TDP-43-immunoreactive pathology in frontotemporal lobar degeneration with TDP proteinopathy (FTLD-TDP) with and without associated motor neuron disease (MND)

A proportion of patients with motor neuron disease (MND) exhibit frontotemporal dementia (FTD) and some patients with FTD develop the clinical features of MND. Frontotemporal lobar degeneration (FTLD) is the pathological substrate of FTD and some forms of this disease (referred to as FTLD-U) share with MND the common feature of ubiquitin-immunoreactive, tau-negative cellular inclusions in the cerebral cortex and hippocampus. Recently, the transactive response (TAR) DNA-binding protein of 43 kDa (TDP-43) has been found to be a major protein of the inclusions of FTLD-U with or without MND and these cases are referred to as FTLD with TDP-43 proteinopathy (FTLD-TDP). To clarify the relationship between MND and FTLD-TDP, TDP-43 pathology was studied in nine cases of FTLD-MND and compared with cases of familial and sporadic FTLD-TDP without associated MND. A principal components analysis (PCA) of the nine FTLD-MND cases suggested that variations in the density of surviving neurons in the frontal cortex and neuronal cytoplasmic inclusions (NCI) in the dentate gyrus (DG) were the major histological differences between cases. The density of surviving neurons in FTLD-MND was significantly less than in FTLD-TDP cases without MND, and there were greater densities of NCI but fewer neuronal intranuclear inclusions (NII) in some brain regions in FTLD-MND. A PCA of all FTLD-TDP cases, based on TDP-43 pathology alone, suggested that neuropathological heterogeneity was essentially continuously distributed. The FTLD-MND cases exhibited consistently high loadings on PC2 and overlapped with subtypes 2 and 3 of FTLD-TDP. The data suggest: (1) FTLD-MND cases have a consistent pathology, variations in the density of NCI in the DG being the major TDP-43-immunoreactive difference between cases, (2) there are considerable similarities in the neuropathology of FTLD-TDP with and without MND, but with greater neuronal loss in FTLD-MND, and (3) FTLD-MND cases are part of the FTLD-TDP 'continuum' overlapping with FTLD-TDP disease subtypes 2 and 3. © 2012 Nova Science Publishers, Inc. All rights reserved.

Antibacterial proteins and peptides in nurse shark ( Ginglymostoma cirratum) peripheral blood leukocytes

In many vertebrate and invertebrate species mediators of innate immunity include antimicrobial peptides (AMPs) such as peptide fragments of histones and other proteins with previously ascribed different functions. Shark AMPs have not been described and this research examines the antibacterial activity of nurse shark (Ginglymostoma cirratum) peripheral blood leukocyte lysates. Screening of lysates prepared by homogenizing unstimulated peripheral blood leukocytes identified muramidase (lysozyme-like) and non-muramidase antibacterial activity. Lysates were tested for lysozyme using the lysoplate assays, and antibacterial (AB) activity was assayed for by a microdilution growth assay that was developed using Planococcus citreus as the target bacterium. Fractionation of crude lysates by ion exchange and affinity chromatography was followed by a combination of SDS-PAGE with LC/MS-MS and/or N-terminal sequence analysis of low molecular weight protein bands (<20 kDa). This yielded several peptides with amino acid sequence similarity to lysozyme, ubiquitin, hemoglobin, human histones H2A, H2B and H4 and to antibacterial histone fragments of the catfish and the Asian toad. Not all peptide sequences corresponded to peptides potentially antibacterial. The correlation of a specific protein band in active lysate fractions was accomplished by employing the acid-urea gel overlay assays in which AB activity was seen as zones of growth inhibition on a lawn of P. citreus at a position corresponding to that of the putative AB protein band. This study is the first to describe putative AMPs in the shark and their potential role in innate immunity.^

Caractérisation de l’activité Nur77 et de ses complexes en essais BRET par complémentation

Dans le système nerveux central, la dopamine joue un rôle crucial dans de nombreuses fonctions physiologiques telles que : l’apprentissage, le mouvement volontaire, la motivation, la cognition et la production hormonale. Il a été aussi démontré que le système de signalisation dopaminergique est altéré dans plusieurs maladies neurologiques et psychiatriques comme la maladie de Parkinson et la schizophrénie. Des études, effectuées dans le laboratoire du Dr.Daniel Lévesque (laboratoire d’accueil), ont montré que les récepteurs nucléaires Nur77 (NR4A1, NGFI-B) et RXRγ (retinoid X receptors γ) sont impliqués dans la régulation des effets de la dopamine dans le système nerveux central. De plus, ces données suggèrent que le complexe Nur77 et RXR joueraient un rôle crucial dans l’effet des médicaments antipsychotiques et antiparkinsoniens. Toutefois, très peu de médicaments ciblant Nur77 ont été identifiés à ce jour et les médicaments agissant sur RXRγ restent mal caractérisés. En outre, les analyses actuellement disponibles ne peuvent pas résumer la complexité des activités des NRs et génèrent des mesures indirectes des activités des drogues. Afin de mieux comprendre comment est régulée l’interaction Nur77/RXRγ dans ces processus, mon projet a été de mettre au point un essai BRET (Bioluminescence Resonance Energy Transfer) et PCA-BRET (Protein Complementation Assay-BRET) basé sur le recrutement d'un motif mimant un co-activateur fusionné avec la YFP. Nos différents essais ont été validés par courbes dose-réponse en utilisant différents composés RXR . Les EC50 (concentration efficace médiane, qui permet de mesurer l'efficacité d'un composé) obtenues étaient très semblables aux valeurs précédemment rapportées dans la littérature. Nous avons aussi pu identifier un composé le SR11237 (BMS649) qui semble posséder une sélectivité pour le complexe Nur77/RXRγ par rapport aux complexes Nurr1/RXRγ et RXRγ /RXRγ. Nos résultats indiquent que ces essais de BRET peuvent être utilisés pour évaluer la sélectivité de nouveaux composés pour les complexes Nur77/RXRγ, Nurr1/RXRγ et RXRγ /RXRγ. Un autre aspect de mon projet de doctorat a été de mettre en évidence par BRET l’importance de la SUMOylation dans la régulation de l'activité de Nur77 dans sa forme monomèrique, homodimèrique et hétérodimèrique. Nous avons ainsi identifié que Nur77 recrute principalement SUMO2 sur sa lysine 577. Il est intéressant de noté que le recrutement de la SUMO2 à Nur77 est potentialisé en présence de la SUMO E3 Ligase PIASγ. Aussi, la perte de la SUMOylation sur la lysine 577 entraîne l'incapacité de Nur77 de recruter divers motifs de co-activation mais pas pour ses formes homo- et hétérodimèrique. Cependant, la présence de PIASγ ne potentialise pas le recrutement du co-activateur, suggérant que cette SUMO E3 Ligase est seulement impliqué dans le processus de recrutement de la SUMO mais pas dans celui du co-activateur. Nous avons ainsi déterminé une nouvelle modification post-traductionnelle sur Nur77 régulant spécifiquement son activité monomérique Ces projets pourraient donc apporter de nouvelles données cruciales pour l’amélioration du traitement de la maladie de Parkinson ou de la schizophrénie, ainsi que d'obtenir une meilleure compréhension sur les mécanismes permettant la régulation de la fonction de Nur77

Expression exogène du récepteur du facteur autocrine de motilité (AMF-R) dans les cellules COS-7

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Caractérisation du domaine cytoplasmique du récepteur du facteur autocrine de motilité et formation du complexe AMFR/p97/ubiquitine

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mitochondrial DNA damage induced autophagy, cell death, and disease.

Mammalian mitochondria contain multiple small genomes. While these organelles have efficient base excision removal of oxidative DNA lesions and alkylation damage, many DNA repair systems that work on nuclear DNA damage are not active in mitochondria. What is the fate of DNA damage in the mitochondria that cannot be repaired or that overwhelms the repair system? Some forms of mitochondrial DNA damage can apparently trigger mitochondrial DNA destruction, either via direct degradation or through specific forms of autophagy, such as mitophagy. However, accumulation of certain types of mitochondrial damage, in the absence of DNA ligase III (Lig3) or exonuclease G (EXOG), can directly trigger cell death. This review examines the cellular effects of persistent damage to mitochondrial genomes and discusses the very different cell fates that occur in response to different kinds of damage.

Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

TRPV4 is necessary for trigeminal irritant pain and functions as a cellular formalin receptor.

Detection of external irritants by head nociceptor neurons has deep evolutionary roots. Irritant-induced aversive behavior is a popular pain model in laboratory animals. It is used widely in the formalin model, where formaldehyde is injected into the rodent paw, eliciting quantifiable nocifensive behavior that has a direct, tissue-injury-evoked phase, and a subsequent tonic phase caused by neural maladaptation. The formalin model has elucidated many antipain compounds and pain-modulating signaling pathways. We have adopted this model to trigeminally innervated territories in mice. In addition, we examined the involvement of TRPV4 channels in formalin-evoked trigeminal pain behavior because TRPV4 is abundantly expressed in trigeminal ganglion (TG) sensory neurons, and because we have recently defined TRPV4's role in response to airborne irritants and in a model for temporomandibular joint pain. We found TRPV4 to be important for trigeminal nocifensive behavior evoked by formalin whisker pad injections. This conclusion is supported by studies with Trpv4(-/-) mice and TRPV4-specific antagonists. Our results imply TRPV4 in MEK-ERK activation in TG sensory neurons. Furthermore, cellular studies in primary TG neurons and in heterologous TRPV4-expressing cells suggest that TRPV4 can be activated directly by formalin to gate Ca(2+). Using TRPA1-blocker and Trpa1(-/-) mice, we found that both TRP channels co-contribute to the formalin trigeminal pain response. These results imply TRPV4 as an important signaling molecule in irritation-evoked trigeminal pain. TRPV4-antagonistic therapies can therefore be envisioned as novel analgesics, possibly for specific targeting of trigeminal pain disorders, such as migraine, headaches, temporomandibular joint, facial, and dental pain, and irritation of trigeminally innervated surface epithelia.

Function of the PDLIM2 protein in oncogenic Wnt signalling pathways

Aberrant regulation of the Wnt signalling pathway is a recurrent theme in cancer biology. Hyper activation due to oncogenic mutations and paracrine activity has been found in both colon cancer and breast cancer, and continues to evolve as a central mechanism in oncogenesis. PDLIM2, a cytoskeletal PDZ protein, is an IGF-1 regulated gene that is highly expressed in cancer cell lines derived from metastatic tumours. Suppression of PDLIM2 inhibits polarized cell migration, reverses the Epithelial to Mesenchymal transition (EMT) phenotype, suppresses the transcription of β-catenin target genes, and regulates gene expression of key transcription factors in EMT. This thesis investigates the mechanism by which PDLIM2 contributes to the maintenance of Wnt signalling in cancer cells. Here we show that PDLIM2 is a critical regulator of the Wnt pathway by regulating β-catenin at the adherens juctions, as also its transcriptional activity by the interaction of PDLIM2 with TCF4 at the nucleus. Evaluation of PDLIM2 in macrophages and co-culture studies with cancer cells and fibroblasts showed the influence exerted on PDLIM2 by paracrine cues. Thus, PDLIM2 integrates cytoskeleton signalling with gene expression by modulating the Wnt signalling pathway and reconciling microenvironmental cues with signals in epithelial cells. Negative correlation of mRNA and protein levels in the triple negative breast cancer cell BT549 suggests that PDLIM2 is part of a more complex mechanism that involves transcription and posttranslational modifications. GST pulldown studies and subsequent mass spectrometry analysis showed that PDLIM2 interacts with 300 proteins, with a high biological function in protein biosynthesis and Ubiquitin/proteasome pathways, including 13 E3 ligases. Overall, these data suggest that PDLIM2 has two distinct functions depending of its location. Located at the cytoplasm mediates cytoskeletal re-arrangements, whereas at the nucleus PDLIM2 acts as a signal transduction adaptor protein mediating transcription and ubiquitination of key transcription factors in cancer development.

Expression exogène du récepteur du facteur autocrine de motilité (AMF-R) dans les cellules COS-7

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Caractérisation du domaine cytoplasmique du récepteur du facteur autocrine de motilité et formation du complexe AMFR/p97/ubiquitine

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

EXAMINATION OF VALPROIC ACID-INDUCED PERTURBATIONS TO DNA REPAIR, NF-ΚB SIGNALING AND CBP/P300 TRANSCRIPTIONAL CO-ACTIVATORS

Exposure to the antiepileptic drug valproic acid (VPA) is associated with an increased risk of congenital malformations including heart, skeletal and most frequently neural tube defects. Although the mechanisms contributing to its teratogenesis are not well understood, VPA was previously shown to increase homologous recombination (HR)-mediated DNA repair and decrease protein expression of the transcription factor NF-κB/p65. The studies in this thesis utilized in vivo and in vitro models to evaluate the expression of HR mediators, investigate the implications of decreased p65 including DNA binding and transcriptional activation, and the expression and histone acetyltransferase activity of Cbp/p300 with an aim to provide mechanistic insight into VPA-mediated alterations. The first study demonstrated that following maternal administration of VPA, mouse embryonic mRNA expression of HR mediators Rad51, Brca1 and Brca2 exhibited temporal and tissue-specific alterations. Protein expression of Rad51 was similarly altered and preceded increased cleavage of caspase-3 and PARP; indicative of apoptosis. The second study confirms previous findings of decreased total cellular p65 protein using P19 cells, but is the first to demonstrate that nuclear p65 protein is unchanged. NF-κB DNA binding was decreased following VPA exposure and maybe mediated by decreased p50 protein, which dimerizes with p65 prior to DNA binding. Transcriptional activity of NF-κB was also increased with VPA exposure which was not due to increased p65 phosphorylation at Ser276. Furthermore, the transcriptional activation capacity was unaffected by VPA exposure as combined exposure to VPA and TNFα additively increased NF-κB activity. The third study demonstrated that VPA exposure in P19 cells decreased Cbp/p300 total cellular and nuclear protein attributed primarily to ubiquitin proteasome-mediated degradation. Histone acetyltransferase (HAT) activity of p300 was decreased proportionately to nuclear protein following VPA exposure. Inhibition of Cbp/p300 HAT activity decreased p65 total cellular protein, increased caspase-3 cleavage and ROS similar to VPA exposures. Furthermore, pre-treatment with the antioxidant enzyme catalase attenuated the increase in caspase-3 cleavage, but not p65 protein. Overall, this thesis demonstrates that VPA exposure impacts the expression and activity of the transcription factor NF-κB and transcriptional co-activators/HATs Cbp/p300, which has implications for downstream VPA targets including Rad51, Brca1 and Brca2.

Cellular and molecular phenotypes depending upon the RNA repair system RtcAB of Escherichia coli

RNA ligases function pervasively across the three kingdoms of life for RNA repair, splicing and can be stress induced. The RtcB protein (also HSPC117, C22orf28, FAAP and D10Wsu52e) is one such conserved ligase, involved in tRNA and mRNA splicing. However, its physiological role is poorly described, especially in bacteria. We now show in Escherichia coli bacteria that the RtcR activated rtcAB genes function for ribosome homeostasis involving rRNA stability. Expression of rtcAB is activated by agents and genetic lesions which impair the translation apparatus or may cause oxidative damage in the cell. Rtc helps the cell to survive challenges to the translation apparatus, including ribosome targeting antibiotics. Further, loss of Rtc causes profound changes in chemotaxis and motility. Together, our data suggest that the Rtc system is part of a previously unrecognised adaptive response linking ribosome homeostasis with basic cell physiology and behaviour.

Brooke-Spiegler Syndrome - an underrecognized cause of multiple familial scalp tumors: report of a new germline mutation

BACKGROUND: Brooke-Spiegler syndrome (BSS) is probably an underdiagnosed genodermatosis that predisposes for the development of cylindromas, spiradenomas and trichoepitheliomas mainly of the head and neck. Wide phenotypic variability regarding the number and type of lesions can be observed within a family. Mutations of the CYLD gene are identified in the vast majority of cases and play a key role in BSS pathogenesis. MAIN OBSERVATIONS: Two first degree relatives with numerous erythematous telangiectatic nodules of the scalp present for decades, with recurring tendency regardless the multiple previous excisions. Histopathological review of the lesions revealed predominantly "spiradenocylindromas" in the proband and cylindromas in her sister. The suspicion of BSS was confirmed after detection of a new nonsense germline mutation of CYLD (c.1783C>T pGln 595*) in the proband. CONCLUSIONS: BSS diagnosis can be challenging and is based on clinical-pathological correlation, positive familial association and identification of CYLD mutations. CYLD exerts antineoplastic effects by downregulating intracellular NF-κB signalling pathways. The reported mutation affecting the ubiquitin-specific protease domain leads to a truncated and catalytically inactive enzyme. Despite the expanding list of CYLD mutations no firm genotype-phenotype correlation is known so far. Early recognition and treatment of BSS avoid disfiguring changes like "turban tumor".

Studies of Protein-Protein and Protein-Water Interactions by Small Angle X-Ray Scattering, Terahertz Spectroscopy, ASMOS, And Computer Simulation

The protein folding problem has been one of the most challenging subjects in biological physics due to its complexity. Energy landscape theory based on statistical mechanics provides a thermodynamic interpretation of the protein folding process. We have been working to answer fundamental questions about protein-protein and protein-water interactions, which are very important for describing the energy landscape surface of proteins correctly. At first, we present a new method for computing protein-protein interaction potentials of solvated proteins directly from SAXS data. An ensemble of proteins was modeled by Metropolis Monte Carlo and Molecular Dynamics simulations, and the global X-ray scattering of the whole model ensemble was computed at each snapshot of the simulation. The interaction potential model was optimized and iterated by a Levenberg-Marquardt algorithm. Secondly, we report that terahertz spectroscopy directly probes hydration dynamics around proteins and determines the size of the dynamical hydration shell. We also present the sequence and pH-dependence of the hydration shell and the effect of the hydrophobicity. On the other hand, kinetic terahertz absorption (KITA) spectroscopy is introduced to study the refolding kinetics of ubiquitin and its mutants. KITA results are compared to small angle X-ray scattering, tryptophan fluorescence, and circular dichroism results. We propose that KITA monitors the rearrangement of hydrogen bonding during secondary structure formation. Finally, we present development of the automated single molecule operating system (ASMOS) for a high throughput single molecule detector, which levitates a single protein molecule in a 10 µm diameter droplet by the laser guidance. I also have performed supporting calculations and simulations with my own program codes.