Regulation of fatty acid delivery to metastases by tumor endothelium.

Tumor metastasis, the main cause of death in cancer patients, requires outgrowth of tumor cells after their dissemination and residence in microscopic niches. Nutrient sufficiency is a determinant of such outgrowth1. Fatty acids (FA) can be metabolized by cancer cells for their energetic and anabolic needs but impair the cytotoxicity of T cells in the tumor microenvironment (TME)2,3, thereby supporting metastatic progression. However, despite the important role of FA in metastatic outgrowth, the regulation of intratumoral FA is poorly understood. In this report, we show that tumor endothelium actively promotes tumor growth and restricts anti-tumor cytolysis by transferring FA into developing metastatic tumors. This process uses transendothelial fatty acid transport via endosome cargo trafficking in a mechanism that requires mTORC1 activity. Thus, tumor burden was significantly reduced upon endothelial-specific targeted deletion of Raptor, a unique component of the mTORC1 complex (RptorECKO). In vivo trafficking of a fluorescent palmitic acid analog to tumor cells and T cells was reduced in RptorECKO lung metastatic tumors, which correlated with improved markers of T cell cytotoxicity. Combination of anti-PD1 with RAD001/everolimus, at a low dose that selectively inhibits mTORC1 in endothelial cells4, impaired FA uptake in T cells and reduced metastatic disease, corresponding to improved anti-tumor immunity. These findings describe a novel mechanism of transendothelial fatty acid transfer into the TME during metastatic outgrowth and highlight a target for future development of therapeutic strategies.

Contractile and Genetic Characterization of Cardiac Constructs Engineered from Human Induced Pluripotent Stem Cells: Modeling of Tuberous Sclerosis Complex and the Effects of Rapamycin.

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.

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.

Activation of mTOR signaling in adult lung microvascular progenitor cells accelerates lung aging.

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.

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.

Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy.

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.

Dephosphorylation of 4EBP1/2 Induces Prenatal Neural Stem Cell Quiescence.

A limiting factor in the regenerative capacity of the adult brain is the abundance and proliferative ability of neural stem cells (NSCs). Adult NSCs are derived from a subpopulation of embryonic NSCs that temporarily enter quiescence during mid-gestation and remain quiescent until postnatal reactivation. Here we present evidence that the mechanistic/mammalian target of rapamycin (mTOR) pathway regulates quiescence entry in embryonic NSCs of the developing forebrain. Throughout embryogenesis, two downstream effectors of mTOR, p-4EBP1/2 T37/46 and p-S6 S240/244, were mutually exclusive in NSCs, rarely occurring in the same cell. While 4EBP1/2 was phosphorylated in stem cells undergoing mitosis at the ventricular surface, S6 was phosphorylated in more differentiated cells migrating away from the ventricle. Phosphorylation of 4EBP1/2, but not S6, was responsive to quiescence induction in cultured embryonic NSCs. Further, inhibition of p-4EBP1/2, but not p-S6, was sufficient to induce quiescence. Collectively, this work offers new insight into the regulation of quiescence entry in embryonic NSCs and, thereby, correct patterning of the adult brain. These data suggest unique biological functions of specific posttranslational modifications and indicate that the preferential inhibition of such modifications may be a useful therapeutic approach in neurodevelopmental diseases where NSC numbers, proliferation, and differentiation are altered.

Rescue of Impaired Blood-Brain Barrier in Tuberous Sclerosis Complex Patient Derived Neurovascular Unit.

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. 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). We generated disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. Using these microphysiological systems, we demonstrate that the BBB generated from TSC2 heterozygous mutant cells shows increased permeability which can be rescued by wild type astrocytes and with treatment with rapamycin, an mTOR kinase inhibitor. Our results further demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of the cell lineages contributing to TSC pathogenesis.

Genomic, transcriptomic, and metabolomic profiles of hiPSC-derived dopamine neurons from clinically discordant brothers with identical PRKN deletions.

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.

Use of COVID-19 Vaccines After Reports of Adverse Events Among Adult Recipients of Janssen (Johnson & Johnson) and mRNA COVID-19 Vaccines (Pfizer-BioNTech and Moderna): Update from the Advisory Committee on Immunization Practices - United States, July 2021.

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.

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

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.

Practical Approaches and Knowledge Gaps in the Care for Children With Leukodystrophies.

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.

DEPDC5 haploinsufficiency drives increased mTORC1 signaling and abnormal morphology in human iPSC-derived cortical neurons.

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.

Neuronal modeling of alternating hemiplegia of childhood reveals transcriptional compensation and replicates a trigger-induced phenotype.

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.

Comparisons of dual isogenic human iPSC pairs identify functional alterations directly caused by an epilepsy associated SCN1A mutation.

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.

Inactivation of Tsc2 in Abcg2 lineage-derived cells drives the appearance of polycystic lesions and fibrosis in the adult kidney.

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 Tsc2fl36-37 in the Abcg2pos 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.

A Simplified, Fully Defined Differentiation Scheme for Producing Blood-Brain Barrier Endothelial Cells from Human iPSCs.

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 × cm2 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.

Cerebral aquaporin-4 expression is independent of seizures in tuberous sclerosis complex.

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.