Michael J. Gambello - US grants

DESCRIPTION (provided by applicant): The goal of this application is to initiate genetic studies of a cohort of well characterized autistic patients already enrolled in a program project, titled "Orbitofrontal-limbic dysfunction in autism". Autism is a neurodevelopmental disorder with considerable heterogeneity among affected children. This heterogeneity is due, to a large extent, to many known and unknown genetic factors. Therefore the neurobehavioral development and dysfunction in autism must be understood within a genetic context. Consequently this application will focus on recalling autistic patients and their families so a thorough medical genetics evaluation can be performed. Medical histories and physical examinations will enable the diagnosis of known and novel genetic syndromes. Specifically the frequency of macrocephaly, a physical finding associated with autism, will be assessed and correlated with cognitive functioning and genetic testing. Routine genetic studies will be performed, including G-banded karyotypes at the 550-600 band resolution, fragile X syndrome molecular testing, and plasma amino and urine organic acid analyses. Array Comparative Genomic Hybridization (CGH) will be performed on all mentally retarded and/or dysmorphic patients to screen for subtle, unrecognized genomic deletions or duplications. Metabolic studies will include the novel assessment of creatine metabolism. To further genetic studies, lymphoblastoid cell lines from patients and DNA from patients and parents will be banked. Patient DNA will be used to screen for novel mutations in the tuberous sclerosis complex genes TSC1 and TSC2. The specific role of these genes in autism is still unclear. Results from these studies will further delineate autistic phenotypes, allow the correlation of specific genetic lesions with data from other aspects of the program project, and provide a foundation for larger genetic studies of autism.

[unreadable] DESCRIPTION (provided by applicant): Tuberous sclerosis complex (TSC) is an autosomal dominant tumor predisposition disorder affecting approximately 1/6000 individuals. Patients have mutations in either the TSC1 or TSC2 gene, encoding the proteins hamartin and tuberin, and have similar, but not identical, phenotypes. Substantial morbidity and mortality are caused by brain lesions such as tubers, subependymal nodules, and other neuronal heterotopias. These lesions are associated with seizures, autism, and other developmental disabilities. Remarkably, little is known about the pathogenesis of these lesions or how they lead to developmental disabilities. The overall goal of this project is to model, identify and study the neurodevelopmental defects that lead to the brain lesions of TSC. We have engineered a novel mouse model of the neuropathology of TSC by selectively deleting the Tsc2 gene in radial glial progenitor cells of the developing brain. These mice die from seizures between 3 weeks and a month of age and have enlarged brains, neuronal migration defects composed of giant cells, and increased numbers of astrocytes. These brain lesions are very similar to those described in humans with TSC and provide a novel model in which to study the in vivo neurodevelopmental aspects of TSC. The specific aims are: 1) to investigate how loss of function of Tsc2 in radial glia affects proliferation, differentiation and apoptosis in the developing and adult brain; 2) to understand the in vivo effects of loss of tuberin on neuronal morphology and migration; and, 3) to identify separate functions of the Tsc1 and Tsc2 genes in vivo by generating a similar Tsc1 based model and comparing its brain phenotype to the Tsc2 model. These studies will expand the understanding of tuberin and hamartin's role in the developing brain in TSC neuropathology, and in other TSC-associated diseases such as autism and epilepsy. Knowledge of independent in vivo functions of each gene may lead to targeted therapeutic interventions for patients with disease due to TSC1 mutations versus TSC2 mutations. The goal of this research project is to use mouse genetics to understand the developmental abnormalities that lead to the brain lesions of the genetic disease tuberous sclerosis complex. Well characterized models will establish a foundation for the testing of future targeted therapies and establish developmental neurobiologic differences between subsets of patients with TSC. [unreadable] [unreadable] [unreadable]

Project Summary Tuberous sclerosis complex (TSC) is a genetic disorder that often causes neurologic pathology such as epilepsy, developmental disability, or neuropsychiatric disease. TSC is caused by heterozygous, inactivating mutations in the TSC1 or TSC2 genes that encode protein binding partners hamartin and tuberin. Dimerized hamartin and tuberin negatively regulate mTORC1, the mechanistic target of rapamycin complex 1. In response to anabolic signals, activated mTORC1 stimulates protein synthesis, cell growth and cell cycle progression. The pathogenesis of TSC is largely due to constitutive mTORC1 activation and consequent cellular anabolism. Despite advances in our knowledge about the molecular basis of TSC, the downstream events causing TSC pathology remain unclear. Using an unbiased, global metabolomic profiling approach, we assayed hippocampal tissue from a well-characterized mouse model of TSC in which the Tsc2 gene is conditionally disrupted in radial glial precursor cells (Tsc2-RG). These and subsequent data revealed an upregulation of the polyamine putrescine and increase in ornithine decarboxylase (ODC) activity, the rate limiting enzyme in polyamine synthesis, in Tsc2-RG mutants. Polyamines are essential for cell proliferation and survival and evidence suggests an interaction between polyamines and mTORC signaling. In this proposal, we will determine if directly reducing ODC activity ameliorates TSC-associated neuropathology and dampens mTORC signaling in mice. We will use complementary genetic and pharmacological approaches to reduce ODC activity and concomitant putrescine levels and polyamine flux. We propose to generate Tsc2-RG; Odc+/- compound mutants and determine if Odc haploinsufficiency affects (1) ODC activity and polyamine levels in brain lysates; (2) markers of neuropathology typical of TSC; and (3) levels of TSC/mTORC1 signaling proteins. Similarly, we will treat Tsc2-RG and control mice from postnatal days 1-21 with the ODC inhibitor difluoromethylornithine (DFMO) and determine its effects on these same phenotypic parameters. This project is expected to expand our understanding of the molecular basis of TSC by establishing a novel function for polyamines in determining TSC pathology. Additionally, these studies may define novel targets for TSC therapeutics.