bims-tubesc Biomed News
on Molecular mechanisms in tuberous sclerosis
Issue of 2021‒06‒27
nine papers selected by
Marti Cadena Sandoval

  1. Hum Pathol. 2021 Jun 18. pii: S0046-8177(21)00107-6. [Epub ahead of print]
      Renal cell tumors with oncocytic phenotypes represent a daily challenge with several novel, emerging, and provisional entities enriching the diagnostic repertoire. Eosinophilic solid and cystic renal cell carcinoma (ESC-RCC), low-grade oncocytic tumor (LOT), and eosinophilic vacuolated tumor (EVT) have been recognized as unique entities, although their distinctive nature remains controversial. Although most of these tumors are sporadic, rare reports of similar tumors in tuberous sclerosis complex (TSC) have been published. We describe multifocal, often bilateral, tumors in 6 patients without personal or family history of syndromic diseases. Over 60 tumors in various combinations were identified in 10 nephrectomies and 1 biopsy encompassing ESC-RCC (n=6), LOT (n=14), EVT (n=1), clear cell RCC with fibromyomatous stroma (n=12), clear cell RCC (n=2), angiomyolipomas (n>20), unclassified renal cell tumors (n=2), papillary adenomas (n=4), and renomedullary interstitial cell tumor (n=1). TSC1 germline pathogenic mutations were confirmed in two patients. A tumor without germline testing in a third patient revealed TSC1 biallelic inactivation. Two additional patients had molecular testing which excluded common renal mutations and syndromes. We provide the first evidence of co-existence in the same organ and unequivocal relatedness of ESC-RCC, EVT and LOT. End-stage renal disease was present in three of six patients with precursor lesions to all above tumors within adjacent renal parenchyma. In conclusion, identification of multifocal tumors with TSC-like morphology, especially in association with AMLs, could be the first manifestation of clinically silent TSC guiding clinical recommendations for further genetic testing and/or treatment recommendations.
    Keywords:  Carcinoma, Renal Cell/genetics; Carcinoma, Renal Cell/pathology; Kidney Neoplasms/genetics; Kidney Neoplasms/pathology; Tuberous Sclerosis
  2. Neuropathol Appl Neurobiol. 2021 Jun 25.
      AIMS: To evaluate if the myelin pathology observed in epilepsy-associated focal cortical dysplasia type 2B (FCD2B) and - histologically indistinguishable - cortical tubers of tuberous sclerosis complex (TSC) is primarily related to the underlying malformation or constitutes a secondary phenomenon due to the toxic microenvironment created by epileptic seizures. To investigate the possible beneficial effect of the mTOR pathway regulator everolimus on white matter pathology.METHODS: Primary mixed glial cell cultures derived from epilepsy surgery specimens of one TSC and seven FCD2B patients were grown on polycaprolactone fibre matrices and analysed using immunofluorescence and electron microscopy. Unaffected white matter from three age-matched epilepsy patients with mild malformations of cortical development (mMCD) and one with FCD3D served as controls. Additionally, TSC2 knock-out was performed using an oligodendroglial cell line. Myelination capacities of nanofibre grown cells in an inflammatory environment after mTOR-inhibitor treatment with everolimus was further investigated.
    RESULTS: Reduced oligodendroglial turnover, directly related to a lower myelin content was found in the patients´ primary cells. In our culture model of myelination dynamics, primary cells grown under "inflammatory condition" showed decreased myelination, that was repaired by treatment with everolimus.
    CONCLUSIONS: Results obtained in patient-derived primary oligodendroglial and TSC2 knock-out cells suggest that maturation of oligodendroglia and production of a proper myelin sheath seem to be impaired as a result of mTOR pathway disturbance. Hence, oligodendroglial pathology may reflect a more direct effect of the abnormal genetic program rather than to be an inactive bystander of chronic epilepsy.
    Keywords:  Focal cortical dysplasia 2B; myelination; nanofibres; oligodendrocyte; tuberous sclerosis complex
  3. Orphanet J Rare Dis. 2021 Jun 21. 16(1): 282
      BACKGROUND: Tuberous sclerosis complex (TSC), a multisystem genetic disorder, affects many organs and systems, characterized by benign growths. This German multicenter study estimated the disease-specific costs and cost-driving factors associated with various organ manifestations in TSC patients.METHODS: A validated, three-month, retrospective questionnaire was administered to assess the sociodemographic and clinical characteristics, organ manifestations, direct, indirect, out-of-pocket, and nursing care-level costs, completed by caregivers of patients with TSC throughout Germany.
    RESULTS: The caregivers of 184 patients (mean age 9.8 ± 5.3 years, range 0.7-21.8 years) submitted questionnaires. The reported TSC disease manifestations included epilepsy (92%), skin disorders (86%), structural brain disorders (83%), heart and circulatory system disorders (67%), kidney and urinary tract disorders (53%), and psychiatric disorders (51%). Genetic variations in TSC2 were reported in 46% of patients, whereas 14% were reported in TSC1. Mean total direct health care costs were EUR 4949 [95% confidence interval (95% CI) EUR 4088-5863, median EUR 2062] per patient over three months. Medication costs represented the largest direct cost category (54% of total direct costs, mean EUR 2658), with mechanistic target of rapamycin (mTOR) inhibitors representing the largest share (47%, EUR 2309). The cost of anti-seizure drugs (ASDs) accounted for a mean of only EUR 260 (5%). Inpatient costs (21%, EUR 1027) and ancillary therapy costs (8%, EUR 407) were also important direct cost components. The mean nursing care-level costs were EUR 1163 (95% CI EUR 1027-1314, median EUR 1635) over three months. Total indirect costs totaled a mean of EUR 2813 (95% CI EUR 2221-3394, median EUR 215) for mothers and EUR 372 (95% CI EUR 193-586, median EUR 0) for fathers. Multiple regression analyses revealed polytherapy with two or more ASDs and the use of mTOR inhibitors as independent cost-driving factors of total direct costs. Disability and psychiatric disease were independent cost-driving factors for total indirect costs as well as for nursing care-level costs.
    CONCLUSIONS: This study revealed substantial direct (including medication), nursing care-level, and indirect costs associated with TSC over three months, highlighting the spectrum of organ manifestations and their treatment needs in the German healthcare setting.
    TRIAL REGISTRATION: DRKS, DRKS00016045. Registered 01 March 2019,
    Keywords:  Anti-seizure medication; Epilepsy; Everolimus; Rhabdomyoma; Seizure; mTOR inhibitor
  4. J Biol Chem. 2021 Jun 16. pii: S0021-9258(21)00684-0. [Epub ahead of print] 100884
      The mechanistic target of rapamycin (mTOR) is often referred to as a master regulator of cellular metabolism that can integrate growth factor and nutrient signaling. Fasting suppresses hepatic mTORC1 activity via the activity of the Tuberous Sclerosis Complex (TSC), a negative regulator of mTORC1, in order to suppress anabolic metabolism. The loss of TSC1 in the liver locks the liver in a constitutively anabolic state even during fasting, which was suggested to regulate PPARα signaling and ketogenesis, but the molecular determinants of this regulation are unknown. Here, we examined if the activation of the mTORC1 complex in mice by the liver-specific deletion of TSC1 (TSC1L-/-) is sufficient to suppress PPARα signaling and therefore ketogenesis in the fasted state. We found that the activation of mTORC1 in the fasted state is not sufficient to repress PPARα-responsive genes or ketogenesis. Further, we examined whether the activation of the anabolic program mediated by mTORC1 complex activation in the fasted state could suppress the robust catabolic programming and enhanced PPARα transcriptional response of mice with a liver-specific defect in mitochondrial long-chain fatty acid oxidation using Cpt2L-/- mice. We generated liver-specific Cpt2L-/-; Tsc1L-/- double knockout mice and showed that the activation of mTORC1 by deletion of TSC1 could not suppress the catabolic PPARα-mediated phenotype of Cpt2L-/- mice. These data demonstrate that the activation of mTORC1 by the deletion of TSC1 is not sufficient to suppress a PPARα transcriptional program or ketogenesis following fasting.
    Keywords:  Ketogenesis; carnitine palmitoyltransferase 2 (Cpt2); fatty acid oxidation; mTOR; metabolism; peroxisome proliferator-activated receptor alpha (PPARα); β-hydroxybutyrate (βHB)
  5. Life Sci. 2021 Jun 22. pii: S0024-3205(21)00731-1. [Epub ahead of print] 119745
      The evolutionarily conserved mechanistic target of rapamycin (mTOR) forms two functionally distinct complexes, -the mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)-which differ in their subunit composition. Although the function of mTORC1 has been studied extensively, the interaction between mTORC1 and the ubiquitin-proteasome system (UPS) remains unclear. To facilitate a thorough understanding of the mechanismby which UPS regulates mTORC1 activity, steady isotope labeling with amino acids in cell culture (SILAC) technology was used to screen for potential mTORC1-interacting UPS members. Fourteen previously unknown proteins bound to mTOR in HEK293 cells with a SILAC ratio (heavy/light, H/L) above 2, five of which are components of the UPS. Subsequent immunoprecipitation analysis confirmed that ubiquitin-relevant protein 2-like (UBAP2L, also known as NICE-4) binds to both mTOR and Raptor, but not Rictor, suggesting that NICE-4 specifically interacts with mTORC1, but not mTORC2. Interestingly, NICE-4 is essential for basic mTORC1 activity in both HeLa cancer cells and HEK293 cells. In addition, NICE-4 depletion markedly suppressed proliferation of both HeLa and HEK293 cells as well as survival of HeLa cells. Collectively, these results revealed the identity of novel mTOR-interacting UPS proteins and established NICE-4 as a critical UPS member that maintains mTORC1 activity.
    Keywords:  Cancer; HeLa cells; NICE-4; SILAC; Ubiquitin-proteasome system; mTORC1
  6. Hum Pathol. 2021 Jun 18. pii: S0046-8177(21)00110-6. [Epub ahead of print]
      Molecular findings in ovarian, fallopian tube, and peritoneal high-grade serous carcinoma (HGSCa) are emerging as potential prognostic indicators. The chemotherapy response score (CRS) has been proposed as a histologic based prognostic factor in patients with HGSCa treated with neoadjuvant chemotherapy (NACT). No study details the relationship between the mutational landscape of HGSCa and the CRS. This study addresses this issue using Next-Generation Sequencing (NGS).We retrospectively identified 25 HGSCas treated with NACT and pathology material available to calculate the CRS. All cases had NGS on the primary debulking specimen post-NACT. The 3-tier Böhm CRS was applied to the omentum, adnexa and calculated as a combined score. Tumor mutation burden (TMB) and TP53 variant allele frequency (VAF) were calculated and used in correlative analysis. All cases had at least 1 mutation, most commonly TP53 (25 cases, 100%). Other mutations were: BRCA2 (1 case, 4%), ARID1A (2 cases, 8%), and 1 (4%) of each of the following: ERBB2, NTRK3, STK11, NTRK2, TSC1, PIK3CA, NF1, NOTCH3, CDK2, SMAD4 and PMS2. TMB ranged from 2.58 to 7.75 (median 3.84). There was no statistically significant relationship between the TMB and omental CRS, R-squared = 0.011 (p=0.62); adnexal CRS, R-squared = 0.005 (p=0.74); or with the combined CRS, R-squared = 0.009 (p=0.65). Statistically significant correlation was found between the TP53 VAF and the omental CRS (R-squared = 0.28, p=0.007), adnexal CRS (R-squared = 0.26, p=0.01), and the combined CRS (R-squared = 0.33, p=0.0026). The TP53 VAF was adjusted for percent of tumor present on the slide resulting in an average per cell TP53 mutational load, resulting in similar results with a statistically significant correlation between the average per cell TP53 mutational load and the omental CRS (R-squared = 0.27, p=0.02), adnexal CRS (R-squared = 0.16, p=0.05) and the combined CRS (R-squared = 0.23, p=0.02).In summary, NGS confirmed TP53 mutations in all cases of HGSCa. TMB showed no correlation with the CRS. TP53 VAF and average per cell TP53 mutational load showed significant correlation with the CRS, whether graded on the adnexa, omentum or as a combined score, indicating concordance between molecular and histological findings following NACT.
    Keywords:  TP53; chemotherapy response; fallopian tube; high-grade serous carcinoma; mutational landscape; neoadjuvant chemotherapy; ovarian; peritoneal; tumor mutation burden
  7. J Bone Miner Res. 2021 Jun 22.
      Tumor necrosis factor receptor-associated factors (TRAFs) are crucial for receptor activator of nuclear factor-κB (RANK) activation in osteoclasts. However, the upstream mechanisms of TRAF members in the osteoclastic lineage remain largely unknown. Here, we demonstrated that Rictor, a key component of mechanistic target of rapamycin complex 2 (mTORC2), was crucial for TRAF6/TRAF3 expression in osteoclasts. Our ex vivo and in vivo studies showed that Rictor ablation from the osteoclastic lineage reduced osteoclast numbers and increased bone mass in mice. Mechanistically, we found that Rictor ablation restricted osteoclast formation which disrupted TRAF6 stability and caused autophagy block in a manner distinct from mTORC1, resulting in reduced TRAF3 degradation. Boosting TRAF6 expression or knockdown of TRAF3 levels in Rictor-deficient cells could both overcome the defect. Moreover, Rictor could interact with TRAF6 upon RANKL stimulation and loss of Rictor impaired TRAF6 stability and promoted its ubiquitinated degradation. These findings established an innovative link between Rictor, TRAF protein levels and autophagic block. More importantly, mTOR complexes in the osteoclastic lineage are likely switches for coordinating TRAF6 and TRAF3 protein levels, and Rictor may function as an essential upstream regulator of TRAF6/TRAF3 that is partially independent of mTORC1 activity. Inhibitors targeting Rictor may therefore be valuable for preventing or treating osteoclast-related diseases.
    Keywords:  Rictor; TRAF3; TRAF6; autophagic flux; osteoclast
  8. Glia. 2021 Jun 22.
      Multiple signals are involved in the regulation of developmental myelination by Schwann cells and in the maintenance of a normal myelin homeostasis throughout adult life, preserving the integrity of the axons in the PNS. Recent studies suggest that Mek/ERK1/2-MAPK and PI3K/Akt/mTOR intracellular signaling pathways play important, often overlapping roles in the regulation of myelination in the PNS. In addition, hyperactivation of these signaling pathways in Schwann cells leads to a late onset of various pathological changes in the sciatic nerves. However, it remains poorly understood whether these pathways function independently or sequentially or converge using a common mechanism to facilitate Schwann cell differentiation and myelin growth during development and in causing pathological changes in the adult animals. To address these questions, we analyzed multiple genetically modified mice using simultaneous loss- and constitutive gain-of-function approaches. We found that during development, the Mek/ERK1/2-MAPK pathway plays a primary role in Schwann cell differentiation, distinct from mTOR. However, during active myelination, ERK1/2 is dependent on mTOR signaling to drive the growth of the myelin sheath and regulate its thickness. Finally, our data suggest that peripheral nerve pathology during adulthood caused by hyperactivation of Mek/ERK1/2-MAPK or PI3K is likely to be independent or dependent on mTOR-signaling in different contexts. Thus, this study highlights the complexities in the roles played by two major intracellular signaling pathways in Schwann cells that affect their differentiation, myelination, and later PNS pathology and predicts that potential therapeutic modulation of these pathways in PNS neuropathies could be a complex process.
    Keywords:  Schwann cells; differentiation; myelin; myelination
  9. Nat Commun. 2021 06 23. 12(1): 3906
      Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aβ, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling-a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to 'nucleotide empty' or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.