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The transcription factor Bcl11b has been linked to neurodevelopmental and neuropsychiatric disorders associated with synaptic dysfunction. Bcl11b is highly expressed in dentate gyrus granule neurons and is required for the structural and functional integrity of mossy fiber-CA3 synapses. The underlying molecular mechanisms, however, remained unclear. We show in mice that the synaptic organizer molecule C1ql2 is a direct functional target of Bcl11b that regulates synaptic vesicle recruitment and long-term potentiation at mossy fiber-CA3 synapses in vivo and in vitro. Furthermore, we demonstrate C1ql2 to exert its functions through direct interaction with a specific splice variant of neurexin-3, Nrxn3(25b+). Interruption of C1ql2-Nrxn3(25b+) interaction by expression of a non-binding C1ql2 mutant or by deletion of Nrxn3 in the dentate gyrus granule neurons recapitulates major parts of the Bcl11b as well as C1ql2 mutant phenotype. Together, this study identifies a novel C1ql2-Nrxn3(25b+)-dependent signaling pathway through which Bcl11b controls mossy fiber-CA3 synapse function. Thus, our findings contribute to the mechanistic understanding of neurodevelopmental disorders accompanied by synaptic dysfunction.
The human brain contains billions of neurons working together to process the vast array of information we receive from our environment. These neurons communicate at junctions known as synapses, where chemical packages called vesicles released from one neuron stimulate a response in another. This synaptic communication is crucial for our ability to think, learn and remember. However, this activity depends on a complex interplay of proteins, whose balance and location within the neuron are tightly controlled. Any disruption to this delicate equilibrium can cause significant problems, including neurodevelopmental and neuropsychiatric disorders, such as schizophrenia and intellectual disability. One key regulator of activity at the synapse is a protein called Bcl11b, which has been linked to conditions affected by synaptic dysfunction. It plays a critical role in maintaining specific junctions known as mossy fibre synapses, which are important for learning and memory. One of the genes regulated by Bcl11b is C1ql2, which encodes for a synaptic protein. However, it is unclear what molecular mechanisms Bcl11b uses to carry out this role. To address this, Koumoundourou et al. explored the role of C1ql2 in mossy fibre synapses of adult mice. Experiments to manipulate the production of C1ql2 independently of Bcl11b revealed that C1ql2 is vital for recruiting vesicles to the synapse and strengthening synaptic connections between neurons. Further investigation showed that C1ql2's role in this process relies on interacting with another synaptic protein called neurexin-3. Disrupting this interaction reduced the amount of C1ql2 at the synapse and, consequently, impaired vesicle recruitment. These findings will help our understanding of how neurodevelopmental and neuropsychiatric disorders develop. Bcl11b, C1ql2 and neurexin-3 have been independently associated with these conditions, and the now-revealed interactions between these proteins offer new insights into the molecular basis of synaptic faults. This research opens the door to further study of how these proteins interact and their roles in brain health and disease.
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Fibras Musgosas Hipocampais , Sinapses , Animais , Camundongos , Fatores de Transcrição , Vesículas Sinápticas , Proteínas Supressoras de Tumor , Proteínas RepressorasRESUMO
Heterogeneous astrocyte populations are defined by diversity in cellular environment, progenitor identity or function. Yet, little is known about the extent of the heterogeneity and how this diversity is acquired during development. To investigate the impact of TGF (transforming growth factor) ß-signaling on astrocyte development in the telencephalon we deleted the TGFBR2 (transforming growth factor beta receptor 2) in early neural progenitor cells in mice using a FOXG1 (forkhead box G1)-driven CRE-recombinase. We used quantitative proteomics to characterize TGFBR2-deficient cells derived from the mouse telencephalon and identified differential protein expression of the astrocyte proteins GFAP (glial fibrillary acidic protein) and MFGE8 (milk fat globule-EGF factor 8). Biochemical and histological investigations revealed distinct populations of astrocytes in the dorsal and ventral telencephalon marked by GFAP or MFGE8 protein expression. The two subtypes differed in their response to TGFß-signaling. Impaired TGFß-signaling affected numbers of GFAP astrocytes in the ventral telencephalon. In contrast, TGFß reduced MFGE8-expression in astrocytes deriving from both regions. Additionally, lineage tracing revealed that both GFAP and MFGE8 astrocyte subtypes derived partly from FOXG1-expressing neural precursor cells.
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In mesial temporal lobe epilepsy (mTLE) an initial precipitating injury can trigger aberrant wiring of neuronal circuits causing seizure activity. While circuit reorganization is known to be largely activity-dependent, the interactions between neuronal activity and synaptic plasticity during the development of mTLE remain poorly understood. Therefore, the present study aimed at delineating the spatiotemporal relationship between epileptic activity, activity-dependent gene expression and synaptic plasticity during kainic acid-induced epileptogenesis in mice. We show that during epileptogenesis the sclerotic hippocampus differed from non-sclerotic regions by displaying a consistently lower power of paroxysmal discharges. However, the power of these discharges steadily increased during epileptogenesis. This increase was paralleled by the upregulation of the activity-related cytoskeleton protein (Arc) gene expression in dentate granule cells (DGCs) of the sclerotic hippocampus. Importantly, we found that Arc mRNA-upregulating DGCs exhibited increased spine densities and spine sizes, but at the same time decreased AMPA-type glutamate receptor (AMPAR) densities. Finally, we show that in vivo optogenetic stimulation of DGC synapses evoked robust seizure activity in epileptic mice, but failed to induce dendritic translocation of Arc mRNA as under healthy conditions, supporting the theory of a breakdown of the dentate gate in mTLE. We conclude that during epileptogenesis epileptic activity emerges early and persists in the whole hippocampus, however, only the sclerotic part shows modulation of discharge amplitudes accompanied by plasticity of DGCs. In this context, we identified Arc as a putative mediator between seizure activity and synaptic plasticity.
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While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. We address these questions using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. We report that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. Our findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases.
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Drosophila melanogaster/fisiologia , Células-Tronco Pluripotentes Induzidas/patologia , Mitocôndrias/patologia , NAD/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Niacinamida/análogos & derivados , Doença de Parkinson/patologia , Animais , Autofagia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Estresse do Retículo Endoplasmático , Glucosilceramidase/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Atividade Motora , Niacinamida/metabolismo , Doença de Parkinson/fisiopatologia , Compostos de Piridínio , Resposta a Proteínas não DobradasRESUMO
Structural and functional plasticity of synapses are critical neuronal mechanisms underlying learning and memory. While activity-dependent regulation of synaptic strength has been extensively studied, much less is known about the transcriptional control of synapse maintenance and plasticity. Hippocampal mossy fiber (MF) synapses connect dentate granule cells to CA3 pyramidal neurons and are important for spatial memory formation and consolidation. The transcription factor Bcl11b/Ctip2 is expressed in dentate granule cells and required for postnatal hippocampal development. Ablation of Bcl11b/Ctip2 in the adult hippocampus results in impaired adult neurogenesis and spatial memory. The molecular mechanisms underlying the behavioral impairment remained unclear. Here we show that selective deletion of Bcl11b/Ctip2 in the adult mouse hippocampus leads to a rapid loss of excitatory synapses in CA3 as well as reduced ultrastructural complexity of remaining mossy fiber boutons (MFBs). Moreover, a dramatic decline of long-term potentiation (LTP) of the dentate gyrus-CA3 (DG-CA3) projection is caused by adult loss of Bcl11b/Ctip2. Differential transcriptomics revealed the deregulation of genes associated with synaptic transmission in mutants. Together, our data suggest Bcl11b/Ctip2 to regulate maintenance and function of MF synapses in the adult hippocampus.
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Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content.
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Ácidos Nucleicos Livres/genética , Vesículas Extracelulares/genética , Mastocitose Sistêmica/diagnóstico , Melanoma/diagnóstico , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/genética , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/sangue , Variações do Número de Cópias de DNA , Humanos , Mastocitose Sistêmica/genética , Melanoma/genéticaRESUMO
MicroRNAs (miRNAs) are class of small RNA molecules with major impact on gene regulation. We analyzed the potential of miRNAs secreted from pre-implantation embryos into the embryonic culture media as biomarkers to predict successful pregnancy. Using microarray analysis, we profiled the miRNome of the 56 spent culture media (SCM) after embryos transfer and found a total of 621 miRNAs in the SCM. On average, we detected 163 miRNAs in SCM of samples with failed pregnancies, but only 149 SCM miRNAs of embryos leading to pregnancies. MiR-634 predicted an embryo transfer leading to a positive pregnancy with an accuracy of 71% and a sensitivity of 85%. Among the 621 miRNAs, 102 (16.4%) showed a differential expression between positive and negative outcome of pregnancy with miR-29c-3p as the most significantly differentially expressed miRNA. The number of extracellular vehicles was lower in SCM with positive outcomes (3.8 × 109/mL EVs), as compared to a negative outcome (7.35 × 109/mL EVs) possibly explaining the reduced number of miRNAs in the SCM associated with failed pregnancies. The analysis of the miRNome in the SCM of couples undergoing fertility treatment lays the ground towards development of biomarkers to predict successful pregnancy and towards understanding the role of embryonic miRNAs found in the SCM.
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Meios de Cultura/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Adulto , Transferência Embrionária , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Fertilização in vitro , Redes Reguladoras de Genes , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The hippocampus is reciprocally connected with the entorhinal cortex. Although several studies emphasized a role for the entorhinal cortex in mesial temporal lobe epilepsy (MTLE), it remains uncertain whether its synaptic connections with the hippocampus are altered. To address this question, we traced hippocampo-entorhinal and entorhino-hippocampal projections, assessed their connectivity with the respective target cells and examined functional alterations in a mouse model for MTLE. We show that hippocampal afferents to the dorsal entorhinal cortex are lost in the epileptic hippocampus. Conversely, entorhino-dentate projections via the medial perforant path (MPP) are preserved, but appear substantially altered on the synaptic level. Confocal imaging and 3D-reconstruction revealed that new putative contacts are established between MPP fibers and dentate granule cells (DGCs). Immunohistochemical identification of pre- and postsynaptic elements indicated that these contacts are functionally mature synapses. On the ultrastructural level, pre- and postsynaptic compartments of MPP synapses were strongly enlarged. The length and complexity of postsynaptic densities were also increased pointing to long-term potentiation-related morphogenesis. Finally, whole-cell recordings of DGCs revealed an enhancement of evoked excitatory postsynaptic currents. In conclusion, the synaptic rearrangement of excitatory inputs to DGCs from the medial entorhinal cortex may contribute to the epileptogenic circuitry in MTLE.
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Córtex Entorrinal/patologia , Epilepsia do Lobo Temporal/patologia , Plasticidade Neuronal , Sinapses/patologia , Animais , Giro Denteado/patologia , Giro Denteado/fisiopatologia , Modelos Animais de Doenças , Córtex Entorrinal/fisiopatologia , Epilepsia do Lobo Temporal/fisiopatologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ácido Caínico , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/patologia , Vias Neurais/fisiopatologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Técnicas de Cultura de TecidosRESUMO
In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia.
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Micropartículas Derivadas de Células/patologia , Vesículas Extracelulares/patologia , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Espectrofotometria Infravermelho/métodos , Análise Espectral Raman/métodos , Micropartículas Derivadas de Células/química , Vesículas Extracelulares/química , Humanos , Masculino , Projetos Piloto , Próstata/patologia , Hiperplasia Prostática/sangue , Hiperplasia Prostática/diagnóstico , Hiperplasia Prostática/patologia , Neoplasias da Próstata/química , Neoplasias da Próstata/patologiaRESUMO
The human small heat shock proteins (HSPBs) form a family of molecular chaperones comprising ten members (HSPB1-HSPB10), whose functions span from protein quality control to cytoskeletal dynamics and cell death control. Mutations in HSPBs can lead to human disease and particularly point mutations in HSPB1 and HSPB8 are known to lead to peripheral neuropathies. Recently, a missense mutation (R7S) in yet another member of this family, HSPB3, was found to cause an axonal motor neuropathy (distal hereditary motor neuropathy type 2C, dHMN2C). Until now, HSPB3 protein localization and function in motoneurons (MNs) have not yet been characterized. Therefore, we studied the endogenous HSPB3 protein distribution in the spinal cords of chicken and mouse embryos and in the postnatal nervous system (central and peripheral) of chicken, mouse and human. We further investigated the impact of wild-type and mutated HSPB3 on MN cell death via overexpressing these genes in ovo in an avian model of MN degeneration, the limb-bud removal. Altogether, our findings represent a first step for a better understanding of the cellular and molecular mechanisms leading to dHMN2C.
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Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Choque Térmico/metabolismo , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/patologia , Degeneração Neural/patologia , Fatores Etários , Animais , Animais Recém-Nascidos , Sobrevivência Celular/genética , Embrião de Galinha , Modelos Animais de Doenças , Embrião de Mamíferos , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Neurônios Motores/ultraestrutura , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Mutação/genética , Degeneração Neural/etiologia , Neuroblastoma/patologia , Medula Espinal/patologia , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestruturaRESUMO
Growing evidence suggests that the lysine methyltransferase DOT1L/KMT4 has important roles in proliferation, survival, and differentiation of stem cells in development and in disease. We investigated the function of DOT1L in neural stem cells (NSCs) of the cerebral cortex. The pharmacological inhibition and shRNA-mediated knockdown of DOT1L impaired proliferation and survival of NSCs. DOT1L inhibition specifically induced genes that are activated during the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Chromatin-immunoprecipitation analyses revealed that two genes encoding for central molecules involved in the ER stress response, Atf4 and Ddit3 (Chop), are marked with H3K79 methylation. Interference with DOT1L activity resulted in transcriptional activation of both genes accompanied by decreased levels of H3K79 dimethylation. Although downstream effectors of the UPR, such as Ppp1r15a/Gadd34, Atf3, and Tnfrsf10b/Dr5 were also transcriptionally activated, this most likely occurred in response to increased ATF4 expression rather than as a direct consequence of altered H3K79 methylation. While stem cells are particularly vulnerable to stress, the UPR and ER stress have not been extensively studied in these cells yet. Since activation of the ER stress program is also implicated in directing stem cells into differentiation or to maintain a proliferative status, the UPR must be tightly regulated. Our and published data suggest that histone modifications, including H3K4me3, H3K14ac, and H3K79me2, are implicated in the control of transcriptional activation of ER stress genes. In this context, the loss of H3K79me2 at the Atf4- and Ddit3-promoters appears to mark a point-of-no-return that activates the death program in NSCs.
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Fator 4 Ativador da Transcrição/metabolismo , Córtex Cerebral/citologia , Estresse do Retículo Endoplasmático , Metiltransferases/metabolismo , Células-Tronco Neurais/citologia , Neuroproteção , Fator de Transcrição CHOP/metabolismo , Animais , Benzimidazóis/farmacologia , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Lisina , Metilação/efeitos dos fármacos , Metiltransferases/antagonistas & inibidores , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neuroproteção/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Proper synaptic function depends on a finely-tuned balance between events such as protein synthesis and structural organization. In particular, the functional loss of just one synaptic-related protein can have a profound impact on overall neuronal network function. To this end, we used a mutant mouse model harboring a mutated form of the presynaptic scaffolding protein Bassoon (Bsn), which is phenotypically characterized by: (i) spontaneous generalized epileptic seizure activity, representing a chronically-imbalanced neuronal network; and (ii) a dramatic increase in hippocampal brain-derived neurotrophic factor (BDNF) protein concentration, a key player in synaptic plasticity. Detailed morphological and neurochemical analyses revealed that the increased BDNF levels are associated with: (i) modified neuropeptide distribution; (ii) perturbed expression of selected markers of synaptic activation or plasticity; (iii) subtle changes to microglial structure; and (iv) morphological alterations to the mossy fiber (MF) synapse. These findings emphasize the important contribution of Bassoon protein to normal hippocampal function, and further characterize the Bsn-mutant as a useful model for studying the effects of chronic changes to network activity.
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The role of microglia in amyloid-ß (Aß) deposition is controversial. In the present study, an organotypic hippocampal slice culture (OHSC) system with an in vivo-like microglial-neuronal environment was used to investigate the potential contribution of microglia to Aß plaque formation. We found that microglia ingested Aß, thereby preventing plaque formation in OHSCs. Conversely, Aß deposits formed rapidly in microglia-free wild-type slices. The capacity to prevent Aß plaque formation was absent in forebrain microglia from young adult but not juvenile 5xFamilial Alzheimer's disease (FAD) mice. Since no loss of Aß clearance capacity was observed in both wild-type and cerebellar microglia from 5xFAD animals, the high Aß1-42 burden in the forebrain of 5xFAD animals likely underlies the exhaustion of microglial Aß clearance capacity. These data may therefore explain why Aß plaque formation has never been described in wild-type mice, and point to a beneficial role of microglia in AD pathology. We also describe a new method to study Aß plaque formation in a cell culture setting.
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Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Hipocampo/patologia , Microglia/patologia , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/patologia , Prosencéfalo/patologia , Fatores Etários , Doença de Alzheimer/metabolismo , Doença de Alzheimer/prevenção & controle , Animais , Transporte Biológico , Modelos Animais de Doenças , Resistência à Doença/fisiologia , Feminino , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Microglia/ultraestrutura , Microtomia , Placa Amiloide/metabolismo , Placa Amiloide/prevenção & controle , Prosencéfalo/metabolismo , Prosencéfalo/ultraestrutura , Técnicas de Cultura de TecidosRESUMO
Cilia are microtubule-based organelles that are present on most cells and are required for normal tissue development and function. Defective cilia cause complex syndromes with multiple organ manifestations termed ciliopathies. A crucial step during ciliogenesis in multiciliated cells (MCCs) is the association of future basal bodies with the apical plasma membrane, followed by their correct spacing and planar orientation. Here, we report a novel role for ELMO-DOCK1, which is a bipartite guanine nucleotide exchange factor complex for the small GTPase Rac1, and for the membrane-cytoskeletal linker Ezrin, in regulating centriole/basal body migration, docking and spacing. Downregulation of each component results in ciliopathy-related phenotypes in zebrafish and disrupted ciliogenesis in Xenopus epidermal MCCs. Subcellular analysis revealed a striking impairment of basal body docking and spacing, which is likely to account for the observed phenotypes. These results are substantiated by showing a genetic interaction between elmo1 and ezrin b. Finally, we provide biochemical evidence that the ELMO-DOCK1-Rac1 complex influences Ezrin phosphorylation and thereby probably serves as an important molecular switch. Collectively, we demonstrate that the ELMO-Ezrin complex orchestrates ciliary basal body migration, docking and positioning in vivo.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Corpos Basais/metabolismo , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Axonema/metabolismo , Axonema/ultraestrutura , Membrana Celular/metabolismo , Cílios/ultraestrutura , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Fosforilação , Ligação Proteica , Xenopus laevis , Peixe-Zebra/embriologia , Proteínas rac de Ligação ao GTPRESUMO
Camillo Golgi's "Reazione Nera" led to the discovery of dendritic spines, small appendages originating from dendritic shafts. With the advent of electron microscopy (EM) they were identified as sites of synaptic contact. Later it was found that changes in synaptic strength were associated with changes in the shape of dendritic spines. While live-cell imaging was advantageous in monitoring the time course of such changes in spine structure, EM is still the best method for the simultaneous visualization of all cellular components, including actual synaptic contacts, at high resolution. Immunogold labeling for EM reveals the precise localization of molecules in relation to synaptic structures. Previous EM studies of spines and synapses were performed in tissue subjected to aldehyde fixation and dehydration in ethanol, which is associated with protein denaturation and tissue shrinkage. It has remained an issue to what extent fine structural details are preserved when subjecting the tissue to these procedures. In the present review, we report recent studies on the fine structure of spines and synapses using high-pressure freezing (HPF), which avoids protein denaturation by aldehydes and results in an excellent preservation of ultrastructural detail. In these studies, HPF was used to monitor subtle fine-structural changes in spine shape associated with chemically induced long-term potentiation (cLTP) at identified hippocampal mossy fiber synapses. Changes in spine shape result from reorganization of the actin cytoskeleton. We report that cLTP was associated with decreased immunogold labeling for phosphorylated cofilin (p-cofilin), an actin-depolymerizing protein. Phosphorylation of cofilin renders it unable to depolymerize F-actin, which stabilizes the actin cytoskeleton. Decreased levels of p-cofilin, in turn, suggest increased actin turnover, possibly underlying the changes in spine shape associated with cLTP. The findings reviewed here establish HPF as an appropriate method for studying the fine structure and molecular composition of synapses on dendritic spines.
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Anterior gradient 2 (AGR2) is a gene predominantly expressed in mucus-secreting tissues or in endocrine cells. Its expression is drastically increased in tumors including prostate cancer. Here we investigated whether AGR2 transcript levels can be used as a biomarker to detect prostate cancer (PCa). Using a PCR-based approach, we could show that in addition to the wild-type (AGRwt long and short) transcripts, five other AGR2 splice variants (SV) (referred to as AGR2 SV-C, -E, -F, -G and -H) were present in cancer cell lines. In tissue biopsies, SV-H and AGR2wt (short) distinguished between benign and PCa (p ≤ 0.05 n = 32). In urine exosomes, AGR2 SV-G and SV-H outperformed serum PSA. Receiver operating characteristic (ROC) curves showed the highest discriminatory power of SV-G and SV-H in predicting PCa. AGR2 SV-G and SV-H are potential diagnostic biomarkers for the non-invasive detection of PCa using urine exosomes.
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Processamento Alternativo , Biomarcadores Tumorais/genética , Neoplasias da Próstata/genética , Proteínas/genética , Idoso , Área Sob a Curva , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/urina , Biópsia , Linhagem Celular Tumoral , Exossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Calicreínas/sangue , Masculino , Pessoa de Meia-Idade , Mucoproteínas , Proteínas Oncogênicas , Projetos Piloto , Valor Preditivo dos Testes , Prognóstico , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Neoplasias da Próstata/urina , RNA Mensageiro/urina , Curva ROCRESUMO
Electron microscopy (EM) allows for the simultaneous visualization of all tissue components at high resolution. However, the extent to which conventional aldehyde fixation and ethanol dehydration of the tissue alter the fine structure of cells and organelles, thereby preventing detection of subtle structural changes induced by an experiment, has remained an issue. Attempts have been made to rapidly freeze tissue to preserve native ultrastructure. Shock-freezing of living tissue under high pressure (high-pressure freezing, HPF) followed by cryosubstitution of the tissue water avoids aldehyde fixation and dehydration in ethanol; the tissue water is immobilized in â¼50 ms, and a close-to-native fine structure of cells, organelles and molecules is preserved. Here we describe a protocol for HPF that is useful to monitor ultrastructural changes associated with functional changes at synapses in the brain but can be applied to many other tissues as well. The procedure requires a high-pressure freezer and takes a minimum of 7 d but can be paused at several points.
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Encéfalo/citologia , Microscopia Crioeletrônica/métodos , Neurônios/ultraestrutura , Pressão , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
Despite the vast abundance of glial progenitor cells in the mouse brain parenchyma, little is known about the molecular mechanisms driving their proliferation in the adult. Here we unravel a critical role of the G1 cell cycle regulator cyclin D1 in controlling cell division of glial cells in the cortical grey matter. We detect cyclin D1 expression in Olig2-immunopositive (Olig2+) oligodendrocyte progenitor cells, as well as in Iba1+ microglia and S100ß+ astrocytes in cortices of 3-month-old mice. Analysis of cyclin D1-deficient mice reveals a cell and stage-specific molecular control of cell cycle progression in the various glial lineages. While proliferation of fast dividing Olig2+ cells at early postnatal stages becomes gradually dependent on cyclin D1, this particular G1 regulator is strictly required for the slow divisions of Olig2+/NG2+ oligodendrocyte progenitors in the adult cerebral cortex. Further, we find that the population of mature oligodendrocytes is markedly reduced in the absence of cyclin D1, leading to a significant decrease in the number of myelinated axons in both the prefrontal cortex and the corpus callosum of 8-month-old mutant mice. In contrast, the pool of Iba1+ cells is diminished already at postnatal day 3 in the absence of cyclin D1, while the number of S100ß+ astrocytes remains unchanged in the mutant.
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Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Ciclina D1/biossíntese , Neuroglia/metabolismo , Células-Tronco/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Divisão Celular/fisiologia , Córtex Cerebral/crescimento & desenvolvimento , Feminino , Masculino , Camundongos , Camundongos KnockoutRESUMO
Little is known about the molecular mechanisms driving proliferation of glial cells after an insult to the central nervous system (CNS). To test the hypothesis that the G1 regulator cyclin D1 is critical for injury-induced cell division of glial cells, we applied an injury model that causes brain damage within a well-defined region. For this, we injected the neurotoxin ibotenic acid into the prefrontal cortex of adult mice, which leads to a local nerve cell loss but does not affect the survival of glial cells. Here, we show that cyclin D1 immunoreativity increases drastically after neurotoxin injection. We find that the cyclin D1-immunopositive (cyclin D1+) cell population within the lesioned area consists to a large extent of Olig2+ oligodendrocyte progenitor cells. Analysis of cyclin D1-deficient mice demonstrates that the proliferation rate of Olig2+ cells diminishes upon loss of cyclin D1. Further, we show that cyclin-dependent kinase (cdk) 4, but not cdk6 or cdk2, is essential for driving cell division of Olig2-expressing cells in our injury model. These data suggest that distinct cell cycle proteins regulate proliferation of Olig2+ progenitor cells following a CNS insult.