RESUMO
Primary cilia project from the surface of most vertebrate cells and are key in sensing extracellular signals and locally transducing this information into a cellular response. Recent findings show that primary cilia are not merely static organelles with a distinct lipid and protein composition. Instead, the function of primary cilia relies on the dynamic composition of molecules within the cilium, the context-dependent sensing and processing of extracellular stimuli, and cycles of assembly and disassembly in a cell- and tissue-specific manner. Thereby, primary cilia dynamically integrate different cellular inputs and control cell fate and function during tissue development. Here, we review the recently emerging concept of primary cilia dynamics in tissue development, organization, remodeling, and function.
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Cílios , Organelas , Cílios/metabolismo , Diferenciação CelularRESUMO
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.
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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.
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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.
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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 prime objective of modeling optical fibers is capturing mode confinement losses correctly. This paper demonstrates that specific modeling choices, especially regarding the outer fiber cladding regions and the placement of the computational boundary, have significant impacts on the calculated mode losses. This sensitivity of the computed mode losses is especially high for microstructure fibers that do not guide light by total internal reflection. Our results illustrate that one can obtain disparate mode confinement loss profiles for the same optical fiber design simply by moving the boundary to a new material region. We conclude with new recommendations for how to better model these losses.
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Recent developments in 3D cultures exploiting the self-organization ability of pluripotent stem cells have enabled the generation of powerful in vitro systems termed brain organoids. These 3D tissues recapitulate many aspects of human brain development and disorders occurring in vivo. When combined with improved differentiation methods, these in vitro systems allow the generation of more complex "assembloids," which are able to reveal cell diversities, microcircuits, and cell-cell interactions within their 3D organization. Here, the ways in which human brain organoids have contributed to demystifying the complexities of brain development and modeling of developmental disorders is reviewed and discussed. Furthermore, challenging questions that are yet to be addressed by emerging brain organoid research are discussed.
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Encéfalo/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Organoides , Animais , Encéfalo/crescimento & desenvolvimento , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/fisiopatologia , Técnicas de Cultura de Células , Descoberta de Drogas/métodos , Humanos , Microcefalia/fisiopatologia , Modelos Neurológicos , Transtornos do Neurodesenvolvimento/fisiopatologiaRESUMO
Bicaudal D2 (BICD2) encodes a highly conserved motor adaptor protein that regulates the dynein-dynactin complex in different cellular processes. Heterozygous mutations in BICD2 cause autosomal dominant lower extremity-predominant spinal muscular atrophy-2 (SMALED2). Although, various BICD2 mutations have been shown to alter interactions with different binding partners or the integrity of the Golgi apparatus, the specific pathological effects of BICD2 mutations underlying SMALED2 remain elusive. Here, we show that the fibroblasts derived from individuals with SMALED2 exhibit stable microtubules. Importantly, this effect was observed regardless of where the BICD2 mutation is located, which unifies the most likely cellular mechanism affecting microtubules. Significantly, overexpression of SMALED2-causing BICD2 mutations in the disease-relevant cell type, motor neurons, also results in an increased microtubule stability which is accompanied by axonal aberrations such as collateral branching and overgrowth. To study the pathological consequences of BICD2 mutations in vivo, and to address the controversial debate whether two of these mutations are neuron or muscle specific, we generated the first Drosophila model of SMALED2. Strikingly, neuron-specific expression of BICD2 mutants resulted in reduced neuromuscular junction size in larvae and impaired locomotion of adult flies. In contrast, expressing BICD2 mutations in muscles had no obvious effect on motor function, supporting a primarily neurological etiology of the disease. Thus, our findings contribute to the better understanding of SMALED2 pathology by providing evidence for a common pathomechanism of BICD2 mutations that increase microtubule stability in motor neurons leading to increased axonal branching and to impaired neuromuscular junction development.
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Proteínas de Drosophila/genética , Proteínas Associadas aos Microtúbulos/genética , Junção Neuromuscular/genética , Atrofias Musculares Espinais da Infância/genética , Animais , Modelos Animais de Doenças , Drosophila , Complexo Dinactina/genética , Dineínas/genética , Complexo de Golgi/genética , Complexo de Golgi/patologia , Humanos , Microtúbulos/genética , Microtúbulos/patologia , Mutação , Mutação de Sentido Incorreto/genética , Junção Neuromuscular/patologia , Linhagem , Ligação Proteica , Atrofias Musculares Espinais da Infância/fisiopatologiaRESUMO
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.
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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
Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4's tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of ß-sheets fold. This unique feature of the TCP domain suggests that it could provide an "extended surface-like" platform to tether the Sas-4-PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9-10th ß-strands (ß9-10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 ß9-10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the ß9-10 surface in mediating protein-protein interactions for efficient Sas-4-PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication.
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Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/patologia , Citoplasma/metabolismo , Drosophila melanogaster/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Microcefalia/genética , Proteínas Associadas aos Microtúbulos , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Testículo/metabolismoRESUMO
Cilia are fascinating organelles that act as cellular antennae, sensing the cellular environment. Cilia gained significant attention in the late 1990s after their dysfunction was linked to genetic diseases known as ciliopathies. Since then, several breakthrough discoveries have uncovered the mechanisms underlying cilia biogenesis and function. Like most cells in the animal kingdom, neurons also harbor cilia, which are enriched in neuromodulatory receptors. Yet, how neuronal cilia modulate neuronal physiology and animal behavior remains poorly understood. By comparing ciliary biology between the sensory and central nervous systems (CNS), we provide new perspectives on the functions of cilia in brain physiology.
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Cílios , Neurônios , Cílios/fisiologia , Animais , Humanos , Neurônios/fisiologia , Encéfalo/fisiologiaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure, yet therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via "centrosome clustering," a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with 2 inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, decreased interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.
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Cistos , Rim Policístico Autossômico Dominante , Humanos , Camundongos , Animais , Rim Policístico Autossômico Dominante/patologia , Proliferação de Células , Rim/patologia , Centrossomo/metabolismo , Fibrose , Cistos/metabolismo , Cistos/patologiaRESUMO
Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compile a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generate and analyze 5 kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map thousands of standalone and complex structural variants (SVs) and the multitude of neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can relate to patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.
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Neoplasias Encefálicas , Cromatina , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Cromatina/metabolismo , Cromatina/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Heterogeneidade Genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica , Elementos Facilitadores Genéticos/genética , Cromossomos Humanos/genéticaRESUMO
Cortical damage is irreparable and poses a challenge to regenerative medicine. Whether brain organoids can compensate for injured brain regions remains unclear. In this issue of Cell Stem Cell, Jgamadze et al. report that human forebrain organoids transplanted into the rat visual system show long-term structural connectivity and the restoration of visual function following lesions.
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Encéfalo , Prosencéfalo , Ratos , Animais , Humanos , Organoides/patologiaRESUMO
DNA-damaging agents and endogenous DNA damage constantly harm genome integrity. Under genotoxic stress conditions, the DNA damage response (DDR) machinery is crucial in repairing lesions and preventing mutations in the basic structure of the DNA. Different repair pathways are implicated in the resolution of such lesions. For instance, the non-homologous DNA end joining and homologous recombination pathways are central cellular mechanisms by which eukaryotic cells maintain genome integrity. However, defects in these pathways are often associated with neurological disorders, indicating the pivotal role of DDR in normal brain development. Moreover, the brain is the most sensitive organ affected by DNA-damaging agents compared to other tissues during the prenatal period. The accumulation of lesions is believed to induce cell death, reduce proliferation and premature differentiation of neural stem and progenitor cells, and reduce brain size (microcephaly). Microcephaly is mainly caused by genetic mutations, especially genes encoding proteins involved in centrosomes and DNA repair pathways. However, it can also be induced by exposure to ionizing radiation and intrauterine infections such as the Zika virus. This review explains mammalian cortical development and the major DNA repair pathways that may lead to microcephaly when impaired. Next, we discuss the mechanisms and possible exposures leading to DNA damage and p53 hyperactivation culminating in microcephaly.
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Background: Sleeping sickness is caused by the extracellular parasite Trypanosoma brucei and is associated with neuroinflammation and neuropsychiatric disorders, including disruption of sleep/wake patterns, and is now recognised as a circadian disorder. Sleeping sickness is traditionally studied using murine models of infection due to the lack of alternative in vitro systems that fully recapitulate the cellular diversity and functionality of the human brain. The aim of this study is to develop a much-needed in vitro system that reduces and replaces live animals for the study of infections in the central nervous system, using sleeping sickness as a model infection. Methods: We developed a co-culture system using induced pluripotent stem cell (iPSC)-derived cortical human brain organoids and the human pathogen T. b. gambiense to model host-pathogen interactions in vitro. Upon co-culture, we analysed the transcriptional responses of the brain organoids to T. b. gambiense over two time points. Results: We detected broad transcriptional changes in brain organoids exposed to T. b. gambiense, mainly associated with innate immune responses, chemotaxis, and blood vessel differentiation compared to untreated organoids. Conclusions: Our co-culture system provides novel, more ethical avenues to study host-pathogen interactions in the brain as alternative models to experimental infections in mice. Although our data support the use of brain organoids to model host-pathogen interactions during T. brucei infection as an alternative to in vivo models, future work is required to increase the complexity of the organoids ( e.g., addition of microglia and vasculature). We envision that the adoption of organoid systems is beneficial to researchers studying mechanisms of brain infection by protozoan parasites. Furthermore, organoid systems have the potential to be used to study other parasites that affect the brain significantly reducing the number of animals undergoing moderate and/or severe protocols associated with the study of neuroinflammation and brain infections.
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Células-Tronco Pluripotentes Induzidas , Tripanossomíase Africana , Humanos , Animais , Camundongos , Trypanosoma brucei gambiense , Doenças Neuroinflamatórias , Encéfalo , OrganoidesRESUMO
BACKGROUND: DYX1C1 (DNAAF4) and DCDC2 are two of the most replicated dyslexia candidate genes in genetic studies. They both have demonstrated roles in neuronal migration, in cilia growth and function and they both are cytoskeletal interactors. In addition, they both have been characterized as ciliopathy genes. However, their exact molecular functions are still incompletely described. Based on these known roles, we asked whether DYX1C1 and DCDC2 interact on the genetic and the protein level. RESULTS: Here, we report the physical protein-protein interaction of DYX1C1 and DCDC2 as well as their respective interactions with the centrosomal protein CPAP (CENPJ) on exogenous and endogenous levels in different cell models including brain organoids. In addition, we show a synergistic genetic interaction between dyx1c1 and dcdc2b in zebrafish exacerbating the ciliary phenotype. Finally, we show a mutual effect on transcriptional regulation among DYX1C1 and DCDC2 in a cellular model. CONCLUSIONS: In summary, we describe the physical and functional interaction between the two genes DYX1C1 and DCDC2. These results contribute to the growing understanding of the molecular roles of DYX1C1 and DCDC2 and set the stage for future functional studies.
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Cílios , Chaperonas Moleculares , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Movimento Celular/genética , Regulação da Expressão Gênica , Fenótipo , Peixe-Zebra/genética , Chaperonas Moleculares/genética , Proteínas de Peixe-Zebra/genética , Cílios/patologiaRESUMO
Glioblastoma ranks among the most aggressive and lethal of all human cancers. Self-renewing, highly tumorigenic glioblastoma stem cells (GSCs) contribute to therapeutic resistance and maintain cellular heterogeneity. Here, we interrogated superenhancer landscapes of primary glioblastoma specimens and patient-derived GSCs, revealing a kelch domain-containing gene, specifically Kelch domain containing 8A (KLHDC8A) with a previously unknown function as an epigenetically driven oncogene. Targeting KLHDC8A decreased GSC proliferation and self-renewal, induced apoptosis, and impaired in vivo tumor growth. Transcription factor control circuitry analyses revealed that the master transcriptional regulator SOX2 stimulated KLHDC8A expression. Mechanistically, KLHDC8A bound chaperonin-containing TCP1 (CCT) to promote the assembly of primary cilia to activate hedgehog signaling. KLHDC8A expression correlated with Aurora B/C Kinase inhibitor activity, which induced primary cilia and hedgehog signaling. Combinatorial targeting of Aurora B/C kinase and hedgehog displayed augmented benefit against GSC proliferation. Collectively, superenhancer-based discovery revealed KLHDC8A as what we believe to be a novel molecular target of cancer stem cells that promotes ciliogenesis to activate the hedgehog pathway, offering insights into therapeutic vulnerabilities for glioblastoma treatment.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Glioblastoma/patologia , Glioma/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Células-Tronco Neoplásicas/patologia , Transdução de SinaisRESUMO
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.
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Células-Tronco Pluripotentes Induzidas , Humanos , Diferenciação Celular/fisiologia , Prosencéfalo , Organoides , Desenvolvimento EmbrionárioRESUMO
Nifuroxazide (NAZ), a nitrofuran derivative used to treat diarrhea, has been recently shown to possess anticancer activity. However, its pharmacokinetic profile is poorly known. The pharmacokinetic profile of NAZ was thus investigated in mice using a newly developed method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). We determined the concentrations of NAZ in the plasma and brain tissue of mice treated with the drug. The method proved to be specific, reproducible, precise, and accurate. It also demonstrated high sensitivity, reaching an LOQ in the order of ppb for both matrices, using samples of 100 µL or 0.2 g. The new HPLC-MS/MS assay was successfully applied to study the pharmacokinetics of NAZ after chronic intraperitoneal administration in mice at a dose of 30 mg/kg. One hour after treatment, plasma concentrations of NAZ were in the range of 336-2640 ng/mL. Moreover, unlike the brains of healthy mice or those with healed mechanical injuries, we found that NAZ was able to cross the injured blood-brain barrier of tumor-infiltrated brains. Thus, following i.p. administration, NAZ reaches systemic levels suitable for testing its efficacy in preclinical models of glioblastoma. Overall, these pharmacokinetic data provide robust evidence supporting the repositioning of NAZ as an antitumor drug.
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Colchicine, the main active alkaloid from Colchicum autumnale L., is a potent tubulin binder and represents an interesting lead structure for the development of potential anticancer chemotherapeutics. We report on the synthesis and investigation of potentially reactive colchicinoids and their surprising biological activities. In particular, the previously undescribed colchicinoid PT-100, a B-ring contracted 6-exo-methylene colchicinoid, exhibits extraordinarily high antiproliferative and apoptosis-inducing effects on various types of cancer cell lines like acute lymphoblastic leukemia (Nalm6), acute myeloid leukemia (HL-60), Burkitt-like lymphoma (BJAB), human melanoma (MelHO), and human breast adenocarcinoma (MCF7) cells at low nanomolar concentrations. Apoptosis induction proved to be especially high in multidrug-resistant Nalm6-derived cancer cell lines, while healthy human leukocytes and hepatocytes were not affected by the concentration range studied. Furthermore, caspase-independent initiation of apoptosis via an intrinsic pathway was observed. PT-100 also shows strong synergistic effects in combination with vincristine on BJAB and Nalm6 cells. Cocrystallization of PT-100 with tubulin dimers revealed its (noncovalent) binding to the colchicine-binding site of ß-tubulin at the interface to the α-subunit. A pronounced effect of PT-100 on the cytoskeleton morphology was shown by fluorescence microscopy. While the reactivity of PT-100 as a weak Michael acceptor toward thiols was chemically proven, it remains unclear whether this contributes to the remarkable biological properties of this unusual colchicinoid.