Добірка наукової літератури з теми "WDR41"

C9orf72 mutations are a major cause of amyotrophic lateral sclerosis and frontotemporal dementia. The C9orf72 protein undergoes regulated recruitment to lysosomes and has been broadly implicated in control of lysosome homeostasis. However, although evidence strongly supports an important function for C9orf72 at lysosomes, little is known about the lysosome recruitment mechanism. In this study, we identify an essential role for WDR41, a prominent C9orf72 interacting protein, in C9orf72 lysosome recruitment. Analysis of human WDR41 knockout cells revealed that WDR41 is required for localization of the protein complex containing C9orf72 and SMCR8 to lysosomes. Such lysosome localization increases in response to amino acid starvation but is not dependent on either mTORC1 inhibition or autophagy induction. Furthermore, WDR41 itself exhibits a parallel pattern of regulated association with lysosomes. This WDR41-dependent recruitment of C9orf72 to lysosomes is critical for the ability of lysosomes to support mTORC1 signaling as constitutive targeting of C9orf72 to lysosomes relieves the requirement for WDR41 in mTORC1 activation. Collectively, this study reveals an essential role for WDR41 in supporting the regulated binding of C9orf72 to lysosomes and solidifies the requirement for a larger C9orf72 containing protein complex in coordinating lysosomal responses to changes in amino acid availability.

PQLC2, a lysosomal cationic amino acid transporter, also serves as a sensor that responds to scarcity of its substrates by recruiting a protein complex composed of C9orf72, SMCR8, and WDR41 to the surface of lysosomes. This protein complex controls multiple aspects of lysosome function. Although it is known that this response to changes in cationic amino acid availability depends on an interaction between PQLC2 and WDR41, the underlying mechanism for the regulated interaction is not known. In this study, we present evidence that the WDR41–PQLC2 interaction is mediated by a short peptide motif in a flexible loop that extends from the WDR41 β-propeller and inserts into a cavity presented by the inward-facing conformation of PQLC2. The data support a transceptor model wherein conformational changes in PQLC2 related to substrate transport regulate the availability of the WDR41-binding site on PQLC2 and mediate recruitment of the WDR41-SMCR8-C9orf72 complex to the surface of lysosomes.

A massive intronic hexanucleotide repeat (GGGGCC) expansion in C9ORF72 is a genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recently, C9ORF72, together with SMCR8 and WDR41, has been shown to regulate autophagy and function as Rab GEF. However, the precise function of C9ORF72 remains unclear. Here, we report the cryogenic electron microscopy (cryo-EM) structure of the human C9ORF72–SMCR8–WDR41 complex at a resolution of 3.2 Å. The structure reveals the dimeric assembly of a heterotrimer of C9ORF72–SMCR8–WDR41. Notably, the C-terminal tail of C9ORF72 and the DENN domain of SMCR8 play critical roles in the dimerization of the two protomers of the C9ORF72–SMCR8–WDR41 complex. In the protomer, C9ORF72 and WDR41 are joined by SMCR8 without direct interaction. WDR41 binds to the DENN domain of SMCR8 by the C-terminal helix. Interestingly, the prominent structural feature of C9ORF72–SMCR8 resembles that of the FLNC–FNIP2 complex, the GTPase activating protein (GAP) of RagC/D. Structural comparison and sequence alignment revealed that Arg147 of SMCR8 is conserved and corresponds to the arginine finger of FLCN, and biochemical analysis indicated that the Arg147 of SMCR8 is critical to the stimulatory effect of the C9ORF72–SMCR8 complex on Rab8a and Rab11a. Our study not only illustrates the basis of C9ORF72–SMCR8–WDR41 complex assembly but also reveals the GAP activity of the C9ORF72–SMCR8 complex.

In the present study, the effect of genetic polymorphism of WD-repeat containing protein 41 (WDR41) and Ankyrin repeat domain containing protein 31 (ANKRD31) gene on 17 traits related to udder and teat type and mastitis in 123 Karan Fries cows was studied. Restriction Fragment Length Polymorphism (RFLP) was used to identify the SNP (410 bp and 475 bp) in PCR amplified product of intron 4 and exon 10 in WDR41 gene. Both of them were polymorphic with Guanine to Adenine transition, and three genotypes namely AA, AG and GG were observed. In ANKRD31 gene, RFLP was used to identify the SNP in 513 bp PCR amplified product of intron 24 and two SNPs were found. We report for the first time that intron 4 and exon 10 of WDR41 gene is significantly associated with udder depth (UD), udder balance (UB), central ligament (CL), teat circumference (TC), SFF and tear diameter (TD), rear udder width (RUW), and mastitis. SNP rs110012582 in intron 24 of ANKRD31 gene is associated significantly with UD, distance between left and right teat (DLR), SFF, and SNP rs473512406 with udder length (UL), udder width (UW), udder circumference (UC), fore teat length (FTL), rear teat length (RTL), distance between fore and rear teat (DFR), DLR and mastitis. This information can augment future studies to determine the role of WDR41 and ANKRD31 genes as a candidate gene marker with desired udder and teat conformation and mastitis. Thus, it is essential to work for better udder health and prevent the incidence of mastitis in the herd.

The SMCR8-WDR41-C9ORF72 complex is a regulator of autophagy and lysosomal function. Autoimmunity and inflammatory disease have been ascribed to loss-of-function mutations of Smcr8 or C9orf72 in mice. In humans, autoimmunity has been reported to precede amyotrophic lateral sclerosis caused by mutations of C9ORF72. However, the cellular and molecular mechanisms underlying autoimmunity and inflammation caused by C9ORF72 or SMCR8 deficiencies remain unknown. Here, we show that splenomegaly, lymphadenopathy, and activated circulating T cells observed in Smcr8−/− mice were rescued by triple knockout of the endosomal Toll-like receptors (TLRs) TLR3, TLR7, and TLR9. Myeloid cells from Smcr8−/− mice produced excessive inflammatory cytokines in response to endocytosed TLR3, TLR7, or TLR9 ligands administered in the growth medium and in response to TLR2 or TLR4 ligands internalized by phagocytosis. These defects likely stem from prolonged TLR signaling caused by accumulation of LysoTracker-positive vesicles and by delayed phagosome maturation, both of which were observed in Smcr8−/− macrophages. Smcr8−/− mice also showed elevated susceptibility to dextran sodium sulfate-induced colitis, which was not associated with increased TLR3, TLR7, or TLR9 signaling. Deficiency of WDR41 phenocopied loss of SMCR8. Our findings provide evidence that excessive endosomal TLR signaling resulting from prolonged ligand–receptor contact causes inflammatory disease in SMCR8-deficient mice.

Abnormal nucleotide insertions of C9orf72, which forms a complex with Smith–Magenis syndrome chromosomal region candidate gene 8 (SMCR8) protein and WD repeat-containing protein 41 (WDR41) protein, are associated with an autosomal-dominant neurodegenerative frontotemporal dementia and/or amyotrophic lateral sclerosis type 1 (FTDALS1). The differentially expressed in normal and neoplastic cells (DENN) domain-containing C9orf72 and its complex with SMCR8 and WDR41 function as a guanine-nucleotide exchange factor for Rab GTP/GDP-binding proteins (Rab GEF, also called Rab activator). Among Rab proteins serving as major effectors, there exists Rab11a. However, it remains to be established which Rab protein is related to promoting or sustaining neuronal morphogenesis or homeostasis. In this study, we describe that the knockdown of Rab11a decreases the expression levels of neuronal differentiation marker proteins, as well as the elongation of neurite-like processes, using N1E-115 cells, a well-utilized neuronal differentiation model. Similar results were obtained in primary cortical neurons. In contrast, the knockdown of Rab11b, a Rab11a homolog, did not significantly affect their cell morphological changes. It is of note that treatment with hesperetin, a citrus flavonoid (also known as Vitamin P), recovered the neuronal morphological phenotypes induced by Rab11a knockdown. Also, the knockdown of Rab11a or Rab11b led to a decrease in glial marker expression levels and in morphological changes in FBD-102b cells, which serve as the oligodendroglial differentiation model. Rab11a is specifically involved in the regulation of neuronal morphological differentiation. The knockdown effect mimicking the loss of function of C9orf72 is reversed by treatment with hesperetin. These findings may reveal a clue for identifying one of the potential molecular and cellular phenotypes underlying FTDALS1.

Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion.

Successful initiation of infection by many different viruses requires their uptake into the endosomal compartment. While some viruses exit this compartment early, others must reach the degradative, acidic environment of the late endosome. Mammalian orthoreovirus (reovirus) is one such late penetrating virus. To identify host factors that are important for reovirus infection, we performed a CRISPR-Cas9 knockout (KO) screen that targets over 20,000 genes in fibroblasts derived from the embryos of C57/BL6 mice. We identified seven genes (WDR81, WDR91, RAB7, CCZ1, CTSL, GNPTAB, and SLC35A1) that were required for the induction of cell death by reovirus. Notably, CRISPR-mediated KO of WD repeat-containing protein 81 (WDR81) rendered cells resistant to reovirus infection. Susceptibility to reovirus infection was restored by complementing KO cells with human WDR81. Although the absence of WDR81 did not affect viral attachment efficiency or uptake into the endosomal compartments for initial disassembly, it reduced viral gene expression and diminished infectious virus production. Consistent with the role of WDR81 in impacting the maturation of endosomes, WDR81-deficiency led to the accumulation of reovirus particles in dead-end compartments. Though WDR81 was dispensable for infection by VSV (vesicular stomatitis virus), which exits the endosomal system at an early stage, it was required for VSV-EBO GP (VSV that expresses the Ebolavirus glycoprotein), which must reach the late endosome to initiate infection. These results reveal a previously unappreciated role for WDR81 in promoting the replication of viruses that transit through late endosomes.

La sclérose latérale amyotrophique (SLA ou maladie de Charcot) est la troisième maladie neurodégénérative la plus répandue. La principale cause génétique de la SLA est une expansion de répétitions GGGGCC dans le gène C9ORF72, dont la protéine forme un complexe avec les protéines SMCR8 et WDR41. Afin de mieux comprendre ses fonctions moléculaires, résoudre sa structure était un objectif principal de ma thèse. En parallèle, nous avons découvert que C9ORF72 régule un mécanisme nouvellement décrit de biogenèse de nouveaux lysosomes nommé reformation autophagique des lysosomes (ALR). Ce processus a largement été investigué dans cette thèse afin de mieux comprendre sa régulation, notamment pour la régénération des lysosomes en conditions basales et de privation d’acides aminés. Mon travail révèle un nouveau partenaire du complexe C9ORF72 et une nouvelle fonction de ce complexe dans la biogenèse des lysosomes. Ces résultats pourraient ainsi expliquer le dysfonctionnement des lysosomes et la neurodégénérescence observés dans la SLA, ce qui pourrait ainsi ouvrir de nouvelles voies thérapeutiques pour cette maladie dévastatrice
Amyotrophic lateral sclerosis (ALS or Charcot disease) is the third most common neurodegenerative disease. The main genetic cause of ALS is an expansion of GGGGCC repeats in the C9ORF72 gene which protein forms a complex with the SMCR8 and WDR41 proteins. To better understand its molecular functions, solving its structure was a main goal of my thesis. In parallel, we discovered that C9ORF72 regulates a newly described mechanism of biogenesis of newly-formed lysosomes, called autophagic lysosome reformation (ALR). This process has been extensively investigated during my thesis, in order to better understand its regulation, particularly for the regeneration of lysosomes in basal conditions and amino acid deprivation. My work reveals a new partner of the C9ORF72 complex as a novel function in lysosome biogenesis. These results could thus explain the dysfunction of lysosomes and neurodegeneration observed in ALS, which open new therapeutic ways for this devastating disease

Nos travaux sur 26 gènes de la famille des WDR a permis d’en identifier sept (Atg16l1, Coro1c, Dmxl2, Herc1, Kif21b, Wdr47, Wdr89) associés à des anomalies cérébrales majeures. Cette grande famille de protéines reste pourtant peu explorée quant à ses rôles dans le développement du système nerveux central. Nous avons choisi d’étudier WDR47, dont la fonction est totalement inconnue en dépit d’une très grande similarité structurale avec LIS1, protéine à l’origine de la lissencéphalie. En combinant trois modèles expérimentaux (souris, siRNA et levure), nous avons démontré que Wdr47 est essentiel pour la survie de l’organisme et est impliqué dans la coordination motrice et le maintien de l’homéostasie énergétique avec une origine probablement centrale. Au niveau cellulaire, Wdr47 assure un rôle clé dans la dynamique des microtubules et la stabilisation du cône de croissance au travers d’interaction protéiques avec Reelin et SCG10. En outre, Wdr47 est aussi impliqué dans la prolifération neuronale et la macroautophagie. Ces résultats ont permis d’établir un lien de causalité entre une duplication de 200 kb contenant Wdr47 et des troubles de coordination motrice et une obésité hyperphagique chez un jeune patient
WD40-repeat (WDR) proteins are one of largest eukaryotic family, however little is known about their role in neurodevelopment. We investigated 26 WDR genes, and found 7 (Atg16l1, Coro1c, Dmxl2, Herc1, Kif21b, Wdr47, Wdr89) with a major impact in brain structure when inactivated in mice. We chose WDR47 for further investigation, as it is a completely unknown protein that shares striking domain similarity with LIS1. Using three independent model systems (mice, siRNA and yeast), we found an essential role of Wdr47 in survival, and key neuronal processes involving microtubule dynamics such as proliferation, autophagy and growth cone stabilization. Next we identified Reelin and superior cervical ganglion 10 (SCG10) as top interacting proteins of WDR47. Interestingly, a 200-kb duplication encompassing WDR47 was linked to poor coordination in one patient, recapitulating mouse behavioural anomalies. Together our data help unravel for the first time a key role of Wdr47 in brain

La microlissencéphalie est une malformation cérébrale congénitale caractérisée par la juxtaposition d’un phénotype de microcéphalie (petit cerveau) et de lissencéphalie (cerveau lisse). Nos collaborateurs ont récemment identifié des mutations responsables de cette pathologie dans le gène WDR81 humain. WDR81 est une protéine peu caractérisée et impliquée dans la maturation des endosomes, via la régulation de Pi3K. L'objectif de mon projet de thèse est de caractériser la fonction de WDR81 dans le cerveau en développement et d'identifier comment la mutation de ce gène peut conduire à un développement cérébral pathologique.J'ai généré une souris WDR81 KO qui présente, comme chez l’Homme, de forts défauts de migration neuronale ainsi qu’une microcéphalie. J'ai démontré que cette réduction de la taille du cerveau n'était pas due à une apoptose accrue, mais à une altération de la prolifération des cellules souches neurales, suggérant un défaut d’intégration de signaux prolifératifs. Le suivi de l’internalisation d’EGF fluorescent par des fibroblastes issus de patients m’a permis de montrer un défaut de dégradation d’EGF/EGFR qui s’ accumulent au sein de larges endosomes aberrants. De façon cohérente, mes résultats préliminaires indiquent que les cellules mutantes répondent moins efficacement à l'EGF.J'ai ensuite étudié la possibilité qu'une croissance cérébrale réduite (microcéphalie) et une croissance cérébrale accrue (mégalencéphalie) puissent représenter les deux faces d'une même médaille. Parce que mes données indiquent des effets opposés sur des voies similaires, j'ai surexprimé les facteurs mutants provoquant la mégalencéphalie dans les cerveaux WDR81 KO, et testé s'ils étaient capables de sauver les défauts de prolifération provoquant la microcéphalie. Mon résultat montre qu'en effet une mutation provoquant une mégalencéphalie peut surmonter l'effet d'une mutation provoquant une microcéphalie sur la prolifération des progéniteurs gliaux radiaux. Ces deux pathologies peuvent donc résulter d'une cause fortement liée: un déséquilibre dans la régulation du cycle cellulaire conduisant soit à une croissance cérébrale réduite, soit à une surcroissance cérébrale
Neocortex development is highly regulated and mutations in genes involved in this process may lead to genetic diseases. Microlissencephaly is a congenital and poorly understood brain malformation characterized by the presence of both microcephaly (small brain) and lissencephaly (smooth brain). Our collaborators recently identified compound heterozygous mutations causing this pathology in the human WD repeat domain 81 (WDR81) gene. WDR81 is a poorly characterized protein involved in endosomal maturation through Pi3K regulation. The goal of my PhD project is to characterize the function of WDR81 in the developing brain, and identify how mutations in this gene may lead to pathological brain development.Using CRISPR/Cas9, I have generated a WDR81 knock-out mouse, which shows strong neuronal migration defects and microcephaly, mimicking the human phenotype. I demonstrated that the reduced brain size was not due to increased apoptosis or altered cell fate, but to reduced neural stem cell proliferation. I also observe this phenotype in patient-derived fibroblasts, altogether suggesting altered processing of proliferative signals. Fluorescent EGF uptake assay reveals a strong intracellular accumulation of EGF in early phases endosomes that fails to be processed and cleared giving as well defects in the activation of the receptor itself. My results indicate that mutant cells indeed poorly respond to EGF stimulation.I then investigated the exciting possibility that reduced brain growth (microcephaly) and increased brain growth (megalencephaly) may represent two sides of the same coin. Because my data point to opposite effects on similar pathways, I overexpressed megalencephaly-causing mutated factors in WDR81 KO brains, and tested whether they were able to rescue the microcephaly-causing proliferation defects. My result show that indeed a megalencephaly-causing mutation can overcome the effect of a microcephaly-causing mutation on the proliferation of radial glial progenitors. These two pathologies can therefore arise from a highly related cause: an imbalance in cell cycle regulation leading either to reduced brain growth or to brain overgrowth

Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics. Medical Biochemistry))--University of Stellenbosch, 2009.
The mammalian neocortex contributes to the increasing functional complexity of the mammalian brain, partly because of its striking organisation into distinct neuronal layers. The development of the neocortex has been well studied because disrupted neurodevelopment results in several human diseases. The basic principles of neocortical development have been well established for some time; however the molecular mechanisms have only recently been identified. One major advance in our understanding of these molecular mechanisms was the discovery of Reelin, an extracellular matrix protein that directs the migration of neurons to their final positions in the developing neocortex. Reelin is a large multi-domain protein that exerts its functions by binding to its ligands on the cell surface and initiating a signal transduction cascade that ultimately results in cytoskeletal rearrangements. Several investigations have been undertaken to elucidate the functions of each of these domains to gain a better understanding reelin’s functions. We have previously identified the WR40 repeat protein 47 (WDR47), a protein of unknown function, as a novel putative ligand for the N-terminal reeler domain of reelin. To gain better understanding into the functional significance of this interaction, the present study sought to identify novel WDR47- interacting proteins. In order to achieve this, a cDNA encoding a polypeptide that contains the two N-terminal domains of WDR47, i.e. the Lis homology and the C-terminal Lis homology domain (CTLH) was used as bait in a Y2H screen of a foetal brain cDNA library. Putative WDR47 ligands were subsequently verified using 3D in vivo co-localisation. Results of these analyses showed that SCG10, a microtubule destabilizing protein belonging to the stathmin family of proteins, interacted with the N-terminal of WDR47. The identification of SCG10 as a novel WDR47 interacting protein not only sheds some light on the role and function of WDR47 but also aids in a better understanding of the reelin pathway and cortical lamination. Moreover, the data presented here, may also provide researchers with new avenues of research into molecular mechanisms involved in neuronal migration disorders.

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Introduction Normal cerebral cortex development depends on extensive neuronal migration during embryogenesis, permitting the formation of accurate synaptic circuits and a highly ordered laminar neocortex. The motility of a migrating neuron is achieved by a dynamic microtubule cytoskeleton that alternates between states of stabilization/lengthening and destabilization/shortening. This dynamic instability of the microtubule cytoskeleton is controlled by numerous microtubule-stabilizing and -destabilising proteins that bind directly to microtubules. Autophagy (“self-eating”), a major bulk intracellular degradation system, involves the fusion of autophagosomes with lysosomes, followed by proteolysis and recycling of cellular constituents. Like neuronal migration, autophagy is a microtubule-dependent process. The dynamic microtubule network serves as a track for autophagosomes to be transported to the lysosomes. WDR47 is a protein that is expressed in the brain during development, but of which the function is largely unknown. Novel interactions have recently been identified between Reelin and WDR47 and between the microtubule-destabilising protein superior cervical ganglion 10 (SCG10) and WDR47. These findings suggest that WDR47 may be regulating microtubule-dependent processes such as neuronal migration and autophagy. We hypothesize that WDR47 may play a role in regulating neuronal migration and/or autophagy, and that this regulation may be mediated by a tubulin stability-regulating role of WDR47. Aims and Methods Our aims are to assess the cellular localization of WDR47 in GT1-7 cells and to determine whether WDR47 is able to influence neuronal migration, filopodia extension, surface adhesion, ultra-structure, autophagy, tubulin stability, and tau or SCG10 protein levels. GT1-7 neuronal cells were cultured under normal conditions and transfected with WDR47 siRNA for 24 hours, followed by western blot verification of the knock-down. A 36 hour neuronal in vitro cell migration assay was performed and images of the wound were captured every 6 hours; the migration distances and the wound areas for the different time points were measured and analysed. A 24 hour migration assay was performed, capturing images every hour, and the direction of migration was determined. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to analyse neuronal surface morphology and ultra-structure. Western blot analysis of SCG10, acetylated α-tubulin, Tau, LC3, and Sequestosome 1/p62 (SQTM1) protein levels was performed. Super-resolution structured Illumination microscopy (SR-SIM) three-dimensional (3-D) imaging of WDR47-YFP transfected cells, confocal microscopy of LC3 and acetylated tubulin, co-localization analysis of WDR47 and acetylated tubulin, and fluorescence recovery after photo-bleaching (FRAP) analysis were performed. Results WDR47 siRNA treatment significantly reduced the average migration distance and the migration velocity, resulted in fewer filopodia-like extensions as well as perturbed surface adhesion of migrating neurons, and lead to an increased presence of endoplasmic reticulum (ER) structures as well as an expanded nuclear envelope. LC3-II protein levels were significantly lower with WDR47 siRNA treatment, but were significantly increased with WDR47 siRNA treatment in conjunction with Bafilomycin A1 treatment, indicating increased autophagic flux. SCG10 protein levels were significantly decreased with WDR47 siRNA treatment. SR-SIM and confocal microscopy of WDR47 siRNA treated cells revealed a robust presence of highly convoluted acetylated tubulin in the perinuclear region as well as decreased LC3 fluorescence signal. Confocal microscopy revealed co-localization of WDR47 with acetylated tubulin. - Discussion and Conclusion: The results suggest that WDR47 is involved in regulating neuronal migration, neuronal surface adhesion and filopodia formation, microtubule dynamics, and likely also autophagic flux. Taken together, we propose that WDR47 is regulating microtubule dynamics by facilitating assembly of microtubule-regulating proteins such as SCG10, thereby affecting microtubule-dependent processes such as neuronal migration and autophagy.
AFRIKAANSE OPSOMMING: Inleiding Normale serebrale korteks ontwikkeling is hoogs afhanklik van neuronale migrasie tydens embriogenese, en is belanrik vir die vorming van akkurate sinaptiese netwerke en 'n hoogs geordende laminêre neokorteks. Die vermoё van 'n neuron om te migreer berus op 'n hoogs dinamiese mikrotubulien sitoskelet wat verleng/stabiliseer of verkort/destabiliseer soos tubulien-eenhede begevoeg of verwyder word. Hierdie dinamiese onstabiliteit van die mikrotubulien sitoskelet word beheer deur verskeie mikrotubulien-stabiliserende en - destabiliserende proteïene wat direk bind aan mikrotubuliene. Autofagie ("self-eet"), 'n grootmaat intrasellulêre degradasie stelsel, behels die fussie van autofagosome met lisosome, gevolg deur proteolitiese afbraak van sellulêre organelle en proteine. Soos neuronale migrasie is autofagie 'n mikrotubulien-afhanklike proses. Die dinamiese mikrotubulien netwerk dien as 'n spoor vir die vervoer van autofagosome na lisosome. WDR47 is 'n proteïen wat voorkom in die brein tydens ontwikkeling, maar waarvan die funksie grootliks onbekend is. Interaksies was onlangs geïdentifiseer tussen beide Reelin en WDR47 en die mikrotubulien-destabiliserende proteïen SCG10 en WDR47. Hierdie bevindinge dui daarop aan dat WDR47 n rol speel in die regulering van tubulienstabiliteit en sodoende mikrotubulien-afhanklike prosesse. Ons veronderstel dat WDR47 'n rol kan speel in die regulering van neuronale migrasie en/of autofagie en dat hierdie regulasie moontlik afhanklik is van 'n tubulien-stabiliteit-regulerende rol van WDR47. - Doelwitte en Metodes: Ons doelwitte is om die sellulêre lokalisering van WDR47 in GT1-7 neurone te evallueer en om te bepaal of WDR47 n effek het op neuronale migrasie, oppervlak adhesie en filopodia formasie, ultra-struktuur, autofagie, tubulien-netwerke en -stabiliteit, en Tau of SCG10 proteïenvlakke. GT1-7 neuronale selle is gekweek onder normale omstandighede en vir 24 uur getransfekteer met WDR47 siRNA, gevolg deur verifikasie met Western-blot analise. 'n 36 uur neuronale in vitro sel migrasie toets is uitgevoer en fotos van die wond is elke 6 uur geneem. Die migrasie afstande en die wondareas vir die verskillende tydpunte is gemeet en ontleed. 'N 24-uur-migrasie toets is uitgevoer, 'n foto van die wond is elke uur geneem, en die rigting van migrasie is bepaal. Skandering elektronmikroskopie (SEM) en transmissieelektronmikroskopie (TEM) is uitgevoer om neuronale oppervlakmorfologie en ultrastruktuur te observeer. Western blot analise van SCG10, geasetieleerde α-tubulien, Tau, LC3 en Sequestosome 1/p62 (SQTM1) proteïenvlakke is uitgevoer. Super-resolusie gestruktureerde verligting mikroskopie (SR-SIM) driedimensionele (3-D) beelding van WDR47-YFP getransfekteerde selle, konfokale mikroskopie vir visualisering van LC3 en tubulien, co-lokalisering analise van beide WDR47 en LC3 en WDR47 en tubulien, asook fluorescentie hersteling na foto-bleek (FRAP) analise is uitgevoer. Resultate Die gemiddelde migrasie-afstand en die migrasiesnelheid (μm/min) het beduidend afgeneem met WDR47 siRNA behandeling. SEM analise van WD47 siRNA-behandelde neurone het minder filopodia en veranderde oppervlak adhesie vertoon, en TEM analise het 'n verhoogde teenwoordigheid van endoplasmiese retikulum (ER) strukture, en 'n uitgebreide kernmembraan vertoon. LC3-II proteïenvlakke was beduidend laer met slegs WDR47 siRNA behandeling, maar beduidend hoёr met WDR47 siRNA behandeling in samewerking met Bafilomycin A1 behandeling. Hierdie resultate dui aan op toeneemende autofagie met WDR47 siRNA behandeling. Verder, beduidend laer vlakke van SCG10 proteïenvlakke is waargeneem met WDR47 siRNA behandeling. SR-SIM en konfokale mikroskopie van WDR47 siRNA behandelde selle het 'n robuuste teenwoordigheid van hoogs buigende geasetieleerdetubulien in die area rondom die nukleus, 'n afgeneemde LC3 Bespreking en Gevolgtrekking Die resultate dui daarop aan dat WDR47 betrokke is by die regulering van neuronale migrasie, filopodia vormasie, oppervlak adhesie, mikrotubuliendinamika, en waarskynlik ook autofagie. Ons stel voor dat WDR47 mikrotubuliendinamika afekteer deur die regulering van proteïene soos SCG10, en sodoende mikrotubulienafhanklike prosesse soos neuronale migrasie en autofagie fasiliteer.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Odontologia, Florianópolis, 2016.
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Para inibir a diferenciação e manter as características das células-tronco, proteínas inibidoras da ligação de DNA (ID) antagonizam os fatores de transcrição basic helix-loop-helix (bHLH). A ubiquitinização da proteína ID ocorre em diferentes tecidos, mas em muitos neoplasmas esta consegue escapar da degradação. O complexo proteico USP1/WDR48 pode estar envolvido nesse processo. Paralelamente, a proteína p53, guardiã do genoma humano, controla a auto renovação neoplásica. Este estudo objetivou associar a expressão imuno-histoquímica das proteínas p53, USP1 e WDR48 aos dados clínico-histopatológicos de Carcinomas Epidermóides Intrabucais (CEI). Trinta casos de CEI, grupo teste, e 40 casos de Hiperplasia Fibrosa (HF), grupo controle, foram utilizados para as análises imuno-histoquímicas (anticorpos anti-p53, anti-USP1 e anti-WDR48). A expressão destes marcadores foi dividida em 4 categorias (núcleo+, citoplasma+, núcleo e citoplasma+ e células-). As fichas de biópsia, laudos e prontuários foram avaliados. A classificação histopatológica (Bryne et al., 1992) foi realizada por meio de lâminas com coloração HE. Após, os dados foram submetidos a análise estatística (Kruskal Wallis). Os resultados imuno-histoquímicos apresentaram diferença estatisticamente significante entre os três marcadores (p53, USP1 e WDR48) (p=0,0001 para todos os grupos), e também entre as lesões (CEI ou HF) em 5 grupos (p=0,0000; 0,0028; 0,0010; 0,0000; 0,0413), sendo que, na maiora das vezes, a expressão em HF foi inferior à expressão em CEI. Nenhuma associação com os achados clínicos foi identificada (TNM), mas observou-se com os histopatológicos. Os CEIs bem diferenciados foram os que obtiveram as menores médias de expressão dos marcadores. Conclui-se que parece haver uma associação entre a expressão das proteínas investigadas e CEI, bem como com o grau de malignidade deste tipo de lesão, porém não com as suas características clínicas. Deste modo, os marcadores p53, USP1 e WDR48 têm potencial determinante de prognóstico e tratamento de CEI.

Abstract: To inhibit differentiation and maintain stem cell fate, Inhibitors of DNA binding (IDs) antagonize basic helix-loop-helix (bHLH) transcription factors. ID ubiquitination occurs in differentiated tissues, but IDs in many neoplasms appear to escape degradation. The protein complex USP1/WDR48 can be involved in this process. At the same time, p53 protein, human genome guardian, controls the neoplastic self renewal. When occurs loss of function of the tumor suppressor gene TP53 there is the possibility of cancer development. This study aimed to associate the immunohistochemical expression of p53, USP1, WDR48 and clinical-histopathological characteristic of OSCC (oral squamous cell carcinoma). Thirty OSCC cases, test group, and 40 FH cases (fibrous hyperplasia), control group, were used for immunohistochemical analysis (anti-p53, anti-USP1 and anti-WDR48). The expression of these markers was divided into 4 categories (nucleus+; cytoplasm+, nucleus and cytoplasm+ and cells-). Biopsy form, histopathological results and patients? records were evaluated. The histopathological classification (Bryne et al., 1992) was performed using HE staining. After that, data were subjected to statistical analysis (Kruskal Wallis). The immunohistochemical results revealed a statistically significant difference among the three markers (p53, USP1 and WDR48) (p=0,0001 to all groups), and also between the lesions (OSCC or FH) in 5 groups (p=0,0000; 0,0028; 0,0010; 0,0000; 0,0413). The expression in FH was less commonly found than expression in OSCC. No association with clinical findings was identified (TNM), but it was noted regarding histopathological issues. Well differentiated OSCC achieved the lowest average of expression of the markers. We may conclude that, possibly, exist an association between these proteins and OSCC, its immunolocalization can be connected with malignant grading. However, the association with clinical findings cannot be determined. The p53, USP1 and WDR48 have the potential to define OSCC prognosis and treatment.

Les Encéphalopathies Épileptiques Précoces sont des pathologies rares et sévères caractérisées par des crises fréquentes commençant dans les trois premiers mois de vie accompagnées d’un EEG intercritique altéré et un pronostic très défavorable. Au cours de la caractérisation génétique d’une cohorte de 402 patients, nous avons mis en évidence une délétion de 19,9 kb localisée en Xp11.23 chez un garçon et 34 mutations de novo du gène KCNQ2. La première partie de mon projet a consisté en l’étude de la pathogénicité de la délétion Xp11.23, qui implique trois gènes dont WDR45. Les mutations de WRD45 ont été décrites dans une dégénérescence neuronale avec accumulation de fer et presque exclusivement chez des patients de sexe féminin. Le diagnostic initial, chez ce patient, montre une IRM normale avec un phénotype d'EEP et l'accumulation de fer a été détectée à partir de 5 ans. Ce travail m’a permis de décrire le premier patient atteint d’EEP porteur d’une délétion de WDR45. La deuxième partie de mon projet a concerné le gène KCNQ2. Nos résultats ont montré que les mutations sont impliquées dans deux mécanismes physiopathologiques, une délocalisation subcellulaire et un gain de fonction. Ces résultats ouvrent de nouvelles perspectives en terme de compréhension de la pathologie et de thérapies qui peuvent être proposées. Une dernière partie de ce projet a consisté en l’élaboration de nouveaux modèles in vitro, j’ai mis au point des lignées stables exprimant KCNQ2 qui permettront le criblage de molécules thérapeutiques à haut-débit, ainsi que des progéniteurs neuronaux différenciés à partir de cellules iPS issues de la reprogrammation de fibroblastes de patients
Early onset epileptic encephalopathies are rare and severe disorders, characterized by frequent motor seizures occurring before three months of age associated with an altered interictal EEG pattern. The prognosis is poor. During the course of the genetic characterization of a cohort of 402 EOEE patients, we identified a de novo deletion located at Xp11.23 in a male patient and 34 KCNQ2 de novo mutations. The first part of my project consisted in the study of the pathogenicity of the Xp11.23 deletion that encompasses three genes including WDR45. Mutations in the WDR45 gene been have recently identified in patients suffering from neurodegeneration with brain iron accumulation. WDR45 mutations have been almost exclusively found in females. Our patient with the Xp11.23 deletion presented a normal MRI and the EOEE phenotype was predominant. Iron accumulation began only at 5 years. My work reveals that deletions of WDR45 are viable in males and can be diagnosed as EOEE. The second part of my project was aimed at the functional study of two KCNQ2 gene mutations. During this work, my results showed that those mutations were involved in new pathological mechanisms, namely a mislocalization or gain of function. Those results provide new perspectives in term of disease knowledge and therapy. The last part of my project consisted in the development of two new in vitro models for the study of KCNQ2 mutations: stable cell lines expressing the Kv7.2 channel for high-throughput screening of drugs and the production of neurons from induced pluripotent stem cells arising from reprogrammed patient fibroblasts

Les malformations du cortex cérébral (MDC) représentent une cause importante de handicap et d'épilepsie pharmaco-résistante. Le séquençage à haut débit a permis une amélioration considérable de l'identification des bases moléculaires des MDC non syndromiques. Toutefois, certaines formes, notamment les MDC complexes, demeurent inexpliquées. Mon projet de thèse a pour objectif de progresser dans la compréhension des MDC complexes en utilisant deux modèles : les microlissencéphalies (MLIS) et le syndrome d'Aicardi (AIC), une forme syndromique particulière associant des malformations de l'oeil et du cerveau uniquement rapporté chez les filles. L'étude par séquençage d'exome en trios de 16 familles MLIS m'a permis d'identifier et de caractériser un nouveau gène, WDR81, impliqué dans le cycle cellulaire. Par la même stratégie, j'ai pu identifier un variant homozygote pathogène dans TLE1, un partenaire majeur de FOXG1 dans la balance prolifération/différenciation de progéniteurs neuronaux, dans une famille consanguine de microcéphalie postnatale dont le phénotype est proche du syndrome FOXG1. En parallèle, mes travaux ont permis de préciser les spectres phénotypiques associés à RTTN, EPG5, COL4A1, COL4A2, TBR1, KIF5C, KIF2A et FOXG1. La deuxième partie de mon projet avait pour objet l'identification des bases moléculaires du syndrome d'Aicardi à partir d'une cohorte internationale de 19 patientes. Après avoir exclu un biais d'inactivation du chromosome X et la présence de microremaniements chromosomiques, j'ai réalisé un séquençage d'exome en trio. Aucun variant récurrent n'a été retrouvé dans les séquences codantes. Dans un second temps, j'ai testé une approche combinant les données du séquençage de génome et l'analyse du transcriptome (RNA-Seq) sur fibroblastes, me permettant d'identifier des transcrits dérégulés qui étaient impliqués dans le développement du cerveau et de l'oeil. J'ai comparé les résultats de cette analyse avec ceux de l'analyse du génome dans le but d'identifier des variants dans ces gènes candidats. En conclusion, mon travail de thèse a permis d'améliorer la connaissance des bases moléculaires des MDC complexes et d'ouvrir des perspectives de nouveaux mécanismes tels que ceux engageant les gènes WDR81 et EPG5, et le rôle des endosomes et de l'autophagie dans les MDC, et aussi TLE1 comme nouvelle cause de microcéphalies postnatales. Mes travaux ont également permis de générer une collection de données de séquençage haut débit (WES, WGS et RNA-Seq) qui seront mises en commun dans le cadre d'un consortium international afin de développer des nouvelles stratégies d'analyse en particulier pour les séquences non codantes. Cette approche permettra également d'ouvrir la voie vers la compréhension des mécanismes cellulaires impliqués dans la formation du cerveau et de l' œil
Malformations of cortical development (MCD) are a major cause of intellectual disability and drug-resistant epilepsy. Next Generation Sequencing (NGS) has considerably improved the identification of the molecular basis of non-syndromic MCD. However, certain forms, including complex MCD, remain unexplained. My PhD project aimed to improve the understanding of complex MCD using two disorders: Microlissencephaly (MLIS) and Aicardi Syndrome (AIC), the latter associating brain and eye malformations and only reported in girls. Trio Whole Exome Sequencing (WES) performed in 16 MLIS families allowed me to identify and functionally characterize a new MLIS gene, WDR81, in which mutations lead to cell cycle alteration. Moreover, using the same strategy, I was able to identify a pathogenic homozygous variant in TLE1 in a patient from consanguineous family with a postnatal microcephaly, suggestive of a FOXG1-like presentation. Interestingly, TLE1 is a major partner of FOXG1, a gene involved in maintaining the balance between progenitor proliferation and differentiation. In parallel, my work allowed me to redefine the phenotypic spectrum associated with RTTN, EPG5, COL4A1 and COL4A2, TBR1, KIF5C, KIF2A and FOXG1. The second part of my PhD program was aimed at identifying the genetic basis of AIC in an international cohort of 19 patients. After excluding a skewed X chromosome inactivation and the presence of chromosomal rearrangements, I performed WES in trios. The analysis of the data from WES did not allow me to identify any recurrent variants. I therefore tested a new approach combining Whole Genome Sequencing (WGS) and RNA-Sequencing (RNA-Seq) on fibroblast cells. I identified a number of deregulated transcripts implicated in brain and eye development. I compared the results of this analysis with the WGS analysis in order to find variants in these candidate genes. In conclusion, these studies have improved the knowledge of the molecular basis of complex MCD, such as TLE1 in postnatal microcephaly, and revealed the pathogenic mechanisms such as WDR81 in cell cycle progression and EPG5 in endosomes and autophagy. My work has also generated a collection of NGS data (WES, WGS and RNA-Seq) that will be shared in an international consortium to develop new analytical strategies, in particular for the non-coding DNA regions. This novel strategy provides opportunities to improve understanding of the cellular mechanisms involved in brain and eye development

Trans/cis photoisomerization of azobenzene (Az) molecular films on the substrate is capable of inducing homeotropic/planar alignment changes of nematic liquid crystals (LCs). These photofunctional surfaces are called "command surfaces." (Fig.1)1 The Langmuir-Blodgett (LB) technique provides importance information on the design of the surface layer. The LB film preparation condition should influence, to a large extent, the aligning behavior of the nematic LC as formerly proposed by Hiltrop and Stegemeyer.2 The present work is undertaken to elucidate the relationship between the conditions of the LB film conditions and the LC aligning behaviors. For Az LB film preparation, two sets of amphiphilic Az side chain polymers are employed, the backbone being (i) poly(vinyl alcohol) and (ii) poly(ethyleneimine). Polymeric materials are required to obtain highly photoreactive Az LB films. Single monolayers are prepared as the Az LB films throughout the investigation to avoid complexity of the film structure.