Oxidised LDL-lipids alter redox ratio, lipid raft formation and increase amyloid beta production by SHSY-5Y cells

Elevated cholesterol in mid-life has been associated with increased risk of dementia in later life. We have previously shown that low density lipoprotein (LDL) is more oxidised in the plasma of dementia patients although total cholesterol levels remained unchanged [1]. We have investigated the hypothesis that amyloid beta production and neurodegeneration can be driven by oxidised lipids derived from LDL following the loss of blood brain barrier integrity with ageing. Therefore, we have investigated amyloid beta formation in SHSY5Y cells treated with LDL, minimally modified (ox) LDL, and lipids extracted from both forms of LDL. LDL-treated SHSY-5Y cell viability was not significantly decreased with up to 8 μg LDL/2 × 104 cells compared to untreated cells. However, 8 μg oxLDL protein/2 × 104 cells decreased the cell viability significantly by 33.7% (P < 0.05). A more significant decrease in cell viability was observed when treating cells with extracted lipids from 8 μg of LDL (by 32.7%; P < 0.01) and oxLDL (by 41%; P < 0.01). In parallel, the ratio of reduced to oxidised GSH was decreased; GSH concentrations were significantly decreased following treatment with 0.8 μg/ml oxLD-L (7.35 ± 0.58;P < 0.01), 1.6 μg/ml (5.27 ± 0.23; P < 0.001) and 4 μg/ml (5.31 ± 0.31; P < 0.001). This decrease in redox potential was associated with an increase acid sphingomyelinase activity and lipid raft formation which could be inhibited by desipramine; SHSY5Y cells treated with oxLDL, and lipids from LDL and oxLDL for 16 h showed significantly increased acid sphingomyelinase activity (5.32 ± 0.35; P < 0.05, 5.21 ± 0.6; P < 0.05, and 5.58 ± 0.44; P < 0.01, respectively) compared to control cells (2.96 ± 0.34). As amyloid beta production is driven by the activity of beta secretase and its association with lipid rafts, we investigated whether lipids from ox-LDL can influence amyloid beta by SHSY-5Y cells in the presence of oxLDL. Using ELISA and Western blot, we confirmed that secretion of amyloid beta oligomers is increased by SHSY-5Y cells in the presence of oxLDL lipids. These data suggest a mechanism whereby LDL, and more significantly oxLDL lipids, can drive amyloid beta production and cytotoxicity in neuronal cells. [1] Li L, Willets RS, Polidori MC, Stahl W, Nelles G, Sies H, Griffiths HR. Oxidative LDL modification is increased in vascular dementia and is inversely associated with cognitive performance. Free Radic Res. 2010 Mar; 44(3): 241–8.

Spatial patterns of neuronal cytoplasmic inclusions in the tauopathies: A comparative study of five disorders

The tauopathies are a major molecular group of neurodegenerative disorders characterised by the deposition of abnormal cellular aggregates of the microtubule associated protein (MAP) tau in the form of neuronal cytoplasmic inclusions (NCI). Recent research suggests that cell to cell propagation of pathogenic tau may be involved in the neurodegeneration of these disorders. If pathogenic tau spreads along anatomical pathways it may give rise to specific spatial patterns of the NCI in brain tissue. To test this hypothesis, the spatial patterns of NCI in cerebral cortical regions were compared in tissue sections taken from five major tauopathies: (1) argyrophilic grain disease (AGD), (2) Alzheimer's disease (AD), (3) corticobasal degeneration (CBD), (4) Pick's disease (PiD), and (5) progressive supranuclear palsy (PSP). In the cerebral cortex of these disorders, NCI were frequently aggregated into clusters and the clusters were regularly distributed parallel to the pia mater. In a significant proportion of regions, the mean size of the regularly distributed clusters of NCI was in the range 400 – 800 m, measured parallel to the pia mater, approximating to the dimension of cell columns associated with the cortico-cortical anatomical pathways. Hence, the data suggest that cortical NCI in the tauopathies exhibit a spatial pattern in the cortex which could result from the spread of pathogenic tau along anatomical pathways. Treatments designed to protect the cortex from tau propagation may therefore be applicable across several different disorders within this molecular group.

Caractérisation des interactions établies par la région riche en prolines de la ligase de l’ubiquitine Itch.

Itch est un membre de la famille des ligases de l’ubiquitine de type CWH (C2-WW- HECT) impliqué dans le contrôle de la signalisation inflammatoire, des facteurs de transcription et le tri des récepteurs membranaires. La fonction d’Itch implique généralement sa capacité à induire la dégradation de ses substrats. Pour accomplir cette fonction, Itch doit d’abord interagir avec ses cibles. Itch possède quatre domaines WW lui permettant d’accomplir la majorité de ses fonctions. En plus de ces domaines, Itch possède une PRR (région riche en prolines) unique parmi les ligases CWH. Cette région est bien conservée chez les vertébrés, ce qui suggère son importance. Cette région permet à Itch d’interagir avec des protéines contenant un domaine SH3 (Src homology 3). Plusieurs partenaires SH3 furent identifiés, cependant l’on connait peu de choses concernant la fonction et l’établissement de ces complexes. Dans ce projet, nous avons analysé les propriétés de liaison d’un sous-groupe de protéines à domaine SH3 impliquées dans l’endocytose et la signalisation cellulaire. Nos travaux ont permis d’identifier de nouveaux partenaires et aussi de déterminer que différents domaines SH3 ciblent la même région riche en prolines, mais impliquent des résidus distincts. Ces résultats démontrent la variété des propriétés de liaison démontrées par la PRR d’Itch et sa préférence marquée pour l’Endophiline. Parmi les partenaires identifiés, Grb2 (Growth factor receptor-bound protein 2) est particulièrement intéressant en raison de son rôle crucial dans la signalisation cellulaire. Nous avons démontré ici qu’Itch ubiquityle Grb2, mais ne cause pas sa dégradation, contrairement à l’Endophiline. Nos travaux démontrent que la PRR d’Itch est versatile quant à ses interactions et leurs conséquences.

Renal Drug Transporters – Clinical Significance and Role in Drug-Drug Interactions

Thesis (Ph.D.)--University of Washington, 2016-07

Improving interfaces for nerve repair

Autologous nerve grafts are the current gold standard for the repair of peripheral nerve injuries. However, there is a need to develop an alternative to this technique, as donor-site morbidities such as neuroma formation and permanent loss of function are a few of the limitations concerned with this technique. Artificial nerve conduits have therefore emerged as an alternative for the repair of short peripheral nerve defects of less than 30 mm, however they do not surpass autologous nerve grafts clinically. To develop a nerve conduit that supports regeneration over long nerve gaps and in large diameter nerves, researchers have focused on functionalizing of the conduits by studying the components that enhance nerve regeneration such as micro/nano-topography, growth factor delivery systems, supportive cells and extracellular matrix (ECM) proteins as well as understanding the complex biological reactions that take place during peripheral nerve regeneration. This thesis presents strategies to improve peripheral nerve interfaces to better the regenerative potential by using dorsal root ganglions (DRGs) isolated from neonatal rats as an in vitro model of nerve regeneration. The work started off by investigating the usefulness of a frog foam protein Ranaspumin-2 (Rsn2) to coat biomaterials for compatibility, this lead to the discovery of temporary cell adhesion on polydimethylsiloxane (PDMS), which was investigated as a suitable tool to derive cell-sheets for nerve repair. The influence of Rsn2 anchored to specific adhesion peptide sequences, such as isoleucine-lysine-valine-alanine-valine (IKVAV), a sequence derived from laminin proven to promote cell adhesion and neurite outgrowth, was tested as a useful means to influence nerve regeneration. This approach improves the axonal outgrowth and maintains outgrowth long term. Based on the hypothesis that combinational modulation of substrate topography, stiffness and neurotrophic support, affects axonal outgrowth in whole DRGs, dissociated DRGs were used to assess if these factors similarly act at the single cell level. Rho associated protein kinase (ROCK) and myosin II inhibitors, which affect cytoskeletal contractility, were used to influence growth cone traction forces and have shown that these factors work in combination by interfering with growth cone dynamic creating a different response in axonal outgrowth at the single cell level.

Caractérisation des interactions établies par la région riche en prolines de la ligase de l’ubiquitine Itch

Itch est un membre de la famille des ligases de l’ubiquitine de type CWH (C2-WW- HECT) impliqué dans le contrôle de la signalisation inflammatoire, des facteurs de transcription et le tri des récepteurs membranaires. La fonction d’Itch implique généralement sa capacité à induire la dégradation de ses substrats. Pour accomplir cette fonction, Itch doit d’abord interagir avec ses cibles. Itch possède quatre domaines WW lui permettant d’accomplir la majorité de ses fonctions. En plus de ces domaines, Itch possède une PRR (région riche en prolines) unique parmi les ligases CWH. Cette région est bien conservée chez les vertébrés, ce qui suggère son importance. Cette région permet à Itch d’interagir avec des protéines contenant un domaine SH3 (Src homology 3). Plusieurs partenaires SH3 furent identifiés, cependant l’on connait peu de choses concernant la fonction et l’établissement de ces complexes. Dans ce projet, nous avons analysé les propriétés de liaison d’un sous-groupe de protéines à domaine SH3 impliquées dans l’endocytose et la signalisation cellulaire. Nos travaux ont permis d’identifier de nouveaux partenaires et aussi de déterminer que différents domaines SH3 ciblent la même région riche en prolines, mais impliquent des résidus distincts. Ces résultats démontrent la variété des propriétés de liaison démontrées par la PRR d’Itch et sa préférence marquée pour l’Endophiline. Parmi les partenaires identifiés, Grb2 (Growth factor receptor-bound protein 2) est particulièrement intéressant en raison de son rôle crucial dans la signalisation cellulaire. Nous avons démontré ici qu’Itch ubiquityle Grb2, mais ne cause pas sa dégradation, contrairement à l’Endophiline. Nos travaux démontrent que la PRR d’Itch est versatile quant à ses interactions et leurs conséquences.

Large national series of patients with Xq28 duplication involving MECP2: Delineation of brain MRI abnormalities in 30 affected patients.

Xq28 duplications encompassing MECP2 have been described in male patients with a severe neurodevelopmental disorder associated with hypotonia and spasticity, severe learning disability, stereotyped movements, and recurrent pulmonary infections. We report on standardized brain magnetic resonance imaging (MRI) data of 30 affected patients carrying an Xq28 duplication involving MECP2 of various sizes (228201;kb to 11.7201;Mb). The aim of this study was to seek recurrent malformations and attempt to determine whether variations in imaging features could be explained by differences in the size of the duplications. We showed that 93% of patients had brain MRI abnormalities such as corpus callosum abnormalities (n201;=201;20), reduced volume of the white matter (WM) (n201;=201;12), ventricular dilatation (n201;=201;9), abnormal increased hyperintensities on T2-weighted images involving posterior periventricular WM (n201;=201;6), and vermis hypoplasia (n201;=201;5). The occipitofrontal circumference varied considerably between >+2SD in five patients and <-2SD in four patients. Among the nine patients with dilatation of the lateral ventricles, six had a duplication involving L1CAM. The only patient harboring bilateral posterior subependymal nodular heterotopia also carried an FLNA gene duplication. We could not demonstrate a correlation between periventricular WM hyperintensities/delayed myelination and duplication of the IKBKG gene. We thus conclude that patients with an Xq28 duplication involving MECP2 share some similar but non-specific brain abnormalities. These imaging features, therefore, could not constitute a diagnostic clue. The genotype-phenotype correlation failed to demonstrate a relationship between the presence of nodular heterotopia, ventricular dilatation, WM abnormalities, and the presence of FLNA, L1CAM, or IKBKG, respectively, in the duplicated segment.

Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.

Nouveaux inducteurs covalents de la voie de signalisation Keap1/Nrf2/ARE

Le stress électrophile et oxydant est un souci grandissant pour la santé avec l'évolution de nos modes de vie. L'exposition aux ultraviolets, à la pollution, aux substances carcinogènes, à la fumée de cigarette et la pratique intensive d'activités sportives sont autant de causes de stress oxydant pour l'organisme. Ces dommages sont associés à plusieurs maladies et conditions pathologiques telles que cancers, diabètes, infections pulmonaires et maladies neurodégénératives. L'élément de réponse antioxydant (ARE) est un des composants principaux des défenses de la cellule contre ce phénomène. Ce promoteur agit sous le contrôle de Nrf2 (Nuclear factor erythroid 2-related factor 2). Une stratégie populaire pour l'activation de ce mécanisme est l'utilisation d'inducteurs covalents. Ces molécules agissent par la formation de liens covalents avec les nombreux résidus cystéine de Keap1 (Kelch-like ECH-associated protein 1), une protéine chaperonne qui contrôle l'activité de Nrf2. Cette thèse présente la synthèse, les propriétés biologiques et l'étude des relations structure-activité d'une librairie d'électrophiles capables d'induire la transcription des gènes cibles de la voie de signalisation Keap1/Nrf2/ARE. Le premier volet fait état de la comparaison d'une variété d'électrophiles simples pour étudier les préférences de la cible. Le deuxième volet montre que la présence d'une seconde fonction capable de piéger un résidu cystéine fournit des analogues très puissants.

Actitudes hacia la Enfermedad del Alzheimer según el género y la edad de adultos colombianos

La enfermedad de Alzheimer (EA) es la demencia más frecuente y su prevalencia continúa en aumento tanto en Colombia como en el mundo. Esta investigación tuvo como objetivo explorar si las actitudes hacia la EA varían según la edad y género de 450 personas adultas colombianas. Se realizó un estudio exploratorio de corte transversal en el que se aplicó un cuestionario autodiligenciado. Se encontró que efectivamente hay algunas diferencias según la edad y el género en el componente cognoscitivo (creencias y conocimiento) y conductual (intención conductual y conducta) de las actitudes; y diferencias según el género en el componente afectivo. Se concluye que los conocimientos sobre la EA son escasos, que la tristeza es la emoción predominante hacia la EA y que es un tema de interés en el que predomina la idea de que afecta especialmente la memoria. Se discutieron los resultados reconociendo que esta es una aproximación inicial a las actitudes hacia la EA.

EXPRESSION OF GENES RELATED TO MYOSTATIN SIGNALING DURING RAT SKELETAL MUSCLE LONGITUDINAL GROWTH

In this study we investigated the gene expression of proteins related to myostatin (MSTN) signaling during skeletal muscle longitudinal growth. To promote muscle growth, Wistar male rats were submitted to a stretching protocol for different durations (12, 24, 48, and 96 hours). Following this protocol, soleus weight and length and sarcomere number were determined. In addition, expression levels of the genes that encode MSTN, follistatin isoforms 288 and 315 (FLST288 and FLST315), follistatin-like 3 protein (FLST-L3), growth and differentiation factor-associated protein-1 (GASP-1), activin IIB receptor (ActIIB), and SMAD-7 were determined by real-time polymerase chain reaction. Prolonged stretching increased soleus weight, length, and sarcomere number. In addition, MSTN gene expression was increased at 12-24 hours, followed by a decrease at 96 hours when compared with baseline values. FLST isoforms, FLST-L3, and GASP-1 mRNA levels increased significantly over all time-points. ActIIB gene expression decreased quickly at 12-24 hours. SMAD-7 mRNA levels showed a late increase at 48 hours, which peaked at 96 hours. The gene expression pattern of inhibitory proteins related to MSTN signaling suggests a strong downregulation of this pathway in response to prolonged stretching. Muscle Nerve 40: 992-999, 2009

Impact of cholesterol on ABC and SLC transporters expression and function and its role in disposition variability to lipid-lowering drugs

This report focuses on the effects of cholesterol on the expression and function of the ATP-binding cassette (ABCB1, ABCG2 and ABCC2) and solute-linked carrier (SLCO1B1 and SLCO2B1) drug transporters with a particular focus on the potential impact of cholesterol on lipid-lowering drug disposition. Statins are the most active agents in the treatment of hypercholesterolemia. However, considerable interindividual variation exists in the response to statin therapy. Therefore, it would be huge progress if factors were identified that reliably differentiate between responders and nonresponders. Many studies have suggested that plasma lipid concentrations can affect drug disposition of compounds, such as ciclosporin and amphotericin B. Both compounds are able to affect the expression and function of ABC transporters. Although still speculative, these effects might be owing to the regulation of drug transporters by plasma cholesterol levels. Studies with normo- and hyper-cholesterolemic individuals, before and after atorvastatin treatment, have demonstrated that plasma cholesterol levels are correlated with drug transporter expression, as well as being related to atorvastatin`s cholesterol-lowering effect. The mechanism influencing the correlation between cholesterol levels and the expression and function of drug transporters remains unclear. Some studies provide strong evidence that nuclear receptors, such as the pregnane X receptor and the constitutive androstane receptor, mediate this effect. In the near future, pharmacogenomic studies with individuals in a pathological state should be performed in order to identify whether high plasma cholesterol levels might be a factor contributing to interindividual oral drug bioavailability.

Biomolecular interaction analysis of IFN gamma-induced signaling events in whole-cell lysates: Prevalence of latent STAT1 in high-molecular weight complexes

The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA, The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell Lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immune-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor ct-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) nonactivated STAT 1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.

Mutations in the DLG3 gene cause nonsyndromic X-linked mental retardation

We have identified truncating mutations in the human DLG3 ( neuroendocrine dlg) gene in 4 of 329 families with moderate to severe X-linked mental retardation. DLG3 encodes synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase protein family. Neuronal SAP102 is expressed during early brain development and is localized to the postsynaptic density of excitatory synapses. It is composed of three amino-terminal PDZ domains, an src homology domain, and a carboxyl-terminal guanylate kinase domain. The PDZ domains interact directly with the NR2 subunits of the NMDA glutamate receptor and with other proteins responsible for NMDA receptor localization, immobilization, and signaling. The mutations identified in this study all introduce premature stop codons within or before the third PDZ domain, and it is likely that this impairs the ability of SAP102 to interact with the NMDA receptor and/or other proteins involved in downstream NMDA receptor signaling pathways. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity, such as long-term potentiation and long-term depression, and these changes in synaptic efficacy have been proposed as neural mechanisms underlying memory and learning. The disruption of NMDA receptor targeting or signaling, as a result of the loss of SAP102, may lead to altered synaptic plasticity and may explain the intellectual impairment observed in individuals with DLG3 mutations.

GIGYF2 mutations are not a frequent cause of familial Parkinson`s disease

Mutations in the Grb10-interacting GYF protein 2 (GIGYF2) gene, within the PARK11 locus, have been nominated as a cause of Parkinson`s disease in Italian and French populations. By sequencing the whole GIGYF2 coding region in forty-six probands (thirty-seven Italians) with familial Parkinson`s disease compatible with an autosomal dominant inheritance, we identified no mutations. Our data add to a growing body of evidence suggesting that GIGYF2 mutations are not a frequent cause of PD. (C) 2009 Elsevier Ltd. All rights reserved.