in a variety of organisms from yeast to human, and inhibition of the mTORC1 pathway has the prospect to treat cancer or achieve longevity. The tuberous sclerosis protein complex (TSCC) is a master negative regulator of the mTORC1 signaling pathway through hydrolyzing the GTP loaded on the small GTPase Rheb, which is a key activator of mTOR. However, the large size (~700 kDa) and complex structural organization of TSCC render it vulnerable to degradation and inactivation, thus limiting its potential application. In this work, based on thorough analysis and understanding of the structural mechanism of how the stabilization domain of TSC2 secures the association of TSC2-GAP with Rheb and thus enhances its GAP activity, we designed two proteins, namely SSG-MTM (short stabilization domain and GAP domain-membrane targeting motif) and SSG-TSC1N, which were able to function like TSCC to negatively regulate Rheb and mTORC1, but with much-reduced sizes (~1/15 and ~ 1/9 of the size of TSCC, respectively). Biochemical and cell biological assays demonstrated that these designed proteins indeed could promote the GTPase activity of Rheb to hydrolyze GTP, inhibit the kinase activity of mTORC1, and prevent mTORC1 from down-regulating catabolism and autophagy.

Arginine depletion attenuates renal cystogenesis in tuberous sclerosis complex model.

Cystic kidney disease is a leading cause of morbidity in patients with tuberous sclerosis complex (TSC). We characterize the misregulated metabolic pathways using cell lines, a TSC mouse model, and human kidney sections. Our study reveals a substantial perturbation in the arginine biosynthesis pathway in TSC models with overexpression of argininosuccinate synthetase 1 (ASS1). The rise in ASS1 expression is dependent on the mechanistic target of rapamycin complex 1 (mTORC1) activity. Arginine depletion prevents mTORC1 hyperactivation and cell cycle progression and averts cystogenic signaling overexpression of c-Myc and P65. Accordingly, an arginine-depleted diet substantially reduces the TSC cystic load in mice, indicating the potential therapeutic effects of arginine deprivation for the treatment of TSC-associated kidney disease.

Tuberous Sclerosis Complex cell-derived EVs have an altered protein cargo capable of regulating their microenvironment and have potential as disease biomarkers.

Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) is a feature of many solid tumours and is a key pathogenic driver in the inherited condition Tuberous Sclerosis Complex (TSC). Modulation of the tumour microenvironment by extracellular vesicles (EVs) is known to facilitate the development of various cancers. The role of EVs in modulating the tumour microenvironment and their impact on the development of TSC tumours, however, remains unclear. This study, therefore, focuses on the poorly defined contribution of EVs to tumour growth in TSC. We characterised EVs secreted from TSC2-deficient and TSC2-expressing cells and identified a distinct protein cargo in TSC2-deficient EVs, containing an enrichment of proteins thought to be involved in tumour-supporting signalling pathways. We show EVs from TSC2-deficient cells promote cell viability, proliferation and growth factor secretion from recipient fibroblasts within the tumour microenvironment. Rapalogs (mTORC1 inhibitors) are the current therapy for TSC tumours. Here, we demonstrate a previously unknown intercellular therapeutic effect of rapamycin in altering EV cargo and reducing capacity to promote cell proliferation in the tumour microenvironment. Furthermore, EV cargo proteins have the potential for clinical applications as TSC biomarkers, and we reveal three EV-associated proteins that are elevated in plasma from TSC patients compared to healthy donor plasma.

Tyrosine Kinase Inhibitors Diminish Renal Neoplasms in a Tuberous Sclerosis Model Via Induction of Apoptosis.

Tuberous sclerosis complex (TSC) tumors are presently incurable despite a cytostatic response to mTOR pathway inhibition because recurrence of disease occurs after treatment is discontinued. Here, we explored the hypothesis that inhibiting tyrosine kinase activity in mesenchymal lineage-specific platelet-derived growth factor receptor ß (PDGFRß) signaling in TSC tumors is cytocidal and attenuates tumorigenesis at significantly higher levels than treatment with an mTOR inhibitor. Rapamycin-induced versus tyrosine kinase inhibitor (TKI)-induced renal angiomyolipoma (AML) and pulmonary lymphangioleiomyomatosis (LAM) tumor cells were comparatively analyzed using cell survival assays, RNA sequencing, and bioinformatics to distinguish tumoricidal mechanisms adopted by each drug type. The efficacy of imatinib therapy was validated against spontaneously developing renal cystadenomas in tuberous sclerosis Tsc2+/- mouse models (C57BL/6J mice; N = 6; 400 mg/kg/d; oral gavage) compared with Tsc2+/- mice treated with PBS (C57BL/6J mice; N = 6). Our study revealed that TKIs imatinib and nilotinib were cytocidal to both pulmonary LAM and renal AML cell cultures through the downregulation of the glycoprotein GPVI pathway and resultant disruption in mitochondrial permeability, increased cytosolic cytochrome C, and caspase 3 activation. Importantly, renal tumor growth was significantly attenuated in imatinib-treated Tsc2+/- mice compared with PBS treatment. The preclinical studies reported here provide evidence documenting the effectiveness of TKIs in limiting LAM and AML cell growth and viability with important clinical potential. Furthermore, these drugs elicit their effects by targeting a PDGF pathway-dependent apoptotic mechanism supporting the investigation of these drugs as a novel class of TSC therapeutics.

Proteomic analysis of murine Tsc1-deficient neural stem progenitor cells.

Tuberous sclerosis complex (TSC) is a rare, multisystem genetic disorder that leads to the development of benign tumors in multiple organs and neurological symptoms. TSC clinical manifestations show a great heterogenicity, with most patients presenting severe neuropsychiatric and neurological disorders. TSC is caused by loss-of-function mutations in either TSC1 or TSC2 genes, leading to overexpression of the mechanistic target of rapamycin (mTOR) and, consequently, abnormal cellular growth, proliferation and differentiation as well as to cell migration defects. Beside the growing interest, TSC remains a disorder poorly understood, with limited perspectives in the field of therapeutic strategies. Here we used murine postnatal subventricular zone (SVZ) neural stem progenitor cells (NSPCs) deficient of Tsc1 gene as a TSC model to unravel novel molecular aspects of the pathophysiology of this disease. 2D-DIGE-based proteomic analysis detected 55 differently represented spots in Tsc1-deficient cells, compared to wild-type counterparts, which were associated with 36 protein entries after corresponding trypsinolysis and nanoLC-ESI-Q-Orbitrap-MS/MS analysis. Proteomic results were validated using various experimental approaches. Bioinformatics associated differently represented proteins with oxidative stress and redox pathways, methylglyoxal biosynthesis, myelin sheath, protein S-nitrosylation and carbohydrate metabolism. Because most of these cellular pathways have already been linked to TSC features, these results were useful to clarify some molecular aspects of TSC etiopathogenesis and suggested novel promising therapeutic protein targets. SIGNIFICANCE: Tuberous Sclerosis Complex (TSC) is a multisystemic disorder caused by inactivating mutations of TSC1 or TSC2 genes, which induce overactivation of the mTOR component. The molecular mechanisms underlying the pathogenesis of TSC remain unclear, probably due to complexity of mTOR signaling network. To have a picture of protein abundance changes occurring in TSC disorder, murine postnatal subventricular zone (SVZ) neural stem progenitor cells (NSPCs) deficient of Tsc1 gene were used as a model of disease. Thus, Tsc1-deficient SVZ NSPCs and wild-type cells were comparatively evaluated by proteomics. This analysis evidenced changes in the abundance of proteins involved in oxidative/nitrosative stress, cytoskeleton remodelling, neurotransmission, neurogenesis and carbohydrate metabolism. These proteins might clarify novel molecular aspects of TSC etiopathogenesis and constitute putative molecular targets for novel therapeutic management of TSC-related disorders.

Microtubule Cytoskeletal Network Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders.

Tuberous sclerosis complex (TSC) is a rare genetic multisystem disorder caused by loss-of-function mutations in the tumour suppressors TSC1/TSC2, both of which are negative regulators of the mammalian target of rapamycin (mTOR) kinase. Importantly, mTOR hyperactivity seems to be linked with the pathobiology of autism spectrum disorders (ASD). Recent studies suggest the potential involvement of microtubule (MT) network dysfunction in the neuropathology of "mTORopathies", including ASD. Cytoskeletal reorganization could be responsible for neuroplasticity disturbances in ASD individuals. Thus, the aim of this work was to study the effect of Tsc2 haploinsufficiency on the cytoskeletal pathology and disturbances in the proteostasis of the key cytoskeletal proteins in the brain of a TSC mouse model of ASD. Western-blot analysis indicated significant brain-structure-dependent abnormalities in the microtubule-associated protein Tau (MAP-Tau), and reduced MAP1B and neurofilament light (NF-L) protein level in 2-month-old male B6;129S4-Tsc2tm1Djk/J mice. Alongside, pathological irregularities in the ultrastructure of both MT and neurofilament (NFL) networks as well as swelling of the nerve endings were demonstrated. These changes in the level of key cytoskeletal proteins in the brain of the autistic-like TSC mice suggest the possible molecular mechanisms responsible for neuroplasticity alterations in the ASD brain.

Tuberous sclerosis complex-1 (TSC1) contributes to selective neuronal vulnerability in Alzheimer's disease.

AIMS: Selective neuronal vulnerability of hippocampal Cornu Ammonis (CA)-1 neurons is a pathological hallmark of Alzheimer's disease (AD) with an unknown underlying mechanism. We interrogated the expression of tuberous sclerosis complex-1 (TSC1; hamartin) and mTOR-related proteins in hippocampal CA1 and CA3 subfields. METHODS: A human post-mortem cohort of mild (n = 7) and severe (n = 10) AD and non-neurological controls (n = 9) was used for quantitative and semi-quantitative analyses. We also developed an in vitro TSC1 knockdown model in rat hippocampal neurons, and transcriptomic analyses of TSC1 knockdown neuronal cultures were performed. RESULTS: We found a selective increase of TSC1 cytoplasmic inclusions in human AD CA1 neurons with hyperactivation of one of TSC1's downstream targets, the mammalian target of rapamycin complex-1 (mTORC1), suggesting that TSC1 is no longer active in AD. TSC1 knockdown experiments showed accelerated cell death independent of amyloid-beta toxicity. Transcriptomic analyses of TSC1 knockdown neuronal cultures revealed signatures that were significantly enriched for AD-related pathways. CONCLUSIONS: Our combined data point to TSC1 dysregulation as a key driver of selective neuronal vulnerability in the AD hippocampus. Future work aimed at identifying targets amenable to therapeutic manipulation is urgently needed to halt selective neurodegeneration, and by extension, debilitating cognitive impairment characteristic of AD.

Bcat1 is controlled by Tsc2/mTORC1 pathway at expression levels and its deficiency together with Bcat2 inactivation suppresses the growth of a Tsc2-/- tumor cell line.

The tuberous sclerosis complex (TSC) gene products (TSC1/TSC2) negatively regulate mTORC1. Although mTORC1 inhibitors are used for the treatment of TSC, incomplete tumor elimination and the adverse effects from long-term administration are problems that need to be solved. Branched-chain amino acid (BCAA) metabolism is involved in the growth of many tumor cells via the mTORC1 pathway. However, it remains unclear how BCAA metabolism affects the growth of mTORC1-dysregulated tumors. We show here that the expression of branched-chain amino transferase1 (Bcat1) was suppressed in Tsc2-deficient murine renal tumor cells either by treatment with rapamycin or restoration of Tsc2 expression suggesting that Bcat1 is located downstream of Tsc2-mTORC1 pathway. We also found that gabapentin, a Bcat1 inhibitor suppressed the growth of Tsc2-deficient tumor cells and increased efficacy when combined with rapamycin. We investigate the functional importance of Bcat1 and the mitochondrial isoform Bcat2 by inhibiting each enzyme separately or both together by genome editing and shRNA in Tsc2-deficient cells. We found that deficiency of both enzymes, but not either alone, inhibited cell growth, indicating that BCAA-metabolic reactions support Tsc2-deficient cell proliferation. Our results indicate that inhibition of Bcat1 and Bcat2 by specific drugs should be a useful method for TSC treatment.

NFATc2-dependent epigenetic downregulation of the TSC2/Beclin-1 pathway is involved in neuropathic pain induced by oxaliplatin.

Neuropathic pain is a common dose-limiting side effect of oxaliplatin, which hampers the effective treatment of tumors. Here, we found that upregulation of transcription factor NFATc2 decreased the expression of Beclin-1, a critical molecule in autophagy, in the spinal dorsal horn, and contributed to neuropathic pain following oxaliplatin treatment. Meanwhile, manipulating autophagy levels by intrathecal injection of rapamycin (RAPA) or 3-methyladenine (3-MA) differentially altered mechanical allodynia in oxaliplatin-treated or naïve rats. Utilizing chromatin immunoprecipitation-sequencing (ChIP-seq) assay combined with bioinformatics analysis, we found that NFATc2 negatively regulated the transcription of tuberous sclerosis complex protein 2 (TSC2), which contributed to the oxaliplatin-induced Beclin-1 downregulation. Further assays revealed that NFATc2 regulated histone H4 acetylation and methylation in the TSC2 promoter site 1 in rats' dorsal horns with oxaliplatin treatment. These results suggested that NFATc2 mediated the epigenetic downregulation of the TSC2/Beclin-1 autophagy pathway and contributed to oxaliplatin-induced mechanical allodynia, which provided a new therapeutic insight for chemotherapy-induced neuropathic pain.

Extension of microglial activation is associated with epilepsy and cognitive dysfunction in Tuberous sclerosis complex: A TSPO-PET study.

BACKGROUND AND OBJECTIVES: Neuroinflammation contributes to the severity of various neurological disorders, including epilepsy. Tuberous sclerosis complex (TSC) is a condition that results in the overactivation of the mammalian target of rapamycin (mTOR) pathway, which has been linked to the activation of microglia responsible for neuroinflammation. To clarify the involvement of neuroinflammation in the neuropathophysiology of TSC, we performed a positron emission tomography (PET) study using the translocator protein (TSPO) radioligand, [11C] DPA713, and investigated microglial activation in relation to neurological manifestations, especially epilepsy and cognitive function. METHODS: This cross-sectional study included 18 patients with TSC (6 in the no-seizure group, 6 in the refractory seizure group, and 6 in the mTOR-inhibitor [mTOR-i] group). All participants underwent [11C] DPA713-PET. PET results were superimposed with a 3D T2-weighted fluid-attenuated inversion-recovery (FLAIR) and T1-weighted image (T1WI) to evaluate the location of cortical tubers. Microglial activation was assessed using the standardized uptake value ratio (SUVr) of DPA713 binding. The volume ratio of the DPA713-positive area to the intracranial volume (volume ratio of DPA713/ICV) was calculated to evaluate the extent of microglial activation. A correlation analysis was performed to examine the relationship between volume ratio of DPA713/ICV and severity of epilepsy and cognitive function. RESULTS: Most cortical tubers with hyperintensity on FLAIR and hypo- or isointensity on T1WI showed microglial activation. The extent of microglial activation was significantly greater in the refractory seizure group than in the no-seizure or mTOR-i groups (p < 0.001). The extent of microglial activation in subjects without mTOR-i treatment correlated positively with epilepsy severity (r = 0.822, P = 0.001) and negatively with cognitive function (r = -0.846, p = 0.001), but these correlations were not present in the mTOR-i group (r = 0.232, P = 0.658, r = 0.371, P = 0.469, respectively). CONCLUSION: Neuroinflammation is associated with the severity of epilepsy and cognitive dysfunction in brains with TSC. mTOR-i may suppress the extent of neuroinflammation in TSC. Investigating the spread of microglial activation using TSPO-PET in these patients may help to predict the progression of neuropathy by assessing the degree of neuroinflammation and therefore be useful for determining how aggressive the treatment should be and in assessing the effectiveness of such treatment in patients with TSC.