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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.

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.

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.

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.

Microcephaly is neurological condition within which the brain fails to develop to a normal size resulting in the appearance of a smaller head. Microcephaly often accompanies various neurodevelopmental disorders. The corpus callosum is the largest white matter structure in the brain, comprised primarily of heavily myelinated axons. The corpus callosum connects the left and right hemisphere and allows for communication to occur between hemispheres. Using MRI measurements from a sample of 18 microcephalic patients, I analyzed whether the corpus callosum was impacted as a result of microcephaly. When compared to normocephalic controls, the corpus callosum was generally smaller in relation to overall cerebral hemispheric volume, suggesting that white matter brain tissues may be affected by microcephaly. A deeper understanding of the brain through research on the underlying mechanisms responsible for brain evolution and development is critical to our ability to detect, treat and prevent neurodevelopmental, neurodegenerative and psychiatric disorders. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Orientador: Ana Cláudia Figueiredo Frizzo / Resumo: Introdução: O Zica vírus tornou-se uma epidemia no Brasil a partir do início de 2015, tendo os primeiros casos de microcefalia com suspeita de causa pelo Zica Virus confirmados nos estados do Rio Grande do Norte e Pernambuco. Desde então, pesquisas evidenciam a necessidade de maiores investigações acerca da fisiologia dos indivíduos afetados pela doença, sintomas e prognósticos a fim de aprimorar o tratamento das crianças com a chamada “síndrome congênita do zica vírus”. Desta forma, para estudar e minimizar os impactos que uma possível alteração no córtex auditivo possa acarretar no desenvolvimento comunicativo destas crianças, este estudo tem como objetivo principal descrever os valores do potencial cortical auditivo de crianças que provavelmente foram infectadas pela síndrome congênita do Zica vírus. Métodos: Trata-se de um estudo multicêntrico, transversal descritivo, o qual foi desenvolvido no Setor de Audiologia do Centro de Estudos da Educação e da Saúde (CEES), da Universidade Estadual Paulista (UNESP), e na Universidade Estadual de Ciências da Saúde de Alagoas – UNCISAL, Laboratório de Audiologia do Centro Especializado em Reabilitação (CER III), com aprovação no comitê de ética CAAE nº 68684117.8.1001.5406. Participaram do estudo 30 crianças, de ambos os gêneros, de seis a 38 meses de vida, compondo o grupo estudo, com microcefalia e provável síndrome congênita do zica vírus e grupo controle, nos quais foram realizados exames de potencial evocado auditivo cortical e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: The Zika virus became an epidemic in Brazil from the beginning of 2015, with the first cases of microcephaly with suspected cause by Zika Virus confirmed in the states of Rio Grande do Norte and Pernambuco. Since then, research has shown the need for further research into the physiology of individuals affected by the disease, symptoms and prognosis in order to improve the treatment of children with the so-called congenital zygote virus syndrome. Thus, in order to study and minimize the impacts that a possible alteration in the auditory cortex may have on the communicative development of these children, this study has as main objective to describe the cortical auditory potential values of children who were probably infected by the congenital syndrome of Zika virus. Methods: This is a multicenter, cross-sectional descriptive study, which was developed in the Audiology Sector of the Center for Education and Health Studies (CEES), Universidade Estadual Paulista (UNESP), and at the State University of Health Sciences of Alagoas - UNCISAL, Audiology Laboratory of the Specialized Center for Rehabilitation (CER III), with approval in the ethics committee CAAE nº 68684117.8.1001.5406. The study consisted of 30 children of both genders, from six to 38 months of age, making up the study group, with microcephaly and probable congenital syndrome of the virus zika and control group, in which cortical auditory evoked potential tests were performed in children with development.... (Complete abstract click electronic access below) / Mestre

The molecular mechanisms that coordinate postnatal brain enlargement, synaptic properties and cognition remain an enigma. This study demonstrates that neuronal complexity controlled by p21-activated kinases (PAKs) is a key determinant for postnatal brain enlargement and synaptic properties. Double knockout (DK) mice lacking both PAK1 and PAK3 were severely impaired in postnatal brain growth, resulting in a dramatic reduction in brain volume at maturity. Remarkably, the reduced brain was accompanied by minimal changes in total cell count, due to a significant increase in cell density. However, the DK neurons have smaller soma, markedly simplified dendritic arbors/axons and reduced synapse density. Surprisingly, the DK mice were elevated in basal synaptic responses due to enhanced individual synaptic potency, but severely impaired in bi-directional synaptic plasticity. The PAK1/3 action is likely mediated by cofilin-dependent actin regulation because the activity of cofilin and the properties of actin filaments were specifically altered in the DK mice.

The molecular mechanisms that coordinate postnatal brain enlargement, synaptic properties and cognition remain an enigma. This study demonstrates that neuronal complexity controlled by p21-activated kinases (PAKs) is a key determinant for postnatal brain enlargement and synaptic properties. Double knockout (DK) mice lacking both PAK1 and PAK3 were severely impaired in postnatal brain growth, resulting in a dramatic reduction in brain volume at maturity. Remarkably, the reduced brain was accompanied by minimal changes in total cell count, due to a significant increase in cell density. However, the DK neurons have smaller soma, markedly simplified dendritic arbors/axons and reduced synapse density. Surprisingly, the DK mice were elevated in basal synaptic responses due to enhanced individual synaptic potency, but severely impaired in bi-directional synaptic plasticity. The PAK1/3 action is likely mediated by cofilin-dependent actin regulation because the activity of cofilin and the properties of actin filaments were specifically altered in the DK mice.

Chromatin remodeling complexes modulate DNA accessibility permitting neuronal progenitor cells to proliferate and differentiate to form the mammalian neocortex. In the case of BPTF (Bromodomain PHD transcription Factor), the major subunit of a chromatin remodelling complex called NURF (Nucleosome Remodelling Factor), mutations leading to its haploinsufficiency have been linked to cause a recently annotated human neurodevelopmental disorder called NEDDFL (Neurodevelopmental disorder with dysmorphic facies and distal limb anomalies). Patients with this syndrome are mainly characterized with microcephaly and intellectual disability. We conditionally knockout (cKO) the Bptf gene during neocortical neurogenesis to analyze its role during embryonic and postnatal brain development. The Bptf cKO animals reveal significant forebrain hypoplasia. During cortical neurogenesis, the Bptf cKO mice show a reduction in intermediate neuronal progenitor (INP) cells, an increase in apoptosis as well as a prolonged cell cycle within proliferating progenitors. Similarly, the postmitotic pyramidal neurons of the Bptf cKO mice contained lower levels of Ctip2 and Foxp1. Lastly, our RNA-seq analysis delineated gene pathways deregulated by Bptf removal, which are involved in neurogenesis and neuronal differentiation. Our results indicate that Bptf is critical for murine telencephalon neurogenesis. The hypoplasia demonstrated in the mouse model can resemble the microcephaly displayed by the human NEDDFL patients, highlighting the relevance of chromatin remodelling complexes during intricate neural developmental processes.