bims-tubesc 2021-06-13 papers

bims-tubesc

Biomed News

on Molecular mechanisms in tuberous sclerosis

Issue of 2021–06–13
eleven papers selected by

αB-crystallin/HSPB2 is critical for hyperactive mTOR-induced cardiomyopathy.
A bistable, multiport valve enables microformulators creating microclinical analyzers that reveal aberrant glutamate metabolism in astrocytes derived from a tuberous sclerosis patient.
An interdomain hydrogen bond in the Rag GTPases maintains stable mTORC1 signaling in sensing amino acids.
Sirolimus in the treatment of kaposiform lymphangiomatosis.
[A case of tuberous sclerosis complex due to a novel splicing variant of TSC2 gene].
Electroclinical markers to differentiate between focal and generalized epilepsies.
A patent review of mTOR inhibitors for cancer therapy (2011-2020).
The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II.
Altered serum levels of autophagy proteins Beclin-1 and mTOR in patients with exudative age-related macular degeneration.
Mutational Landscape of PI3K-AKT-mTOR Pathway in Breast Cancer: Implications for Targeted Therapeutics.
IGF1-Stimulated Posttraumatic Hippocampal Remodeling Is Not Dependent on mTOR.

J Cell Physiol. 2021 Jun 08.

αB-crystallin/HSPB2 is critical for hyperactive mTOR-induced cardiomyopathy.

Lianmei Wang, Fang Wang, Kemei Liu, Caifeng Long, Yi Chen, Chunjia Li, Li Li, Fangming Liu, Xinyu Zhang, Yanling Jing, Yanan Wang, Aihua Liang, Hongbing Yan, Hongbing Zhang.

  Even though aberrant mechanistic target of rapamycin (mTOR) signaling is known to cause cardiomyopathy, its underlying mechanism remains poorly understood. Because augmentation of αB-crystallin and hspB2 was presented in the cortical tubers and lymphangioleiomyomatosis of tuberous sclerosis complex patients, we deciphered the role of αB-crystallin and its adjacent duplicate gene, hspB2, in hyperactive mTOR-induced cardiomyopathy. Cardiac Tsc1 deletion (T1-hKO) caused mouse mTOR activation and cardiomyopathy. Overexpression of αB-crystallin and hspB2 was presented in the hearts of these mice. Knockout of αB-crystallin/hspB2 reversed deficient Tsc1-mediated fetal gene expression, mTOR activation, mitochondrial damage, cardiomyocyte vacuolar degeneration, cardiomyocyte size, and fibrosis of T1-hKO mice. These cardiac-Tsc1; αB-crystallin; hspB2 triple knockout (tKO) mice had improved cardiac function, smaller heart weight to body weight ratio, and reduced lethality compared with T1-hKO mice. Even though activated mTOR suppressed autophagy in T1-hKO mice, ablation of αB-crystallin and hspB2 failed to restore autophagy in tKO mice. mTOR inhibitors suppressed αB-crystallin expression in T1-hKO mice and rat cardiomyocyte line H9C2. Starvation of H9C2 cells activated autophagy and suppressed αB-crystallin expression. Since inhibition of autophagy restored αB-crystallin expression in starved H9C2 cells, autophagy is a negative regulator of αB-crystallin expression. mTOR thus stimulates αB-crystallin expression through suppression of autophagy. In conclusion, αB-crystallin and hspB2 play a pivotal role in Tsc1 knockout-related cardiomyopathy and are therapeutic targets of hyperactive mTOR-associated cardiomyopathy.

Keywords:  TSC1; cardiomyopathy; hspB2; mTOR; αB-crystallin

DOI:  https://doi.org/10.1002/jcp.30465
Sens Actuators B Chem. 2021 Aug 15. pii: 129972. [Epub ahead of print]341

A bistable, multiport valve enables microformulators creating microclinical analyzers that reveal aberrant glutamate metabolism in astrocytes derived from a tuberous sclerosis patient.

Dusty R Miller, David K Schaffer, M Diana Neely, Ethan S McClain, Adam R Travis, Frank E Block, Jennifer Mckenzie, Erik M Werner, Laura Armstrong, Dmitry A Markov, Aaron B Bowman, Kevin C Ess, David E Cliffel, John P Wikswo.

  There is a need for valves and pumps that operate at the microscale with precision and accuracy, are versatile in their application, and are easily fabricated. To that end, we developed a new rotary planar multiport valve to faithfully select solutions (contamination = 5.22 ± 0.06 ppb) and a rotary planar peristaltic pump to precisely control fluid delivery (flow rate = 2.4 ± 1.7 to 890 ± 77 μL/min). Both the valve and pump were implemented in a planar format amenable to single-layer soft lithographic fabrication. These planar microfluidics were actuated by a rotary motor controlled remotely by custom software. Together, these two devices constitute an innovative microformulator that was used to prepare precise, high-fidelity mixtures of up to five solutions (deviation from prescribed mixture = ±|0.02 ± 0.02| %). This system weighed less than a kilogram, occupied around 500 cm3, and generated pressures of 255 ± 47 kPa. This microformulator was then combined with an electrochemical sensor creating a microclinical analyzer (μCA) for detecting glutamate in real time. Using the chamber of the μCA as an in-line bioreactor, we compared glutamate homeostasis in human astrocytes differentiated from human-induced pluripotent stem cells (hiPSCs) from a control subject (CC-3) and a Tuberous Sclerosis Complex (TSC) patient carrying a pathogenic TSC2 mutation. When challenged with glutamate, TSC astrocytes took up less glutamate than control cells. These data validate the analytical power of the μCA and the utility of the microformulator by leveraging it to assess disease-related alterations in cellular homeostasis.

Keywords:  Tuberous sclerosis complex (TSC); electrochemistry; microclinical analyzer; soft lithography; time-division multiplexing

DOI:  https://doi.org/10.1016/j.snb.2021.129972
J Biol Chem. 2021 Jun 08. pii: S0021-9258(21)00661-X. [Epub ahead of print] 100861

An interdomain hydrogen bond in the Rag GTPases maintains stable mTORC1 signaling in sensing amino acids.

Shawn B Egri, Kuang Shen.

  Cellular growth and proliferation are primarily dictated by the mechanistic target of rapamycin complex 1 (mTORC1), which balances nutrient availability against the cell's anabolic needs. Central to the activity of mTORC1 is the RagA-RagC GTPase heterodimer, which under favorable conditions recruits the complex to the lysosomal surface to promote its activity. The RagA-RagC heterodimer has a unique architecture in that both subunits are active GTPases. To promote mTORC1 activity, the RagA subunit is loaded with GTP and the RagC subunit is loaded with GDP, while the opposite nucleotide loading configuration inhibits this signaling pathway. Despite its unique molecular architecture, how the Rag GTPase heterodimer maintains the oppositely loaded nucleotide state remains elusive. Here, we applied structure-function analysis approach to the crystal structures of the Rag GTPase heterodimer, and identified a key hydrogen bond that stabilizes the GDP-loaded state of the Rag GTPases. This hydrogen bond is mediated by the backbone carbonyl of Asn30 in the nucleotide binding domain (NBD) of RagA or Lys84 of RagC, and the hydroxyl group on the side chain of Thr210 in the C-terminal roadblock domain (CRD) of RagA or Ser266 of RagC, respectively. Eliminating this interdomain hydrogen bond abolishes the ability of the Rag GTPase to maintain its functional state, resulting in a distorted response to amino acid signals. Our results reveal that this long-distance interdomain interaction within the Rag GTPase is required for the maintenance and regulation of the mTORC1 nutrient-sensing pathway.

Keywords:  Rag GTPase; amino acid; enzyme mechanism; hydrogen bond; mTOR complex 1 (mTORC1); nutrient sensing

DOI:  https://doi.org/10.1016/j.jbc.2021.100861
Orphanet J Rare Dis. 2021 Jun 08. 16(1): 260

Sirolimus in the treatment of kaposiform lymphangiomatosis.

Jiangyuan Zhou, Kaiying Yang, Siyuan Chen, Yi Ji.

   BACKGROUND: Kaposiform lymphangiomatosis (KLA), which is a new subtype of generalized lymphatic anomaly, is a rare disease with a poor prognosis. Currently, there is no standard treatment due to the poor understanding of KLA. Sirolimus, which is an inhibitor of mammalian target of rapamycin, has been shown to have promising potential in the treatment of complicated vascular anomalies. The aim of this study was to introduce the use of sirolimus for the treatment of KLA and to highlight the challenges of managing this refractory disease.
RESULTS: We reported seven patients with KLA who received sirolimus therapy in our center. Combined with previously reported cases, 58.3% achieved a partial response, 25.0% had stable disease, and 16.7% experienced disease progression. No severe sirolimus-related adverse events occurred during treatment.
CONCLUSIONS: This study suggests that sirolimus is currently an option for the treatment of KLA, and it is hoped that more specific therapies will be developed in the future. Rapid advances in basic science and clinical practice may facilitate the development of important new treatments for KLA.

Keywords:  Kaposiform lymphangiomatosis; Lymphatic malformation; Mammalian target of rapamycin; Sirolimus; Vascular anomaly

DOI:  https://doi.org/10.1186/s13023-021-01893-3
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2021 Jun 10. 38(6): 553-556

[A case of tuberous sclerosis complex due to a novel splicing variant of TSC2 gene].

Yuping Niu, Sexin Huang, Peiwen Xu, Jie Li, Ming Gao, Xiaowei Chen, Hongxia Chu, Yuan Gao.

OBJECTIVE: To explore the genetic basis for a patient with tuberous sclerosis complex.
METHODS: Genomic DNA was extracted from peripheral blood samples from members of his family and 100 unrelated healthy controls. The proband was subjected to next-generation sequencing, and candidate variant was confirmed by multiple ligation-dependent probe amplification (MLPA) and Sanger sequencing. Reverse transcription-PCR (RT-PCR) was carried out to determine the relative mRNA expression in the proband.
RESULTS: The patient was found to harbor a c.2355+1G>C splicing variant of the TSC2 gene. Sequencing of cDNA confirmed that 62 bases have been inserted into the 3' end of exon 21, which has caused a frameshift producing a truncated protein.
CONCLUSION: The novel splicing variant c.2355+1G>C of the TSC2 gene probably underlay the TSC in the proband. Above finding has expanded the variant spectrum of TSC2 and provided a basis for preimplantation genetic testing and/or prenatal diagnosis.

DOI: https://doi.org/10.3760/cma.j.cn511374-20200211-00072
Epileptic Disord. 2021 Jun 09.

Electroclinical markers to differentiate between focal and generalized epilepsies.

Sharika Raga, Sylvain Rheims, Nicola Specchio, Jo M Wilmshurst.

  Generalized-onset seizures originate at a point within the brain and rapidly engage bilaterally distributed networks, whilst focal-onset seizures originate within networks limited to one hemisphere which may be discretely localized or more widely distributed. Challenges in data capturing have resulted in a lack of consistency across incidence and prevalence studies for estimates of generalized versus focal epilepsies in population groups, with a preponderance of unclassified cases. Infants and young children can further challenge delineation due to variable expression at different stages of brain maturation and impaired ability to express sensory seizure semiology. Focal seizures are divided into those in which the affected person is aware and those with impaired awareness; these events can be motor or non-motor. Generalized seizures are also categorized as motor and non-motor, but loss of awareness occurs with most events. Delineation between generalized and focal epilepsies is important for ongoing management. EEG, neuroimaging and other investigations (genetics, immune markers, etc.) can support the outcome but distinction between focal and generalized epilepsy in isolation is rarely achieved using such techniques. Certain clinical settings such as age, underlying aetiologies, and co-morbidities may be more predisposed to specific types of epilepsy. The differentiation between generalized and focal seizures remains a clinical process based on meticulous clinical history and examination, and is of relevance for the selection of antiseizure medications and identification of patients who may be viable for epilepsy surgery.

Keywords:  electroclinical markers; focal-onset seizures; generalized-onset seizures

DOI:  https://doi.org/10.1684/epd.2021.1291
Expert Opin Ther Pat. 2021 Jun 08.

A patent review of mTOR inhibitors for cancer therapy (2011-2020).

Han-Yue Qiu, Peng-Fei Wang, Min Zhang.

   INTRODUCTION: The mammalian target of rapamycin (mTOR) kinase is a central component in the PI3K/Akt/mTOR pathway and plays a crucial role in tumor biology, making it one appealing therapeutic target. In the past decade, the mTORi (mTOR inhibitor) development field has made great progress, with more agents entering key trials and the proposal of third-generation mTORi concept. Yet to achieve significant clinical success, combined efforts from multiple disciplines are ever needed.
AREAS COVERED: This review focuses on the progress of mTORi development with anticancer potential from the perspective of the patent literature proposed between 2011 to 2020.
EXPERT OPINION: The highly complex regulatory mechanism network of mTOR proposes huge challenges to the development of clinically efficient mTORis. While in-depth biological research and fundamental medchemistry research are of importance to provide guidelines for improving mTORis, new technologies to pre-diagnose applicable populations is another key to provide precise personal cancer treatment. New mTOR agents are ever needed to tackle the common problems of side effects and drug resistance.

Keywords:  cancer treatment; mTOR inhibitors; mTOR/PI3K dualrapalink; rapalogsATP-competitive

DOI:  https://doi.org/10.1080/13543776.2021.1940137
Nat Commun. 2021 06 07. 12(1): 3333

The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II.

Takeshi Nakatani, Kohei Tsujimoto, JeongHoon Park, Tatsunori Jo, Tetsuya Kimura, Yoshitomo Hayama, Hachiro Konaka, Takayoshi Morita, Yasuhiro Kato, Masayuki Nishide, Shyohei Koyama, Shigeyuki Nada, Masato Okada, Hyota Takamatsu, Atsushi Kumanogoh.

Lysosomes are involved in nutrient sensing via the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 is tethered to lysosomes by the Ragulator complex, a heteropentamer in which Lamtor1 wraps around Lamtor2-5. Although the Ragulator complex is required for cell migration, the mechanisms by which it participates in cell motility remain unknown. Here, we show that lysosomes move to the uropod in motile cells, providing the platform where Lamtor1 interacts with the myosin phosphatase Rho-interacting protein (MPRIP) independently of mTORC1 and interferes with the interaction between MPRIP and MYPT1, a subunit of myosin light chain phosphatase (MLCP), thereby increasing myosin II-mediated actomyosin contraction. Additionally, formation of the complete Ragulator complex is required for leukocyte migration and pathophysiological immune responses. Together, our findings demonstrate that the lysosomal Ragulator complex plays an essential role in leukocyte migration by activating myosin II through interacting with MPRIP.

DOI: https://doi.org/10.1038/s41467-021-23654-3
J Physiol Pharmacol. 2021 Feb;72(1):

Altered serum levels of autophagy proteins Beclin-1 and mTOR in patients with exudative age-related macular degeneration.

A Kubicka-Trzaska, K Zuber-Laskawiec, H Plutecka, B Romanowska-Dixon, M Sanak, I Karska-Basta.

Autophagy is a key process in the maintenance of cellular survival and homeostasis. Inhibition of autophagy results in degenerative changes resembling ageing. We wondered if autophagy can contribute to the pathogenesis of age-related macular degeneration (AMD). We aimed to investigate the serum concentrations of two key autophagy regulators, Beclin-1 and mechanistic target of rapamycin (mTOR), in patients with exudative AMD. This retrospective case-control study included 38 patients with exudative AMD and 36 sex- and age-matched controls selected among senile cataract patients. Circulating Beclin-1 and mTOR were assessed using an enzyme-linked immunosorbent assay. The proteins levels were correlated with age, sex, duration of ocular symptoms, as well as angiographic and optical coherence tomography findings. Serum Beclin-1 levels were much lower in patients with AMD than in controls (median, 0.100 ng/ml versus 1.123 ng/ml; p = 0.0033), while mTOR levels did not differ (median, 4.377 ng/ml versus 3.608 ng/ml; p = 0.4522). Participants of the study older than 70 years had lower Beclin-1 levels than younger ones (p = 0.0444). However, this difference was the most evident in patients with AMD (p = 0.0024). Serum mTOR levels increased with age. In patients with AMD, lower mTOR levels were associated with drusen, while higher levels were observed in those with a fibrous scar in the contralateral eye (p = 0.0212). Our findings suggest that circulating Beclin-1 decreases with age and that is downregulated in patients with AMD.

DOI: https://doi.org/10.26402/jpp.2021.1.09
J Cancer. 2021 ;12(14): 4408-4417

Mutational Landscape of PI3K-AKT-mTOR Pathway in Breast Cancer: Implications for Targeted Therapeutics.

Weikai Xiao, Guochun Zhang, Bo Chen, Xiaoqing Chen, Lingzhu Wen, Jianguo Lai, Xuerui Li, Min Li, Hao Liu, Jing Liu, Han Han-Zhang, Analyn Lizaso, Ning Liao.

  Background: Comprehensive analysis of PI3K-AKT-mTOR pathway gene alterations in breast cancer may be helpful for targeted therapy. Methods: We performed targeted sequencing using a panel of 520 cancer-related genes to investigate gene alterations in the PI3K-AKT-mTOR pathway from 589 consecutive Chinese women diagnosed with stage I-III breast cancer. Analyses of overall survival (OS) were performed using the publicly available clinical and genomic data from METABRIC. Results: PI3K-AKT-mTOR pathway gene alterations were detected in 62.6% (369/589) of our cohort. The most commonly altered genes were PIK3CA (45%), PTEN (7.5%), AKT1 (5.9 %), PIK3R1 (2.7%), and PIK3CG (2%). Four PIK3CA mutations (E545K, H1047R, E542K, and H1047L) were detected in all the breast cancer molecular subtypes. Seven PIK3CA mutations (E545G, E418_L422delinsV, E726K, E110del, G1049R, G118D, and D350G) were only detected in HR+ subtypes. Two PIK3CA mutations (C420R and N345K) were only detected in non-triple-negative subtypes. Most cases with PTEN mutation were HR+/HER2- subtype (77.3%), followed by triple-negative subtype (18.2%). In the METABRIC breast cancer dataset, no significant OS difference was observed between the PIK3CA-mutant and wild-type groups. However, patients with multiple PIK3CA mutations (mOS: 131 vs. 159 months, P= 0.029), or PIK3CA mutations located in the C2 domain had significantly shorter OS (mOS, 130 vs. 154 months, P=0.020) than those without the mutations. Conclusions: Our study reveals the heterogeneity in PI3K-AKT-mTOR pathway among the breast cancer molecular subtypes in our cohort. Moreover, the number and specific sites of PIK3CA mutations have distinct prognostic impact.

Keywords:  Breast cancer; PI3K-AKT-mTOR pathway; gene alteration; molecular subtypes; prognosis

DOI:  https://doi.org/10.7150/jca.52993
Front Cell Dev Biol. 2021 ;9 663456

IGF1-Stimulated Posttraumatic Hippocampal Remodeling Is Not Dependent on mTOR.

Erica L Littlejohn, Anthony J DeSana, Hannah C Williams, Rudy T Chapman, Binoy Joseph, Jelena A Juras, Kathryn E Saatman.

  Adult hippocampal neurogenesis is stimulated acutely following traumatic brain injury (TBI). However, many hippocampal neurons born after injury develop abnormally and the number that survive long-term is debated. In experimental TBI, insulin-like growth factor-1 (IGF1) promotes hippocampal neuronal differentiation, improves immature neuron dendritic arbor morphology, increases long-term survival of neurons born after TBI, and improves cognitive function. One potential downstream mediator of the neurogenic effects of IGF1 is mammalian target of rapamycin (mTOR), which regulates proliferation as well as axonal and dendritic growth in the CNS. Excessive mTOR activation is posited to contribute to aberrant plasticity related to posttraumatic epilepsy, spurring preclinical studies of mTOR inhibitors as therapeutics for TBI. The degree to which pro-neurogenic effects of IGF1 depend upon upregulation of mTOR activity is currently unknown. Using immunostaining for phosphorylated ribosomal protein S6, a commonly used surrogate for mTOR activation, we show that controlled cortical impact TBI triggers mTOR activation in the dentate gyrus in a time-, region-, and injury severity-dependent manner. Posttraumatic mTOR activation in the granule cell layer (GCL) and dentate hilus was amplified in mice with conditional overexpression of IGF1. In contrast, delayed astrocytic activation of mTOR signaling within the dentate gyrus molecular layer, closely associated with proliferation, was not affected by IGF1 overexpression. To determine whether mTOR activation is necessary for IGF1-mediated stimulation of posttraumatic hippocampal neurogenesis, wildtype and IGF1 transgenic mice received the mTOR inhibitor rapamycin daily beginning at 3 days after TBI, following pulse labeling with bromodeoxyuridine. Compared to wildtype mice, IGF1 overexpressing mice exhibited increased posttraumatic neurogenesis, with a higher density of posttrauma-born GCL neurons at 10 days after injury. Inhibition of mTOR did not abrogate IGF1-stimulated enhancement of posttraumatic neurogenesis. Rather, rapamycin treatment in IGF1 transgenic mice, but not in WT mice, increased numbers of cells labeled with BrdU at 3 days after injury that survived to 10 days, and enhanced the proportion of posttrauma-born cells that differentiated into neurons. Because beneficial effects of IGF1 on hippocampal neurogenesis were maintained or even enhanced with delayed inhibition of mTOR, combination therapy approaches may hold promise for TBI.

Keywords:  dendritic outgrowth; insulin-like growth factor-1; mTOR; neurogenesis; neuronal differentiation; pS6; rapamycin; traumatic brain injury

DOI:  https://doi.org/10.3389/fcell.2021.663456