Developmental critical period in genetic redox dysregulation: animal and human studies in schizophrenia

Converging evidence favors an abnormal susceptibility to oxidative stress in schizophrenia. Decreased levels of glutathione (GSH), the major cellular antioxidant and redox regulator, was observed in cerebrospinal-fluid and prefrontal cortex of patients. Importantly, abnormal GSH synthesis of genetic origin was observed: Two case-control studies showed an association with a GAG trinucleotide repeat (TNR) polymorphism in the GSH key synthesizing enzyme glutamate-cysteine-ligase (GCL) catalytic subunit (GCLC) gene. The most common TNR genotype 7/7 was more frequent in controls, whereas the rarest TNR genotype 8/8 was three times more frequent in patients. The disease associated genotypes (35% of patients) correlated with decreased GCLC protein, GCL activity and GSH content. Similar GSH system anomalies were observed in early psychosis patients. Such redox dysregulation combined with environmental stressors at specific developmental stages could underlie structural and functional connectivity anomalies. In pharmacological and knock-out (KO) models, GSH deficit induces anomalies analogous to those reported in patients. (a) morphology: spine density and GABA-parvalbumine immunoreactivity (PV-I) were decreased in anterior cingulate cortex. KO mice showed delayed cortical PV-I at PD10. This effect is exacerbated in mice with increased DA from PD5-10. KO mice exhibit cortical impairment in myelin and perineuronal net known to modulate PV connectivity. (b) physiology: In cultured neurons, NMDA response are depressed by D2 activation. In hippocampus, NMDA-dependent synaptic plasticity is impaired and kainate induced g-oscillations are reduced in parallel to PV-I. (c) cognition: low GSH models show increased sensitivity to stress, hyperactivity, abnormal object recognition, olfactory integration and social behavior. In a clinical study, GSH precursor N-acetyl cysteine (NAC) as add on therapy, improves the negative symptoms and decreases the side effects of antipsychotics. In an auditory oddball paradigm, NAC improves the mismatched negativity, an evoked potential related to pre-attention and to NMDA receptors function. In summary, clinical and experimental evidence converge to demonstrate that a genetically induced dysregulation of GSH synthesis combined with environmental insults in early development represent a major risk factor contributing to the development of schizophrenia

An update of the mechanisms of resistance to EGFR-tyrosine kinase inhibitors in breast cancer: gefitinib (Iressatrade mark)-induced changes in the expression and nucleo-cytoplasmic trafficking of HER-ligands (Review)

Intrinsic resistance to the epidermal growth factor receptor (EGFR; HER1) tyrosine kinase inhibitor (TKI) gefitinib, and more generally to EGFR TKIs, is a common phenomenon in breast cancer. The availability of molecular criteria for predicting sensitivity to EGFR-TKIs is, therefore, the most relevant issue for their correct use and for planning future research. Though it appears that in non-small-cell lung cancer (NSCLC) response to gefitinib is directly related to the occurrence of specific mutations in the EGFR TK domain, breast cancer patients cannot be selected for treatment with gefitinib on the same basis as such EGFR mutations have beenreported neither in primary breast carcinomas nor in several breast cancer cell lines. Alternatively, there is a generalagreement on the hypothesis that the occurrence of molecular alterations that activate transduction pathways downstreamof EGFR (i.e., MEK1/MEK2 - ERK1/2 MAPK and PI-3'K - AKT growth/survival signaling cascades) significantly affect the response to EGFR TKIs in breast carcinomas. However,there are no studies so far addressing a role of EGF-related ligands as intrinsic breast cancer cell modulators of EGFR TKIefficacy. We recently monitored gene expression profiles andsub-cellular localization of HER-1/-2/-3/-4 related ligands (i.e., EGF, amphiregulin, transforming growth factor-α, ß-cellulin,epiregulin and neuregulins) prior to and after gefitinib treatment in a panel of human breast cancer cell lines. First, gefitinibinduced changes in the endogenous levels of EGF-related ligands correlated with the natural degree of breast cancer cellsensitivity to gefitinib. While breast cancer cells intrinsically resistant to gefitinib (IC50 ≥15 μM) markedly up-regulated(up to 600 times) the expression of genes codifying for HERspecific ligands, a significant down-regulation (up to 106 times)of HER ligand gene transcription was found in breast cancer cells intrinsically sensitive to gefitinib (IC50 ≤1 μM). Second,loss of HER1 function differentially regulated the nuclear trafficking of HER-related ligands. While gefitinib treatment induced an active import and nuclear accumulation of the HER ligand NRG in intrinsically gefitinib-resistant breastcancer cells, an active export and nuclear loss of NRG was observed in intrinsically gefitinib-sensitive breast cancer cells.In summary, through in vitro and pharmacodynamic studies we have learned that, besides mutations in the HER1 gene,oncogenic changes downstream of HER1 are the key players regulating gefitinib efficacy in breast cancer cells. It now appears that pharmacological inhibition of HER1 functionalso leads to striking changes in both the gene expression and the nucleo-cytoplasmic trafficking of HER-specific ligands,and that this response correlates with the intrinsic degree of breast cancer sensitivity to the EGFR TKI gefitinib. Therelevance of this previously unrecognized intracrine feedback to gefitinib warrants further studies as cancer cells could bypassthe antiproliferative effects of HER1-targeted therapeutics without a need for the overexpression and/or activation of other HER family members and/or the activation of HER-driven downstream signaling cascades

Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

Protein oligomers studied by solid-state NMR the case of the full-length nucleoid-associated protein histone-like nucleoid structuring protein

Members of the histone-like nucleoid structuring protein (H-NS) family play roles both as architectural proteins and as modulators of gene expression in Gram-negative bacteria. The H-NS protein participates in modulatory processes that respond to environmental changes in osmolarity, pH, or temperature. H-NS oligomerization is essential for its activity. Structural models of different truncated forms are available. However, high-resolution structural details of full-length H-NS and its DNA-bound state have largely remained elusive. We report on progress in characterizing the biologically active H-NS oligomers with solid-state NMR. We compared uniformly ((13)C,(15)N)-labeled ssNMR preparations of the isolated N-terminal region (H-NS 1-47) and full-length H-NS (H-NS 1-137). In both cases, we obtained ssNMR spectra of good quality and characteristic of well-folded proteins. Analysis of the results of 2D and 3D (13)C-(13)C and (15)N-(13)C correlation experiments conducted at high magnetic field led to assignments of residues located in different topological regions of the free full-length H-NS. These findings confirm that the structure of the N-terminal dimerization domain is conserved in the oligomeric full-length protein. Small changes in the dimerization interface suggested by localized chemical shift variations between solution and solid-state spectra may be relevant for DNA recoginition.

Electrical pump & probe and injected carrier losses quantification in Er doped Si slot waveguides

Electrically driven Er3+ doped Si slot waveguides emitting at 1530 nm are demonstrated. Two different Er3+ doped active layers were fabricated in the slot region: a pure SiO2 and a Si-rich oxide. Pulsed polarization driving of the waveguides was used to characterize the time response of the electroluminescence (EL) and of the signal probe transmission in 1 mm long waveguides. Injected carrier absorption losses modulate the EL signal and, since the carrier lifetime is much smaller than that of Er3+ ions, a sharp EL peak was observed when the polarization was switched off. A time-resolved electrical pump & probe measurement in combination with lock-in amplifier techniques allowed to quantify the injected carrier absorption losses. We found an extinction ratio of 6 dB, passive propagation losses of about 4 dB/mm, and a spectral bandwidth > 25 nm at an effective d.c. power consumption of 120 μW. All these performances suggest the usage of these devices as electro-optical modulators.

Nuclear receptor peroxisome proliferator activated receptor (PPAR) beta/delta in skin wound healing and cancer

We review the functions of peroxisome proliferator activated receptor (PPAR) beta/delta in skin wound healing and cancer. In particular, we highlight the roles of PPAR beta/delta in inhibiting keratinocyte apoptosis at wound edges via activation of the PI3K/PKB alpha/Akt1 pathway and its role during re-epithelialization in regulating keratinocyte adhesion and migration. In fibroblasts, PPAR beta/delta controls IL-1 signalling and thereby contributes to the homeostatic control of keratinocyte proliferation. We discuss its therapeutic potential for treating diabetic wounds and inflammatory skin diseases such as psoriasis and acne vulgaris. PPAR beta/delta is classified as a tumour growth modifier; it is activated by chronic low-grade inflammation, which promotes the production of lipids that, in turn, enhance PPAR beta/delta transcription activity. Our earlier,work unveiled a cascade of events triggered by PPAR beta/delta that involve the oncogene Src, which promotes ultraviolet-induced skin cancer in mice via enhanced EGFR/Erk1/2 signalling and the expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, PPAR beta/delta expression is correlated with the expression of SRC and EMT markers in human skin squamous cell carcinoma. Furthermore, there is a positive interaction between PPAR beta/delta, SRC, and TGF beta 1 at the transcriptional level in various human epithelial cancers. Taken together, these observations suggest the need for evaluating PPAR beta/delta modulators that attenuate or increase its activity, depending on the therapeutic target.

A highly expressed miR-101 isomiR is a functional silencing small RNA

Background MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression usually producing translational repression and gene silencing. High-throughput sequencing technologies have revealed heterogeneity at length and sequence level for the majority of mature miRNAs (IsomiRs). Most isomiRs can be explained by variability in either Dicer1 or Drosha cleavage during miRNA biogenesis at 5" or 3" of the miRNA (trimming variants). Although isomiRs have been described in different tissues and organisms, their functional validation as modulators of gene expression remains elusive. Here we have characterized the expression and function of a highly abundant miR-101 5"-trimming variant (5"-isomiR-101). Results The analysis of small RNA sequencing data in several human tissues and cell lines indicates that 5"-isomiR-101 is ubiquitously detected and a highly abundant, especially in the brain. 5"- isomiR-101 was found in Ago-2 immunocomplexes and complementary approaches showed that 5"-isomiR-101 interacted with different members of the silencing (RISC) complex. In addition, 5"-isomiR-101 decreased the expression of five validated miR-101 targets, suggesting that it is a functional variant. Both the binding to RISC members and the degree of silencing were less efficient for 5"-isomiR-101 compared with miR-101. For some targets, both miR-101 and 5"-isomiR-101 significantly decreased protein expression with no changes in the respective mRNA levels. Although a high number of overlapping predicted targets suggest similar targeted biological pathways, a correlation analysis of the expression profiles of miR-101 variants and predicted mRNA targets in human brains at different ages, suggest specific functions for miR-101- and 5"-isomiR-101. Conclusions These results suggest that isomiRs are functional variants and further indicate that for a given miRNA, the different isomiRs may contribute to the overall effect as quantitative and qualitative fine-tuners of gene expression.

Allosteric conversation in the androgen receptor ligand-binding domain surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.

Synthesis of highly focused fields with circular polarization at any transverse plane

We develop a method for generating focused vector beams with circular polarization at any transverse plane. Based on the Richards-Wolf vector model, we derive analytical expressions to describe the propagation of these set of beams near the focal area. Since the polarization and the amplitude of the input beam are not uniform, an interferometric system capable of generating spatially-variant polarized beams has to be used. In particular, this wavefront is manipulated by means of spatial light modulators displaying computer generated holograms and subsequently focused using a high numerical aperture objective lens. Experimental results using a NA=0.85 system are provided: irradiance and Stokes images of the focused field at different planes near the focal plane are presented and compared with those obtained by numerical simulation.

Optical trapping: a review of essential concepts

Optical tweezers are an innovative technique for the non-contact, all-optical manipulation of small material samples, which has extraordinarily expanded and evolved since its inception in the mid-80s of the last century. Nowadays, the potential of optical tweezers has been clearly proven and a wide range of applications both from the physical and biological sciences have solidly emerged, turning the early ideas and techniques into a powerful paradigm for experimentation in the micro- and nanoworld. This review aims at highlighting the fundamental concepts that are essential for a thorough understanding of optical trapping, making emphasis on both its manipulation and measurement capabilities, as well as on the vast array of important biological applications appeared in the last years.

The central histaminergic neurons in vitro, and their neuroprotective role in excitotoxic cell death in the postnatal rat hippocampus.

Histamine acts as a neurotransmitter in the central nervous system. Brain histamine in synthesized in neurons located to the posterior hypothalamus, from where these neurons send their projections to different parts of the brain. Released histamine participates in the regulation of several physiological functions such as arousal, attention and body homeostasis. Disturbances in the histaminergic system have been detected in diseases such as epilepsy, sleep disorders, anxiety, depression, Alzheimer’s disease, and schizophrenia. The purpose of this thesis was to develop optimal culture conditions for the histaminergic neurons, to study their detailed morphology, and to find out their significance in the kainic acid (KA)-induced neuronal death in the immature rat hippocampus. The morphology of the histaminergic neurons in vitro was comparable with the earlier findings. Histamine-containing vesicles were found in the axon but also in the cell body and dendrites suggesting a possibility for the somatodendritic release. Moreover, histamine was shown to be colocalized with the vesicular monoamine transporter 2 (VMAT2) suggesting that VMAT2 transports histamine to the subcellular storage vesicles. Furthermore, histamine was localized with γ-aminobutyric acid (GABA) in distinct storage vesicles and with neuropeptide galanin partly in the same storage vesicles suggesting different corelease mechanisms for GABA and galanin with histamine. In the organotypic hippocampal slice cultures, KA-induced neuronal death was first detected 12 h after the treatment being restricted mainly to the CA3 subregion. Moreover, cell death was irreversible, since the 48 h recovery period did not save the cells, but instead increased the damage. Finally, neuronal death was suggested to be necrotic, since intracellular apoptotic pathways were not activated, and the morphological changes detected with the electron microscopy were characteristic for necrosis. In the coculture system of the hippocampal and posterior hypothalamic slices, histaminergic neurons significantly decreased epileptiform burst activity and neuronal death in the hippocampal slices, this effect being mediated by histamine 1 (H1) and 3 (H3) receptors. In conclusion, the histaminergic neurons were maintained succesfully in the in vitro conditions exhibiting comparable morphological characteristics as detected earlier in vivo. Moreover, they developed functional innervations within the hippocampal slices in the coculture system. Finally, the KA-induced regionspecific, irreversible and necrotic hippocampal pyramidal cell damage was significantly decreased by the histaminergic neurons through H1 and H3 receptors.

Structural and functional properties of two human FXYD3 (Mat-8) isoforms.

Six of 7 FXYD proteins have been shown to be tissue-specific modulators of Na,K-ATPase. In this study, we have identified two splice variants of human FXYD3, or Mat-8, in CaCo-2 cells. Short human FXYD3 has 72% sequence identity with mouse FXYD3, whereas long human FXYD3 is identical to short human FXYD3 but has a 26-amino acid insertion after the transmembrane domain. Short and long human FXYD3 RNAs and proteins are differentially expressed during differentiation of CaCo-2 cells. Long human FXYD3 is mainly expressed in nondifferentiated cells and short human FXYD3 in differentiated cells and both FXYD3 variants can be co-immunoprecipitated with a Na,K-ATPase antibody. In contrast to mouse FXYD3, which has two transmembrane domains for lack of cleavage of the signal peptide, human FXYD3 has a cleavable signal peptide and adopts a type I topology. After co-expression in Xenopus oocytes, both human FXYD3 variants associate stably only with Na,K-ATPase isozymes but not with H,K-ATPase or Ca-ATPase. Similar to mouse FXYD3, short human FXYD3 decreases the apparent K(+) and Na(+) affinity of Na,K-ATPase over a large range of membrane potentials. On the other hand, long human FXYD3 decreases the apparent K(+) affinity only at slightly negative and positive membrane potentials and increases the apparent Na(+) affinity of Na,K-ATPase. Finally, both short and long human FXYD3 induce a hyperpolarization activated current, similar to that induced by mouse FXYD3. Thus, we have characterized two human FXYD3 isoforms that are differentially expressed in differentiated and non-differentiated cells and show different functional properties.

Gamma oscillations in V1 are correlated with GABAA receptor density: A multi-modal MEG and Flumazenil-PET study.

High-frequency oscillations in the gamma-band reflect rhythmic synchronization of spike timing in active neural networks. The modulation of gamma oscillations is a widely established mechanism in a variety of neurobiological processes, yet its neurochemical basis is not fully understood. Modeling, in-vitro and in-vivo animal studies suggest that gamma oscillation properties depend on GABAergic inhibition. In humans, search for evidence linking total GABA concentration to gamma oscillations has led to promising -but also to partly diverging- observations. Here, we provide the first evidence of a direct relationship between the density of GABAA receptors and gamma oscillatory gamma responses in human primary visual cortex (V1). By combining Flumazenil-PET (to measure resting-levels of GABAA receptor density) and MEG (to measure visually-induced gamma oscillations), we found that GABAA receptor densities correlated positively with the frequency and negatively with amplitude of visually-induced gamma oscillations in V1. Our findings demonstrate that gamma-band response profiles of primary visual cortex across healthy individuals are shaped by GABAA-receptor-mediated inhibitory neurotransmission. These results bridge the gap with in-vitro and animal studies and may have future clinical implications given that altered GABAergic function, including dysregulation of GABAA receptors, has been related to psychiatric disorders including schizophrenia and depression.

Modifier Genes as Therapeutics: The Nuclear Hormone Receptor Rev Erb Alpha (Nr1d1) Rescues Nr2e3 Associated Retinal Disease

Nuclear hormone receptors play a major role in many important biological processes. Most nuclear hormone receptors are ubiquitously expressed and regulate processes such as metabolism, circadian function, and development. They function in these processes to maintain homeostasis through modulation of transcriptional gene networks. In this study we evaluate the effectiveness of a nuclear hormone receptor gene to modulate retinal degeneration and restore the integrity of the retina. Currently, there are no effective treatment options for retinal degenerative diseases leading to progressive and irreversible blindness. In this study we demonstrate that the nuclear hormone receptor gene Nr1d1 (Rev-Erba) rescues Nr2e3- associated retinal degeneration in the rd7 mouse, which lacks a functional Nr2e3 gene. Mutations in human NR2E3 are associated with several retinal degenerations including enhanced S cone syndrome and retinitis pigmentosa. The rd7 mouse, lacking Nr2e3, exhibits an increase in S cones and slow, progressive retinal degeneration. A traditional genetic mapping approach previously identified candidate modifier loci. Here, we demonstrate that in vivo delivery of the candidate modifier gene, Nr1d1 rescues Nr2e3 associated retinal degeneration. We observed clinical, histological, functional, and molecular restoration of the rd7 retina. Furthermore, we demonstrate that the mechanism of rescue at the molecular and functional level is through the re-regulation of key genes within the Nr2e3-directed transcriptional network. Together, these findings reveal the potency of nuclear receptors as modulators of disease and specifically of NR1D1 as a novel therapeutic for retinal degenerations.

Maturation and Functional Integration of New Granule Cells into the Adult Hippocampus.

The adult hippocampus generates functional dentate granule cells (GCs) that release glutamate onto target cells in the hilus and cornus ammonis (CA)3 region, and receive glutamatergic and γ-aminobutyric acid (GABA)ergic inputs that tightly control their spiking activity. The slow and sequential development of their excitatory and inhibitory inputs makes them particularly relevant for information processing. Although they are still immature, new neurons are recruited by afferent activity and display increased excitability, enhanced activity-dependent plasticity of their input and output connections, and a high rate of synaptogenesis. Once fully mature, new GCs show all the hallmarks of neurons generated during development. In this review, we focus on how developing neurons remodel the adult dentate gyrus and discuss key aspects that illustrate the potential of neurogenesis as a mechanism for circuit plasticity and function.