Expression of a Constitutively Active Human Insulin Receptor in Hippocampal Neurons Does Not Alter VGCC Currents.

Memory and cognitive decline are the product of numerous physiological changes within the aging brain. Multiple theories have focused on the oxidative, calcium, cholinergic, vascular, and inflammation hypotheses of brain aging, with recent evidence suggesting that reductions in insulin signaling may also contribute. Specifically, a reduction in insulin receptor density and mRNA levels has been implicated, however, overcoming these changes remains a challenge. While increasing insulin receptor occupation has been successful in offsetting cognitive decline, alternative molecular approaches should be considered as they could bypass the need for brain insulin delivery. Moreover, this approach may be favorable to test the impact of continued insulin receptor signaling on neuronal function. Here we used hippocampal cultures infected with lentivirus with or without IRß, a constitutively active, truncated form of the human insulin receptor, to characterize the impact continued insulin receptor signaling on voltage-gated calcium channels. Infected cultures were harvested between DIV 13 and 17 (48 h after infection) for Western blot analysis on pAKT and AKT. These results were complemented with whole-cell patch-clamp recordings of individual pyramidal neurons starting 96 h post-infection. Results indicate that while a significant increase in neuronal pAKT/AKT ratio was seen at the time point tested, effects on voltage-gated calcium channels were not detected. These results suggest that there is a significant difference between constitutively active insulin receptors and the actions of insulin on an intact receptor, highlighting potential alternate mechanisms of neuronal insulin resistance and mode of activation.

Exciton size and binding energy limitations in one-dimensional organic materials.

In current organic photovoltaic devices, the loss in energy caused by the charge transfer step necessary for exciton dissociation leads to a low open circuit voltage, being one of the main reasons for rather low power conversion efficiencies. A possible approach to avoid these losses is to tune the exciton binding energy to a value of the order of thermal energy, which would lead to free charges upon absorption of a photon, and therefore increase the power conversion efficiency towards the Shockley-Queisser limit. We determine the size of the excitons for different organic molecules and polymers by time dependent density functional theory calculations. For optically relevant transitions, the exciton size saturates around 0.7 nm for one-dimensional molecules with a size longer than about 4 nm. For the ladder-type polymer poly(benzimidazobenzophenanthroline), we obtain an exciton binding energy of about 0.3 eV, serving as a lower limit of the exciton binding energy for the organic materials investigated. Furthermore, we show that charge transfer transitions increase the exciton size and thus identify possible routes towards a further decrease of the exciton binding energy.

PDGF-A promoter and enhancer elements provide efficient and selective antineoplastic gene therapy in multiple cancer types.

Development of antineoplastic gene therapies is impaired by a paucity of transcription control elements with efficient, cancer cell-specific activity. We investigated the utility of promoter (AChP) and 5'-distal enhancer (ACE66) elements from the platelet-derived growth factor-A (PDGF-A) gene, which are hyperactive in many human cancers. Efficacy of these elements was tested in multiple tumor cell lines, both in cell culture and as tumor explants in athymic nude mice. Plasmid and viral vectors were constructed with the AChP promoter alone or in fusion with three copies of the ACE66 enhancer for expression of the prototype suicide gene, thymidine kinase (TK). ACE/AChP and AChP cassettes elicited ganciclovir (GCV)-induced cytotoxicity in multiple tumor cell lines. The ACE enhancer element also exhibited synergism with placental and liver-specific promoter elements. An adenovirus containing the AChP-TK cassette produced striking increases in GCV sensitivity in cultured tumor cell lines, as well as GCV-induced regression of U87 MG glioblastoma explants in vivo. TK expression was distributed throughout tumors receiving the therapeutic virus, whereas TdT-mediated dUTP nick end labeling (TUNEL) analysis revealed numerous regions undergoing apoptosis. Vascularization and reticulin fiber networks were less pronounced in virus-GCV-treated tumors, suggesting that both primary and stromal cell types may have been targeted. These studies provide proof-of-principle for utility of the PDGF-A promoter and ACE66 enhancer in antineoplastic gene therapy for a diverse group of human cancers.

Harnessing the power of gene microarrays for the study of brain aging and Alzheimer's disease: statistical reliability and functional correlation.

During normal brain aging, numerous alterations develop in the physiology, biochemistry and structure of neurons and glia. Aging changes occur in most brain regions and, in the hippocampus, have been linked to declining cognitive performance in both humans and animals. Age-related changes in hippocampal regions also may be harbingers of more severe decrements to come from neurodegenerative disorders such as Alzheimer's disease (AD). However, unraveling the mechanisms underlying brain aging, AD and impaired function has been difficult because of the complexity of the networks that drive these aging-related changes. Gene microarray technology allows massively parallel analysis of most genes expressed in a tissue, and therefore is an important new research tool that potentially can provide the investigative power needed to address the complexity of brain aging/neurodegenerative processes. However, along with this new analytic power, microarrays bring several major bioinformatics and resource problems that frequently hinder the optimal application of this technology. In particular, microarray analyses generate extremely large and unwieldy data sets and are subject to high false positive and false negative rates. Concerns also have been raised regarding their accuracy and uniformity. Furthermore, microarray analyses can result in long lists of altered genes, most of which may be difficult to evaluate for functional relevance. These and other problems have led to some skepticism regarding the reliability and functional usefulness of microarray data and to a general view that microarray data should be validated by an independent method. Given recent progress, however, we suggest that the major problem for current microarray research is no longer validity of expression measurements, but rather, the reliability of inferences from the data, an issue more appropriately redressed by statistical approaches than by validation with a separate method. If tested using statistically defined criteria for reliability/significance, microarray data do not appear a priori to require more independent validation than data obtained by any other method. In fact, because of added confidence from co-regulation, they may require less. In this article we also discuss our strategy of statistically correlating individual gene expression with biologically important endpoints designed to address the problem of evaluating functional relevance. We also review how work by ourselves and others with this powerful technology is leading to new insights into the complex processes of brain aging and AD, and to novel, more comprehensive models of aging-related brain change.

Congenital myasthenic syndrome of Brahman cattle in South Africa.

A congenital myasthenic syndrome in Brahman cattle is caused by a homozygous 20 base pair deletion (470del20) in the gene coding for the epsilon subunit of the acetylcholine receptor at the neuromuscular junction. It causes a progressive muscle weakness starting either at birth or within the first month. A PCR-based DNA test, using blood or semen stored on FTA paper, was developed and validated; the test makes it possible to differentiate rapidly and accurately between homozygous wild-type, heterozygous and homozygous affected animals. Preliminary testing of Brahman cattle in South Africa has revealed several carrier animals, some of them influential animals in the breeding population.

Immature end-plates and utrophin deficiency in congenital myasthenic syndrome caused by epsilon-AChR subunit truncating mutations.

Congenital myasthenic syndromes (CMS) are inborn disorders due to presynaptic, synaptic, or postsynaptic defects of neuromuscular transmission. Some previously described kinships with typical signs of CMS showed a marked deficiency of acetylcholine receptors (AChR) and utrophin at the neuromuscular junctions. Additionally, the end-plate ultrastructure was immature, with reduced enfolding of the postsynaptic membrane. In two such families, we found truncating mutations of the epsilon-AChR subunit. In family 1, both affected siblings were heteroallelic for a epsilon911delT and a epsilonIVS4+1G-->A mutation within the AChR epsilon-subunit gene (CHRNE). In the affected member of family 2, a epsilon1030delC mutation and a previously described epsilonR64X mutation were found. These deleterious epsilonAChR mutations not only result in AChR deficiency, but also affect end-plate maturation, including the formation of secondary synaptic clefts during ontogenesis.

Severe congenital myasthenic syndrome due to homozygosity of the 1293insG epsilon-acetylcholine receptor subunit mutation.

Recently, a congenital myasthenic syndrome (CMS) with end-plate acetylcholine receptor (AChR) deficiency due to missense mutations in the genes for the AChR subunit was described. The first observed patient with this CMS was heteroallelic for the two epsilon-AChR subunit mutations epsilon1101insT and epsilon1293insG. This patient had only a moderate phenotype with mild muscle weakness and abnormal fatigue. We have now found homozygosity for the epsilon1293insG mutation in a severely affected CMS patient, who lost the ability to walk in midchildhood and shows profound weakness and muscle wasting. Our observation allows a genotype-phenotype correlation illustrating how differences in the AChR mutation haplotype can profoundly influence disease severity.

Altered gene expression in steroid-treated denervated muscle.

In rats treated with high-dose corticosteroids, skeletal muscle that is denervated in vivo (steroid-denervated) develops electrical inexcitability similar to that seen in patients with acute quadriplegic myopathy. To determine whether changes in muscle gene transcription might underlie inexcitability of steroid-denervated muscle we performed RNase protection assays to quantitate adult (SkM1) and embryonic (SkM2) sodium channel isoforms and chloride channel (CLC-1) mRNA levels in control, denervated, steroid-innervated, and steroid-denervated skeletal muscle. While SkM1 mRNA levels were relatively unaffected by denervation or steroid treatment, SkM2 mRNA levels were increased by both. These effects were synergistic and high levels of SkM2 mRNA were expressed in denervated muscle exposed to corticosteroids. Skeletal muscle CLC-1 mRNA levels were decreased by denervation. To better understand the marked upregulation of SkM2 in steroid-denervated muscle we examined changes in myogenin and glucocorticoid receptor mRNA levels. However, changes in these mRNA levels cannot account for the upregulation of SkM2 in steroid-denervated muscle.

Disruption of Trkb-mediated signaling induces disassembly of postsynaptic receptor clusters at neuromuscular junctions.

Neurotrophins and tyrosine receptor kinase (Trk) receptors are expressed in skeletal muscle, but it is unclear what functional role Trk-mediated signaling plays during postnatal life. Full-length TrkB (trkB.FL) as well as truncated TrkB (trkB.t1) were found to be localized primarily to the postsynaptic acetylcholine receptor- (AChR-) rich membrane at neuromuscular junctions. In vivo, dominant-negative manipulation of TrkB signaling using adenovirus to overexpress trkB.t1 in mouse sternomastoid muscle fibers resulted in the disassembly of postsynaptic AChR clusters at neuromuscular junctions, similar to that observed in mutant trkB+/- mice. When TrkB-mediated signaling was disrupted in cultured myotubes in the absence of motor nerve terminals and Schwann cells, agrin-induced AChR clusters were also disassembled. These results demonstrate a novel role for neurotrophin signaling through TrkB receptors on muscle fibers in the ongoing maintenance of postsynaptic AChR regions.

Interaction between the skeletal muscle type 1 Na+ channel promoter E-box and an upstream repressor element. Release of repression by myogenin.

We have defined how four elements that regulate expression of the rat skeletal muscle type 1 sodium channel (SkM1) gene cooperate to yield specific expression in differentiated muscle. A basal promoter region containing within it a promoter E-box (-31/-26) is broadly expressed in many cells, including myoblasts and myotubes; mutations within the promoter E-box that disrupt binding of the myogenic basic helix-loop-helix (bHLH) factors reduce expression in all cell types only slightly. Sequential addition of upstream elements to the wild-type promoter confer increasing specificity of expression in differentiated cells, even though all three upstream elements, including a positive element (-85/-57), a repressor E-box (-90/-85), and upstream repressor sequences (-135/-95), bind ubiquitously expressed transcription factors. Mutations in the promoter E-box that disrupt the binding of the bHLH factors counteract the specificity conferred by addition of the upstream elements, with the greatest interaction observed between the upstream repressor sequences and the promoter E-box. Forced expression of myogenin in myoblasts releases repression exerted by the upstream repressor sequences in conjunction with the wild-type, but not mutant, promoter E-box, and also initiates expression of the endogenous SkM1 protein. Our data suggest that particular myogenic bHLH proteins bound at the promoter E-box control expression of SkM1 by releasing repression exerted by upstream repressor sequences in differentiated muscle cells.

Inactivation and secondary structure in the D4/S4-5 region of the SkM1 sodium channel.

The D4/S4-5 interhelical region plays a role in sodium channel fast inactivation. Examination of S4-5 primary structure in all domains suggests a possible amphipathic helical conformation in which a conserved group of small hydrophobic residues occupies one contiguous surface with a more variable complement of nonpolar and polar residues on the opposite face. We evaluated this potential structure by replacing each residue in D4/S4-5 of the rat SkM1 skeletal muscle sodium channel with substitutions having different side chain properties. Of the 63 mutations analyzed, 44 produced functional channels. P1473 was intolerant of substitutions. Nonpolar substitutions in the conserved hydrophobic region were functionally similar to wild type, while charged mutations in this region before P1473 were nonfunctional. Charged mutations at F1466, M1469, M1470, and A1474, located on the opposite surface of the predicted helix, produced functional channels with pronounced slowing of inactivation, shifted voltage dependence of steady-state inactivation, and increased rate of recovery from inactivation. The substituted-cysteine-accessibility method was used to probe accessibility at each position. Residues L1465, F1466, A1467, M1469, M1470, L1472, A1474, and F1476C were easily accessible for modification by sulfhydryl reagents; L1464, L1468, S1471, and L1475 were not accessible within the time frame of our measurements. Molecular dynamics simulations of residues A1458 to N1477 were then used to explore energetically favorable local structures. Based on mutagenesis, substituted-cysteine-accessibility method, and modeling results, we suggest a secondary structure for the D4/S4-5 region in which the peptide chain is alpha-helical proximal to P1473, bends at this residue, and may continue beyond this point as a random coil. In this configuration, the entire resultant loop is amphipathic; four residues on one surface could form part of the binding site for the inactivation particle.

Two E-boxes are the focal point of muscle-specific skeletal muscle type 1 Na+ channel gene expression.

We have characterized a group of cis-regulatory elements that control muscle-specific expression of the rat skeletal muscle type 1 sodium channel (SkM1) gene. These elements are located within a 3. 1-kilobase fragment that encompasses the 5'-flanking region, first exon, and part of the first intron of SkM1. We sequenced the region between -1062 and +311 and determined the start sites of transcription; multiple sites were identified between +1 and +30. The basal promoter (-65/+11) lacks cell-type specificity, while an upstream repressor (-174/-65) confers muscle-specific expression. A positive element (+49/+254) increases muscle-specific expression. Within these broad elements, two E boxes play a pivotal role. One E box at -31/-26 within the promoter, acting in part through its ability to bind the myogenic basic helix-loop-helix proteins, recruits additional factor(s) that bind elsewhere within the SkM1 sequence to control positive expression of the gene. A second E box at -90/-85 within the repressor controls negative regulation of the gene and acts through a different complex of proteins. Several of these cis-regulatory elements share both sequence and functional similarities with cis-regulatory elements of the acetylcholine receptor delta-subunit; the different arrangement of these elements may contribute to unique expression patterns for the two genes.

Analysis of local structure in the D2/S1-S2 region of the rat skeletal muscle type 1 sodium channel using insertional mutagenesis.

A reporter epitope was inserted at 11 positions in a region encompassing proposed transmembrane segments S1 and S2 in the second repeat domain (D2) of the rat skeletal muscle type 1 sodium channel. All mutations produced full-length membrane-associated protein following transfection into cultured cells, although the level of expression varied with insertion position. Characterization of cognate cRNAs for each mutation in Xenopus oocytes by two-electrode voltage clamp defined a permissive region between the proposed transmembrane regions in which these large insertions did not interfere with channel function. Two of the mutations, in which the point of insertion was within the proposed S1-S2 loop, demonstrated extracellular membrane labeling when studied either by antibody binding in oocytes or by confocal analysis following transfection into primary muscle cells. Our results define the likely boundaries of an extramembrane region linking the S1 and S2 transmembrane segments in D2 and confirm the extracellular location of this S1-S2 loop predicted by current models of channel tertiary structure.

Loss of electrical excitability in an animal model of acute quadriplegic myopathy.

In rats treated with high-dose corticosteroids, skeletal muscle that is denervated in vivo (steroid-denervated [S-D]) develops electrical inexcitability similar to that seen in patients with acute quadriplegic myopathy. In studies of affected muscles in vitro, the majority of S-D fibers failed to generate action potentials in response to intracellular stimulation although the average resting potential of these fibers was no different from that of control denervated muscle. The downregulation of membrane chloride conductance (G[Cl]) seen in normal muscle after denervation did not occur in S-D muscle. Although block of chloride channels in S-D muscle produced high specific membrane resistance, comparable to similarly treated control denervated muscle, and partially restored excitability in many fibers, action potential amplitude was still reduced in S-D fibers, suggesting a concomitant reduction in sodium current. 3H-saxitoxin binding measurements revealed a reduction in the density of the adult muscle sodium channel isoform in S-D muscle, suggesting that a decrease in the number of sodium channels present may play a role in the reduction of sodium current, although altered properties of channels may also contribute. The weakness seen in S-D muscle may involve the interaction of a number of factors that modify membrane excitability, including membrane depolarization, persistence of G(Cl), and reduced voltage-gated sodium currents.

REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons.

Expression of the type II voltage-dependent sodium channel gene is restricted to neurons by a silencer element active in nonneuronal cells. We have cloned cDNA coding for a transcription factor (REST) that binds to this silencer element. Expression of a recombinant REST protein confers the ability to silence type II reporter genes in neuronal cell types lacking the native REST protein, whereas expression of a dominant negative form of REST in nonneuronal cells relieves silencing mediated by the native protein. REST transcripts in developing mouse embryos are detected ubiquitously outside of the nervous system. We propose that expression of the type II sodium channel gene in neurons reflects a default pathway that is blocked in nonneuronal cells by the presence of REST.

Silencing the type II sodium channel gene: a model for neural-specific gene regulation.

Neural-specific expression of a sodium channel mini-gene has been shown to be mediated by a 28 bp silencer element, RE1, located in the 5' flanking region of the gene. This element is active exclusively in cell lines that do not express the endogenous brain type II sodium channel gene, including fibroblast, skeletal muscle, and certain neuronal cell lines. All of these non-type II expressing cells contain RE1-binding complexes. On the basis of mutational analysis and in vivo "repressor trap" experiments, we propose that cell-specific RE1-binding proteins are responsible, at least in part, for restricting expression of the type II sodium channel gene to specific neurons in the vertebrate nervous system.

Helping your patients who smoke quit for good. A quick and easy approach.

Cigarette smoking is the single most important behavior contributing to illness, disability, and death in the United States. However, by using appropriate techniques, physicians can help patients quit smoking, even when office visits are limited. Physicians who adopt the "differential diagnosis approach" (which categorizes smokers into stages of smoking cessation and offers specific counseling techniques) have increased confidence in their ability to help their patients. They thus can have a significant impact on the large percentage of smokers who visit physicians' offices each year.

Neuron-specific expression of the rat brain type II sodium channel gene is directed by upstream regulatory elements.

Genetic elements involved in cell-specific expression of the type II sodium channel gene were revealed using transient expression assays. A chimeric reporter gene containing 1051 bp of the sodium channel 5' flanking region was active in neuroblastoma and PC12 cells, but inactive in nonneuronal cell types. Deletion of upstream sequences resulted in an 80-fold increase in reporter gene activity in skeletal muscle cells, suggesting the presence of negative elements. Although no homologies were found between sequences in the type II 5' flanking region and other negative elements or "silencers," a small region common to the type II gene and other genes expressed in the nervous system was identified and may be involved in transcriptional regulation of neuronal genes.