Mouse Model Of Tuberous Sclerosis Research Articles

Premature cognitive decline in a mouse model of tuberous sclerosis.

Little is known about the influence of (impaired) neurodevelopment on cognitive aging. We here used a mouse model for tuberous sclerosis (TS) carrying a heterozygous deletion of the Tsc2 gene. Loss of Tsc2 function leads to mTOR hyperactivity in mice and patients. In a longitudinal behavioral analysis, we found premature decline of hippocampus-based cognitive functions together with a significant reduction of immediate early gene (IEG) expression. While we did not detect any morphological changes of hippocampal projections and synaptic contacts, molecular markers of neurodegeneration were increased and the mTOR signaling cascade was downregulated in hippocampal synaptosomes. Injection of IGF2, a molecule that induces mTOR signaling, could fully rescue cognitive impairment and IEG expression in aging Tsc2+/- animals. This data suggests that TS is an exhausting disease that causes erosion of the mTOR pathway over time and IGF2 is a promising avenue for treating age-related degeneration in mTORopathies.

Postnatal immune activation causes social deficits in a mouse model of tuberous sclerosis: Role of microglia and clinical implications.

There is growing evidence that prenatal immune activation contributes to neuropsychiatric disorders. Here, we show that early postnatal immune activation resulted in profound impairments in social behavior, including in social memory in adult male mice heterozygous for a gene responsible for tuberous sclerosis complex (Tsc2+/−), a genetic disorder with high prevalence of autism. Early postnatal immune activation did not affect either wild-type or female Tsc2+/− mice. We demonstrate that these memory deficits are caused by abnormal mammalian target of rapamycin–dependent interferon signaling and impairments in microglia function. By mining the medical records of more than 3 million children followed from birth, we show that the prevalence of hospitalizations due to infections in males (but not in females) is associated with future development of autism spectrum disorders (ASD). Together, our results suggest the importance of synergistic interactions between strong early postnatal immune activation and mutations associated with ASD.

Ribosome profiling in mouse hippocampus: plasticity-induced regulation and bidirectional control by TSC2 and FMRP

BackgroundMutations in TSC2 are the most common cause of tuberous sclerosis (TSC), a disorder with a high incidence of autism and intellectual disability. TSC2 regulates mRNA translation required for group 1 metabotropic glutamate receptor-dependent synaptic long-term depression (mGluR-LTD) and behavior, but the identity of mRNAs responsive to mGluR-LTD signaling is largely unknown.MethodsWe utilized Tsc2+/− mice as a mouse model of TSC and prepared hippocampal slices from these animals. We induced mGluR-LTD synaptic plasticity in slices and processed the samples for RNA-seq and ribosome profiling to identify differentially expressed genes in Tsc2+/− and following mGluR-LTD synaptic plasticity.ResultsRibosome profiling reveals that in Tsc2+/− mouse hippocampal slices, the expression of several mRNAs was dysregulated: terminal oligopyrimidine (TOP)-containing mRNAs decreased, while FMRP-binding targets increased. Remarkably, we observed the opposite changes of FMRP binding targets in Fmr1−/y hippocampi. In wild-type hippocampus, induction of mGluR-LTD caused rapid changes in the steady-state levels of hundreds of mRNAs, many of which are FMRP targets. Moreover, mGluR-LTD failed to promote phosphorylation of eukaryotic elongation factor 2 (eEF2) in TSC mice, and chemically mimicking phospho-eEF2 with low cycloheximide enhances mGluR-LTD in TSC mice.ConclusionThese results suggest a molecular basis for bidirectional regulation of synaptic plasticity and behavior by TSC2 and FMRP. Our study also suggests that altered mGluR-regulated translation elongation contributes to impaired synaptic plasticity in Tsc2+/− mice.

NitroSynapsin for the treatment of neurological manifestations of tuberous sclerosis complex in a rodent model.

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by heterozygous mutations in the TSC1 or TSC2 gene. TSC is often associated with neurological, cognitive, and behavioral deficits. TSC patients also express co-morbidity with anxiety and mood disorders. The mechanism of pathogenesis in TSC is not entirely clear, but TSC-related neurological symptoms are accompanied by excessive glutamatergic activity and altered synaptic spine structures. To address whether extrasynaptic (e)NMDA-type glutamate receptor (NMDAR) antagonists, as opposed to antagonists that block physiological phasic synaptic activity, can ameliorate the synaptic and behavioral features of this disease, we utilized the Tsc2+/- mouse model of TSC to measure biochemical, electrophysiological, histological, and behavioral parameters in the mice. We found that antagonists that preferentially block tonic activity as found at eNMDARs, particularly the newer drug NitroSynapsin, provide biological and statistically significant improvement in Tsc2+/- phenotypes. Accompanying this improvement was correction of activity in the p38 MAPK-TSC-Rheb-mTORC1-S6K1 pathway. Deficits in hippocampal long-term potentiation (LTP), histological loss of synapses, and behavioral fear conditioning in Tsc2+/- mice were all improved after treatment with NitroSynapsin. Taken together, these results suggest that amelioration of excessive excitation, by limiting aberrant eNMDAR activity, may represent a novel treatment approach for TSC.

In vivo synaptic transmission and morphology in mouse models of Tuberous sclerosis, Fragile X syndrome, Neurofibromatosis type 1, and Costello syndrome.

Defects in the rat sarcoma viral oncogene homolog (Ras)/extracellular-signal-regulated kinase and the phosphatidylinositol 3-kinase-mammalian target of rapamycin (mTOR) signaling pathways are responsible for several neurodevelopmental disorders. These disorders are an important cause for intellectual disability; additional manifestations include autism spectrum disorder, seizures, and brain malformations. Changes in synaptic function are thought to underlie the neurological conditions associated with these syndromes. We therefore studied morphology and in vivo synaptic transmission of the calyx of Held synapse, a relay synapse in the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem, in mouse models of tuberous sclerosis complex (TSC), Fragile X syndrome (FXS), Neurofibromatosis type 1 (NF1), and Costello syndrome. Calyces from both Tsc1+/- and from Fmr1 knock-out (KO) mice showed increased volume and surface area compared to wild-type (WT) controls. In addition, in Fmr1 KO animals a larger fraction of calyces showed complex morphology. In MNTB principal neurons of Nf1+/- mice the average delay between EPSPs and APs was slightly smaller compared to WT controls, which could indicate an increased excitability. Otherwise, no obvious changes in synaptic transmission, or short-term plasticity were observed during juxtacellular recordings in any of the four lines. Our results in these four mutants thus indicate that abnormalities of mTOR or Ras signaling do not necessarily result in changes in in vivo synaptic transmission.

196. AAV-Mediated Gene Replacement Therapy in Mouse Model of Tuberous Sclerosis

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder due to mutations in either TSC1 or TSC2 that affects many organs with hamartomas and tumors. TSC-associated brain lesions include cortical heterotopias, tubers, subependymal nodules, and decreased myelination. Neurologic manifestations include epilepsy, hydrocephalus, mental retardation and autism. Here, we describe a new mouse model of TSC brain lesions in which complete loss of Tsc1 is achieved in multiple cell types in the brain in a stochastic pattern starting from the time of birth. Injection of an adeno-associated virus (AAV) vector encoding Cre recombinase into the cerebral ventricles of mice homozygous for a floxed Tsc1 conditional allele on P0 led to reduced survival, and pathologic findings of enlarged neurons, cortical heterotopias, subependymal nodules, and hydrocephalus. Although several other models of TSC brain disease exist, this model is unique in that the pathology reflects a variety of TSC-associated brain lesions involving different numbers and types of cells in different animals. This model provides a valuable and unique addition for therapeutic assessment.In addition, we have assessed the benefit of gene therapy in a related mouse model of Tsc1 in which the encoded gene product, hamartin is lost in most brain neurons at embryonic day 12. An adeno-associated virus (AAV) vector expressing a tagged form of hamartin was injected into the cerebral ventricles of newborn pups with the genotype Tsc1cc (homozygous for a conditional floxed Tsc1 allele) Syn-Cre+ (with Cre under the synapsin promoter). The AAV rh8 serotype gave widespread delivery of the tagged hamartin to brain cells. Treated mice showed an improvement in survival, from a mean of 22 days in non-injected Tsc1ccSynIcre+ mice to 52 days in hamartin AAV vector-injected, with improved weight gain and normalized behavior. Pathologic studies showed a reduction in neuron enlargement in comparison to untreated mutant littermates. Hence, we show that gene therapy is an effective approach in this mouse model of TSC. Our strategy for delivery has the advantage of single application, and AAV vectors are known to have minimal to nil toxicity in clinical trials for other neurologic conditions.

Equivalent Benefit of Rapamycin and a Potent mTOR ATP-Competitive Inhibitor, MLN0128 (INK128), in a Mouse Model of Tuberous Sclerosis

Tuberous sclerosis complex (TSC) is a hamartoma syndrome in which brain, renal, and lung tumors develop and cause both morbidity and death. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1. Rapamycin and related drugs have been shown to have clinical benefit for these tumors in patients with TSC and those with sporadic forms of TSC-related neoplasms. However, lifelong therapy seems to be required, as tumors are not eliminated by this treatment. We examined the potential benefit of MLN0128, a novel potent mTOR ATP-competitive inhibitor, as a therapeutic strategy for renal cystadenomas that develop in A/J Tsc2(+/-) mice. Rapamycin given by intraperitoneal injection at 3 mg/kg 3 times per week, and MLN0128 given by gavage at 0.75 mg/kg 5 times per week had equivalent effects in suppressing tumor development during a 4-week treatment period, with an approximate 99% reduction in microscopic tumor cell volume. Marked reduction in activation of mTOR complex (mTORC)1 and blockade of cell growth was seen with both drugs, whereas only MLN0128 treatment had effects in blocking mTORC2 and 4EBP1 phosphorylation. However, when either drug was discontinued and mice were observed for two additional months, there was dramatic recovery of tumor growth, with extensive proliferation. Hence, longlasting tumor growth control is not achieved with transient treatment with either drug, and MLN0128 and rapamycin have equivalent therapeutic benefit in this mouse model. Differences in side-effect profiles might make MLN0128 more attractive for treatment of patients with TSC-related tumors, but will require additional study in humans.

Excitatory/Inhibitory Synaptic Imbalance Leads to Hippocampal Hyperexcitability in Mouse Models of Tuberous Sclerosis

Neural circuits are regulated by activity-dependent feedback systems that tightly control network excitability and which are thought to be crucial for proper brain development. Defects in the ability to establish and maintain network homeostasis may be central to the pathogenesis of neurodevelopmental disorders. Here, we examine the function of the tuberous sclerosis complex (TSC)-mTOR signaling pathway, a common target of mutations associated with epilepsy and autism spectrum disorder, in regulating activity-dependent processes in the mouse hippocampus. We find that the TSC-mTOR pathway is a central component of a positive feedback loop that promotes network activity by repressing inhibitory synapses onto excitatory neurons. In Tsc1 KO neurons, weakened inhibition caused by deregulated mTOR alters the balance of excitatory and inhibitory synaptic transmission, leading to hippocampal hyperexcitability. These findings identify the TSC-mTOR pathway as a regulator of neural network activity and have implications for the neurological dysfunction in disorders exhibiting deregulated mTOR signaling.

Renal tumours in a Tsc1+/– mouse model show epigenetic suppression of organic cation transporters Slc22a1, Slc22a2 and Slc22a3, and do not respond to metformin

Metformin, a substrate of several poly-specific organic cation transporters, is a widely used biguanide for the treatment of type II diabetes. Recent studies suggest that metformin attenuates mTORC1 signalling by the activation of 5’ adenosine monophosphate-activated protein kinase (AMPK) in the presence or absence of a functional hamartin/tuberin (TSC1/TSC2) complex. Metformin has also been reported to inhibit mTORC1 independent of AMPK through p53-dependent regulated in development and DNA damage responses 1 (REDD1) or by inhibiting Rag GTPases. These observations suggest that metformin could have therapeutic potential for tuberous sclerosis, an inherited disorder characterised by the aberrant activation of mTORC1 and the development of tumours in many organs, including the kidneys. In this study, we investigated the effect of metformin on renal lesions in a Tsc1+/– mouse model of tuberous sclerosis. Continuous treatment of metformin for 9months at doses of up to 600mg/kg/day had no significant effect on renal lesions in nine treated mice compared to 10 controls. Metformin treatment appeared to attenuate mTORC1 signalling in Tsc1+/– kidney tissues but not in renal tumours. Surprisingly, the expression of the organic cation transporters Slc22a1, Slc22a2 and Slc22a3 essential for the cellular uptake of metformin was highly suppressed in renal tumours. Treatment of cultured cells derived from a Tsc1-associated renal tumour with 5-aza-2-deoxycytidine or trichostatin A greatly increased the expression of these genes. These data suggest that the epigenetic suppression of the organic cation transporters in Tsc-associated mouse renal tumours may contribute to the lack of response to metformin treatment.

Patent Highlights

Patent Highlights

T2 weighted MRI for assessing renal lesions in transgenic mouse models of tuberous sclerosis

ObjectiveTransgenic mouse models of tuberous sclerosis (TSC) develop renal cysts, cystadenomas, solid adenomas and carcinomas. Identification and characterisation of these lesions in vivo may help in TSC pre-clinical trials. This study was to evaluate T2 weighted MRI for assessment of renal lesions in two Tsc mouse models. Materials and MethodsTsc1+/−, Tsc2+/− and wild type mice were subjected to a first MRI scan at 12 months of age and a second scan 2 months later. One Tsc2+/− mouse was treated with rapamycin for two months after the initial scan. Immediately following the second scan, mice were sacrificed and MRI images were compared to renal histological findings. ResultsMRI identified all types of Tsc-associated renal lesions in both Tsc1+/− and Tsc2+/− mice. The smallest detectable lesions were <0.1mm3. Eighty three percent of all renal lesions detected in the first scan were re-identified in the second scan. By MRI, these lesions demonstrated significant growth in the 9 untreated Tsc1+/− and Tsc2+/− mice but shrinkage in the rapamycin treated Tsc2+/− mouse. Between the two scans, MRI also revealed significant increase in both the total number and volume of lesions in untreated mice and decrease in the rapamycin treated mouse, respectively. In comparison to histological analysis MRI detected most cysts and cystadenomas (66%) but only a minority of solid tumours (29%). ConclusionThese results suggest that T2 weighted MRI may be a useful tool for assessing some renal lesions in pre-clinical studies using Tsc mouse models. However, improved sensitivity for T2 weighted MRI is required, particularly for solid renal lesions.

Naturally Secreted Amyloid-β Increases Mammalian Target of Rapamycin (mTOR) Activity via a PRAS40-mediated Mechanism

Reducing the mammalian target of rapamycin (mTOR) activity increases lifespan and health span in a variety of organisms. Alterations in protein homeostasis and mTOR activity and signaling have been reported in several neurodegenerative disorders, including Alzheimer disease (AD); however, the causes of such deregulations remain elusive. Here, we show that mTOR activity and signaling are increased in cell lines stably transfected with mutant amyloid precursor protein (APP) and in brains of 3xTg-AD mice, an animal model of AD. In addition, we show that in the 3xTg-AD mice, mTOR activity can be reduced to wild type levels by genetically preventing Aβ accumulation. Similarly, intrahippocampal injections of an anti-Aβ antibody reduced Aβ levels and normalized mTOR activity, indicating that high Aβ levels are necessary for mTOR hyperactivity in 3xTg-AD mice. We also show that the intrahippocampal injection of naturally secreted Aβ is sufficient to increase mTOR signaling in the brains of wild type mice. The mechanism behind the Aβ-induced mTOR hyperactivity is mediated by the proline-rich Akt substrate 40 (PRAS40) as we show that the activation of PRAS40 plays a key role in the Aβ-induced mTOR hyperactivity. Taken together, our data show that Aβ accumulation, which has been suggested to be the culprit of AD pathogenesis, causes mTOR hyperactivity by regulating PRAS40 phosphorylation. These data further indicate that the mTOR pathway is one of the pathways by which Aβ exerts its toxicity and further support the idea that reducing mTOR signaling in AD may be a valid therapeutic approach.

Gestational immune activation and Tsc2 haploinsufficiency cooperate to disrupt fetal survival and may perturb social behavior in adult mice

Approximately 40-50% of individuals affected by tuberous sclerosis (TSC) develop autism spectrum disorders (ASDs). One possible explanation for this partial penetrance is an interaction between TSC gene mutations and other risk factors such as gestational immune activation. In this study, we report the interactive effects of these two ASD risk factors in a mouse model of TSC. Combined, but not single, exposure had adverse effects on intrauterine survival. Additionally, provisional results suggest that these factors synergize to disrupt social approach behavior in adult mice. Moreover, studies in human populations are consistent with an interaction between high seasonal flu activity in late gestation and TSC mutations in ASD. Taken together, our studies raise the possibility of a gene × environment interaction between heterozygous TSC gene mutations and gestational immune activation in the pathogenesis of TSC-related ASD.

Increased Levels of Anxiety-related Behaviors in a Tsc2 Dominant Negative Transgenic Mouse Model of Tuberous Sclerosis

Tuberous sclerosis (TSC) is a single-gene disorder caused by heterozygous mutations in the TSC1 or TSC2 gene. TSC is often associated with neurological (e.g., epilepsy), cognitive (intellectual disabilities, specific neuropsychological impairments) and behavioral pathologies (e.g., autism, attention deficit hyperactivity disorder). In addition, there is a high prevalence of psychiatric problems in TSC populations, including anxiety and mood disorders. To date, little is known about the pathogenetic bases of these associated psychiatric symptoms; for instance, it is unclear whether they are rooted in TSC-associated neurobiological alterations or whether they are secondary psychological phenomena (e.g., because individuals have to cope with the burden of the disease). Here, we report elevated levels of anxiety-related behaviors and mild deficits in two hippocampal-dependent learning tasks in a Tsc2 dominant negative transgenic mouse model of TSC. These findings establish a mouse model for TSC-related anxiety phenotypes and suggest that anxiety disorders in TSC have a biological foundation.

How DISC1 Regulates Postnatal Brain Development: Girdin Gets In on the AKT

In this issue of Neuron, Kim et al. and Enomoto et al. show that DISC1 plays a key role in regulating postnatal brain development though interaction with Girdin. Girdin in turn regulates AKT signaling. Thus, another facet of the role of DISC1 is established, shedding more light on fundamental brain processes and the developmental basis of major psychiatric disorders.

Equivalent benefit of mTORC1 blockade and combined PI3K-mTOR blockade in a mouse model of tuberous sclerosis

BackgroundTuberous sclerosis (TSC) is a hamartoma syndrome in which renal and lung tumors cause the greatest morbidity. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1 and suppression of AKT. Recent studies indicate that inhibition of mTORC1 with RAD001 (everolimus) leads to rebound activation of AKT, which could protect tumors from drug-induced cell death. Here we examine the potential benefit of inhibition of both mTOR and AKT signaling in a mouse model of TSC, using a dual pan class I PI3K/mTOR catalytic small molecule inhibitor NVP-BEZ235.ResultsUsing ENU to enhance Tsc2+- kidney tumor development, both RAD001 (10 mg/kg PO 5 d/week) and NVP-BEZ235 (45 mg/kg PO QD) had equivalent effects in suppressing tumor development during a 4 week treatment period, with a 99% reduction in tumor cell mass. Marked reduction in activation of mTORC1, induction of cell cycle arrest, and absence of apoptotic cell death was seen in mice treated with either drug. However, when either was discontinued, there was prompt recovery of tumor growth, with extensive proliferation.ConclusionBoth mTORC1 blockade alone and combined PI3K-mTOR blockade lead to suppression of tumor development but not tumor elimination in this TSC model.

Critical Role for Hypothalamic mTOR Activity in Energy Balance

The mammalian target of rapamycin (mTOR) promotes anabolic cellular processes in response to growth factors and metabolic cues. The TSC1 and TSC2 tumor suppressors are major upstream inhibitory regulators of mTOR signaling. Mice with Rip2/Cre-mediated deletion of Tsc1 (Rip-Tsc1cKO mice) developed hyperphagia and obesity, suggesting that hypothalamic disruption (for which Rip2/Cre is well known) of Tsc1 may dysregulate feeding circuits via mTOR activation. Indeed, Rip-Tsc1cKO mice displayed increased mTOR signaling and enlarged neuron cell size in a number of hypothalamic populations, including Pomc neurons. Furthermore, Tsc1 deletion with Pomc/Cre (Pomc-Tsc1cKO mice) resulted in dysregulation of Pomc neurons and hyperphagic obesity. Treatment with the mTOR inhibitor, rapamycin, ameliorated the hyperphagia, obesity, and the altered Pomc neuronal morphology in developing or adult Pomc-Tsc1cKO mice, and cessation of treatment reinstated these phenotypes. Thus, ongoing mTOR activation in Pomc neurons blocks the catabolic function of these neurons to promote nutrient intake and increased adiposity.

Reversal of learning deficits in a Tsc2+/− mouse model of tuberous sclerosis

Tuberous sclerosis is a single-gene disorder caused by heterozygous mutations in the TSC1 (9q34) or TSC2 (16p13.3) gene and is frequently associated with mental retardation, autism and epilepsy. Even individuals with tuberous sclerosis and a normal intelligence quotient (approximately 50%) are commonly affected with specific neuropsychological problems, including long-term and working memory deficits. Here we report that mice with a heterozygous, inactivating mutation in the Tsc2 gene (Tsc2(+/-) mice) show deficits in learning and memory. Cognitive deficits in Tsc2(+/-) mice emerged in the absence of neuropathology and seizures, demonstrating that other disease mechanisms are involved. We show that hyperactive hippocampal mammalian target of rapamycin (mTOR) signaling led to abnormal long-term potentiation in the CA1 region of the hippocampus and consequently to deficits in hippocampal-dependent learning. These deficits included impairments in two spatial learning tasks and in contextual discrimination. Notably, we show that a brief treatment with the mTOR inhibitor rapamycin in adult mice rescues not only the synaptic plasticity, but also the behavioral deficits in this animal model of tuberous sclerosis. The results presented here reveal a biological basis for some of the cognitive deficits associated with tuberous sclerosis, and they show that treatment with mTOR antagonists ameliorates cognitive dysfunction in a mouse model of this disorder.

A Mouse Model of Tuberous Sclerosis: Neuronal Loss of Tsc1 Causes Dysplastic and Ectopic Neurons, Reduced Myelination, Seizure Activity, and Limited Survival

Tuberous sclerosis (TSC) is a hamartoma syndrome caused by mutations in TSC1 or TSC2 in which cerebral cortical tubers and seizures are major clinical issues. We have engineered mice in which most cortical neurons lose Tsc1 expression during embryonic development. These Tsc1 mutant mice display several neurological abnormalities beginning at postnatal day 5 with subsequent failure to thrive and median survival of 35 d. The mice also display clinical and electrographic seizures both spontaneously and with physical stimulation, and some seizures end in a fatal tonic phase. Many cortical and hippocampal neurons are enlarged and/or dysplastic in the Tsc1 mutant mice, strongly express phospho-S6, and are ectopic in multiple sites in the cortex and hippocampus. There is a striking delay in myelination in the mutant mice, which appears to be caused by an inductive neuronal defect. This new TSC brain model replicates several features of human TSC brain lesions and implicates an important function of Tsc1/Tsc2 in neuronal development.

Magnetic Resonance Imaging and Characterization of Spontaneous Lesions in a Transgenic Mouse Model of Tuberous Sclerosis as a Model for Endothelial Cell-Based Transgene Delivery

Tuberous sclerosis (TSC) is an autosomal dominant genetic disorder characterized by abnormalities in cellular migration, proliferation, and differentiation in many tissues. Benign hamartomas develop in multiple organs, believed to be caused by somatic mutation in addition to germ line mutation to cause loss of both alleles of either the TSC1 or TSC2 tumor suppressor gene, with resultant dysregulated growth due to loss of hamartin or tuberin function, respectively. This study focuses on detecting spontaneous lesions in a knockout mouse model of TSC2 by magnetic resonance imaging (MRI) and exploring the efficiency of introducing gene products into lesions, using transduced endothelial cells as gene vehicles. MRI was shown to be effective in detecting spontaneous lesions in multiple tissues as a means of assessing the prevalence of tumors. Tsc(2+/) heterozygous mice were screened at 12-24 months of age. MRI detected 100% of the renal lesions (cystadenomas, renal cell carcinomas) and 75% of the hepatic lesions (hemangiosarcomas), later identified by histology. Cell-mediated gene delivery was evaluated by immunohistochemical analysis of renal, hepatic, and lung lesions after intravenous delivery of MS1 mouse endothelial cells, transduced to express an enhanced form of green fluorescent protein (EGFP). Preliminary immunohistochemical analysis, using a polyclonal antibody to EGFP and a horseradish peroxidase-diaminobenzidine detection system, revealed these cells throughout liver, kidney, and lung sections from injected animals, organs that are frequently affected in TSC2 patients, as well as within the lesions themselves.