bims-tubesc 2022-01-02 papers

bims-tubesc

Biomed News

on Molecular mechanisms in tuberous sclerosis

Issue of 2022–01–02
ten papers selected by

[Analysis of a patient with tuberous sclerosis complex due to mosaicism TSC2 mutation].
Classical neurocutaneous manifestations of tuberous sclerosis complex.
Adults with tuberous sclerosis complex: A distinct patient population.
Dorsal telencephalon-specific Nprl2- and Nprl3-knockout mice: novel mouse models for GATORopathy.
Long-term cannabidiol treatment for seizures in patients with tuberous sclerosis complex: An open-label extension trial.
Simultaneous loss of TSC1 and DEPDC5 in skeletal and cardiac muscles produces early-onset myopathy and cardiac dysfunction associated with oxidative damage and SQSTM1/p62 accumulation.
GPCR Signaling and mTORC1 Regulation.
A Highly Sensitive Fluorescent Akt Biosensor Reveals Lysosome-Selective Regulation of Lipid Second Messengers and Kinase Activity.
Autophagy and lysosomal defects in cells expressing disease-associated tau.
The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride.

Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2022 Jan 10. 39(1): 68-71

[Analysis of a patient with tuberous sclerosis complex due to mosaicism TSC2 mutation].

Weiliang Liu, Fang Li, Zhixu He, Rong Ai.

OBJECTIVE: To analyze variants of TSC1 and TSC2 genes in a Chinese patient with tuberous sclerosis complex (TSC).
METHODS: Peripheral blood samples were collected from the patient and her parents with informed consent. Following extraction of genomic DNA, potential variants of the TSC1 and TSC2 genes was detected by using targeted capture next-generation sequencing (NGS) and Sanger sequencing.
RESULTS: The patient was found to harbor a de novo mosaicism variant c.3295_3298delG (Val1100CysfsTer3) of the TSC2 gene, with the proportion of the mutant allele determined as 13.4%, which was confirmed by Sanger sequencing. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.3295_3298delG (Val1100CysfsTer3) variant was predicted to be pathogenic (PVS1+PS2+PM2).
CONCLUSION: The mosaicism heterozygous variant of c.3295_3298delG of the TSC2 gene, as detected by both NGS and Sanger sequencing, probably underlay the TSC2 in this patient.

DOI: https://doi.org/10.3760/cma.j.cn511374-20201013-00716
QJM. 2021 Dec 28. pii: hcab332. [Epub ahead of print]

Classical neurocutaneous manifestations of tuberous sclerosis complex.

Uddalak Chakraborty, Kaushiki Hajra, Atanu Chandra.

DOI: https://doi.org/10.1093/qjmed/hcab332
Epilepsia. 2021 Dec 29.

Adults with tuberous sclerosis complex: A distinct patient population.

Kenan Bachour, Andrew A House, Danielle M Andrade, Mary Connolly, Dereck B Debicki, Richard Desbiens, Tadeu A Fantaneanu, Paulina Kyriakopoulos, Jean-Baptiste Lattouf, Ana Suller-Marti, Paula T Marques, Mark R Keezer.

   OBJECTIVES: There are few data on adults living with tuberous sclerosis complex (TSC), with most studies focusing on pediatric populations. The objective of our study was to examine a large national cohort of adults with TSC, and to describe the clinical characteristics of these adults and the nature of the multidisciplinary care that they receive.
METHODS: Six Canadian medical centers collaborated in this study. Data were collected using a standardized form, and descriptive statistics were used for the analyses.
RESULTS: Our study included 181 adults with definite TSC (mean age = 33.6 years [SD = 13.7]). More than 40% (n = 75) had family members affected by TSC. Forty-six percent (n = 83) of individuals had intellectual disability. Nearly 30% (n = 52) of individuals reported living alone or with a partner/spouse. Seventy-six percent (n = 138) of people had epilepsy, 43% (n = 59) of whom had drug-resistant epilepsy, and 21% (n = 29) had undergone epilepsy surgery. Neuropsychiatric disease (n = 128) and renal angiomyolipomas (n = 130) were both present in approximately 70% of people. Renal imaging was performed in 75.7% (n = 137) of participants within the past 3 years. Renal and pulmonary function tests, as well as electrocardiograms, were recently performed in a minority of individuals.
SIGNIFICANCE: Our cohort of adults with TSC showed that an important proportion have a milder phenotype, and are more frequently familial, as compared to children with TSC (and differing from prior reports in adult cohorts). Drug-resistant epilepsy, neuropsychiatric comorbidities, and renal angiomyolipoma are challenging factors in adults with TSC. Our participating medical centers generally followed recommended screening strategies, but there remain important gaps in care. Multidisciplinary and structured TSC care centers offering service to adults may help to improve the health of this important patient population.

Keywords:  epilepsy; health services research; neurocutaneous syndrome; seizures

DOI:  https://doi.org/10.1111/epi.17159
Hum Mol Genet. 2021 Nov 20. pii: ddab337. [Epub ahead of print]

Dorsal telencephalon-specific Nprl2- and Nprl3-knockout mice: novel mouse models for GATORopathy.

Saeko Ishida, Di Zhao, Yuta Sawada, Yuichi Hiraoka, Tomoji Mashimo, Kohichi Tanaka.

The most frequent genetic cause of focal epilepsies is variations in the GAP activity toward RAGs 1 complex genes DEP domain containing 5 (DEPDC5), nitrogen permease regulator 2-like protein (NPRL2) and nitrogen permease regulator 3-like protein (NPRL3). Because these variations are frequent and associated with a broad spectrum of focal epilepsies, a unique pathology categorized as GATORopathy can be conceptualized. Animal models recapitulating the clinical features of patients are essential to decipher GATORopathy. Although several genetically modified animal models recapitulate DEPDC5-related epilepsy, no models have been reported for NPRL2- or NPRL3-related epilepsies. Here, we conditionally deleted Nprl2 and Nprl3 from the dorsal telencephalon in mice [Emx1cre/+; Nprl2f/f (Nprl2-cKO) and Emx1cre/+; Nprl3f/f (Nprl3-cKO)] and compared their phenotypes with Nprl2+/-, Nprl3+/- and Emx1cre/+; Depdc5f/f (Depdc5-cKO) mice. Nprl2-cKO and Nprl3-cKO mice recapitulated the major abnormal features of patients-spontaneous seizures, and dysmorphic enlarged neuronal cells with increased mechanistic target of rapamycin complex 1 signaling-similar to Depdc5-cKO mice. Chronic postnatal rapamycin administration dramatically prolonged the survival period and inhibited seizure occurrence but not enlarged neuronal cells in Nprl2-cKO and Nprl3-cKO mice. However, the benefit of rapamycin after withdrawal was less durable in Nprl2- and Nprl3-cKO mice compared with Depdc5-cKO mice. Further studies using these conditional knockout mice will be useful for understanding GATORopathy and for the identification of novel therapeutic targets.

DOI: https://doi.org/10.1093/hmg/ddab337
Epilepsia. 2021 Dec 27.

Long-term cannabidiol treatment for seizures in patients with tuberous sclerosis complex: An open-label extension trial.

Elizabeth A Thiele, E Martina Bebin, Francis Filloux, Patrick Kwan, Rachael Loftus, Farhad Sahebkar, Steven Sparagana, James Wheless.

   OBJECTIVE: To evaluate the long-term safety and efficacy of add-on cannabidiol (CBD) in patients with seizures associated with tuberous sclerosis complex (TSC) in the open-label extension (OLE) of the randomized, placebo-controlled phase 3 trial GWPCARE6 (NCT02544763). Results of an interim (February 2019 data cut) analysis are reported.
METHODS: Patients who completed the randomized trial enrolled to receive CBD (Epidiolex® in the United States; Epidyolex® in the EU; 100 mg/mL oral solution). The initial target dose was 25 mg/kg/day, which, based on response and tolerability, could be decreased or increased up to 50 mg/kg/day. The primary end point was safety. Key secondary end points included percentage reduction in TSC-associated (countable focal and generalized) seizures, responder rates, and Subject/Caregiver Global Impression of Change (S/CGIC).
RESULTS: Of 201 patients who completed the randomized phase, 199 (99%) entered the OLE. Mean age was 13 years (range, 1-57). At the time of analysis, 5% of patients had completed treatment, 20% had withdrawn, and 75% were ongoing. One-year retention rate was 79%. Median treatment time was 267 days (range, 18-910) at a 27 mg/kg/day mean modal dose. Most patients (92%) had an adverse event (AE). Most common AEs were diarrhea (42%), seizure (22%), and decreased appetite (20%). AEs led to permanent discontinuation in 6% of patients. There was one death that was deemed treatment unrelated by the investigator. Elevated liver transaminases occurred in 17 patients (9%) patients; 12 were taking valproate. Median percentage reductions in seizure frequency (12-week windows across 48 weeks) were 54%-68%. Seizure responder rates (≥50%, ≥75%, 100% reduction) were 53%-61%, 29%-45%, and 6%-11% across 12-week windows for 48 weeks. Improvement on the S/CGIC scale was reported by 87% of patients/caregivers at 26 weeks.
SIGNIFICANCE: In patients with TSC, long-term add-on CBD treatment was well tolerated and sustainably reduced seizures through 48 weeks, with most patients/caregivers reporting global improvement.

Keywords:  antiseizure medication; cannabidiol; epilepsy; focal seizures; treatment-resistant epilepsy; tuberous sclerosis complex

DOI:  https://doi.org/10.1111/epi.17150
Autophagy. 2021 Dec 29. 1-20

Simultaneous loss of TSC1 and DEPDC5 in skeletal and cardiac muscles produces early-onset myopathy and cardiac dysfunction associated with oxidative damage and SQSTM1/p62 accumulation.

Chun-Seok Cho, Yongsung Kim, Sung-Rye Park, Boyoung Kim, Carol Davis, Irene Hwang, Susan V Brooks, Jun Hee Lee, Myungjin Kim.

  By promoting anabolism, MTORC1 is critical for muscle growth and maintenance. However, genetic MTORC1 upregulation promotes muscle aging and produces age-associated myopathy. Whether MTORC1 activation is sufficient to produce myopathy or indirectly promotes it by accelerating tissue aging is elusive. Here we examined the effects of muscular MTORC1 hyperactivation, produced by simultaneous depletion of TSC1 and DEPDC5 (CKM-TD). CKM-TD mice produced myopathy, associated with loss of skeletal muscle mass and force, as well as cardiac failure and bradypnea. These pathologies were manifested at eight weeks of age, leading to a highly penetrant fatality at around twelve weeks of age. Transcriptome analysis indicated that genes mediating proteasomal and macroautophagic/autophagic pathways were highly upregulated in CKM-TD skeletal muscle, in addition to inflammation, oxidative stress, and DNA damage signaling pathways. In CKM-TD muscle, autophagosome levels were increased, and the AMPK and ULK1 pathways were activated; in addition, autophagy induction was not completely blocked in CKM-TD myotubes. Despite the upregulation of autolysosomal markers, CKM-TD myofibers exhibited accumulation of autophagy substrates, such as SQSTM1/p62 and ubiquitinated proteins, suggesting that the autophagic activities were insufficient. Administration of a superoxide scavenger, tempol, normalized most of these molecular pathologies and subsequently restored muscle histology and force generation. However, CKM-TD autophagy alterations were not normalized by rapamycin or tempol, suggesting that they may involve non-canonical targets other than MTORC1. These results collectively indicate that the concomitant muscle deficiency of TSC1 and DEPDC5 can produce early-onset myopathy through accumulation of oxidative stress, which dysregulates myocellular homeostasis.Abbreviations: AMPK: AMP-activated protein kinase; CKM: creatine kinase, M-type; COX: cytochrome oxidase; DEPDC5: DEP domain containing 5, GATOR1 subcomplex subunit; DHE: dihydroethidium; EDL: extensor digitorum longus; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GAP: GTPase-activating protein; GTN: gastrocnemius; MTORC1: mechanistic target of rapamycin kinase complex 1; PLA: plantaris; QUAD: quadriceps; RPS6KB/S6K: ribosomal protein S6 kinase beta; SDH: succinate dehydrogenase; SOL: soleus; SQSTM1: sequestosome 1; TA: tibialis anterior; TSC1: TSC complex subunit 1; ULK1: unc-51 like autophagy activating kinase 1.

Keywords:  MTORC1; ULK1; myopathy; oxidative stress; tempol

DOI:  https://doi.org/10.1080/15548627.2021.2016255
Mol Pharmacol. 2021 Dec 28. pii: MOLPHARM-MR-2021-000302. [Epub ahead of print]

GPCR Signaling and mTORC1 Regulation.

Chase H Melick, Tshering D Lama-Sherpa, Adna Curukovic, Jenna L Jewell.

  The mammalian target of rapamycin (mTOR) senses upstream stimuli to regulate numerous cellular functions such as metabolism, growth, and autophagy. The activation of mTOR complex 1 (mTORC1) is typically observed in human disease and continues to be an important therapeutic target. Understanding the upstream regulators of mTORC1 will provide a crucial link to targeting mTORC1 hyperactivated diseases. In this review, we will discuss the regulation of mTORC1 by upstream stimuli, with a specific focus on G-protein coupled receptor (GPCR) signaling to mTORC1. Significance Statement mTORC1 is a master regulator of many cellular processes and is often hyperactivated in human disease. Therefore, understanding the molecular underpinnings of these pathways will undoubtedly be promising to the mTORC1 field and human disease.

Keywords:  G protein coupled signaling; G proteins; Mammalian target of rapamycin (mTOR); Protein Kinase A (PKA); g protein-coupled receptors (GPCRS)

DOI:  https://doi.org/10.1124/molpharm.121.000302
ACS Cent Sci. 2021 Dec 22. 7(12): 2009-2020

A Highly Sensitive Fluorescent Akt Biosensor Reveals Lysosome-Selective Regulation of Lipid Second Messengers and Kinase Activity.

Mingyuan Chen, Tengqian Sun, Yanghao Zhong, Xin Zhou, Jin Zhang.

The serine/threonine protein kinase Akt regulates a wide range of cellular functions via phosphorylation of various substrates distributed throughout the cell, including at the plasma membrane and endomembrane compartments. Disruption of compartmentalized Akt signaling underlies the pathology of many diseases such as cancer and diabetes. However, the specific spatial organization of Akt activity and the underlying regulatory mechanisms, particularly the mechanism controlling its activity at the lysosome, are not clearly understood. We developed a highly sensitive excitation-ratiometric Akt activity reporter (ExRai-AktAR2), enabling the capture of minute changes in Akt activity dynamics at subcellular compartments. In conjunction with super-resolution expansion microscopy, we found that growth factor stimulation leads to increased colocalization of Akt with lysosomes and accumulation of lysosomal Akt activity. We further showed that 3-phosphoinositides (3-PIs) accumulate on the lysosomal surface, in a manner dependent on dynamin-mediated endocytosis. Importantly, lysosomal 3-PIs are needed for growth-factor-induced activities of Akt and mechanistic target of rapamycin complex 1 (mTORC1) on the lysosomal surface, as targeted depletion of 3-PIs has detrimental effects. Thus, 3-PIs, a class of critical lipid second messengers that are typically found in the plasma membrane, unexpectedly accumulate on the lysosomal membrane in response to growth factor stimulation, to direct the multifaceted kinase Akt to organize lysosome-specific signaling.

DOI: https://doi.org/10.1021/acscentsci.1c00919
Alzheimers Dement. 2021 Dec;17 Suppl 2 e058299

Autophagy and lysosomal defects in cells expressing disease-associated tau.

Saskia J Pollack, Dina Dakkak, Tong Guo, Patricia Gómez-Suaga, Wendy Noble, Diane P Hanger.

BACKGROUND: Intracellular accumulation of insoluble tau is an important hallmark of Alzheimer's disease (AD) and related tauopathies. We have previously identified in human tauopathy brain a truncated tau species (Tau35), comprising the C-terminal half with four microtubule-binding repeats. Minimal Tau35 expression in transgenic mice results in a progressive tauopathy phenotype including tau phosphorylation and aggregation, cognitive and behavioural abnormalities and impaired protein clearance. The autophagy-lysosomal pathway (ALP) plays a crucial role in the clearance of protein aggregates and defects in ALP are associated with the pathogenesis of AD. We sought to explore the effect of Tau35 expression on the ALP and whether autophagy is disrupted due to lysosomal dysfunction.
METHOD: Chinese hamster ovary (CHO) cells stably expressing Tau35 (CHO-Tau35) or full-length human 2N4R tau (CHO-FL) were generated. Primary cortical neurons from Tau35 transgenic and wild-type mice were cultured for 14 days in vitro(DIV) and brain homogenates were prepared from mice aged 4 and 12 months. The effect of Tau35 on the ALP was examined using immunofluorescence and western blots.
RESULT: Nuclear translocation of transcription factor EB (TFEB), a key mediator of lysosomal biogenesis, was significantly reduced in both CHO-FL and CHO-Tau35 cells. However, only CHO-Tau35 cells exhibited disrupted mammalian target of rapamycin complex 1 (mTORC1) activity and autophagic flux. Expression of ALP markers, including LC3-I/II, LAMP1, LAMP2 and cathepsin D, were also reduced in CHO-Tau35 cells.
CONCLUSION: Our findings suggest that N-terminally cleaved tau damages both lysosomal clearance of cellular proteins and lysosomal biogenesis. The Tau35-expressing cultured neurons will provide a useful tool to explore molecular mechanisms underlying tau-induced lysosomal dysfunction, which may lead to the identification of novel therapeutic targets for dementia.

DOI: https://doi.org/10.1002/alz.058299
PLoS One. 2021 ;16(12): e0262180

The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride.

Rossana Segreto, Hoda Bazafkan, Julia Millinger, Martina Schenk, Lea Atanasova, Maria Doppler, Christoph Büschl, Mélanie Boeckstaens, Silvia Soto Diaz, Ulrike Schreiner, Fabiano Sillo, Raffaella Balestrini, Rainer Schuhmacher, Susanne Zeilinger.

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

DOI: https://doi.org/10.1371/journal.pone.0262180