RESUMO
Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1-5' UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.
Assuntos
Proteínas de Ligação ao Cálcio , Infertilidade Masculina , Animais , Humanos , Masculino , Camundongos , Divisão Celular , Proteínas do Citoesqueleto/genética , Sequenciamento do Exoma , Fertilidade/genética , Infertilidade Masculina/genética , Espermatogênese/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genéticaRESUMO
COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within 2 days of exposure. We identified that SARS-CoV-2 preferably targets neurons of brain organoids. Imaging neurons of organoids reveal that SARS-CoV-2 exposure is associated with altered distribution of Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Our studies, therefore, provide initial insights into the potential neurotoxic effect of SARS-CoV-2 and emphasize that brain organoids could model CNS pathologies of COVID-19.
Assuntos
Betacoronavirus/fisiologia , Encéfalo/virologia , Neurônios/virologia , Animais , Morte Celular , Chlorocebus aethiops , Humanos , Doenças do Sistema Nervoso/virologia , Organoides , SARS-CoV-2 , Células Vero , Proteínas tau/metabolismoRESUMO
Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP-dependent peri-centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP-tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP-tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de-clustering, prolonged multipolar mitosis, and cell death. 3D-organotypic invasion assays reveal that CCB02 has broad anti-invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)-resistant EGFR-mutant non-small-cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug-resistant cancers exhibiting high incidence of centrosome amplification.
Assuntos
Centrossomo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/administração & dosagem , Tubulina (Proteína)/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HeLa , Humanos , Camundongos , Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms ofNPCs maintenance remain unclear. Here, we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, andOFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.
Assuntos
Cílios/metabolismo , Microcefalia/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/patologia , Aurora Quinase A/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Cílios/genética , Cílios/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação , Células-Tronco Neurais/metabolismo , Proteínas/metabolismo , SíndromeRESUMO
Induced pluripotent stem cell-derived brain organoids enable the developmental complexities of the human brain to be deconstructed. During embryogenesis, optic vesicles (OVs), the eye primordium attached to the forebrain, develop from diencephalon. However, most 3D culturing methods generate either brain or retinal organoids individually. Here we describe a protocol to generate organoids with both forebrain entities, which we call OV-containing brain organoids (OVB organoids). In this protocol, we first induce neural differentiation (days 0-5) and collect neurospheres, which we culture in a neurosphere medium to initiate their patterning and further self-assembly (days 5-10). Then, upon transfer to spinner flasks containing OVB medium (days 10-30), neurospheres develop into forebrain organoids with one or two pigmented dots restricted to one pole, displaying forebrain entities of ventral and dorsal cortical progenitors and preoptic areas. Further long-term culture results in photosensitive OVB organoids constituting complementary cell types of OVs, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections and electrically active neuronal networks. OVB organoids provide a system to help dissect interorgan interactions between the OVs as sensory organs and the brain as a processing unit, and can help model early eye patterning defects, including congenital retinal dystrophy. To conduct the protocol, experience in sterile cell culture and maintenance of human induced pluripotent stem cells is essential; theoretical knowledge of brain development is advantageous. Furthermore, specialized expertise in 3D organoid culture and imaging for the analysis is needed.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Diferenciação Celular/fisiologia , Prosencéfalo , Organoides , Desenvolvimento EmbrionárioRESUMO
COVID-19, caused by SARS-CoV-2, is a socioeconomic burden, which exhibits respiratory illness along with unexpected neurological complications. Concerns have been raised about whether the observed neurological symptoms are due to direct effects on CNS or associated with the virus's systemic effect. Recent SARS-CoV-2 infection studies using human brain organoids revealed that SARS-CoV-2 targets human neurons. Human brain organoids are stem cell-derived reductionist experimental systems that have highlighted the neurotropic effects of SARS-CoV-2. Here, we summarize the neurotoxic effects of SARS-CoV-2 using brain organoids and comprehensively discuss how brain organoids could further improve our understanding when they are fine-tuned.
Assuntos
Encéfalo/virologia , COVID-19/virologia , Neurônios/virologia , Organoides/virologia , SARS-CoV-2/patogenicidade , Humanos , Células-Tronco/virologiaRESUMO
During embryogenesis, optic vesicles develop from the diencephalon via a multistep process of organogenesis. Using induced pluripotent stem cell (iPSC)-derived human brain organoids, we attempted to simplify the complexities and demonstrate formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day 30, brain organoids attempt to assemble optic vesicles, which develop progressively as visible structures within 60 days. These optic vesicle-containing brain organoids (OVB-organoids) constitute a developing optic vesicle's cellular components, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections, and electrically active neuronal networks. OVB-organoids also display synapsin-1, CTIP-positive myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photosensitive activity of OVB-organoids, and light sensitivities could be reset after transient photobleaching. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow interorgan interaction studies within a single organoid.
Assuntos
Células-Tronco Pluripotentes Induzidas , Organoides , Diferenciação Celular , Desenvolvimento Embrionário , Humanos , Organogênese , ProsencéfaloRESUMO
Brain organoids are stem cell-based self-assembling 3D structures that recapitulate early events of human brain development. Recent improvements with patient-specific 3D brain organoids have begun to elucidate unprecedented details of the defective mechanisms that cause neurodevelopmental disorders of congenital and acquired microcephaly. In particular, brain organoids derived from primary microcephaly patients have uncovered mechanisms that deregulate neural stem cell proliferation, maintenance, and differentiation. Not only did brain organoids reveal unknown aspects of neurogenesis but also have illuminated surprising roles of cellular structures of centrosomes and primary cilia in regulating neurogenesis during brain development. Here, we discuss how brain organoids have started contributing to decoding the complexities of microcephaly, which are unlikely to be identified in the existing non-human models. Finally, we discuss the yet unresolved questions and challenges that can be addressed with the use of brain organoids as in vitro models of neurodevelopmental disorders.
RESUMO
Glioblastoma (GBM) possesses glioma stem cells (GSCs) that exhibit aggressive invasion behavior in the brain. Current preclinical GBM invasion assays using mouse brain xenografts are time consuming and less efficient. Here, we demonstrate an array of methods that allow rapid and efficient assaying of GSCs invasion in human brain organoids. The assays are versatile to characterize various aspects of GSCs, such as invasion, integration, and interaction with mature neurons of brain organoids. Tissue clearing and quantitative 3D imaging of GSCs in host organoids reveal that invasiveness is inversely correlated with the organoids' age. Importantly, the described invasion assays can distinguish the invasive behaviors of primary and recurrent GSCs. The assays are also amenable to test pharmacological agents. As an example, we show that GI254023X, an inhibitor of ADAM10, could prevent the integration of GSCs into the organoids.
Assuntos
Encéfalo/fisiopatologia , Glioblastoma/fisiopatologia , Organoides/fisiopatologia , HumanosRESUMO
Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4's localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion.
Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Proteínas de Drosophila/química , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Larva/citologia , Masculino , Meiose , Proteínas Associadas aos Microtúbulos , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Espermatócitos/citologia , Espermatócitos/metabolismoRESUMO
The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.
Assuntos
Encéfalo/patologia , Diferenciação Celular , Células-Tronco Neurais/patologia , Células-Tronco Neurais/virologia , Organoides/patologia , Zika virus/isolamento & purificação , Zika virus/fisiologia , Centrossomo/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mitose , Células-Tronco Neurais/ultraestrutura , Zika virus/ultraestruturaRESUMO
Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets ß-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-ß surface of ß-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAP(F375A), with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAP(EE343RR) that unmasks the ß-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a 'clutch-like' mechanism.