Centrosome defects cause microcephaly by activating the 53BP1-USP28-TP53 mitotic surveillance pathway

Mutations in centrosome genes deplete neural progenitor cells (NPCs) during brain development, causing microcephaly. While NPC attrition is linked to TP53-mediated cell death in several microcephaly models, how TP53 is activated remains unclear. In cultured cells, mitotic delays resulting from centrosome loss prevent the growth of unfit daughter cells by activating a pathway involving 53BP1, USP28, and TP53, termed the mitotic surveillance pathway. Whether this pathway is active in the developing brain is unknown. Here, we show that the depletion of centrosome proteins in NPCs prolongs mitosis and increases TP53-mediated apoptosis. Cell death after a delayed mitosis was rescued by inactivation of the mitotic surveillance pathway. Moreover, 53BP1 or USP28 deletion restored NPC proliferation and brain size without correcting the upstream centrosome defects or extended mitosis. By contrast, microcephaly caused by the loss of the non-centrosomal protein SMC5 is also TP53-dependent but is not rescued by loss of 53BP1 or USP28. Thus, we propose that mutations in centrosome genes cause microcephaly by delaying mitosis and pathologically activating the mitotic surveillance pathway in the developing brain

GENOME STABILITY DURING NEURODEVELOPMENT: THE IMPORTANCE OF THE STRUCTURAL MAINTENANCE OF CHROMOSOMES COMPLEX, SMC5/6

Structural maintenance of chromosome (SMC) complexes are highly conserved multisubunit proteins that regulate structural and functional chromosome homeostasis in all domains of life. There are three classes of SMC complexes expressed in all eukaryotes; cohesin, condensin, and SMC5/6. Cohesin is canonically known to mediate sister chromatid cohesion and regulate gene expression. Condensins play a central role in chromosome organization, condensation, and segregation. SMC5/6 is implicated in DNA damage repair and genome stability. Mutations in subunits of all SMC complexes are linked to human diseases, ranging from developmental abnormalities to malignancies. Molecular functions and associated pathologies of cohesin and condensin have been extensively studied and previously reviewed. Therefore, in Chapter I we focus on the discussion of the current knowledge of the less well characterized member of the SMC complex family, SMC5/6. The research presented in this thesis emphasizes the importance of the SMC5/6 complex during neurodevelopment. In Chapter II we use mouse to model neurodevelopmental defects that ensue due to mutation of Smc5. We find that Smc5 cKO mice exhibited neurodevelopmental defects due to neural progenitor cell apoptosis, which led to a reduction in cortical layer neurons. SMC5/6 knockout triggers a CHEK2-p53 DNA damage response, as concurrent deletion of the Trp53 tumor suppressor or Chek2 DNA damage checkpoint kinase alleviated Smc5 cKO neurodevelopmental deficiencies. In Chapter III we investigated the roles of SMC5/6 in mouse behavior and found that Smc5 is imperative for normal sensorimotor function. Chapter IV discusses the findings reported in the prior chapters of the thesis and proposes future experimental routes to delineate the role of SMC5/6 during cell cycle progression, in heterochromatin maintenance, post-mitotic neurons, and during DNA replication and repair. Overall, the findings from this thesis provide critical insights into the cellular and molecular mechanisms that the SMC5/6 complex is crucial for during mammalian neurogenesis

GENOME STABILITY DURING NEURODEVELOPMENT: THE IMPORTANCE OF THE STRUCTURAL MAINTENANCE OF CHROMOSOMES COMPLEX, SMC5/6

Structural maintenance of chromosome (SMC) complexes are highly conserved multisubunit proteins that regulate structural and functional chromosome homeostasis in all domains of life. There are three classes of SMC complexes expressed in all eukaryotes; cohesin, condensin, and SMC5/6. Cohesin is canonically known to mediate sister chromatid cohesion and regulate gene expression. Condensins play a central role in chromosome organization, condensation, and segregation. SMC5/6 is implicated in DNA damage repair and genome stability. Mutations in subunits of all SMC complexes are linked to human diseases, ranging from developmental abnormalities to malignancies. Molecular functions and associated pathologies of cohesin and condensin have been extensively studied and previously reviewed. Therefore, in Chapter I we focus on the discussion of the current knowledge of the less well characterized member of the SMC complex family, SMC5/6. The research presented in this thesis emphasizes the importance of the SMC5/6 complex during neurodevelopment. In Chapter II we use mouse to model neurodevelopmental defects that ensue due to mutation of Smc5. We find that Smc5 cKO mice exhibited neurodevelopmental defects due to neural progenitor cell apoptosis, which led to a reduction in cortical layer neurons. SMC5/6 knockout triggers a CHEK2-p53 DNA damage response, as concurrent deletion of the Trp53 tumor suppressor or Chek2 DNA damage checkpoint kinase alleviated Smc5 cKO neurodevelopmental deficiencies. In Chapter III we investigated the roles of SMC5/6 in mouse behavior and found that Smc5 is imperative for normal sensorimotor function. Chapter IV discusses the findings reported in the prior chapters of the thesis and proposes future experimental routes to delineate the role of SMC5/6 during cell cycle progression, in heterochromatin maintenance, post-mitotic neurons, and during DNA replication and repair. Overall, the findings from this thesis provide critical insights into the cellular and molecular mechanisms that the SMC5/6 complex is crucial for during mammalian neurogenesis

Centrosome defects cause microcephaly by activating the 53BP1-USP28-TP53 mitotic surveillance pathway

Mutations in centrosome genes deplete neural progenitor cells (NPCs) during brain development, causing microcephaly. While NPC attrition is linked to TP53-mediated cell death in several microcephaly models, how TP53 is activated remains unclear. In cultured cells, mitotic delays resulting from centrosome loss prevent the growth of unfit daughter cells by activating a pathway involving 53BP1, USP28, and TP53, termed the mitotic surveillance pathway. Whether this pathway is active in the developing brain is unknown. Here, we show that the depletion of centrosome proteins in NPCs prolongs mitosis and increases TP53-mediated apoptosis. Cell death after a delayed mitosis was rescued by inactivation of the mitotic surveillance pathway. Moreover, 53BP1 or USP28 deletion restored NPC proliferation and brain size without correcting the upstream centrosome defects or extended mitosis. By contrast, microcephaly caused by the loss of the non-centrosomal protein SMC5 is also TP53-dependent but is not rescued by loss of 53BP1 or USP28. Thus, we propose that mutations in centrosome genes cause microcephaly by delaying mitosis and pathologically activating the mitotic surveillance pathway in the developing brain

An international report on bacterial communities in esophageal squamous cell carcinoma

The incidence of esophageal squamous cell carcinoma (ESCC) is disproportionately high in the eastern corridor of Africa and parts of Asia. Emerging research has identified a potential association between poor oral health and ESCC. One possible link between poor oral health and ESCC involves the alteration of the microbiome. We performed an integrated analysis of four independent sequencing efforts of ESCC tumors from patients from high- and low-incidence regions of the world. Using whole genome sequencing (WGS) and RNA sequencing (RNAseq) of ESCC tumors from 61 patients in Tanzania, we identified a community of bacteria, including members of the genera Fusobacterium, Selenomonas, Prevotella, Streptococcus, Porphyromonas, Veillonella and Campylobacter, present at high abundance in ESCC tumors. We then characterized the microbiome of 238 ESCC tumor specimens collected in two additional independent sequencing efforts consisting of patients from other high-ESCC incidence regions (Tanzania, Malawi, Kenya, Iran, China). This analysis revealed similar ESCC-associated bacterial communities in these cancers. Because these genera are traditionally considered members of the oral microbiota, we next explored whether there was a relationship between the synchronous saliva and tumor microbiomes of ESCC patients in Tanzania. Comparative analyses revealed that paired saliva and tumor microbiomes were significantly similar with a specific enrichment of Fusobacterium and Prevotella in the tumor microbiome. Together, these data indicate that cancer-associated oral bacteria are associated with ESCC tumors at the time of diagnosis and support a model in which oral bacteria are present in high abundance in both saliva and tumors of some ESCC patients