A large brain is one of the most distinguishing features of humans compared to other members of the animal kingdom. During mammalian evolution there has been a disproportionate enlargement of the brain relative to body size and this expansion has been particularly prominent during the past 3 million years of human lineage. This must be the consequence of adaptive genetic alterations during mammalian evolution, but the genes and molecular processes altered are essentially unknown. One approach for identifying candidate genes for brain size regulation is through characterisation of Mendelian disorders of brain development. In particular, primary microcephaly has received considerable interest as a model disease for studying brain size regulators because patients present with a profoundly reduced brain size but have no other malformations. Genetic studies have identified mutations in seven genes that can cause primary microcephaly. All the primary microcephaly proteins localise to the centrosome at some stage during the cell cycle and have roles in a diverse range of functions including centrosome maturation, centriole formation and microtubule organisation at the spindle pole. The precise mechanism leading to primary microcephaly is not known but a prevalent hypothesis is that centrosome dysfunction disrupts mitosis of neural progenitor cells. Despite there being strong evidence in support of this hypothesis for most primary microcephaly genes, MCPH1 (the first primary microcephaly gene to be identified) always appeared to be functionally distinct from other primary microcephaly proteins. Most work on MCPH1 has focussed on its role in the DNA damage response and cell cycle timing rather than on its mitotic role. As a result, the aim of this thesis is to perform a detailed analysis of MCPH1 function during mitosis. In this thesis, three isoforms of MCPH1 were characterised and their localisation, expression and stability examined. It was established that MCPH1 is highly regulated during mitosis. MCPH1 transcript and protein levels vary significantly throughout the cell cycle and MCPH1 protein is targeted for degradation late in mitosis. In addition, MCPH1 is hyperphosphorylated during mitosis (in prometaphase-arrested cells) suggesting that phosphorylation could potentially regulate MCPH1 mitotic function. Twelve mitotic phosphorylation sites were identified by phosphopeptide mapping, many of which were CDK1 and PLK1 consensus sites. Both PLK1 and CDK1 also contribute to MCPH1 phosphorylation in vivo. Although MCPH1 non-phosphorylatable mutants localise normally during mitosis, binding to interaction partners may be affected which may have functional consequences. During mitosis MCPH1 localises to the centrosomes and kinetochores. Consistent with this localisation, RNAi-mediated knockdown of MCPH1 leads to metaphase arrest with multipolar spindles, major defects in chromosome alignment and loss of chromatid cohesion. In addition, MCPH1 deficient mouse embryonic fibroblast cells also demonstrate similar chromosome alignment defects, strengthening this finding in an independent system. Live-imaging of MCPH1 depleted cells demonstrate that a normal bipolar spindle and metaphase plate are initially formed, but subsequently chromosomes and chromatids drop off the metaphase plate and eventually the spindle collapses. This suggests that the primary function of MCPH1 is to allow timely progression through metaphase, possibly by mediating kinetochore-microtubule attachments to satisfy the spindle activated checkpoint. Therefore my work describes several roles for MCPH1 in mitosis (centrosome stability, chromosome alignment and metaphase progression) suggesting that its role in mitosis could result in primary microcephaly in a number of different ways.

Congenital microcephaly (conMiC) is a manifestation of severely disrupted prenatal brain development, caused by genetic defects, toxins, severe maternal malnutrition, or infection. The Zika virus outbreak and the devastating impact of Zika infection on the fetal brain have focused much attention on the cellular and molecular pathophysiology of conMiC. Mendelian conMiC disorders offer a unique opportunity for understanding gene and protein networks that direct cellular processes essential for prenatal brain development. Using OMIM and literature searches, I analyzed 68 conMiC disorders and their 65 corresponding genes. ConMiC-disorder phenotypes were characterized by analyzing the co-occurrence of ID, retinal abnormalities, seizures, and short stature. Short stature co-occurred with 70% of conMiC disorders, while seizures and retinopathy co-occurred with 68% and 37%, respectively. In 53% of conMiC disorders, seizures and short stature overlapped, while all features overlapped in 22% of conMiC disorders; only 7% of conMiC disorders lacked one of these co-occurring features. This shows conMiC genes are rarely specialized for brain growth, with generalized functions in overall body growth, retinal development, and/or regulation of neural activity. ConMiC-gene transcript accumulation in the brain is typically greatest during the prenatal period, and then declines postnatally, suggesting active transcriptional repression. Nonetheless, in neurons and glia of the adult brain, 44 conMiC genes had confirmed persistent protein accumulation. Experimental evidence indicates transcription in neural progenitor cells (NPCs) for at least 82% of conMiC genes. The spatiotemporal expression patterns of conMiC genes tend to align well with their biological functions and corresponding mutant phenotypes. Nearly 60% of conMiC gene products have functions in the cell cycle and/or DNA repair. Most conMiC disorders are caused by recessive, loss-of-function mutations. There are direct binding and regulatory interactions amongst many conMiC genes, which interact in larger networks and shared pathways. Depletion of single conMiC gene products can affect the transcript and/or protein levels of other conMiC gene products, which could have a “domino effect”, and disrupt entire networks important for brain development. Further evidence for this model is that 22 conMiC genes are consistently dysregulated in Zika-infected developing human brain tissue. Due to the complexity of conMiC genes and their interactions, there are many unique challenges to developing treatments for conMiC, particularly conMiC caused by maternal Zika-virus infection. However, insights to treatment strategies could be gained by using human genetics to find potential modifiers, screening for drugs that can normalize disrupted cell cycle and DNA-repair processes, or can stabilize protein complexes that are disrupted due to a conMiC gene mutation.

No description available.

Autosomal recessive primary microcephaly (MCPH) is defined by congenital microcephaly and associated mental retardation with head circumference of the affected individual at least 3 standard deviations below age- and sex-means. It is a disorder of abnormal fetal brain growth which is a consequence of impaired neurogenesis. It is genetically heterogeneous with seven known loci and genes for all the seven loci have been identified: MCPH-1-MCPH1, MCPH2-WDR62, MCPH3-CDK5RAP2, MCPH4-CEP152, MCPH5-ASPM, MCPH6-CENPJ, and MCPH7-STIL. All the seven MCPH proteins localize at the centrosome. Apart from MCPH, many other proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. For example, Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. The study of MCPH genes can also provide insights into the basics of neurogenesis that lead to a normal brain size. The most common cause of MCPH is mutations in the ASPM (abnormal spindle-like, microcephaly-associated protein) gene. The main aim of this study was to gain insight into the function of ASPM using the yeast two-hybrid technique. The main findings of the study are listed below. To find novel interacting proteins for SPM, a GAL4 based yeast two-hybrid system was used. The 3,477 amino acid long ASPM was divided into eight different baits and each bait was individually used for screening a human fetal brain cDNA library cloned in the pACT2 vector. To generate baits, the different regions were amplified from human fetal brain cDNA and cloned in-frame with the GAL4-DNA binding domain in the pGBKT7 vector. Screening with a C-terminus ASPM bait (pGBKT7-CTR) identified Angelman syndrome protein ubiquitin protein ligase E3A (UBE3A) as an ASPM interactor. A region of UBE3A from amino acids 639-875 was found to interact with ASPM. The identification of UBE3A as an ASPM interacting partner was interesting as more than 80% of Angleman syndrome patients are reported to have microcephaly. Screening with the baits pGBKT7-1.4 kb ASPM and pGBKT7-2.1 kb ASPM harboring parts of IQ domain identified calmodulin as an ASPM interating partner. The full length calmodulin was found to interact with the IQ domain of ASPM. The interactions identified in the yeast two-hybrid assay were confirmed in vivo by co-immunoprecipitation studies. For this, a rabbit polyclonal anti-ASPM antibody was raised against the N-terminal region of ASPM (from amino acids 544-1059). The specificity of the antibody was tested by Western blot analysis and immunofluorescence microscopy. ASPM antibody recognized the 410 KDa fulllength ASPM protein in lysates from human fetal tissues and different cell lines. Immunofluorescence analysis in HEK293 cells with the antibody revealed centrosomal staining of ASPM throughout mitosis and midbody staining in cytokinesis, as reported previously. Using antibodies against ASPM and UBE3A and human fetal kidney lysate, ASPM and UBE3A interaction was confirmed in vivo by co-immunoprecipitation. The interaction between ASPM and calmodulin was confirmed similarly. The relevance of the interaction between ASPM and UBE3A was pursued further Like ASPM, UBE3A localized to the centrosome throughout mitotic progression. ASPM levels were found to be unaffected upon overexpression of UBE3A in HEK293 cells, indicating that ASPM is not degraded by a UBE3A-dependent proteasomal pathway or the degradation may be spatial-temporal control. Further, immunofluorescence analysis of UBE3A overexpressing HEK293 cells revealed that UBE3A does not affect either the ASPM localization or its protein level at the centrosome. Synchronization of HEK293 cells in different cell cycle phases revealed that UBE3A is a cell cycle dependent protein and its level peaks in mitosis To explore the functional role of UBE3A’s increased level in mitosis, UBE3A was depleted in HEK293 cells with a shRNA construct and stable clones were generated. HEK293- UBE3A shRNA knockdown cells were examined for normal mitotic progession and spindle defects. There was a 3.81- to 5.52-fold increase in the frequency of anaphase/telophase cells with missegregated chromosomes in UBE3A knockdown clones as compared to scrambled clones. Hence, we identified a definitive role of UBE3A in chromosome segregation. Defective chromosome segregation has been reported in many studies associated with microcephaly-related proteins. Interestingly, chromosome malfunctioning has also been reported in Drosophilia asp mutants (ASPM orthologue) and Celegans aspm-1 knockdown cells. Therefore, the loss of both ASPM and UBE3A leading to chromosome segregation defects reveals the existence of a molecular pathway common to both ASPM and UBE3A As a consequence of chromosome missegregation, UBE3A knockdown cells were found to undergo abnormal cytokinesis and apoptosis. The percentage of apoptotic cells in UBE3A knockdown clones was 1.25- to 3.04-fold higher as compared to scrambled clones. Interestingly, an extensive apoptosis has been found in the neural folds of MCPH7 gene STIL null mice embryos. Thus, the present study links Angleman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time.

Autosomal recessive primary microcephaly (MCPH) is a congenital neurodevelopmental disorder characterised by a reduced occipital-frontal head circumference (OFC) of less than -3 SDs below the population mean for age and sex. It is a genetically heterogeneous disorder caused by mutations in one of the following 10 MCPH genes: MCPH1 (microcephalin 1), WDR62 (WD repeat domain 62), CDK5RAP2 (cyclin-dependent kinase 5 regulatory associated protein 2), CASC5 (cancer susceptibility candidate 5), CEP152 (centrosomal protein 152 kDa), ASPM (asp [abnormal spindle] homolog, microcephaly associated [Drosophila]), CENPJ (centromeric protein J), STIL (SCL/TAL1-interrupting locus), CEP135 (centrosomal protein 135 kDa) and CEP63 (centrosomal protein 135 kDa). The MCPH1 (microcephalin 1) gene is located on chromosome 8p23.1. Microsatellite analysis has previously shown LOH at the markers D8S518 and D8S277 flanking the MCPH1 locus in 1/21 oral tumors. Furthermore, LOH at the markers D8S1742 and D8S277 flanking the MCPH1 locus has also been observed in 2/32 hepatocellular carcinomas. MCPH1 has been found to be mutated in breast and endometrial cancers. Additionally, it was found to be downregulated at the transcript level in 19/30 ovarian cancer tissues and the protein level in 93/319 breast cancer tissues. Decreased MCPH1 protein levels are associated with triple negative breast cancers and a lower transcript level of MCPH1 correlates with lesser time for metastasis to occur in breast cancer patients. Interestingly, MCPH1 knockout mice in a null TP53 background show susceptibility to cancer.So far, studies have indicated that MCPH1 is a DNA repair protein. MCPH1 is required for the formation of DNA repair foci, chromatin relaxation, HR and NHEJ. It regulates G1/S and G2/M cell cycle checkpoints. Also, depletion of MCPH1 leads to genomic instability and centrosome amplification. Hence, the defect in the function of MCPH1 can lead to plethora of anomalies including cancer. Based on these observations, we hypothesized that MCPH1 may also function as a tumor suppressor (TS) gene, in addition to its role in the brain development. The purpose of this study was to test if MCPH1 also functions as a TS gene using different approaches in OSCC (oral squamous cell carcinoma). OSCC is the sixth most common type of cancer. It includes the cancer of the lips, anterior 2/3rd of the tongue, buccal mucosa, floor of the mouth, retromolar trigone and gingiva. Despite the advances in the treatment of oral cancer, the five-yr survival rate has not increased. Hence, the effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. LOH, mutations and promoter methylation in tumors are the hallmarks of TS genes. In order to ascertain the TS roles of MCPH1, we carried out LOH analysis in 81 matched blood/normal and tumor oral tissues using D8S1819, D8S277 and D8S1798 markers flanking the MCPH1 locus. The results showed LOH at one or more markers in 14/71 (19.72%) informative samples across the tumor stages from T1 to T4. The entire coding region and the exon-intron junctions of the MCPH1 gene were sequenced for mutations in 15 OSCC samples and 5 cancer cell lines (viz., A549, HeLa, KB, SCC084 and SCC131). In total, three mutations namely c.1561G>T(p.Glu521X), c.321delA(p.Lys107fsX39) and c.1402delA(p.Thr468fsX32) were identified. The expression of MCPH1 was analysed at both the transcript and protein levels by real-time quantitative RT-PCR and immunohistochemistry, respectively, in OSCC samples. MCPH1 was downregulated in 51.22% (21/41) of OSCC samples at the transcript level. The MCPH1 protein was downregulated in 76% (19/25) of the OSCC samples. In order to elucidate if the MCPH1 promoter was methylated in OSCC tissues, we retrieved the MCPH1 promoter from the database TRED (Transcriptional Regulatory Element Database). The promoter was analysed for the presence of CpG islands using the CpG Plot/CpG Report program. Two CpG islands (CpGI and CpGII) were identified within the MCPH1 promoter. Both the CpG islands were analysed for methylation in 40 OSCC samples by COBRA (Combined Bisulfite Restriction Analysis). CpGI showed no methylation in 40 OSCC samples. However, CpGII showed methylation in 4/40 (10%) OSCC samples and the methylation was absent in their corresponding normal oral tissues. To analyse the methylation of the MCPH1 promoter in cancer cell lines, HeLa, KB, SCC084 and SCC131 cells were treated with 5’-2-deoxy azacytidine (AZA), a methyltrasferase inhibitor. HeLa and KB cells did not show any change in the MCPH1 transcript level after the AZA treatment. However, SCC084 and SCC131 cells showed upregulation of MCPH1 after the treatment, suggesting methylation of the MCPH1 promoter. To validate these observations, we examined the methylation status of both the CpG islands in these cell lines. We found methylation of CpGII only in SCC084 cells. HeLa, KB and SCC131 cells showed no methylation of CpGI and CpGII. The results obtained by COBRA in these cell lines were further confirmed by bisulfite sequencing of CpGI and CpGII islands. Further, the upregulation of MCPH1 after azacytidine treatment in SCC131 cells can be attributed to a promoter independent mechanism or due to methylation of the CpG sites not examined by us. To elucidate the biological effects of MCPH1 in a cancer cell line, we generated stable clones overexpressing MCPH1 in KB cells. The results showed that MCPH1 overexpression decreased cellular proliferation, cell invasion, anchorage-independent growth in soft-agar and tumor growth in nude mice. Further, MCPH1 overexpression lead to apoptosis. A low frequency of LOH, mutations and promoter methylation suggested that they might not be the major mechanisms of downregulation of MCPH1 in OSCC. We then speculated that MCPH1 could be regulated by miRNAs. We therefore used five miRNA target prediction softwares to identify miRNAs targeting MCPH1. The programs identified two binding sites for miR-27a within the 5.4 kb region of the 3’-UTR of MCPH1. The luciferase assay showed that both the seed regions of MCPH1 were binding to miR-27a. In addition, transient transfection of the premiR-27a construct in KB cells decreased the protein level of MCPH1. Additionally, in a small panel of 10 OSCC samples, there was a negative correlation between the levels of miR-27a and MCPH1. To the best of our knowledge, this is the first report showing any miRNA regulating the MCPH1 gene. It is important to note that tumor suppressors can serve as potential biomarkers with prognostic value. Hence, we analysed the correlation of the expression levels of MCPH1 with clinico-pathological parameters such as TNM, gender, age and site of the cancer by Fischer’s exact test. No statistical correlation was observed between the transcript or protein levels with any of the clinico-pathological parameters. In summary, the results of the present study have suggested that the primary microcephaly gene MCPH1 shows several hallmarks of TS genes and functions as a tumor suppressor in OSCC, in addition to its role in brain development. We have for the first time shown that miR-27a targets MCPH1 and regulates its level. It is interesting to note that none of the other 10 MCPH genes have been shown to be regulated by any miRNA yet. Our study will be useful in designing novel therapeutic methods for the treatment of OSCC either by overexpression of MCPH1 or reducing the level of miR-27a by an antagomir.

Un des aspects les plus marquants de l'évolution des grands singes est l'augmentation relative du volume du cerveau, et en particulier du néocortex, qui culmine chez Homo sapiens. La microcéphalie primaire est une anomalie congénitale du développement cérébral humain caractérisée par un cerveau normalement formé mais de petit volume. Il en existe une forme isolée, non syndromique, dont la majorité des cas sont d'origine génétique et transmis sur le mode autosomique récessif (MCPH), qui constituent donc un modèle génétiquement simple qui résulte de l'altération d'un seul gène, essentiel dans le développement volumique du cerveau. Une consanguinité parentale est présente dans la majorité des cas, ce qui permet une approche puissante de localisation génomique de la mutation responsable, nommée cartographie d'homozygotie. A ce jour, huit gènes causant cette anomalie ont déjà été identifiés :BRIT1 (MCPH1), ASPM (MCPH5), CDK5RAP2 (MCPH3) et CENPJ (MCPH6), et plus récemment, STIL (MCPH7), CEP152 (MCPH9), WDR62 (MCPH2) et CEP135 (MCPH8). Tous ces gènes jouent un rôle au niveau du cycle cellulaire. Nous avons tenté, au cours de ce doctorat, d’identifier et de caractériser un nouveau gène du locus MCPH4 cartographié au laboratoire et situé sur le bras long du chromosome 15. <p>Dans trois familles MCPH4 originaires de villages voisins du Maroc rural, nous avons affiné la zone de liaison à un segment de 3,7cM, contenant un haplotype commun sur une longueur de 2,7cM suggérant un déséquilibre de liaison autour d’une mutation ancestrale. Le LOD score combiné dans les trois familles était supérieur à 6. Parmi les gènes contenus dans cette région, nous avons sélectionné des candidats que nous avons ensuite analysés par séquençage direct de l’ADN de nos patients. Parmi ces gènes, CASC5 présentait un variant, homozygote chez nos patients, hétérozygote chez leurs parents sains et absent chez 150 contrôles non apparentés. Nous avons utilisé la technologie 454 de séquençage à haut débit de Roche pour séquencer les gènes de l’intervalle de 2.7Mb en une fois. Parmi les mutations identifiées, nous n’avons trouvé qu’une seule variation exonique inconnue qui correspondait à la variation faux-sens déjà identifiée dans le gène CASC5. CASC5 est une protéine centromérique requise pour l’alignement des chromosomes à la métaphase et pour le point de contrôle métaphasique de la progression mitotique. Il était donc potentiellement un très bon candidat causal de la microcéphalie primaire. CASC5 lie directement MIS12, BUB1, BUBR1 et Zwint-1, et fait partie du réseau KMN du kinétochore. Il est nécessaire à l’ancrage des centromères chromosomiques au fuseau mitotique, et est requis pour le contrôle du cycle cellulaire au niveau du Spindle-Assembly Checkpoint.<p>Nous avons ensuite confirmé que la mutation génère un défaut d’épissage chez nos patients consistant en la perte partielle de l’exon 18 dans l’ARNm. La perte de cet exon conduit à un déphasage du cadre de lecture provoquant l’apparition d’un codon STOP prématuré dans l’exon 19. Ceci prédit donc la formation d’une protéine tronquée, ou absente après dégradation par le mécanisme cellulaire de dégradation des ARNm non-sens. Par Western-Blotting nous avons pu révéler, en lignée lymphoblastoïdes, la protéine CASC5 endogène chez tous nos patients, y compris, à notre surprise, chez les sujets atteints. <p>Il est décrit dans la littérature qu’un knockdown de CASC5 provoque un mauvais alignement des chromosomes, une entrée prématurée en mitose et la formation de micronoyaux, conséquence d’un mauvais alignement des chromosomes pendant la métaphase. Les différentes études menées sur le phénotype cellulaire de nos patients en lignées lymphoblastoïdes n’ont pu révéler ces défauts. Notre hypothèse est que l’allèle muté est hypomorphe et que le phénotype cellulaire décrit en boites de culture ne s’observerait in vivo que dans certaines cellules du cerveau en cours de développement.<p>En parallèle de ces travaux, nous avons également contribué à l’identification de la cause d’une microcéphalie primaire syndromique, associée à un diabète insulino-requérant précoce, tansmis sur le mode récessif autosomique et identifié dans une famille d’origine marocaine. Notre laboratoire avait localisé la mutation dans une région de 3 cM du chromosome 4. Parmi les 39 gènes compris dans cette région, nous en avons sélectionné et séquencé plusieurs. Aucun n’a cependant montré de mutation. Un séquençage de l'exome complet de l’un de nos patients, a permis de mettre en évidence une mutation non-sens homozygote dans un gène de l’intervalle critique de liaison. La mutation ségrège avec le phénotype autosomique récessif chez les malades, leurs parents et leurs germains asymptomatiques. L’abondance du transcrit de ce gène a été mesurée en lignées lymphoblastoïdes de patients :il est présent en quantité similaire chez les patients et chez un contrôle non apparenté. <p>En conclusion, notre travail a permis l’identification d’un nouveau gène muté chez des patients atteints de microcéphalie primaire, CASC5, avec un haut degré de preuve de causalité de cette mutation, impliquant ainsi une protéine du réseau KMN du kinétochore dans le développement volumique du cerveau humain. Nous avons par ailleurs contribué à l’identification d’un nouveau gène causant microcéphalie primaire et diabète juvénile, dont le mécanisme biologique est en cours d’investigation.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished