Autophagy Induction Is a Tor- and Tp53-Independent Cell Survival Response in a Zebrafish Model of Disrupted Ribosome Biogenesis

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.This research was funded by the National Health and Medical Research Council of Australia through Project grant 433614 (JKH), Program grant 487922 (JKH), a Senior Research Fellowship (JKH), and a Howard Florey Centenary Fellowship (HV). Operational Infrastructure Support was provided by the Victorian Government, Australia. Additional support was from Australian Research Council grant DP0346823 (GJL); NIH grant DK060322 (DYRS); and CDMRP, Department of Defense, USA W81XWH-10-1-0854 (KCE)

Updated international tuberous sclerosis complex diagnostic criteria and surveillance and management recommendations

Background Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disease affecting multiple body systems with wide variability in presentation. In 2013, Pediatric Neurology published articles outlining updated diagnostic criteria and recommendations for surveillance and management of disease manifestations. Advances in knowledge and approvals of new therapies necessitated a revision of those criteria and recommendations. Methods Chairs and working group cochairs from the 2012 International TSC Consensus Group were invited to meet face-to-face over two days at the 2018 World TSC Conference on July 25 and 26 in Dallas, TX, USA. Before the meeting, working group cochairs worked with group members via e-mail and telephone to (1) review TSC literature since the 2013 publication, (2) confirm or amend prior recommendations, and (3) provide new recommendations as required. Results Only two changes were made to clinical diagnostic criteria reported in 2013: “multiple cortical tubers and/or radial migration lines” replaced the more general term “cortical dysplasias,” and sclerotic bone lesions were reinstated as a minor criterion. Genetic diagnostic criteria were reaffirmed, including highlighting recent findings that some individuals with TSC are genetically mosaic for variants in TSC1 or TSC2. Changes to surveillance and management criteria largely reflected increased emphasis on early screening for electroencephalographic abnormalities, enhanced surveillance and management of TSC-associated neuropsychiatric disorders, and new medication approvals. Conclusions Updated TSC diagnostic criteria and surveillance and management recommendations presented here should provide an improved framework for optimal care of those living with TSC and their families

Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin

Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin) or TSC2 (encodes tuberin) genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242) in the tsc2 gene. This tsc2vu242 allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2vu242 is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2vu242/vu242 homozygous zebrafish, and is moderately increased in tsc2vu242/+ heterozygotes. Forebrain neurons are poorly organized in tsc2vu242/vu242 homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2vu242/vu242 mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC

Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish

SUMMARY Tuberous sclerosis complex (TSC) is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1) kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-α, Hif2-α and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors

Reproducible and efficient generation of functionally active neurons from human hiPSCs for preclinical disease modeling

The use of human induced pluripotent stem cell (hiPSC)-derived neuronal cultures to study the mechanisms of neurological disorders is often limited by low efficiency and high variability in differentiation of functional neurons. Here we compare the functional properties of neurons in cultures prepared with two hiPSC differentiation protocols, both plated on astroglial feeder layers. Using a protocol with an expandable intermediate stage, only a small percentage of cells with neuronal morphology were excitable by 21–23 days in culture. In contrast, a direct differentiation strategy of the same hiPSC line produced cultures in which the majority of neurons fired action potentials as early as 4–5 days. By 35–38 days over 80% of the neurons fired repetitively and many fired spontaneously. Spontaneous post-synaptic currents were observed in ~40% of the neurons at 4–5 days and in ~80% by 21–23 days. The majority (75%) received both glutamatergic and GABAergic spontaneous postsynaptic currents. The rate and degree of maturation of excitability and synaptic activity was similar between multiple independent platings from a single hiPSC line, and between two different control hiPSC lines. Cultures of rapidly functional neurons will facilitate identification of cellular mechanisms underlying genetically defined neurological disorders and development of novel therapeutics. Keywords: Induced pluripotent stem cells, hiPSC-derived neurons, Functional differentiation, Reproducibility, Disease modelin

Non-canonical functions of a mutant TSC2 protein in mitotic division.

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. TSC is caused by mutations in the TSC1 or TSC2 genes, which encode the hamartin/tuberin proteins respectively. These proteins function as a heterodimer that negatively regulates the mechanistic Target of Rapamycin Complex 1 (mTORC1). TSC research has focused on the effects of mTORC1, a critical signaling hub, on regulation of diverse cell processes including metabolism, cell growth, translation, and neurogenesis. However, non-canonical functions of TSC2 are not well studied, and the potential disease-relevant biological mechanisms of mutations affecting these functions are not well understood. We observed aberrant multipolar mitotic division, a novel phenotype, in TSC2 mutant iPSCs. The multipolar phenotype is not meaningfully affected by treatment with the inhibitor rapamycin. We further observed dominant negative activity of the mutant form of TSC2 in producing the multipolar division phenotype. These data expand the knowledge of TSC2 function and pathophysiology which will be highly relevant to future treatments for patients with TSC

Reproducible and efficient generation of functionally active neurons from human hiPSCs for preclinical disease modeling.

The use of human induced pluripotent stem cell (hiPSC)-derived neuronal cultures to study the mechanisms of neurological disorders is often limited by low efficiency and high variability in differentiation of functional neurons. Here we compare the functional properties of neurons in cultures prepared with two hiPSC differentiation protocols, both plated on astroglial feeder layers. Using a protocol with an expandable intermediate stage, only a small percentage of cells with neuronal morphology were excitable by 21-23days in culture. In contrast, a direct differentiation strategy of the same hiPSC line produced cultures in which the majority of neurons fired action potentials as early as 4-5days. By 35-38days over 80% of the neurons fired repetitively and many fired spontaneously. Spontaneous post-synaptic currents were observed in ~40% of the neurons at 4-5days and in ~80% by 21-23days. The majority (75%) received both glutamatergic and GABAergic spontaneous postsynaptic currents. The rate and degree of maturation of excitability and synaptic activity was similar between multiple independent platings from a single hiPSC line, and between two different control hiPSC lines. Cultures of rapidly functional neurons will facilitate identification of cellular mechanisms underlying genetically defined neurological disorders and development of novel therapeutics

A rare cause of posterior reversible encephalopathy syndrome: Acute lymphoblastic leukemia

Key Clinical Message The presentation of posterior reversible encephalopathy syndrome (PRES) as the initial presenting sign of acute lymphoblastic leukemia is unusual, as PRES is more often a complication of therapy. This case highlights the importance of maintaining a broad differential diagnosis for pediatric hypertension and its complications. Abstract A 6‐year‐old male presented with a seizure‐like episode. Evaluation revealed hypertension and brain imaging showed findings consistent with posterior reversible encephalopathy syndrome. Complete blood count showed lymphoblasts, and the cause of his hypertension was determined to be renal infiltration of leukemia cells due to B‐cell acute lymphoblastic leukemia