The autism-associated gene chromodomain helicase DNA-binding protein 8 (CHD8) regulates noncoding RNAs and autism-related genes.

Chromodomain helicase DNA-binding protein 8 (CHD8) was identified as a leading autism spectrum disorder (ASD) candidate gene by whole-exome sequencing and subsequent targeted-sequencing studies. De novo loss-of-function mutations were identified in 12 individuals with ASD and zero controls, accounting for a highly significant association. Small interfering RNA-mediated knockdown of CHD8 in human neural progenitor cells followed by RNA sequencing revealed that CHD8 insufficiency results in altered expression of 1715 genes, including both protein-coding and noncoding RNAs. Among the 10 most changed transcripts, 4 (40%) were noncoding RNAs. The transcriptional changes among protein-coding genes involved a highly interconnected network of genes that are enriched in neuronal development and in previously identified ASD candidate genes. These results suggest that CHD8 insufficiency may be a central hub in neuronal development and ASD risk.

Association of a MET genetic variant with autism-associated maternal autoantibodies to fetal brain proteins and cytokine expression.

The contribution of peripheral immunity to autism spectrum disorders (ASDs) risk is debated and poorly understood. Some mothers of children with ASD have autoantibodies that react to fetal brain proteins, raising the possibility that a subset of ASD cases may be associated with a maternal antibody response during gestation. The mechanism by which the maternal immune system breaks tolerance has not been addressed. We hypothesized that the mechanism may involve decreased expression of the MET receptor tyrosine kinase, an ASD risk gene that also serves as a key negative regulator of immune responsiveness. In a sample of 365 mothers, including 202 mothers of children with ASD, the functional MET promoter variant rs1858830 C allele was strongly associated with the presence of an ASD-specific 37+73-kDa band pattern of maternal autoantibodies to fetal brain proteins (P=0.003). To determine the mechanism of this genetic association, we measured MET protein and cytokine production in freshly prepared peripheral blood mononuclear cells from 76 mothers of ASD and typically developing children. The MET rs1858830 C allele was significantly associated with MET protein expression (P=0.025). Moreover, decreased expression of the regulatory cytokine IL-10 was associated with both the MET gene C allele (P=0.001) and reduced MET protein levels (P=0.002). These results indicate genetic distinction among mothers who produce ASD-associated antibodies to fetal brain proteins, and suggest a potential mechanism for how a genetically determined decrease in MET protein production may lead to a reduction in immune regulation.

Return of collective rotation in 157Er and 158Er at ultrahigh spin.

A new frontier of discrete-line gamma-ray spectroscopy at ultrahigh spin has been opened in the rare-earth nuclei (157,158) Er. Four rotational structures, displaying high moments of inertia, have been identified, which extend up to spin approximately 65 variant Planck's over 2pi and bypass the band-terminating states in these nuclei which occur at approximately 45 variant Planck's over 2pi. Cranked Nilsson-Strutinsky calculations suggest that these structures arise from well-deformed triaxial configurations that lie in a valley of favored shell energy which also includes the triaxial strongly deformed bands in (161-167) Lu.

Bacterial artificial chromosome transgenic analysis of dynamic expression patterns of regulator of G-protein signaling 4 during development. II. Subcortical regions.

A large family of regulator of G protein signaling (RGS) proteins modulates signaling through G-protein-coupled receptors. Previous studies have implicated RGS4 as a vulnerability gene in schizophrenia. To begin to understand structure-function relationships, we have utilized bacterial artificial chromosome (BAC) methods to create transgenic mice that express green fluorescent protein (GFP) under the control of endogenous RGS4 enhancer elements, circumventing the lack of suitable antibodies for analysis of dynamic patterns of expression. This report follows from the accompanying mapping paper in cerebral cortex, with a focus on developmental and mature expression patterns in subcortical telencephalic, diencephalic and brainstem areas. Based on reporter distribution, the data suggest that alterations in RGS4 function will engender a complex phenotype of increased and decreased neuronal output, with developmental, regional, and cellular specificity.

Bacterial artificial chromosome transgenic analysis of dynamic expression patterns of regulator of G-protein signaling 4 during development. I. Cerebral cortex.

Signaling through G-protein-coupled receptors is modulated by a family of regulator of G protein signaling (RGS) proteins that have been implicated in several neurological and psychiatric disorders. Defining the detailed expression patterns and developmental regulation of RGS proteins has been hampered by an absence of antibodies useful for mapping. We have utilized bacterial artificial chromosome (BAC) methods to create transgenic mice that express GFP under the control of endogenous regulator of G-protein signaling 4 (RGS4) enhancer elements. This report focuses on expression patterns in the developing and mature cerebral cortex. Based on reporter distribution, RGS4 is expressed by birth in neurons across all cortical domains, but in different patterns that suggest region- and layer-specific regulation. Peak expression typically occurs before puberty, with complex down-regulation by adulthood. Deep and superficial neurons, in particular, vary in their patterns across developmental age and region and, in primary sensory cortices, layer IV neurons exhibit low or no expression of the GFP reporter. These data suggest that altering RGS4 function will produce a complex neuronal phenotype with cell- and subdomain-specificity in the cerebral cortex.

p-sd Shell gap reduction in neutron-rich systems and cross-shell excitations in 20 O.

Excited states in 20O were populated in the reaction 10Be(14C,alpha) at Florida State University (FSU). Charged particles were detected with a particle telescope consisting of 4 annularly segmented Si surface barrier detectors and gamma radiation was detected with the FSU gamma detector array. Five new states were observed below 6 MeV from the alpha-gamma and alpha-gamma-gamma coincidence data. Shell model calculations suggest that most of the newly observed states are core-excited 1p-1h excitations across the N=Z=8 shell gap. Comparisons between experimental data and calculations for the neutron-rich O and F isotopes imply a steady reduction of the p-sd shell gap as neutrons are added.

High-spin structure beyond band termination in 157Er.

The angular-momentum induced transition from a deformed state of collective rotation to a noncollective configuration has been studied. In 157Er this transition manifests itself as favored band termination near I=45 Planck's. The feeding of these band terminating states has been investigated for the first time using the Gammasphere spectrometer. Many weakly populated states lying at high excitation energy that decay into these special states have been discovered. Cranked Nilsson-Strutinsky calculations suggest that these states arise from weakly collective "core-breaking" configurations.

Binary asteroids in the near-Earth object population.

Radar images of near-Earth asteroid 2000 DP107 show that it is composed of an approximately 800-meter-diameter primary and an approximately 300-meter-diameter secondary revolving around their common center of mass. The orbital period of 1.755 +/- 0.007 days and semimajor axis of 2620 +/- 160 meters constrain the total mass of the system to 4.6 +/- 0.5 x 10(11) kilograms and the bulk density of the primary to 1.7 +/- 1.1 grams per cubic centimeter. This system and other binary near-Earth asteroids have spheroidal primaries spinning near the breakup point for strengthless bodies, suggesting that the binaries formed by spin-up and fission, probably as a result of tidal disruption during close planetary encounters. About 16% of near-Earth asteroids larger than 200 meters in diameter may be binary systems.

Topography of the lunar poles from radar interferometry: a survey of cold trap locations.

Detailed topographic maps of the lunar poles have been obtained by Earth-based radar interferometry with the 3.5-centimeter wavelength Goldstone Solar System Radar. The interferometer provided maps 300 kilometers by 1000 kilometers of both polar regions at 150-meter spatial resolution and 50-meter height resolution. Using ray tracing, these digital elevation models were used to locate regions that are in permanent shadow from solar illumination and may harbor ice deposits. Estimates of the total extent of shadowed areas poleward of 87.5 degrees latitude are 1030 and 2550 square kilometers for the north and south poles, respectively.

L-type calcium channels contribute to the tottering mouse dystonic episodes.

Tottering mice inherit a recessive mutation of the calcium channel alpha1A subunit that causes ataxia, polyspike discharges, and intermittent dystonic episodes. The calcium channel alpha1A subunit gene encodes the pore-forming protein of P/Q-type voltage-dependent calcium channels and is predominantly expressed in cerebellar granule and Purkinje neurons with moderate expression in hippocampus and inferior colliculus. Because calcium misregulation likely underlies the tottering mouse phenotype, calcium channel blockers were tested for their ability to block the motor episodes. Pharmacologic agents that specifically block L-type voltage-dependent calcium channels, but not P/Q-type calcium channels, prevented the inducible dystonia of tottering mutant mice. Specifically, the dihydropyridines nimodipine, nifedipine, and nitrendipine, the benzothiazepine diltiazem, and the phenylalkylamine verapamil all prevented restraint-induced tottering mouse motor episodes. Conversely, the L-type calcium channel agonist Bay K8644 induced stereotypic tottering mouse dystonic at concentrations significantly below those required to induce seizures in control mice. In situ hybridization demonstrated that L-type calcium channel alpha1C subunit mRNA expression was up-regulated in the Purkinje cells of tottering mice. Radioligand binding with [3H]nitrendipine also revealed a significant increase in the density of L-type calcium channels in tottering mouse cerebellum. These data suggest that although a P/Q-type calcium channel mutation is the primary defect in tottering mice, L-type calcium channels may contribute to the generation of the intermittent dystonia observed in these mice. The susceptibility of L-type calcium channels to voltage-dependent facilitation may promote this abnormal motor phenotype.

Tottering mouse motor dysfunction is abolished on the Purkinje cell degeneration (pcd) mutant background.

Tottering (tg) mice inherit a recessive mutation of the calcium channel alpha 1A subunit gene, which encodes the pore-forming protein of P/Q-type voltage-sensitive calcium channels and is predominantly expressed in cerebellar granule and Purkinje neurons. The phenotypic consequences of the tottering mutation include ataxia, polyspike discharges, and an intermittent motor dysfunction best described as paroxysmal dystonia. These dystonic episodes induce c-fos mRNA expression in the cerebellar circuitry, including cerebellar granule and Purkinje neurons, deep cerebellar nuclei, and the postsynaptic targets of the deep nuclei. Cellular abnormalities associated with the mutation include hyperarborization of brainstem nucleus locus ceruleus axons and abnormal expression of L-type calcium channels in cerebellar Purkinje cells. Here, the role of these two distinct neural pathways in the expression of tottering mouse intermittent dystonia was assessed. Lesion of locus ceruleus axons with the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine (DSP-4) did not affect the frequency of tottering mouse dystonic episodes. In contrast, removal of cerebellar Purkinje cells with the Purkinje cell degeneration (pcd) mutation by generation of tg/tg; pcd/pcd double mutant mice completely eliminated tottering mouse dystonia. Further, the c-fos expression pattern of tg/tg; pcd/pcd double mutants following restraint was indistinguishable from that of wild-type mice, suggesting that the pcd lesion eliminated an essential link in this abnormal neural network. These data suggest that the cerebellar cortex, where the mutant gene is abundantly expressed, contributes to the expression of tottering mouse dystonic episodes.

H-cadherin expression inhibits in vitro invasiveness and tumor formation in vivo.

H-cadherin is a newly characterized cadherin molecule whose expression is decreased in a variety of human carcinoma cells, suggesting that it may play a role in maintaining normal cellular phenotype. To investigate how re-expression of H-cadherin could influence the malignant phenotype of human breast carcinoma cells in vivo, we transfected both control and H-cadherin expression vectors into human breast cancer cells (MDAMB435), which do not express H-cadherin constitutively. We found that invasiveness of these cells could be prevented by transfection with H-cadherin. We also compared the ability of control- and H-cadherin-transfected cells to induce subcutaneous tumors after injection into mammary fat pads of nude mice. Our results show that H-cadherin transfection produced a marked inhibition of tumor growth and modified the morphology of tumor cells: tumors from mice injected with control cells were significantly larger and contained larger cells having a higher degree of pleomorphism than those of tumors generated from carcinoma cells expressing H-cadherin. Altogether, these results indicate that H-cadherin expression antagonizes tumor growth in nude mice, presumably by enhancing cell-cell association in a tissue environment. These findings strongly suggest that H-cadherin could provide a possible target for corrective gene therapy against breast cancer.

Cerebellar circuitry is activated during convulsive episodes in the tottering (tg/tg) mutant mouse.

Tottering (tg) is an autosomal recessive mutation of the calcium channel alpha1A subunit in the mouse that results in epileptic spike and wave discharges, mild ataxia and paroxysmal episodes of involuntary spasms of the limbs, trunk and face. These convulsions have been especially difficult to characterize because of their unpredictable occurrence and lack of electroencephalographic correlates. However, it is, in fact, possible to induce these convulsions, making this facet of the tottering phenotype amenable to controlled experimentation for the first time. Here, the neuroanatomical basis of the convulsions in tottering mice has been identified using in situ hybridization for c-fos messenger RNA to chart abnormal neuronal activity. Convulsion-induced c-fos messenger RNA expression was most prominent in the cerebellum of convulsing tottering mice. Additionally, cerebral cortex and principal cerebellar relay nuclei were also activated during a convulsion. The c-fos activation in the cerebellum temporally preceded expression in cerebral cortex, suggesting that cerebral cortex is not driving the expression of convulsions. These results suggest that the cerebellum, a region not classically associated with paroxysmal events, is important in the generation and/or maintenance of the intermittent convulsions in tottering mutant mice.

Tumor cell growth inhibition by caveolin re-expression in human breast cancer cells.

Cancer development is a multistage process that results from the step-wise acquisition of somatic alterations in diverse genes. Recent studies indicate that caveolin-1 expression correlates with the level of oncogenic transformation in NIH3T3 cells, suggesting that caveolin in caveolae may regulate normal cell proliferation. In order to better understand potential functions of caveolin-1 in cancer development, we have studied expression levels of caveolin-1 in human breast cancer cells, and have found that caveolin expression is significantly reduced in human breast cancer cells compared with their normal mammary epithelial counterparts. When the caveolin cDNA linked to the CMV promoter is transfected into human mammary cancer cells having no detectable endogenous caveolin, overexpression of caveolin-1 resulted in substantial growth inhibition, as seen by the 50% decrease in growth rate and by approximately 15-fold reduction in colony formation in soft agar. In addition, characterization of caveolin-1 expression during cell cycle progression indicates that expression of alpha-caveolin-1 is regulated during cell cycle. Furthermore p53-deficient cells showed a loss in caveolin expression. In summary, the overall expression patterns, its ability to inhibit tumor growth in culture, its regulation during the cell cycle, and the loss of expression in p53-deficient cells all are consistent with an important growth regulating function for caveolin-1 in normal human mammary cells, that needs to be repressed in oncogenic transformation and tumor cell growth.

The role of radiopharmacological imaging in streamlining the drug development process.

Radioimaging techniques have found a place in clinical diagnosis, but there has been a hesitancy to use this approach in drug development. This reluctance may have been due to the availability of ligands, the time and cost of synthesis and the number of centres and, although these perceived problems have been largely overcome, for many the benefits are not evident. The use in drug development is potentially large since tomography can measure drug levels, specific binding, blood flow and activity within the human body. In drug discovery, combinational chemistry and high throughput screening, the synthesis of candidate drugs with specific binding properties are dependent on understanding the disease and using appropriate in vitro or animal models. Using small animal tomographs, these can be validated using radioimaging. Pharmacokinetics and metabolic problems, such as the distribution of inhaled gases, drug targeting into tumours of the brain or specific gastrointestinal absorption sites can be investigated within the human rather than relying on animals. The high specific activity allows low doses to be administered to man with limited safety studies, permitting kinetic and metabolic studies to be undertaken early in development. Safety studies and ensuing toxicological endpoints in animals rely on histopathology for gross degenerative in physiological function. Where concern exists, radioimaging could detect early in situ changes in humans, for example hepatic toxicity, before they become hazardous. In clinical studies, the action of drugs can be measured directly at the effector site prior to undertaking longer studies, which is important for many diseases, but particularly for those such as Alzheimer's disease, where improvements may be slow or subtle.