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The advent of high-dimensional imaging offers new opportunities to molecularly characterize diagnostic cells in disorders that have previously relied on histopathological definitions. One example case is found in tuberous sclerosis complex (TSC), a developmental disorder characterized by systemic growth of benign tumors. Within resected brain tissues from patients with TSC, detection of abnormally enlarged balloon cells (BCs) is pathognomonic for this disorder. Though BCs can be identified by an expert neuropathologist, little is known about the specificity and broad applicability of protein markers for these cells, complicating classification of proposed BCs identified in experimental models of this disorder. Here, we report the development of a customized machine learning pipeline (BAlloon IDENtifier; BAIDEN) that was trained to prospectively identify BCs in tissue sections using a histological stain compatible with high-dimensional cytometry. This approach was coupled to a custom 36-antibody panel and imaging mass cytometry (IMC) to explore the expression of multiple previously proposed BC marker proteins and develop a descriptor of BC features conserved across multiple tissue samples from patients with TSC. Here, we present a modular workflow encompassing BAIDEN, a custom antibody panel, a control sample microarray, and analysis pipelines-both open-source and in-house-and apply this workflow to understand the abundance, structure, and signaling activity of BCs as an example case of how high-dimensional imaging can be applied within human tissues., (© 2024 The Author(s). Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, lungs, kidney, and brain are all hallmarks of the disease, the most severe symptoms of TSC are often neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented, it is not yet known how early in neural development TSC1/2 -mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes, we observed aberrant early neurodevelopment in vitro , including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions, including loci associated with key genes in neurodevelopment. Collectively, these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated, with implications for whether and how prenatal treatment should be pursued.

Background: Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene and dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB)., Methods: We generated TSC disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies., Results: Using microphysiological systems, we demonstrate that a BBB generated from TSC2 heterozygous mutant cells shows increased permeability. This can be rescued by wild type astrocytes or by treatment with rapamycin, an mTOR kinase inhibitor., Conclusion: Our results demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of cell lineages contributing to TSC pathogenesis and informs future therapeutics., (© 2024. The Author(s).)

The implementation of three-dimensional tissue engineering concurrently with stem cell technology holds great promise for in vitro research in pharmacology and toxicology and modeling cardiac diseases, particularly for rare genetic and pediatric diseases for which animal models, immortal cell lines, and biopsy samples are unavailable. It also allows for a rapid assessment of phenotype-genotype relationships and tissue response to pharmacological manipulation. Mutations in the TSC1 and TSC2 genes lead to dysfunctional mTOR signaling and cause tuberous sclerosis complex (TSC), a genetic disorder that affects multiple organ systems, principally the brain, heart, skin, and kidneys. Here we differentiated healthy (CC3) and tuberous sclerosis (TSP8-15) human induced pluripotent stem cells (hiPSCs) into cardiomyocytes to create engineered cardiac tissue constructs (ECTCs). We investigated and compared their mechano-elastic properties and gene expression and assessed the effects of rapamycin, a potent inhibitor of the mechanistic target of rapamycin (mTOR). The TSP8-15 ECTCs had increased chronotropy compared to healthy ECTCs. Rapamycin induced positive inotropic and chronotropic effects (i.e., increased contractility and beating frequency, respectively) in the CC3 ECTCs but did not cause significant changes in the TSP8-15 ECTCs. A differential gene expression analysis revealed 926 up- and 439 down-regulated genes in the TSP8-15 ECTCs compared to their healthy counterparts. The application of rapamycin initiated the differential expression of 101 and 31 genes in the CC3 and TSP8-15 ECTCs, respectively. A gene ontology analysis showed that in the CC3 ECTCs, the positive inotropic and chronotropic effects of rapamycin correlated with positively regulated biological processes, which were primarily related to the metabolism of lipids and fatty and amino acids, and with negatively regulated processes, which were predominantly associated with cell proliferation and muscle and tissue development. In conclusion, this study describes for the first time an in vitro TSC cardiac tissue model, illustrates the response of normal and TSC ECTCs to rapamycin, and provides new insights into the mechanisms of TSC.

Reactivation and dysregulation of the mTOR signaling pathway are a hallmark of aging and chronic lung disease; however, the impact on microvascular progenitor cells (MVPCs), capillary angiostasis, and tissue homeostasis is unknown. While the existence of an adult lung vascular progenitor has long been hypothesized, these studies show that Abcg2 enriches for a population of angiogenic tissue-resident MVPCs present in both adult mouse and human lungs using functional, lineage, and transcriptomic analyses. These studies link human and mouse MVPC-specific mTORC1 activation to decreased stemness, angiogenic potential, and disruption of p53 and Wnt pathways, with consequent loss of alveolar-capillary structure and function. Following mTOR activation, these MVPCs adapt a unique transcriptome signature and emerge as a venous subpopulation in the angiodiverse microvascular endothelial subclusters. Thus, our findings support a significant role for mTOR in the maintenance of MVPC function and microvascular niche homeostasis as well as a cell-based mechanism driving loss of tissue structure underlying lung aging and the development of emphysema.

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., Competing Interests: MS reports grant support from Novartis, Biogen, Astellas, Aeovian, Bridgebio, and Aucta. He has served on Scientific Advisory Boards for Novartis, Roche, Regenxbio, SpringWorks Therapeutics, Jaguar Therapeutics and Alkermes. The other authors have no competing interests to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Chalkley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure., Methods: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further., Results: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN , the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes., Conclusions: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

We previously reported on two brothers who carry identical compound heterozygous PRKN mutations yet present with significantly different Parkinson's Disease (PD) clinical phenotypes. Juvenile cases demonstrate that PD is not necessarily an aging-associated disease. Indeed, evidence for a developmental component to PD pathogenesis is accumulating. Thus, we hypothesized that the presence of additional genetic modifiers, including genetic loci relevant to mesencephalic dopamine neuron development, could potentially contribute to the different clinical manifestations of the two brothers. We differentiated human-induced pluripotent stem cells (hiPSCs) derived from the two brothers into mesencephalic neural precursor cells and early postmitotic dopaminergic neurons and performed wholeexome sequencing and transcriptomic and metabolomic analyses. No significant differences in the expression of canonical dopamine neuron differentiation markers were observed. Yet our transcriptomic analysis revealed a significant downregulation of the expression of three neurodevelopmentally relevant cell adhesion molecules, CNTN6, CNTN4 and CHL1, in the cultures of the more severely affected brother. In addition, several HLA genes, known to play a role in neurodevelopment, were differentially regulated. The expression of EN2, a transcription factor crucial for mesencephalic dopamine neuron development, was also differentially regulated. We further identified differences in cellular processes relevant to dopamine metabolism. Lastly, wholeexome sequencing, transcriptomics and metabolomics data all revealed differences in glutathione (GSH) homeostasis, the dysregulation of which has been previously associated with PD. In summary, we identified genetic differences which could potentially, at least partially, contribute to the discordant clinical PD presentation of the two brothers., (© 2022. The Author(s).)

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 cm 3 , 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., Competing Interests: Declaration of interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The technologies presented in this MS are the subject of multiple pending and issued patents. Licenses to some of the Vanderbilt pump and valve technologies have been issued to KIYATEC, Inc. and CN Bio Innovations, which has also licensed the Multiwell MicroFormulator. Our MicroClinical Analyzer patents have been licensed to Agilent. John Wikswo is an Inaugural Member of the Scientific Advisory Board of BiOasis Technologies, Inc.

In December 2020, the Food and Drug Administration (FDA) issued Emergency Use Authorizations (EUAs) for Pfizer-BioNTech and Moderna COVID-19 vaccines, and in February 2021, FDA issued an EUA for the Janssen (Johnson & Johnson) COVID-19 vaccine. After each EUA, the Advisory Committee on Immunization Practices (ACIP) issued interim recommendations for vaccine use; currently Pfizer-BioNTech is authorized and recommended for persons aged ≥12 years and Moderna and Janssen for persons aged ≥18 years (1-3). Both Pfizer-BioNTech and Moderna vaccines, administered as 2-dose series, are mRNA-based COVID-19 vaccines, whereas the Janssen COVID-19 vaccine, administered as a single dose, is a recombinant replication-incompetent adenovirus-vector vaccine. As of July 22, 2021, 187 million persons in the United States had received at least 1 dose of COVID-19 vaccine (4); close monitoring of safety surveillance has demonstrated that serious adverse events after COVID-19 vaccination are rare (5,6). Three medical conditions have been reported in temporal association with receipt of COVID-19 vaccines. Two of these (thrombosis with thrombocytopenia syndrome [TTS], a rare syndrome characterized by venous or arterial thrombosis and thrombocytopenia, and Guillain-Barré syndrome [GBS], a rare autoimmune neurologic disorder characterized by ascending weakness and paralysis) have been reported after Janssen COVID-19 vaccination. One (myocarditis, cardiac inflammation) has been reported after Pfizer-BioNTech COVID-19 vaccination or Moderna COVID-19 vaccination, particularly after the second dose; these were reviewed together and will hereafter be referred to as mRNA COVID-19 vaccination. ACIP has met three times to review the data associated with these reports of serious adverse events and has comprehensively assessed the benefits and risks associated with receipt of these vaccines. During the most recent meeting in July 2021, ACIP determined that, overall, the benefits of COVID-19 vaccination in preventing COVID-19 morbidity and mortality outweigh the risks for these rare serious adverse events in adults aged ≥18 years; this balance of benefits and risks varied by age and sex. ACIP continues to recommend COVID-19 vaccination in all persons aged ≥12 years. CDC and FDA continue to closely monitor reports of serious adverse events and will present any additional data to ACIP for consideration. Information regarding risks and how they vary by age and sex and type of vaccine should be disseminated to providers, vaccine recipients, and the public., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Nicola P. Klein reports institutional research support from Pfizer, Sanofi Pasteur, Merck, GlaxoSmithKline, and Protein Science (now Sanofi Pasteur). No other potential conflicts of interest were disclosed.

Leukodystrophies are a group of neurodegenerative genetic disorders that affect approximately 1 in 7500 individuals. Despite therapeutic progress in individual leukodystrophies, guidelines in neurologic care are sparse and consensus among physicians and caregivers remains a challenge. At patient advocacy meetings hosted by Hunter's Hope from 2016-2018, multidisciplinary experts and caregivers met to conduct a literature review, identify knowledge gaps and summarize best practices regarding neurologic care. Stages of severity in leukodystrophies guided recommendations to address different levels of need based on a newly defined system of disease severity. Four core neurologic domains prioritized by families were identified and became the focus of this guideline: sleep, pain, seizures/epilepsy, and language/cognition. Based on clinical severity, the following categories were used: presymptomatic, early symptomatic, intermediate symptomatic, and advanced symptomatic. Across the leukodystrophies, neurologic care should be tailored to stages of severity while accounting for unique aspects of every disease and multiple knowledge gaps present. Standardized tools and surveys can help guide treatment but should not overburden families.

Mutations in the DEPDC5 gene can cause epilepsy, including forms with and without brain malformations. The goal of this study was to investigate the contribution of DEPDC5 gene dosage to the underlying neuropathology of DEPDC5-related epilepsies. We generated induced pluripotent stem cells (iPSCs) from epilepsy patients harboring heterozygous loss of function mutations in DEPDC5. Patient iPSCs displayed increases in both phosphorylation of ribosomal protein S6 and proliferation rate, consistent with elevated mTORC1 activation. In line with these findings, we observed increased soma size in patient iPSC-derived cortical neurons that was rescued with rapamycin treatment. These data indicate that human cells heterozygous for DEPDC5 loss-of-function mutations are haploinsufficient for control of mTORC1 signaling. Our findings suggest that human pathology differs from mouse models of DEPDC5-related epilepsies, which do not show consistent phenotypic differences in heterozygous neurons, and support the need for human-based models to affirm and augment the findings from animal models of DEPDC5-related epilepsy., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disease caused by heterozygous de novo missense mutations in the ATP1A3 gene that encodes the neuronal specific α3 subunit of the Na,K-ATPase (NKA) pump. Mechanisms underlying patient episodes including environmental triggers remain poorly understood, and there are no empirically proven treatments for AHC. In this study, we generated patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls for the E815K ATP1A3 mutation that causes the most phenotypically severe form of AHC. Using an in vitro iPSC-derived cortical neuron disease model, we found elevated levels of ATP1A3 mRNA in AHC lines compared to controls, without significant perturbations in protein expression. Microelectrode array analyses demonstrated that in cortical neuronal cultures, ATP1A3 +/E815K iPSC-derived neurons displayed less overall activity than neurons differentiated from isogenic mutation-corrected and unrelated control cell lines. However, induction of cellular stress by elevated temperature revealed a hyperactivity phenotype following heat stress in ATP1A3 +/E815K neurons compared to control lines. Treatment with flunarizine, a drug commonly used to prevent AHC episodes, did not impact this stress-triggered phenotype. These findings support the use of iPSC-derived neuronal cultures for studying complex neurodevelopmental conditions such as AHC and provide a platform for mechanistic discovery in a human disease model., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Over 1250 mutations in SCN1A, the Nav1.1 voltage-gated sodium channel gene, are associated with seizure disorders including GEFS+. To evaluate how a specific mutation, independent of genetic background, causes seizure activity we generated two pairs of isogenic human iPSC lines by CRISPR/Cas9 gene editing. One pair is a control line from an unaffected sibling, and the mutated control carrying the GEFS+ K1270T SCN1A mutation. The second pair is a GEFS+ patient line with the K1270T mutation, and the corrected patient line. By comparing the electrophysiological properties in inhibitory and excitatory iPSC-derived neurons from these pairs, we found the K1270T mutation causes cell type-specific alterations in sodium current density and evoked firing, resulting in hyperactive neural networks. We also identified differences associated with genetic background and interaction between the mutation and genetic background. Comparisons within and between dual pairs of isogenic iPSC-derived neuronal cultures provide a novel platform for evaluating cellular mechanisms underlying a disease phenotype and for developing patient-specific anti-seizure therapies., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Tuberous sclerosis complex 2 (TSC2), or tuberin, is a pivotal regulator of the mechanistic target of rapamycin signaling pathway that controls cell survival, proliferation, growth, and migration. Loss of Tsc2 function manifests in organ-specific consequences, the mechanisms of which remain incompletely understood. Recent single cell analysis of the kidney has identified ATP-binding cassette G2 (Abcg2) expression in renal proximal tubules of adult mice as well as a in a novel cell population. The impact in adult kidney of Tsc2 knockdown in the Abcg2-expressing lineage has not been evaluated. We engineered an inducible system in which expression of truncated Tsc2 , lacking exons 36-37 with an intact 3' region and polycystin 1, is driven by Abcg2. Here, we demonstrate that selective expression of Tsc2 fl36-37 in the Abcg2 pos lineage drives recombination in proximal tubule epithelial and rare perivascular mesenchymal cells, which results in progressive proximal tubule injury, impaired kidney function, formation of cystic lesions, and fibrosis in adult mice. These data illustrate the critical importance of Tsc2 function in the Abcg2-expressing proximal tubule epithelium and mesenchyme during the development of cystic lesions and remodeling of kidney parenchyma.

Astrocytes serve many functions in the human brain, many of which focus on maintenance of homeostasis. Astrocyte dysfunction in Tuberous Sclerosis Complex (TSC) has long been appreciated with activation of the mTORC1 signaling pathway resulting in gliosis and possibly contributing to the very frequent phenotype of epilepsy. We hypothesized that aberrant expression of the astrocyte protein aquaporin-4 (AQP4) may be present in TSC and contribute to disease pathology. Characterization of AQP4 expression in epileptic cortex from TSC patients demonstrated a diffuse increase in AQP4. To determine if this was due to exposure to seizures, we examined Aqp4 expression in mouse models of TSC in which Tsc1 or Tsc2 inactivation was targeted to astrocytes or glial progenitors, respectively. Loss of either Tsc1 or Tsc2 from astrocytes resulted in a marked increase in Aqp4 expression which was sensitive to mTORC1 inhibition with rapamycin. Our findings in both TSC epileptogenic cortex and in a variety of astrocyte culture models demonstrate for the first time that AQP4 expression is dysregulated in TSC. The extent to which AQP4 contributes to epilepsy in TSC is not known, though the similarities in AQP4 expression between TSC and temporal lobe epilepsy supports further studies targeting AQP4 in TSC., (Copyright © 2019. Published by Elsevier Inc.)

Human induced pluripotent stem cell (iPSC)-derived developmental lineages are key tools for in vitro mechanistic interrogations, drug discovery, and disease modeling. iPSCs have previously been differentiated to endothelial cells with blood-brain barrier (BBB) properties, as defined by high transendothelial electrical resistance (TEER), low passive permeability, and active transporter functions. Typical protocols use undefined components, which impart unacceptable variability on the differentiation process. We demonstrate that replacement of serum with fully defined components, from common medium supplements to a simple mixture of insulin, transferrin, and selenium, yields BBB endothelium with TEER in the range of 2,000-8,000 Ω × cm 2 across multiple iPSC lines, with appropriate marker expression and active transporters. The use of a fully defined medium vastly improves the consistency of differentiation, and co-culture of BBB endothelium with iPSC-derived astrocytes produces a robust in vitro neurovascular model. This defined differentiation scheme should broadly enable the use of human BBB endothelium for diverse applications., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neural stem/progenitor cells (NSPCs) of the ventricular-subventricular zone (V-SVZ) are candidate cells of origin for many brain tumors. However, whether NSPCs in different locations within the V-SVZ differ in susceptibility to tumorigenic mutations is unknown. Here, single-cell measurements of signal transduction intermediates in the mechanistic target of rapamycin complex 1 (mTORC1) pathway reveal that ventral NSPCs have higher levels of signaling than dorsal NSPCs . These features are linked with differences in mTORC1-driven disease severity: introduction of a pathognomonic Tsc2 mutation only results in formation of tumor-like masses from the ventral V-SVZ. We propose a direct link between location-dependent intrinsic growth properties imbued by mTORC1 and predisposition to tumor development., (© 2019 Rushing et al.)

MRI is a valuable tool to assess myelin during development and demyelinating disease processes. While multiexponential T 2 and quantitative magnetization transfer measures correlate with myelin content, neither provides the total myelin volume fraction. In many cases correlative measures are adequate; but to assess microstructure of myelin, (e.g. calculate the g-ratio using MRI), an accurate measure of myelin volume fraction is imperative. Using a volumetric model of white matter, we relate MRI measures of myelin to absolute measures of myelin volume fraction and compare them to quantitative histology. We assess our approach in control mice along with two models of hypomyelination and one model of hypermyelination and find strong agreement between MRI and histology amongst models. This work investigates the sensitivities of MRI myelin measures to changes in axon geometry and displays promise for estimating g-ratio from MRI., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mutations in ATP1A3 encoding the catalytic subunit of the Na/K-ATPase expressed in mammalian neurons cause alternating hemiplegia of childhood (AHC) as well as an expanding spectrum of other neurodevelopmental syndromes and neurological phenotypes. Most AHC cases are explained by de novo heterozygous ATP1A3 mutations, but the fundamental molecular and cellular consequences of these mutations in human neurons are not known. In this study, we investigated the electrophysiological properties of neurons generated from AHC patient-specific induced pluripotent stem cells (iPSCs) to ascertain functional disturbances underlying this neurological disease. Fibroblasts derived from two subjects with AHC, a male and a female, both heterozygous for the common ATP1A3 mutation G947R, were reprogrammed to iPSCs. Neuronal differentiation of iPSCs was initiated by neurogenin-2 (NGN2) induction followed by co-culture with mouse glial cells to promote maturation of cortical excitatory neurons. Whole-cell current clamp recording demonstrated that, compared with control iPSC-derived neurons, neurons differentiated from AHC iPSCs exhibited a significantly lower level of ouabain-sensitive outward current ('pump current'). This finding correlated with significantly depolarized potassium equilibrium potential and depolarized resting membrane potential in AHC neurons compared with control neurons. In this cellular model, we also observed a lower evoked action potential firing frequency when neurons were held at their resting potential. However, evoked action potential firing frequencies were not different between AHC and control neurons when the membrane potential was clamped to -80 mV. Impaired neuronal excitability could be explained by lower voltage-gated sodium channel availability at the depolarized membrane potential observed in AHC neurons. Our findings provide direct evidence of impaired neuronal Na/K-ATPase ion transport activity in human AHC neurons and demonstrate the potential impact of this genetic defect on cellular excitability., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Published by Elsevier B.V.)

Tuberous sclerosis complex (TSC) is a pediatric disorder of dysregulated growth and differentiation caused by loss of function mutations in either the TSC1 or TSC2 genes, which regulate mTOR kinase activity. To study aberrations of early development in TSC, we generated induced pluripotent stem cells using dermal fibroblasts obtained from patients with TSC. During validation, we found that stem cells generated from TSC patients had a very high rate of integration of the reprogramming plasmid containing a shRNA against TP53. We also found that loss of one allele of TSC2 in human fibroblasts is sufficient to increase p53 levels and impair stem cell reprogramming. Increased p53 was also observed in TSC2 heterozygous and homozygous mutant human stem cells, suggesting that the interactions between TSC2 and p53 are consistent across cell types and gene dosage. These results support important contributions of TSC2 heterozygous and homozygous mutant cells to the pathogenesis of TSC and the important role of p53 during reprogramming., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Myelin abnormalities are increasingly being recognized as an important component of a number of neurologic developmental disorders. The integration of many signaling pathways and cell types are critical for correct myelinogenesis. The PI3-K and mechanistic target of rapamycin (mTOR) pathways have been found to play key roles. mTOR is found within two distinct complexes, mTORC1 and mTORC2. mTORC1 activity has been shown to play a major role during myelination, while the role of mTORC2 is not yet well understood. To determine the role of mTORC2 signaling in myelinogenesis, we generated a mouse lacking the critical mTORC2 component Rictor in oligodendrocyte precursors (OPCs). Targeted deletion of Rictor in these cells decreases and delays the expression of myelin related proteins and reduces the size of cerebral white matter tracts. This is developmentally manifest as a transient reduction in myelinated axon density and g-ratio. OPC cell number is reduced at birth without detectable change in proliferation with proportional reductions in mature oligodendrocyte number at P15. The total number of oligodendrocytes as well as extent of myelination, does improve over time. Adult conditional knock-out (CKO) animals do not demonstrate a behavioral phenotype likely due in part to preserved axonal conduction velocities. These data support and extend prior studies demonstrating an important but transient contribution of mTORC2 signaling to myelin development.

On March 10 to March 12, 2015, the National Institute of Neurological Disorders and Stroke and the Tuberous Sclerosis Alliance sponsored a workshop in Bethesda, Maryland, to assess progress and new opportunities for research in tuberous sclerosis complex with the goal of updating the 2003 Research Plan for Tuberous Sclerosis (http://www.ninds.nih.gov/about_ninds/plans/tscler_research_plan.htm). In addition to the National Institute of Neurological Disorders and Stroke and Tuberous Sclerosis Alliance, participants in the strategic planning effort and workshop included representatives from six other Institutes of the National Institutes of Health, the Department of Defense Tuberous Sclerosis Complex Research Program, and a broad cross-section of basic scientists and clinicians with expertise in tuberous sclerosis complex along with representatives from the pharmaceutical industry. Here we summarize the outcomes from the extensive premeeting deliberations and final workshop recommendations, including (1) progress in the field since publication of the initial 2003 research plan for tuberous sclerosis complex, (2) the key gaps, needs, and challenges that hinder progress in tuberous sclerosis complex research, and (3) a new set of research priorities along with specific recommendations for addressing the major challenges in each priority area. The new research plan is organized around both short-term and long-term goals with the expectation that progress toward specific objectives can be achieved within a five to ten year time frame., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Alterations in DNA damage response and repair have been observed in Huntington's disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown.

Diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), and DKI-derived white matter tract integrity metrics (WMTI) were experimentally evaluated ex vivo through comparisons to histological measurements and established magnetic resonance imaging (MRI) measures of myelin in two knockout mouse models with varying degrees of hypomyelination. DKI metrics of mean and radial kurtosis were found to be better indicators of myelin content than conventional DTI metrics. The biophysical WMTI model based on the DKI framework reported on axon water fraction with good accuracy in cases with near normal axon density, but did not provide additional specificity to myelination. Overall, DKI provided additional information regarding white matter microstructure compared with DTI, making it an attractive method for future assessments of white matter development and pathology., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: While abnormalities in myelin in tuberous sclerosis complex (TSC) have been known for some time, recent imaging-based data suggest myelin abnormalities may be independent of the pathognomonic cortical lesions ("tubers"). Multiple mouse models of TSC exhibit myelination deficits, though the cell types responsible and the mechanisms underlying the myelin abnormalities remain unclear., Methods: To determine the role of alterations in mTOR signaling in myelination, we generated a conditional knockout (CKO) mouse model using Cre-recombinase and the Olig2 promoter to inactivate the Tsc2 gene in oligodendrocyte precursor cells., Results: Characterization of myelin and myelin constituent proteins demonstrated a marked hypomyelination phenotype. Diffusion-based magnetic resonance imaging studies were likewise consistent with hypomyelination. Hypomyelination was due in part to decreased myelinated axon density and myelin thickness as well as decreased oligodendrocyte numbers. Coincident with hypomyelination, an extensive gliosis was seen in both the cortex and white matter tracks, suggesting alterations in cell fate due to changes in mTOR activity in oligodendrocyte precursors. Despite a high-frequency appendicular tremor and altered gait in CKO mice, no significant changes in activity, vocalizations, or anxiety-like phenotypes were seen., Interpretation: Our findings support a known role of mTOR signaling in regulation of myelination and demonstrate that increased mTORC1 activity early in development within oligodendrocytes results in hypomyelination and not hypermyelination. Our data further support a dissociation between decreased Akt activity and increased mTORC1 activity toward hypomyelination. Thus, therapies promoting activation of Akt-dependent pathways while reducing mTORC1 activity may prove beneficial in treatment of human disease.

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo, we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro. In this study, we employed a library of combinatorial polymers comprising of three functional subunits - poly-ε-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) - as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-96%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro. Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions., (Published by Elsevier Ltd.)

MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at ∼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease such as simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis and fibrosis. However, the molecular pathogenesis and genetic variations causing NAFLD are poorly understood. The high prevalence and incidence of NAFLD suggests that genetic variations on a large number of genes might be involved in NAFLD. To identify genetic variants causing inherited liver disease, we used zebrafish as a model system for a large-scale mutant screen, and adopted a whole genome sequencing approach for rapid identification of mutated genes found in our screen. Here, we report on a forward genetic screen of ENU mutagenized zebrafish. From 250 F2 lines of ENU mutagenized zebrafish during post-developmental stages (5 to 8 days post fertilization), we identified 19 unique mutant zebrafish lines displaying visual evidence of hepatomegaly and/or steatosis with no developmental defects. Histological analysis of mutants revealed several specific phenotypes, including common steatosis, micro/macrovesicular steatosis, hepatomegaly, ballooning, and acute hepatocellular necrosis. This work has identified multiple post-developmental mutants and establishes zebrafish as a novel animal model for post-developmental inherited liver disease.

The essential micronutrient manganese is enriched in brain, especially in the basal ganglia. We sought to identify neuronal signaling pathways responsive to neurologically relevant manganese levels, as previous data suggested that alterations in striatal manganese handling occur in Huntington's disease (HD) models. We found that p53 phosphorylation at serine 15 is the most responsive cell signaling event to manganese exposure (of 18 tested) in human neuroprogenitors and a mouse striatal cell line. Manganese-dependent activation of p53 was severely diminished in HD cells. Inhibitors of ataxia telangiectasia mutated (ATM) kinase decreased manganese-dependent phosphorylation of p53. Likewise, analysis of ATM autophosphorylation and additional ATM kinase targets, H2AX and CHK2, support a role for ATM in the activation of p53 by manganese and that a defect in this process occurs in HD. Furthermore, the deficit in Mn-dependent activation of ATM kinase in HD neuroprogenitors was highly selective, as DNA damage and oxidative injury, canonical activators of ATM, did not show similar deficits. We assessed cellular manganese handling to test for correlations with the ATM-p53 pathway, and we observed reduced Mn accumulation in HD human neuroprogenitors and HD mouse striatal cells at manganese exposures associated with altered p53 activation. To determine if this phenotype contributes to the deficit in manganese-dependent ATM activation, we used pharmacological manipulation to equalize manganese levels between HD and control mouse striatal cells and rescued the ATM-p53 signaling deficit. Collectively, our data demonstrate selective alterations in manganese biology in cellular models of HD manifest in ATM-p53 signaling., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Tuberous sclerosis complex is a multisystem genetic disorder with a range of physical manifestations that require evaluation, surveillance, and management. Individuals with tuberous sclerosis complex also have a range of behavioral, psychiatric, intellectual, academic, neuropsychologic, and psychosocial difficulties. These may represent the greatest burden of the disease. Around 90% of individuals with tuberous sclerosis complex will have some of these difficulties during their lifetime, yet only about 20% ever receive evaluation and treatment. The Neuropsychiatry Panel at the 2012 Tuberous Sclerosis Complex International Consensus Conference expressed concern about the significant "treatment gap" and about confusion regarding terminology relating to the biopsychosocial difficulties associated with tuberous sclerosis complex., Methods: The Tuberous Sclerosis Complex Neuropsychiatry Panel coined the term TAND-tuberous sclerosis complex-associated neuropsychiatric disorders-to bring together these multidimensional manifestations of the disorder, and recommended annual screening for TAND. In addition, the Panel agreed to develop a TAND Checklist as a guide for screening., Results: Here, we present an outline of the conceptualization of TAND, rationale for the structure of the TAND Checklist, and include the full US English version of the TAND Checklist., Conclusion: We hope that the unified term TAND and the TAND Checklist will raise awareness of the importance of tuberous sclerosis complex-associated neuropsychiatric disorders and of the major burden of disease associated with it, provide a shared language and a simple tool to describe and evaluate the different levels of TAND, alert clinical teams and families or individuals of the importance of screening, assessment, and treatment of TAND, and provide a shared framework for future studies of tuberous sclerosis complex-associated neuropsychiatric disorders., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Poorly-defined interactions between environmental and genetic risk factors underlie Parkinson's disease (PD) etiology. Here we tested the hypothesis that human stem cell derived forebrain neuroprogenitors from patients with known familial risk for early onset PD will exhibit enhanced sensitivity to PD environmental risk factors compared to healthy control subjects without a family history of PD. Two male siblings (SM and PM) with biallelic loss-of-function mutations in PARK2 were identified. Human induced pluripotent stem cells (hiPSCs) from SM, PM, and four control subjects with no known family histories of PD or related neurodegenerative diseases were utilized. We tested the hypothesis that hiPSC-derived neuroprogenitors from patients with PARK2 mutations would show heightened cell death, mitochondrial dysfunction, and reactive oxygen species generation compared to control cells as a result of exposure to heavy metals (PD environmental risk factors). We report that PARK2 mutant neuroprogenitors showed increased cytotoxicity with copper (Cu) and cadmium (Cd) exposure but not manganese (Mn) or methyl mercury (MeHg) relative to control neuroprogenitors. PARK2 mutant neuroprogenitors also showed a substantial increase in mitochondrial fragmentation, initial ROS generation, and loss of mitochondrial membrane potential following Cu exposure. Our data substantiate Cu exposure as an environmental risk factor for PD. Furthermore, we report a shift in the lowest observable effect level (LOEL) for greater sensitivity to Cu-dependent mitochondrial dysfunction in patients SM and PM relative to controls, correlating with their increased genetic risk for PD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Understanding differences in gene expression that increase risk for pulmonary arterial hypertension (PAH) is essential to understanding the molecular basis for disease. Previous studies on patient samples were limited by end-stage disease effects or by use of nonadherent cells, which are not ideal to model vascular cells in vivo. These studies addressed the hypothesis that pathological processes associated with PAH may be identified via a genetic signature common across multiple cell types. Expression array experiments were initially conducted to analyze cell types at different stages of vascular differentiation (mesenchymal stromal and endothelial) derived from PAH patient-specific induced pluripotent stem (iPS) cells. Molecular pathways that were altered in the PAH cell lines were then compared with those in fibroblasts from 21 patients, including those with idiopathic and heritable PAH. Wnt was identified as a target pathway and was validated in vitro using primary patient mesenchymal and endothelial cells. Taken together, our data suggest that the molecular lesions that cause PAH are present in all cell types evaluated, regardless of origin, and that stimulation of the Wnt signaling pathway was a common molecular defect in both heritable and idiopathic PAH.

Induced pluripotent stem cells (iPSCs) are becoming mainstream tools to study mechanisms of development and disease. They have a broad range of applications in understanding disease processes, in vitro testing of novel therapies, and potential utility in regenerative medicine. Although the techniques for generating iPSCs are becoming more straightforward, scientists can expend considerable resources and time to establish this technology. A major hurdle is the accurate determination of valid iPSC-like colonies that can be selected for further cloning and characterization. In this study, we describe the use of a gammaretroviral vector encoding a fluorescent marker, mRFP1, to not only monitor the efficiency of initial transduction but also to identify putative iPSC colonies through silencing of mRFP1 gene as a consequence of successful reprogramming.

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.

Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa(vu463) ) that has an inactivating mutation in the etfa gene. dxa(vu463) recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa(vu463) zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa(vu463) zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity., Competing Interests: The authors have declared that no competing interests exist.

Tuberous sclerosis complex (TSC) is a genetic disease characterized by multiorgan benign tumors as well as neurological manifestations. Epilepsy and autism are two of the more prevalent neurological complications and are usually severe. TSC is caused by mutations in either the TSC1 (encodes hamartin) or the TSC2 (encodes tuberin) genes with TSC2 mutations being associated with worse outcomes. Tuberin contains a highly conserved GTPase-activating protein (GAP) domain that indirectly inhibits mammalian target of rapamycin complex 1 (mTORC1). mTORC1 dysregulation is currently thought to cause much of the pathogenesis in TSC but mTORC1-independent mechanisms may also contribute. We generated a novel conditional allele of Tsc2 by flanking exons 36 and 37 with loxP sites. Mice homozygous for this knock-in Tsc2 allele are viable and fertile with normal appearing growth and development. Exposure to Cre recombinase then creates an in-frame deletion involving critical residues of the GAP domain. Homozygous conditional mutant mice generated using Emx1(Cre) have increased cortical mTORC1 signaling, severe developmental brain anomalies, seizures, and die within 3 weeks. We found that the normal levels of the mutant Tsc2 mRNA, though GAP-deficient tuberin protein, appear unstable and rapidly degraded. This novel animal model will allow further study of tuberin function including the requirement of the GAP domain for protein stability., (Copyright © 2013 Wiley Periodicals, Inc.)