Vijaya Ramesh

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.

NHE-RF, a Regulatory Cofactor for Na+-H+Exchange, Is a Common Interactor for Merlin and ERM (MERM) Proteins

We have identified the human homologue of a regulatory cofactor of Na+-H+ exchanger (NHE-RF) as a novel interactor for merlin, the neurofibromatosis 2 tumor suppressor protein. NHE-RF mediates protein kinase A regulation of Na+-H+ exchanger NHE3 to which it is thought to bind via one of its two PDZ domains. The carboxyl-terminal region of NHE-RF, downstream of the PDZ domains, interacts with the amino-terminal protein 4.1 domain-containing segment of merlin in yeast two-hybrid assays. This interaction also occurs in affinity binding assays with full-length NHE-RF expressed in COS-7 cells. NHE-RF binds to the related ERM proteins, moesin and radixin. We have localized human NHE-RF to actin-rich structures such as membrane ruffles, microvilli, and filopodia in HeLa and COS-7 cells, where it co-localizes with merlin and moesin. These findings suggest that hNHE-RF and its binding partners may participate in a larger complex (one component of which might be a Na+-H+exchanger) that could be crucial for the actin filament assembly activated by the ERM proteins and for the tumor suppressor function of merlin.

Mutations in transcript isoforms of the neurofibromatosis 2 gene in multiple human tumour types.

The neurofibromatosis 2 gene (NF2) has recently been isolated and predicted to encode a novel protein related to the moesin–ezrin–radixin family of cytoskeleton–associated proteins. Here we describe a novel isoform of the NF2 transcript that shows differential tissue expression and encodes a modified C terminus of the predicted protein. Mutations affecting both isoforms of the NF2 transcript were detected in multiple tumour types including melanoma and breast carcinoma. These findings provide evidence that alterations in the NF2 transcript occur not only in the hereditary brain neoplasms typically associated with NF2, but also as somatic mutations in their sporadic counterparts and in seemingly unrelated tumour types. The NF2 gene may thus constitute a tumour suppressor gene of more general importance in tumorigenesis.

Survey of Somatic Mutations in Tuberous Sclerosis Complex (TSC) Hamartomas Suggests Different Genetic Mechanisms for Pathogenesis of TSC Lesions

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumor-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at loci of both TSC1 and TSC2. LOH has been documented in renal angiomyolipomas (AMLs), but loss of the wild-type allele in cortical tubers appears to be very uncommon. Analysis of second, somatic events in tumors for which the status of both TSC1 and TSC2 is known is essential for exploration of the pathogenesis of TSC-lesion development. We analyzed 24 hamartomas from 10 patients for second-hit mutations, by several methods, including LOH, scanning of all exons of both TSC1 and TSC2, promoter methylation of TSC2, and clonality analysis. Our results document loss of the wild-type allele in six of seven AMLs, without evidence of the inactivation of the second allele in many of the other lesions, including tumors that appear to be clonally derived. Laser-capture microdissection further demonstrated loss of the second allele in all three cellular components of an AML. This study thus provides evidence that, in both TSC1 and TSC2, somatic mutations resulting in the loss of wild-type alleles may not be necessary in some tumor types-and that other mechanisms may contribute to tumorigenesis in this setting.

NF2/Merlin Is a Novel Negative Regulator of mTOR Complex 1, and Activation of mTORC1 Is Associated with Meningioma and Schwannoma Growth

ABSTRACT Inactivating mutations of the neurofibromatosis 2 (NF2) gene, NF2, result predominantly in benign neurological tumors, schwannomas and meningiomas, in humans; however, mutations in murine Nf2 lead to a broad spectrum of cancerous tumors. The tumor-suppressive function of the NF2 protein, merlin, a membrane-cytoskeleton linker, remains unclear. Here, we identify the mammalian target of rapamycin complex 1 (mTORC1) as a novel mediator of merlins tumor suppressor activity. Merlin-deficient human meningioma cells and merlin knockdown arachnoidal cells, the nonneoplastic cell counterparts of meningiomas, exhibit rapamycin-sensitive constitutive mTORC1 activation and increased growth. NF2 patient tumors and Nf2-deficient mouse embryonic fibroblasts demonstrate elevated mTORC1 signaling. Conversely, the exogenous expression of wild-type merlin isoforms, but not a patient-derived L64P mutant, suppresses mTORC1 signaling. Merlin does not regulate mTORC1 via the established mechanism of phosphoinositide 3-kinase-Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase-mediated TSC2 inactivation and may instead regulate TSC/mTOR signaling in a novel fashion. In conclusion, the deregulation of mTORC1 activation underlies the aberrant growth and proliferation of NF2-associated tumors and may restrain the growth of these lesions through negative feedback mechanisms, suggesting that rapamycin in combination with phosphoinositide 3-kinase inhibitors may be therapeutic for NF2.

Admixture mapping of an allele affecting interleukin 6 soluble receptor and interleukin 6 levels.

Circulating levels of inflammatory markers can predict cardiovascular disease risk. To identify genes influencing the levels of these markers, we genotyped 1,343 single-nucleotide polymorphisms (SNPs) in 1,184 African Americans from the Health, Aging and Body Composition (Health ABC) Study. Using admixture mapping, we found a significant association of interleukin 6 soluble receptor (IL-6 SR) with European ancestry on chromosome 1 (LOD 4.59), in a region that includes the gene for this receptor (IL-6R). Genotyping 19 SNPs showed that the effect is largely explained by an allele at 4% frequency in West Africans and at 35% frequency in European Americans, first described as associated with IL-6 SR in a Japanese cohort. We replicate this association (P<<1.0x10-12) and also demonstrate a new association with circulating levels of a different molecule, IL-6 (P<3.4x10-5). After replication in 1,674 European Americans from Health ABC, the combined result is even more significant: P<<1.0x10-12 for IL-6 SR, and P<2.0x10-9 for IL-6. These results also serve as an important proof of principle, showing that admixture mapping can not only coarsely localize but can also fine map a phenotypically important variant.

TorsinA protein and neuropathology in early onset generalized dystonia with GAG deletion

Familial, early onset, generalized torsion dystonia is the most common and severe primary dystonia. Most cases are caused by a 3-bp deletion (GAG) in the coding region of the TOR1A (DYT1) gene, which is widely expressed in human brain and encodes the protein torsinA. This study compares neuropathology and torsinA expression in the normal human brain with that in dystonia cases with and without the GAG deletion. TorsinA-like protein was expressed in neuronal cytoplasm throughout the human brain, including cerebellum, substantia nigra, hippocampus, and neostriatum, with higher levels in specific neurons. This immunostaining pattern was not discernibly different in dystonia and normal brains in midbrain and neostriatal regions. However, nigral dopaminergic neurons appeared to be larger in both GAG-deletion and non-GAG-deletion dystonia brains compared to normal, and may be more closely spaced in GAG-deletion brains. Beyond these apparent changes in neuronal size and spacing in dystonia brains, there was no indication of neuron loss, inflammation, DNA strand breaks, or altered distribution of torsin-like immunoreactivity, supporting a functional rather than degenerative etiology of early onset torsion dystonia.

Analysis of both TSC1 and TSC2 for germline mutations in 126 unrelated patients with tuberous sclerosis

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the development of multiple hamartomas involving many organs. About two‐thirds of the cases are sporadic and appear to represent new mutations. With the cloning of two causative genes, TSC1 and TSC2 it is now possible to analyze both genes in TSC patients and identify germline mutations. Here we report the mutational analysis of the entire coding region of both TSC1 and TSC2 genes in 126 unrelated TSC patients, including 40 familial and 86 sporadic cases, by single‐stranded conformational polymorphism (SSCP) analysis followed by direct sequencing. Mutations were identified in a total of 74 (59%) cases, including 16 TSC1 mutations (5 sporadic and 11 familial cases) and 58 TSC2 mutations (42 sporadic and 16 familial cases). Overall, significantly more TSC2 mutations were found in our population, with a relatively equal distribution of mutations between TSC1 and TSC2 among the familial cases, but a marked underrepresentation of TSC1 mutations among the sporadic cases (P = 0.0035, Fishers exact test). All TSC1 mutations were predicted to be protein truncating. However, in TSC2 13 missense mutations were found, five clustering in the GAP‐related domain and three others occurring in exon 16. Upon comparison of clinical manifestations, including the incidence of intellectual disability, we could not find any observable differences between TSC1 and TSC2 patients. Our data help define the distribution and spectrum of mutations associated with the TSC loci and will be useful for both understanding the function of these genes as well as genetic counseling in patients with the disease. Hum Mutat 14:412–422, 1999.

Neuropathology and Molecular Genetics of Neurofibromatosis 2 and Related Tumors

Neurofibromatosis 2 (NF2) is an uncommon, autosomal dominant disorder in which patients are predisposed to neoplastic and dysplastic lesions of Schwann cells (schwannomas and schwannosis), meningeal cells (meningiomas and meningioan‐giomatosis) and glial cells (gliomas and glial hamar‐tomas). Clinical and genetic criteria that distinguish NF2 from neurofibromatosis 1 have allowed more accurate assignment of specific pathological features to NF2. The NF2 tumor suppressor gene on chromosome 22q12 encodes a widely expressed protein, named merlin, which may link the cytoskeleton and cell membrane. Germline NF2 mutations in NF2 patients and somatic NF2 mutations in sporadic schwannomas and meningiomas have different mutational spectra, but most NF2 alterations result in a truncated, inactivated merlin protein. In NF2 patients, specific mutations do not necessarily correlate with phenotypic severity, although grossly truncating alterations may result in a more severe phenotype. In schwannomas, NF2 mutations are common and may be necessary for tumorigenesis. In meningiomas, NF2 mutations occur more commonly in fibroblastic than meningothelial subtypes, and may cluster in the first half of the gene. In addition, in meningiomas, a second, non‐NF2 meningioma locus is probably also involved. Future efforts in NF2 research will be directed toward elucidating the role of merlin in the normal cell and the sequelae of its inactivation in human tumors.

Frequency and distribution of NF2 mutations in schwannomas

Sporadic and inherited schwannomas were scanned for the nature, frequency, and distribution of mutations in the NF2 locus encoding the merlin tumor suppressor protein on 22q. Of 58 tumors, 47% displayed loss of heterozygosity for NF2, leaving a total of 89 NF2 alleles to be examined. Pathogenic alterations were identified in 62 of these alleles, including 36 frameshifts with premature termination, 14 nonsense mutations, and 12 changes presumed to affect splicing. Effects of ten of the latter were confirmed in the NF2 transcript and indicated that activation of cryptic splice sites in coding sequence is another frequent mechanism leading to truncation of merlin. The mutations were relatively evenly distributed across both the protein 4.1 superfamily (exons 1–9) and the α‐helical (exons 10–15) domains of merlin, but they did not occur at all in exons 16 and 17, which encode the proteins alternative COOH‐termini. The data support the “two‐hit” tumor suppressor model for formation of schwannomas and indicate that loss of merlin function can be achieved by truncation at various locations in the protein. However, the absence of mutations in exons 16 and 17 suggests that an inactivating mutation affecting only one of the merlins alternative termini may not be sufficient to eliminate tumor suppressor function. Genes Chromosom Cancer 17:45–55 (1996).