Alterations in energy metabolism, neuroprotection and visual signal transduction in the retina of parkinsonian, MPTP-treated monkeys

Parkinson disease is mainly characterized by the degeneration of dopaminergic neurons in the central nervous system, including the retina. Different interrelated molecular mechanisms underlying Parkinson disease-associated neuronal death have been put forward in the brain, including oxidative stress and mitochondrial dysfunction. Systemic injection of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to monkeys elicits the appearance of a parkinsonian syndrome, including morphological and functional impairments in the retina. However, the intracellular events leading to derangement of dopaminergic and other retinal neurons in MPTP-treated animal models have not been so far investigated. Here we have used a comparative proteomics approach to identify proteins differentially expressed in the retina of MPTP-treated monkeys. Proteins were solubilized from the neural retinas of control and MPTP-treated animals, labelled separately with two different cyanine fluorophores and run pairwise on 2D DIGE gels. Out of >700 protein spots resolved and quantified, 36 were found to exhibit statistically significant differences in their expression levels, of at least ±1.4-fold, in the parkinsonian monkey retina compared with controls. Most of these spots were excised from preparative 2D gels, trypsinized and subjected to MALDI-TOF MS and LC-MS/MS analyses. Data obtained were used for protein sequence database interrogation, and 15 different proteins were successfully identified, of which 13 were underexpressed and 2 overexpressed. These proteins were involved in key cellular functional pathways such as glycolysis and mitochondrial electron transport, neuronal protection against stress and survival, and phototransduction processes. These functional categories underscore that alterations in energy metabolism, neuroprotective mechanisms and signal transduction are involved in MPTPinduced neuronal degeneration in the retina, in similarity to mechanisms thought to underlie neuronal death in the Parkinson’s diseased brain and neurodegenerative diseases of the retina proper.

PipX, the coactivator of NtcA, is a global regulator in cyanobacteria

To modulate the expression of genes involved in nitrogen assimilation, the cyanobacterial PII-interacting protein X (PipX) interacts with the global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance. PipX can form alternate complexes with NtcA and PII, and these interactions are stimulated and inhibited, respectively, by 2-oxoglutarate, providing a mechanistic link between PII signaling and NtcA-regulated gene expression. Here, we demonstrate that PipX is involved in a much wider interaction network. The effect of pipX alleles on transcript levels was studied by RNA sequencing of S. elongatus strains grown in the presence of either nitrate or ammonium, followed by multivariate analyses of relevant mutant/control comparisons. As a result of this process, 222 genes were classified into six coherent groups of differentially regulated genes, two of which, containing either NtcA-activated or NtcA-repressed genes, provided further insights into the function of NtcA–PipX complexes. The remaining four groups suggest the involvement of PipX in at least three NtcA-independent regulatory pathways. Our results pave the way to uncover new regulatory interactions and mechanisms in the control of gene expression in cyanobacteria.

Le système endocannabinoïde dans la rétine du singe : expression, localisation et fonctions

Le cannabis produit de nombreux effets psychologiques et physiologiques sur le corps humain. Les molécules contenues dans cette plante, désignées comme « phytocannabinoïdes », activent un système endogène qu’on appelle le système endocannabinoïde (eCB). Les effets de la consommation de cannabis sur la vision ont déjà été décrits sans cependant de formulation sur les mécanismes sous-jacents. Ces résultats comportementaux suggèrent, malgré tout, la présence de ce système eCB dans le système visuel, et particulièrement dans la rétine. Cette thèse vise donc à caractériser l’expression, la localisation et le rôle du système eCB dans la rétine du singe vervet, une espèce animale ayant un système visuel semblable à celui de l’humain. Nous avons mis au point un protocole expérimental d’immunohistochimie décrit dans l’article apparaissant dans l’Annexe I que nous avons utilisé pour répondre à notre objectif principal. Dans une première série de quatre articles, nous avons ainsi caractérisé l’expression et la localisation de deux récepteurs eCBs reconnus, les récepteurs cannabinoïdes de type 1 (CB1R) et de type 2 (CB2R), et d’un 3e présumé récepteur aux cannabinoïdes, le récepteur GPR55. Dans l’article 1, nous avons démontré que CB1R et une enzyme clé de ce système, la fatty acid amide hydrolase (FAAH), sont exprimés dans les parties centrale et périphérique de la rétine, et abondamment présents dans la fovéa, une région où l’acuité visuelle est maximale. Dans l’article 2, nous avons localisé le CB2R dans des cellules gliales de la rétine : les cellules de Müller et nous avons proposé un modèle sur l’action de cette protéine dans la fonction rétinienne faisant appel à une cascade chimique impliquant les canaux potassiques. Dans l’article 3, nous avons observé le GPR55 exclusivement dans les bâtonnets qui sont responsables de la vision scotopique et nous avons soumis un deuxième modèle de fonctionnement de ce récepteur par le biais d'une modulation des canaux calciques et sodiques des bâtonnets. Vu que ces 3 récepteurs se retrouvent dans des cellules distinctes, nous avons suggéré leur rôle primordial dans l’analyse de l’information visuelle au niveau rétinien. Dans l’article 4, nous avons effectué une analyse comparative de l’expression du système eCB dans la rétine de souris, de toupayes (petits mammifères insectivores qui sont sont considérés comme l’étape intermédiaire entre les rongeurs et les primates) et de deux espèces de singe (le vervet et le rhésus). Ces résultats nous ont menés à présenter une hypothèse évolutionniste quant à l’apparition et à la fonction précise de ces récepteurs. Dans les articles subséquents, nous avons confirmé notre hypothèse sur le rôle spécifique de ces trois récepteurs par l’utilisation de l’électrorétinographie (ERG) après injection intravitréenne d’agonistes et d’antagonistes de ces récepteurs. Nous avons conclu sur leur influence indéniable dans le processus visuel rétinien chez le primate. Dans l’article 5, nous avons établi le protocole d’enregistrement ERG normalisé sur le singe vervet, et nous avons produit un atlas d’ondes ERG spécifique à cette espèce, selon les règles de l’International Society for Clinical Electrophysiology of Vision (ISCEV). Les patrons électrorétinographiques se sont avérés semblables à ceux de l’humain et ont confirmé la similarité entre ces deux espèces. Dans l’article 6, nous avons démontré que le blocage de CB1R ou CB2R entraine une modification de l’électrorétinogramme, tant au niveau photopique que scotopique, ce qui supporte l’implication de ces récepteurs dans la modulation des ondes de l’ERG. Finalement, dans l’article 7, nous avons confirmé le modèle neurochimique proposé dans l’article 3 pour expliquer le rôle fonctionnel de GPR55, en montrant que l’activation ou le blocage de ce récepteur, respectivement par un agoniste (lysophosphatidylglucoside, LPG) ou un antagoniste (CID16020046), entraine soit une augmentation ou une baisse significative de l’ERG scotopique seulement. Ces données, prises ensemble, démontrent que les récepteurs CB1R, CB2R et GPR55 sont exprimés dans des types cellulaires bien distincts de la rétine du singe et ont chacun un rôle spécifique. L’importance de notre travail se manifeste aussi par des applications cliniques en permettant le développement de cibles pharmacologiques potentielles dans le traitement des maladies de la rétine.

Statistical Hurdle Models for Single Cell Gene Expression: Differential Expression and Graphical Modeling

Thesis (Ph.D.)--University of Washington, 2016-06

Systemic gene expression in arabidopsis during an incompatible interaction with Alternaria brassicicola

Pathogen challenge can trigger an integrated set of signal transduction pathways, which ultimately leads to a state of high alert, otherwise known as systemic or induced resistance in tissue remote to the initial infection. Although large-scale gene expression during systemic acquired resistance, which is induced by salicylic acid or necrotizing pathogens has been previously reported using a bacterial pathogen, the nature of systemic defense responses triggered by an incompatible necrotrophic fungal pathogen is not known. We examined transcriptional changes that occur during systemic defense responses in Arabidopsis plants inoculated with the incompatible fungal pathogen Alternaria brassicicola. Substantial changes (2.00-fold and statistically significant) were demonstrated in distal tissue of inoculated plants for 35 genes (25 up-regulated and 10 down-regulated), and expression of a selected subset of systemically expressed genes was confirmed using real-time quantitative polymerase chain reaction. Genes with altered expression in distal tissue included those with putative functions in cellular housekeeping, indicating that plants modify these vital processes to facilitate a coordinated response to pathogen attack. Transcriptional up-regulation of genes encoding enzymes functioning in the beta-oxidation pathway of fatty acids was particularly interesting. Transcriptional up-regulation was also observed for genes involved in cell wall synthesis and modification and genes putatively involved in signal transduction. The results of this study, therefore, confirm the notion that distal tissue of a pathogen-challenged plant has a heightened preparedness for subsequent pathogen attacks.

Isolation of an imaginal disc growth factor homologue from Pieris rapae and its expression following parasitization by Cotesia rubecula

Endoparasitoid insects introduce maternal factors into the body of their host at oviposition to suppress cellular defences for the protection of the developing parasitoid. We have shown that transient expression of polydnavirus genes from a hymenopteran parasitoid Cotesia rubecula (CrPDV) is responsible for the inactivation of hemocytes from the lepidopteran host Pieris rapae. Since the observed downregulation of CrPDV genes in infected host tissues is not due to cis-regulatory elements at the CrV1 gene locus, we speculated that the termination of CrPDV gene expression may be due to cellular inactivation caused by the CrV1-mediated immune suppression of infected tissues. To test this assumption, we isolated an imaginal disc growth factor (IDGF) that is expressed in fat body and hemocytes, the target of viral infection and expression of CrPDV genes. Time-course experiments showed that the level of P. rapae IDGF is not affected by parasitization and polydnavirus infection. However, the amount of highly expressed genes, such as storage proteins, arylphorin and lipophorin, are significantly reduced following parasitization. (C) 2004 Elsevier Ltd. All rights reserved.

Targeting genetic resource conservation in widespread species: a case study of Cedrela odorata L.

Conservation of genetic resources is a recognised necessity for the long term maintenance of evolutionary potential. Effective assessment and implementation Strategies are required to permit rapid evaluation and protection of resources. Here we use information from the chloroplast, total genome and quantitative characters assayed across wide-ranging populations to assess genetic resources in a Neotropical tree, Cedrela odorata. A major differentiation identified for organelle, total genomic and quantitative variation was found to coincide with an environmental gradient across Costa Rica. However, a major evolutionary divergence between the Yucatan region and Honduras/Nicaragua identified within the chloroplast genome was not differentiated using quantitative characters. Based on these and other results, a three-tiered conservation genetic prioritisation process is recommended. In order of importance, and where information is available, conservation units should be defined using quantitative (expressed genes), nuclear (genetic connectivity) and organellar (evolutionary) measures. Where possible, information from range wide and local scale studies should be combined and emphasis should be placed on coincidental disjunctions for two or more measures. However, if only rapid assessments of diversity are possible, then assessment of organelle variation provides the most cautious assessment of genetic resources, at least for C. odorata, and can be used to propose initial conservation units. When considering effective implementation of genetic resource management strategies a final tier should be considered, that of landuse/geopolitical divisions. (C) 2004 Elsevier B.V. All rights reserved.

The application of proteomics to the human alcoholic brain

Alcoholism results in changes in the human brain that reinforce the cycle of craving and dependency, and these changes are manifest in the pattern of expression of proteins in key cells and brain areas. Described here is a proteomics-based approach aimed at determining the identity of proteins in the superior frontal cortex (SFC) of the human brain that show different levels of expression in autopsy samples taken from healthy and long-term alcohol abuse subjects. Soluble protein fractions constituting pooled samples combined from SFC biopsies of four well-characterized chronic alcoholics (mean consumption > 80 g ethanol/day throughout adulthood) and four matched controls (< 20 g/day) were generated. Two-dimensional electrophoresis was performed in triplicate on alcoholic and control samples and the resultant protein profiles analyzed for differential expression. Overall, 182 proteins differed by the criterion of twofold or more between case and control samples. Of these, 139 showed significantly lower expression in alcoholics, 35 showed significantly higher expression, and 8 were new or had disappeared. To date, 63 proteins have been identified using MALDI-MS and MS-MS. The finding that the expression level of differentially expressed proteins is preponderantly lower in the alcoholic brain is supported by recent results from parallel studies using microarray mRNA transcript.

Temporal and spatial transcriptional programs in murine kidney development

We have performed a systematic temporal and spatial expression profiling of the developing mouse kidney using Compugen long-oligonucleotide microarrays. The activity of 18,000 genes was monitored at 24-h intervals from 10.5-day-postcoitum (dpc) metanephric mesenchyme (MM) through to neonatal kidney, and a cohort of 3,600 dynamically expressed genes was identified. Early metanephric development was further surveyed by directly comparing RNA from 10.5 vs. 11.5 vs. 13.5dpc kidneys. These data showed high concordance with the previously published dynamic profile of rat kidney development (Stuart RO, Bush KT, and Nigam SK. Proc Natl Acad Sci USA 98: 5649-5654, 2001) and our own temporal data. Cluster analyses were used to identify gene ontological terms, functional annotations, and pathways associated with temporal expression profiles. Genetic network analysis was also used to identify biological networks that have maximal transcriptional activity during early metanephric development, highlighting the involvement of proliferation and differentiation. Differential gene expression was validated using whole mount and section in situ hybridization of staged embryonic kidneys. Two spatial profiling experiments were also undertaken. MM (10.5dpc) was compared with adjacent intermediate mesenchyme to further define metanephric commitment. To define the genes involved in branching and in the induction of nephrogenesis, expression profiling was performed on ureteric bud (GFP+) FACS sorted from HoxB7-GFP transgenic mice at 15.5dpc vs. the GFP- mesenchymal derivatives. Comparisons between temporal and spatial data enhanced the ability to predict function for genes and networks. This study provides the most comprehensive temporal and spatial survey of kidney development to date, and the compilation of these transcriptional surveys provides important insights into metanephric development that can now be functionally tested.

Differential regulation of suppressor of cytokine signaling-3 in the liver and adipose tissue of the sheep fetus in late gestation

Differential regulation of suppressor of cytokine signaling-3 in the liver and adipose tissue of the sheep fetus in late gestation. Am J Physiol Regul Integr Comp Physiol 290: R1044 - R1051, 2006. First published November 10, 2005; doi: 10.1152/ajpregu. 00573.2005. - It is unknown whether the JAK/STAT/suppressor of cytokine signaling-3 (SOCS-3) intracellular signaling pathway plays a role in tissue growth and metabolism during fetal life. We investigated whether there is a differential profile of SOCS-3 expression in the liver and perirenal adipose tissue during the period of increased fetal growth in late gestation and the impact of fetal growth restriction on SOCS-3 expression in the fetal liver. We also determined whether basal SOCS-3 expression in the fetal liver and perirenal adipose tissue is regulated by endogenous fetal prolactin (PRL). SOCS-3 mRNA abundance was higher in the liver than in the pancreas, spleen, and kidney of the sheep fetus during late gestation. In the liver, SOCS-3 mRNA expression was increased (P < 0.05) between 125 (n < 4) and 145 days (n < 7) gestation and lower (P < 0.05) in growth-restricted compared with normally grown fetal sheep in late gestation. The relative expression of SOCS-3 mRNA in the fetal liver was directly related to the mean plasma PRL concentrations during a 48-h infusion of either a dopaminergic agonist, bromocriptine (n < 7), or saline (n < 5), such that SOCS-3 mRNA expression was lower when plasma PRL concentrations decreased below similar to 20 ng/ml [y = 0.99 - (2.47/x) + (4.96/x(2)); r(2) = 0.91, P < 0.0001, n < 12]. No relationship was shown between the abundance of phospho-STAT5 in the fetal liver and circulating PRL. SOCS-3 expression in perirenal adipose tissue decreased (P < 0001) between 90 - 91 (n < 6) and 140 - 145 days (n < 9) gestation and was not related to endogenous PRL concentrations. Thus SOCS-3 is differentially expressed and regulated in key fetal tissues and may play an important and tissue-specific role in the regulation of cellular proliferation and differentiation before birth.

The molecular basis of temperature compensation in the Arabidopsis circadian clock

Circadian clocks maintain robust and accurate timing over a broad range of physiological temperatures, a characteristic termed temperature compensation. In Arabidopsis thaliana, ambient temperature affects the rhythmic accumulation of transcripts encoding the clock components TIMING OF CAB EXPRESSION1 (TOC1), GIGANTEA (GI), and the partially redundant genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). The amplitude and peak levels increase for TOC1 and GI RNA rhythms as the temperature increases (from 17 to 27 degrees C), whereas they decrease for LHY. However, as temperatures decrease ( from 17 to 12 degrees C), CCA1 and LHY RNA rhythms increase in amplitude and peak expression level. At 27 degrees C, a dynamic balance between GI and LHY allows temperature compensation in wild-type plants, but circadian function is impaired in Ihy and gi mutant plants. However, at 12 degrees C, CCA1 has more effect on the buffering mechanism than LHY, as the cca1 and gi mutations impair circadian rhythms more than Ihy at the lower temperature. At 17 degrees C, GI is apparently dispensable for free-running circadian rhythms, although partial GI function can affect circadian period. Numerical simulations using the interlocking-loop model show that balancing LHY/CCA1 function against GI and other evening-expressed genes can largely account for temperature compensation in wild-type plants and the temperature-specific phenotypes of gi mutants.

Microarray and proteomic analysis of the human alcoholic brain

The superior frontal cortex (SFC) is selectively damaged in chronic alcohol abuse, with localized neuronal loss and tissue atrophy. Regions such as motor cortex show little neuronal loss except in severe co-morbidity (liver cirrhosis or WKS). Altered gene expression was found in microarray comparisons of alcoholic and control SFC samples [1]. We used Western blots and proteomic analysis to identify the proteins that also show differential expression. Tissue was obtained at autopsy under informed, written consent from uncomplicated alcoholics and age- and sex-matched controls. Alcoholics had consumed 80 g ethanol/day chronically (often, 200 g/day for 20 y). Controls either abstained or were social drinkers ( 20 g/day). All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were homogenized in water and clarified by brief centrifugation (1000g, 3 min) before storage at –80°C. For proteomics the thawed suspensions were centrifuged (15000g, 50 min) to prepare soluble fractions. Aliquots were pooled from SFC samples from the 5 chronic alcoholics and 5 matched controls used in the previous microarray study [1]. 2-Dimensional electrophoresis was performed in triplicate using 18 cm format pH 4–7 and pH 6–11 immobilized pH gradients for firstdimension isoelectric focusing. Following second-dimension SDS-PAGE the proteins were fluorescently stained and the images collected by densitometry. 182 proteins differed by 2-fold between cases and controls. 141 showed lower expression in alcoholics, 33 higher, and 8 were new or had disappeared. To date 63 proteins have been identified using MALDI-MS and MS-MS. Western blots were performed on uncentrifuged individual samples from 76 subjects (controls, uncomplicated alcoholics and cirrhotic alcoholics). A common standard was run on every gel. After transfer, immunolabeling, and densitometry, the intensities of the unknown bands were compared to those of the standards. We focused on proteins from transcripts that showed clear differences in a series of microarray studies, classified into common sets including Regulators of G-protein Signaling and Myelin-associated proteins. The preponderantly lower level of differentially expressed proteins in alcoholics parallels the microarray mRNA analysis in the same samples. We found that mRNA and protein expression do not frequently correspond; this may help identify pathogenic processes acting at the level of transcription, translation, or post-translationally.

Multilevel modelling for inference of genetic regulatory networks

Time-course experiments with microarrays are often used to study dynamic biological systems and genetic regulatory networks (GRNs) that model how genes influence each other in cell-level development of organisms. The inference for GRNs provides important insights into the fundamental biological processes such as growth and is useful in disease diagnosis and genomic drug design. Due to the experimental design, multilevel data hierarchies are often present in time-course gene expression data. Most existing methods, however, ignore the dependency of the expression measurements over time and the correlation among gene expression profiles. Such independence assumptions violate regulatory interactions and can result in overlooking certain important subject effects and lead to spurious inference for regulatory networks or mechanisms. In this paper, a multilevel mixed-effects model is adopted to incorporate data hierarchies in the analysis of time-course data, where temporal and subject effects are both assumed to be random. The method starts with the clustering of genes by fitting the mixture model within the multilevel random-effects model framework using the expectation-maximization (EM) algorithm. The network of regulatory interactions is then determined by searching for regulatory control elements (activators and inhibitors) shared by the clusters of co-expressed genes, based on a time-lagged correlation coefficients measurement. The method is applied to two real time-course datasets from the budding yeast (Saccharomyces cerevisiae) genome. It is shown that the proposed method provides clusters of cell-cycle regulated genes that are supported by existing gene function annotations, and hence enables inference on regulatory interactions for the genetic network.

Specific lipidome signatures in central nervous system from methionine-restricted mice

Membrane lipid composition is an important correlate of the rate of aging of animals. Dietary methionine restriction (MetR) increases lifespan in rodents. The underlying mechanisms have not been elucidated but could include changes in tissue lipidomes. In this work, we demonstrate that 80% MetR in mice induces marked changes in the brain, spinal cord, and liver lipidomes. Further, at least 50% of the lipids changed are common in the brain and spinal cord but not in the liver, suggesting a nervous system-specific lipidomic profile of MetR. The differentially expressed lipids includes (a) specific phospholipid species, which could reflect adaptive membrane responses, (b) sphingolipids, which could lead to changes in ceramide signaling pathways, and (c) the physiologically redox-relevant ubiquinone 9, indicating adaptations in phase II antioxidant response metabolism. In addition, specific oxidation products derived from cholesterol, phosphatidylcholine, and phosphatidylethanolamine were significantly decreased in the brain, spinal cord, and liver from MetR mice. These results demonstrate the importance of adaptive responses of membrane lipids leading to increased stress resistance as a major mechanistic contributor to the lowered rate of aging in MetR mice. © 2013 American Chemical Society.

Transcriptome and Functional Analysis of Carotid Body Glomus Cells

The carotid body (CB) is a major arterial chemoreceptor containing glomus cells that are activated by changes in arterial blood contents including oxygen. Despite significant advancement in the characterization of their physiological properties, our understanding on the underlying molecular machinery and signaling pathway in CB glomus cells is still limited.

To overcome these limitations, in chapter 1, I demonstrated the first transcriptome profile of CB glomus cells using single cell sequencing technology, which allowed us to uncover a set of abundantly expressed genes, including novel glomus cell-specific transcripts. These results revealed involvement of G protein-coupled receptor (GPCR) signaling pathway, various types of ion channels, as well as atypical mitochondrial subunits in CB function. I also identified ligands for the mostly highly expressed GPCR (Olfr78) in CB glomus cells and examined this receptor’s role in CB mediated hypoxic ventilatory response.

Current knowledge of CB suggest glomus cells rely on unusual mitochondria for their sensitivity to hypoxia. I previously identified the atypical mitochondrial subunit Ndufa4l2 as a highly over-represented gene in CB glomus cells. In chapter 2, to investigate the functional significance of Ndufa4l2 in CB function, I phenotyped both Ndufa4l2 knockout mice and mice with conditional Ndufa4l2 deletion in CB glomus cells. I found that Ndufa4l2 is essential to the establishment of regular breathing after birth. Ablating Ndufa4l2 in postnatal CB glomus cells resulted in defective CB sensitivity to hypoxia as well as CB mediated hypoxic ventilatory response. Together, our data showed that Ndufa4l2 is critical to respiratory control and the oxygen sensitivity of CB glomus cells.