bims-tubesc Biomed News
on Molecular mechanisms in tuberous sclerosis
Issue of 2021‒07‒18
twenty papers selected by
Marti Cadena Sandoval

  1. Nat Commun. 2021 07 12. 12(1): 4245
      Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/- mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.
  2. Am J Med Genet A. 2021 Jul 10.
      Cutaneous angiofibroma is part of the classic triad of tuberous sclerosis complex (TSC). Angiofibroma is rarely reported to affect the mucous membranes of the trachea and bronchus. Tracheobronchial angiofibroma is also a hamartomatous manifestation of TSC. Considering the paucity of literature describing tracheal lesions in TSC, more case reports are needed to guide treatment planning. This case report adds to the existing clinical literature and provides a reference for clinical diagnosis.
    Keywords:  case report; tracheobronchial angiofibroma; tuberous sclerosis complex
  3. Seizure. 2021 Jul 05. pii: S1059-1311(21)00231-4. [Epub ahead of print]91 273-277
      OBJECTIVE: To describe the first large population (n = 297) with tuberous sclerosis complex (TSC) in China and to examine the relationship between variants (type and location) and epilepsy.METHODS: All exons and intron-exon boundaries of TSC1/TSC2 were sequenced with next-generation sequencing, and the distribution of several variants and associations between variant types and epilepsy were investigated.
    RESULTS: Epilepsy occurred in 83.5% (248/297) of the individuals. The TSC1/TSC2 gene variant detection rate was 89.6% (266/297). The rate of epilepsy was significantly higher in the TSC2 group than in the TSC1 (p = 0.02) and no mutation identified (NMI) groups (p = 0.0005). TSC2 individuals are more likely to have spasms than TSC1 individuals (p =0.03). The age at epilepsy onset of individuals in the TSC2 group was younger than that of individuals in the TSC1 group (p = 0.008) and NMI group (p = 0.01). The age at epilepsy onset with truncated variants in the TSC1 group was significantly younger than that of individuals with nontruncated variants (p = 0.0001). The rate of epilepsy was significantly higher if variants occurred in the functional domain than in the nonfunctional domain in TSC2 individuals (p = 0.02).
    CONCLUSION: This was the first large cohort study of the Chinese TSC population in which a comparative analysis of genotype and epilepsy was conducted. Individuals with TSC2 variants appeared to have more severe epileptic phenotypes, such as younger age at epilepsy onset, than those with TSC1 variants and NMI, and individuals with variants that occurred in TSC2 functional domains were more prone to epilepsy and had a younger age at epilepsy onset.
    Keywords:  Child; Domain; Epilepsy; Genotype; TSC
  4. Epilepsia Open. 2021 Jul 16.
      OBJECTIVE: To investigate resting state functional connectivity (FC) in pediatric patients with tuberous sclerosis complex and intractable epilepsy requiring surgery.METHODS: Resting state-functional MRI was utilized to investigate functional connectivity in 13 pediatric patients with tuberous sclerosis complex (TSC) and intractable epilepsy requiring surgery.
    RESULTS: The majority of patients demonstrated a resting state network architecture similar to those reported in healthy individuals. However, pre-operative differences were evident between patients with high versus low tuber burden, as well as those with good versus poor neurodevelopmental outcomes, most notably in the cingulo-opercular and visual resting state networks. One patient with high tuber burden and poor pre-operative development and seizure control had nearly normal development and seizure resolution after surgery. This was accompanied by significant improvement in resting state network architecture just one day post-operatively.
    SIGNIFICANCE: Although many patients with tuberous sclerosis complex and medically refractory epilepsy demonstrate functional connectivity patterns similar to healthy children, relationships within and between RSNs demonstrate clear differences in patients with higher tuber burden and worse outcomes. Improvements in resting state network organization post-operatively may be related to epilepsy surgery outcomes, providing candidate biomarkers for clinical management in this high-risk population.
    Keywords:  Epilepsy; Epilepsy surgery; Functional MRI; Functional connectivity; Tuberous sclerosis complex
  5. Front Physiol. 2021 ;12 630933
      In tuberous sclerosis complex (TSC), Tsc2 mutations are associated with more severe disease manifestations than Tsc1 mutations and the role of extracellular vesicles (EVs) in this context is not yet studied. We report a comparative analysis of EVs derived from isogenic renal cells except for Tsc1 or Tsc2 gene status and hypothesized that in spite of having similar physical characteristics, EVs modulate signaling pathways differently, thus leading to TSC heterogenicity. We used mouse inner medullary collecting duct (mIMCD3) cells with the Tsc1 (T1G cells) or Tsc2 (T2J cells) gene disrupted by CRISPR/CAS9. EVs were isolated from the cell culture media by size-exclusion column chromatography followed by detailed physical and chemical characterization. Physical characterization of EVs was accessed by tunable resistive pulse sensing and dynamic light scattering, revealing similar average sizes and zeta potentials (at pH 7.4) for EVs from mIMCD3 (123.5 ± 5.7 nm and -16.3 ± 2.1 mV), T1G cells (131.5 ± 8.3 nm and -19.8 ± 2.7 mV), and T2J cells (127.3 ± 4.9 nm and -20.2 ± 2.1 mV). EVs derived from parental mIMCD3 cells and both mutated cell lines were heterogeneous (>90% of EVs < 150 nm) in nature. Immunoblotting detected cilial Hedgehog signaling protein Arl13b; intercellular proteins TSG101 and Alix; and transmembrane proteins CD63, CD9, and CD81. Compared to Tsc2 deletion, Tsc1 deletion cells had reduced EV production and release rates. EVs from Tsc1 mutant cells altered mTORC1, autophagy, and β-catenin pathways differently than EVs from Tsc2-mutated cells. Quantitative PCR analysis revealed the down regulation of miR-212a-3p and miR-99a-5p in EVs from Tsc2-mutated cells compared to EVs from Tsc1-mutant cells. Thus, EV-derived miR-212-3p and mIR-99a-5p axes may represent therapeutic targets or biomarkers for TSC disease.
    Keywords:  autophagy; cell signaling; extracellular vesicles; miRNA; tuberous sclerosis complex
  6. Seizure. 2021 Jun 29. pii: S1059-1311(21)00214-4. [Epub ahead of print]91 287-295
      PURPOSE: 10-year retrospective study to assess burden of illness in individuals with tuberous sclerosis complex (TSC) identified from German healthcare data.METHODS: Patients with TSC were identified by International Classification of Diseases code Q85.1. Patients with epilepsy were identified by epilepsy diagnosis or antiseizure medication (ASM) prescription after TSC diagnosis.
    RESULTS: Using data from 2016 (final study year), 100 patients with TSC were identified (mean [range] age: 38 [1-86] years; male: 40%); prevalence: 7.9 per 100,000 (TSC), 2.2 per 100,000 (TSC with epilepsy). During the 10-year study period (2007-2016), 256 patients with TSC were identified and followed up for 1,784 patient-years (epilepsy: 36%, 616 patient-years). TSC manifestations/comorbidities (apart from epilepsy) were identified more frequently in patients with epilepsy than without. Mean annual healthcare costs for patients with TSC were €6,139 per patient-year (PPY), mostly attributable to medication (35%) and inpatient care (29%). Patients with epilepsy incurred costs more than double those without. Mean (standard deviation [SD]) annual hospitalisation rate (AHR) and length of stay (LOS) PPY: 0.5 (1.0) and 5.9 (18.6) days for TSC. AHR and LOS were greater in patients with epilepsy than without. Mean (SD) number of ASMs prescribed (TSC with epilepsy): 3.0 (2.3) over the entire observable time per patient. Mortality rates (vs. control): 5.08% (vs. 1.69%, p<0.001) for TSC, 7.53% (vs. 0.98%, p<0.001) for TSC with epilepsy, 3.68% (vs. 2.03%, p = 0.003) for TSC without epilepsy.
    CONCLUSION: Healthcare costs, resource utilisation, and mortality were greater in patients with TSC and epilepsy than those without epilepsy.
    Keywords:  Burden of illness; Epilepsy; Healthcare costs; Healthcare resource utilisation; Prevalence; Tuberous sclerosis complex
  7. Hum Genomics. 2021 Jul 09. 15(1): 43
      OBJECTIVE: Tuberous sclerosis complex (TSC) is a rare autosomal dominant disease characterized by lesions throughout the body. Our previous study showed the abnormal up-regulation of miRNAs plays an important part in the pathogenesis of TSC-related renal angiomyolipoma (TSC-RAML). circRNAs were known as important regulators of miRNA, but little is known about the circRNAs in TSC-RAMLs.METHODS: Microarray chips and RNA sequencing were used to identify the circRNAs and mRNAs that were differently expressed between the TSC-RAML and normal kidney tissue. A competitive endogenous RNA (ceRNA) regulatory network was constructed to reveal the regulation of miRNAs and mRNAs by the circRNAs. The biological functions of circRNA and mRNA were analyzed by pathway analysis. Microenvironmental cell types were estimated with the MCP-counter package.
    RESULTS: We identified 491 differentially expressed circRNAs (DECs) and 212 differentially expressed genes (DEGs), and 6 DECs were further confirmed by q-PCR. A ceRNA regulatory network which included 6 DECs, 5 miRNAs, and 63 mRNAs was established. Lipid biosynthetic process was significantly up-regulated in TSC-RAML, and the humoral immune response and the leukocyte chemotaxis pathway were found to be down-regulated. Fibroblasts are enriched in TSC-RAML, and the up-regulation of circRNA_000799 and circRNA_025332 may be significantly correlated to the infiltration of the fibroblasts.
    CONCLUSION: circRNAs may regulate the lipid metabolism of TSC-RAML by regulation of the miRNAs. Fibroblasts are enriched in TSC-RAMLs, and the population of fibroblast may be related to the alteration of circRNAs of TSC-RAML. Lipid metabolism in fibroblasts is a potential treatment target for TSC-RAML.
    Keywords:  Circular RNAs; Renal angiomyolipoma; Tuberous sclerosis
  8. Elife. 2021 Jul 14. pii: e67399. [Epub ahead of print]10
      Tuberous sclerosis complex (TSC) is a genetic disorder that is associated with multiple neurological manifestations. Previously, we demonstrated that Tsc1 loss in cerebellar Purkinje cells (PCs) can cause altered social behavior in mice. Here, we performed detailed transcriptional and translational analyses of Tsc1-deficient PCs to understand the molecular alterations in these cells. We found that target transcripts of the Fragile X Mental Retardation Protein (FMRP) are reduced in mutant PCs with evidence of increased degradation. Surprisingly, we observed unchanged ribosomal binding for many of these genes using translating ribosome affinity purification. Finally, we found that multiple FMRP targets, including SHANK2, were reduced, suggesting that compensatory increases in ribosomal binding efficiency may be unable to overcome reduced transcript levels. These data further implicate dysfunction of FMRP and its targets in TSC and suggest that treatments aimed at restoring the function of these pathways may be beneficial.
    Keywords:  FMRP; Tuberous Sclerosis; autism; mTOR; mouse; neuroscience; ribosome; translation
  9. Seizure. 2021 Jul 05. pii: S1059-1311(21)00230-2. [Epub ahead of print]91 271-272
    Keywords:  COVID-19; Everolimus; Tuberous sclerosis complex; Vaccination
  10. Neurology. 2021 Jul 15. pii: 10.1212/WNL.0000000000012511. [Epub ahead of print]
      OBJECTIVE: Compare the effectiveness of initial treatment for infantile spasms.METHODS: The National Infantile Spasms Consortium prospectively followed children with new onset infantile spasms that began at age 2-24 months at 23 US centers (2012-2018). Freedom from treatment failure at 60 days required no second treatment for infantile spasms and no clinical spasms after 30 days of treatment initiation. We managed treatment selection bias with propensity score weighting and within-center correlation with generalized estimating equations.
    RESULTS: Freedom from treatment failure rates were: ACTH 88/190 (46%), oral steroids 42/95 (44%), vigabatrin 32/87 (37%), and non-standard therapy 4/51 (8%). Changing from oral steroids to ACTH was not estimated to affect response (observed 44% estimated to change to 44% [95% CI 34-54]). Changing from non-standard therapy to ACTH would improve response from 8% to 39 [17-67]%, and to oral steroids from 8% to 38 [15-68]%. There were large but not statistically significant estimated effects of changing from vigabatrin to ACTH (29% to 42 [15-75]%), vigabatrin to oral steroids (29% to 42 [28-57]%), and non-standard therapy to vigabatrin (8% to 20 [6-50]%). Among children treated with vigabatrin, those with tuberous sclerosis complex (TSC) responded more often than others (62% vs 29%; p<0.05) CONCLUSION: Compared to non-standard therapy, ACTH and oral steroids are superior for initial treatment of infantile spasms. The estimated effectiveness of vigabatrin is between ACTH / oral steroids and non-standard therapy, though the sample was underpowered for statistical confidence. When used, vigabatrin worked best for TSC.
    CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that for children with new onset infantile spasms, ACTH or oral steroids were superior to non-standard therapies.
  11. Mol Cell. 2021 Jul 05. pii: S1097-2765(21)00497-4. [Epub ahead of print]
      Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.
    Keywords:  ECM; GORASP2; GRASP55; Golgi; MMP2; Rapamycin; Tuberous Sclerosis Complex (TSC); cellular stress response; mTORC1; unconventional protein secretion (UPS)
  12. Adv Sci (Weinh). 2021 07;8(13): 2100209
      Phenotypic polarization of macrophages is regulated by a milieu of cues in the local tissue microenvironment. Currently, little is known about how the intrinsic regulators modulate proinflammatory (M1) versus prohealing (M2) macrophages activation. Here, it is observed that insulin-like growth factor 2 messenger RNA (mRNA)-binding protein 2 (IGF2BP2)-deleted macrophages exhibit enhanced M1 phenotype and promote dextran sulfate sodium induced colitis development. However, the IGF2BP2-/- macrophages are refractory to interleukin-4 (IL-4) induced activation and alleviate cockroach extract induced pulmonary allergic inflammation. Molecular studies indicate that IGF2BP2 switches M1 macrophages to M2 activation by targeting tuberous sclerosis 1 via an N6-methyladenosine (m6A)-dependent manner. Additionally, it is also shown a signal transducer and activators of transcription 6 (STAT6)-high mobility group AT-hook 2-IGF2BP2-peroxisome proliferator activated receptor-γ axis involves in M2 macrophages differentiation. These findings highlight a key role of IGF2BP2 in regulation of macrophages activation and imply a potential therapeutic target of macrophages in the inflammatory diseases.
    Keywords:  IGF2BP2; TSC1; inflammatory diseases; m6A reader; macrophage polarization
  13. Anticancer Drugs. 2021 Jul 12.
      Everolimus, an oral mammalian target of rapamycin complex 1 (mTORC1) inhibitor, presents a therapeutic option in metastatic renal cell carcinoma (RCC) patients who were intolerant to, or previously failed, immune- and vascular endothelial growth factor-targeted therapies. However, the onset of drug resistance limits its clinical use. One possible mechanism underpinning the resistance is that inhibiting mTORC1 by everolimus results in mTORC2-dependent activation of v-Akt murine thymoma viral oncogene (AKT) and upregulation of hypoxia-inducible transcription factors (HIF). Norcantharidin (NCTD) is a demethylated derivative of cantharidin with antitumor properties which is an active ingredient of the traditional Chinese medicine Mylabris. In this study, everolimus-resistant RCC cells (786-O-R) obtained by chronic everolimus treatment revealed higher level of HIF2α and over-activated mTORC2 pathway and NCTD inhibits cell proliferation in both everolimus-resistant and -sensitive RCC cells by arresting cell cycle in G0/G1 phase and reducing cell cycle-related proteins of C-Myc and cyclin D. Furthermore, NCTD shows synergistic anticancer effects combined with everolimus in everolimus-resistant 786-O-R cells. Mechanically, NCTD repressed both mTORC1 and mTORC2 signaling pathways as well as downstream molecular signaling pathways, such as p-4EBP1, p-AKT, HIF1α and HIF2α. Our findings provide sound evidence that combination of NCTD and everolimus is a potential therapeutic strategy for treating RCC and overcoming everolimus resistance by dual inhibition of mTORC1 and mTORC2.
  14. Aging Cell. 2021 Jul 12. e13431
      The mechanistic target of rapamycin (mTOR) has gathered significant attention as a ubiquitously expressed multimeric kinase with key implications for cell growth, proliferation, and survival. This kinase forms the central core of two distinct complexes, mTORC1 and mTORC2, which share the ability of integrating environmental, nutritional, and hormonal cues but which regulate separate molecular pathways that result in different cellular responses. Particularly, mTORC1 has been described as a major negative regulator of endosomal biogenesis and autophagy, a catabolic process that degrades intracellular components and organelles within the lysosomes and is thought to play a key role in human health and disease. In contrast, the role of mTORC2 in the regulation of autophagy has been considerably less studied despite mounting evidence this complex may regulate autophagy in a different and perhaps complementary manner to that of mTORC1. Genetic ablation of unique subunits is currently being utilized to study the differential effects of the two mTOR complexes. RICTOR is the best-described subunit specific to mTORC2 and as such has become a useful tool for investigating the specific actions of this complex. The development of complex-specific inhibitors for mTORC2 is also an area of intense interest. Studies to date have demonstrated that mTORC1/2 complexes each signal to a variety of exclusive downstream molecules with distinct biological roles. Pinpointing the particular effects of these downstream effectors is crucial toward the development of novel therapies aimed at accurately modulating autophagy in the context of human aging and disease.
    Keywords:  AKT; FOXOs; SGK-1; autophagy; mTORC2
  15. Nat Commun. 2021 07 09. 12(1): 4227
      Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma.
  16. Cell Death Dis. 2021 Jul 15. 12(7): 710
      Alcohol-related liver disease (ALD), a condition caused by alcohol overconsumption, occurs in three stages of liver injury including steatosis, hepatitis, and cirrhosis. DEP domain-containing protein 5 (DEPDC5), a component of GAP activities towards Rags 1 (GATOR1) complex, is a repressor of amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. In the current study, we found that aberrant activation of mTORC1 was likely attributed to the reduction of DEPDC5 in the livers of ethanol-fed mice or ALD patients. To further define the in vivo role of DEPDC5 in ALD development, we generated Depdc5 hepatocyte-specific knockout mouse model (Depdc5-LKO) in which mTORC1 pathway was constitutively activated through loss of the inhibitory effect of GATOR1. Hepatic Depdc5 ablation leads to mild hepatomegaly and liver injury and protects against diet-induced liver steatosis. In contrast, ethanol-fed Depdc5-LKO mice developed severe hepatic steatosis and inflammation. Pharmacological intervention with Torin 1 suppressed mTORC1 activity and remarkably ameliorated ethanol-induced hepatic steatosis and inflammation in both control and Depdc5-LKO mice. The pathological effect of sustained mTORC1 activity in ALD may be attributed to the suppression of peroxisome proliferator activated receptor α (PPARα), the master regulator of fatty acid oxidation in hepatocytes, because fenofibrate (PPARα agonist) treatment reverses ethanol-induced liver steatosis and inflammation in Depdc5-LKO mice. These findings provide novel insights into the in vivo role of hepatic DEPDC5 in the development of ALD.
  17. J Biol Chem. 2021 Jul 07. pii: S0021-9258(21)00742-0. [Epub ahead of print] 100942
      TBK1 responds to microbes to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) on S2159 to increase mTOR complex 1 (mTORC1) signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. While TBK1 has been linked to Akt phosphorylation, a direct relationship between TBK1 and mTORC2, an Akt kinase, has not been described. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (MtorA/A) using in vitro kinase assays and cell-based approaches, we demonstrate here that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation. We find that TBK1 and mTOR S2159 phosphorylation promote mTOR-dependent phosphorylation of Akt in response to several growth factors and poly(I:C). Mechanistically, TBK1 co-immunoprecipitates with mTORC2 and phosphorylates mTOR S2159 within mTORC2 in cells. Kinase assays demonstrate that TBK1 and mTOR S2159 phosphorylation increase mTORC2 intrinsic catalytic activity. Growth factors failed to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal crosstalk between TBK1 and mTOR, key regulatory nodes within two major signaling networks. As TBK1 and mTOR contribute to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.
    Keywords:  Akt; TBK1; mTOR; mTORC2; phosphorylation
  18. Epilepsia Open. 2021 Jul 16.
      This is a summary report of clinical and regulatory issues discussed at the 2018 NINDS workshop, entitled "Accelerating Therapies for Antiepileptogenesis and Disease Modification". The intent of the workshop was to optimize and accelerate development of therapies for anti-epileptogenesis (AEG) and disease modification in the epilepsies. The working group discussed nomenclature for antiepileptogenic therapies, subdividing them into antiepileptogenic therapies" and "disease modifying therapies", both of which are urgently needed. We use the example of traumatic brain injury to explain issues and complexities in designing a trial for disease preventing antiepileptogenic therapies, including identifying timing of intervention, selecting the appropriate dose, and the need for biomarkers. We discuss the recent trials of vigabatrin to prevent onset and modify epilepsy outcome in children with tuberous sclerosis (Epistop and PreVeNT). We describe a potential approach to a disease modification trial in adults, using patients with Temporal Lobe Epilepsy. Finally, we discuss regulatory hurdles for antiepileptogenesis and disease modifying trials.
  19. Mol Brain. 2021 Jul 12. 14(1): 112
      Memory and long term potentiation require de novo protein synthesis. A key regulator of this process is mTORC1, a complex comprising the mTOR kinase. Growth factors activate mTORC1 via a pathway involving PI3-kinase, Akt, the TSC complex and the GTPase Rheb. In non-neuronal cells, translocation of mTORC1 to late endocytic compartments (LEs), where Rheb is enriched, is triggered by amino acids. However, the regulation of mTORC1 in neurons remains unclear. In mouse hippocampal neurons, we observed that BDNF and treatments activating NMDA receptors trigger a robust increase in mTORC1 activity. NMDA receptors activation induced a significant recruitment of mTOR onto lysosomes even in the absence of external amino acids, whereas mTORC1 was evenly distributed in neurons under resting conditions. NMDA receptor-induced mTOR translocation to LEs was partly dependent on the BDNF receptor TrkB, suggesting that BDNF contributes to the effect of NMDA receptors on mTORC1 translocation. In addition, the combination of Rheb overexpression and artificial mTORC1 targeting to LEs by means of a modified component of mTORC1 fused with a LE-targeting motif strongly activated mTOR. To gain spatial and temporal control over mTOR localization, we designed an optogenetic module based on light-sensitive dimerizers able to recruit mTOR on LEs. In cells expressing this optogenetic tool, mTOR was translocated to LEs upon photoactivation. In the absence of growth factor, this was not sufficient to activate mTORC1. In contrast, mTORC1 was potently activated by a combination of BDNF and photoactivation. The data demonstrate that two important triggers of synaptic plasticity, BDNF and NMDA receptors, synergistically power the two arms of the mTORC1 activation mechanism, i.e., mTORC1 translocation to LEs and Rheb activation. Moreover, they unmask a functional link between NMDA receptors and mTORC1 that could underlie the changes in the synaptic proteome associated with long-lasting changes in synaptic strength.
    Keywords:  BDNF; Endo-lysosomes; NMDA receptors; Optogenetics; Synaptic plasticity; mTOR