RESUMO
VGLL proteins are transcriptional co-factors that bind TEAD family transcription factors to regulate events ranging from wing development in fly, to muscle fibre composition and immune function in mice. Here, we characterise Vgll3 in skeletal muscle. We found that mouse Vgll3 was expressed at low levels in healthy muscle but that its levels increased during hypertrophy or regeneration; in humans, VGLL3 was highly expressed in tissues from patients with various muscle diseases, such as in dystrophic muscle and alveolar rhabdomyosarcoma. Interaction proteomics revealed that VGLL3 bound TEAD1, TEAD3 and TEAD4 in myoblasts and/or myotubes. However, there was no interaction with proteins from major regulatory systems such as the Hippo kinase cascade, unlike what is found for the TEAD co-factors YAP (encoded by YAP1) and TAZ (encoded by WWTR1). Vgll3 overexpression reduced the activity of the Hippo negative-feedback loop, affecting expression of muscle-regulating genes including Myf5, Pitx2 and Pitx3, and genes encoding certain Wnts and IGFBPs. VGLL3 mainly repressed gene expression, regulating similar genes to those regulated by YAP and TAZ. siRNA-mediated Vgll3 knockdown suppressed myoblast proliferation, whereas Vgll3 overexpression strongly promoted myogenic differentiation. However, skeletal muscle was overtly normal in Vgll3-null mice, presumably due to feedback signalling and/or redundancy. This work identifies VGLL3 as a transcriptional co-factor operating with the Hippo signal transduction network to control myogenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Muscular , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Neoplasias/metabolismo , Ligação Proteica , Fatores de Transcrição de Domínio TEA , Transcriptoma/genéticaRESUMO
During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.
Assuntos
Epilepsia/genética , Proteínas/genética , Espasmos Infantis/genética , Transmissão Sináptica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Modelos Animais de Doenças , Epilepsia/diagnóstico , Fibroblastos/metabolismo , Técnicas de Genotipagem , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Proteínas/metabolismo , Células de Purkinje/metabolismo , Espasmos Infantis/diagnóstico , Vesículas Sinápticas/metabolismo , Transcriptoma , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Feedback control is a key mechanism in signal transduction, intimately involved in regulating the outcome of the cellular response. Here, we report a novel mechanism by which PHLDA1, Pleckstrin homology-like domain, family A, member 1, negatively regulates ErbB receptor signaling by inhibition of receptor oligomerization. We have found that the ErbB3 ligand, heregulin, induces PHILDA1 expression in MCF-7 cells. Transcriptionally-induced PHLDA1 protein directly binds to ErbB3, whereas knockdown of PHLDA1 increases complex formation between ErbB3 and ErbB2. To provide insight into the mechanism for our time-course and single-cell experimental observations, we performed a systematic computational search of network topologies of the mathematical models based on receptor dimer-tetramer formation in the ErbB activation processes. Our results indicate that only a model in which PHLDA1 inhibits formation of both dimers and tetramer can explain the experimental data. Predictions made from this model were further validated by single-molecule imaging experiments. Our studies suggest a unique regulatory feature of PHLDA1 to inhibit the ErbB receptor oligomerization process and thereby control the activity of receptor signaling network.
Assuntos
Receptor ErbB-3/metabolismo , Fatores de Transcrição/metabolismo , Humanos , Células MCF-7 , Modelos Químicos , Neuregulina-1/metabolismo , Multimerização Proteica , Transdução de Sinais , Imagem Individual de Molécula , Análise de Célula Única , Fatores de Transcrição/fisiologia , Transcrição GênicaRESUMO
Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analyzed Taz in vivo and ex vivo in comparison with Yap. Small interfering RNA knockdown or retroviral-mediated expression of wild-type human or constitutively active TAZ mutants in satellite cells showed that TAZ promoted proliferation, a function shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene Wwtr1-/- ) knockout mice, there were no overt effects on regeneration. Conversely, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/+ : Yapfl °x/fl °x :Rosa26Lacz mice produced a regeneration deficit. To identify potential mechanisms, microarray analysis showed many common TAZ/YAP target genes, but TAZ also regulates some genes independently of YAP, including myogenic genes such as Pax7, Myf5, and Myod1 (ArrayExpress-E-MTAB-5395). Proteomic analysis revealed many novel binding partners of TAZ/YAP in myogenic cells, but TAZ also interacts with proteins distinct from YAP that are often involved in myogenesis and aspects of cytoskeleton organization (ProteomeXchange-PXD005751). Neither TAZ nor YAP bind members of the Wnt destruction complex but both regulated expression of Wnt and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to enhance myogenic differentiation. Stem Cells 2017;35:1958-1972.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Músculo Esquelético/citologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/metabolismo , Animais , Proteínas de Ciclo Celular , Diferenciação Celular/genética , Fusão Celular , Proliferação de Células , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Via de Sinalização Hippo , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Células-Tronco/citologia , Transativadores , Via de Sinalização Wnt/genética , Proteínas de Sinalização YAPRESUMO
Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.
Assuntos
Cílios/metabolismo , Cílios/fisiologia , Proteínas/metabolismo , Animais , Dineínas do Axonema , Axonema/genética , Axonema/metabolismo , Sítios de Ligação/genética , Linhagem Celular , Pré-Escolar , Cílios/genética , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Drosophila/genética , Drosophila/metabolismo , Dineínas/genética , Dineínas/metabolismo , Feminino , Humanos , Síndrome de Kartagener/genética , Síndrome de Kartagener/metabolismo , Masculino , Mutação/genética , Linhagem , Fenótipo , Proteínas/genética , Transcrição Gênica/genéticaRESUMO
Epithelial to mesenchymal transition (EMT) is a fundamental cell differentiation/dedifferentiation process which is associated with dramatic morphological changes. Formerly polarized and immobile epithelial cells which form cell junctions and cobblestone-like cell sheets undergo a transition into highly motile, elongated, mesenchymal cells lacking cell-to-cell adhesions. To explore how the proteome is affected during EMT we profiled protein expression and tracked cell biological markers in Madin-Darby kidney epithelial cells undergoing hepatocyte growth factor (HGF) induced EMT. We were able to identify and quantify over 4000 proteins by mass spectrometry. Enrichment analysis of this revealed that expression of proteins associated with the ubiquitination machinery was induced, whereas expression of proteins regulating apoptotic pathways was suppressed. We show that both the mammalian Hippo/MST2 and the ISG15 pathways are regulated at the protein level by ubiquitin ligases. Inhibition of the Hippo pathway by overexpression of either ITCH or A-Raf promotes HGF-induced EMT. Conversely, ISG15 overexpression is sufficient to induce cell scattering and an elongated morphology without external stimuli. Thus, we demonstrate for the first time that the Hippo/MST2 and ISG15 pathways are regulated during growth-factor induced EMT.
Assuntos
Transição Epitelial-Mesenquimal , Fator de Crescimento de Hepatócito/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Ubiquitinas/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular , Cães , Fator de Crescimento de Hepatócito/farmacologia , Integrinas/metabolismo , Células Madin Darby de Rim Canino , Proteoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Migrating cells need to coordinate distinct leading and trailing edge dynamics but the underlying mechanisms are unclear. Here, we combine experiments and mathematical modeling to elaborate the minimal autonomous biochemical machinery necessary and sufficient for this dynamic coordination and cell movement. RhoA activates Rac1 via DIA and inhibits Rac1 via ROCK, while Rac1 inhibits RhoA through PAK. Our data suggest that in motile, polarized cells, RhoA-ROCK interactions prevail at the rear, whereas RhoA-DIA interactions dominate at the front where Rac1/Rho oscillations drive protrusions and retractions. At the rear, high RhoA and low Rac1 activities are maintained until a wave of oscillatory GTPase activities from the cell front reaches the rear, inducing transient GTPase oscillations and RhoA activity spikes. After the rear retracts, the initial GTPase pattern resumes. Our findings show how periodic, propagating GTPase waves coordinate distinct GTPase patterns at the leading and trailing edge dynamics in moving cells.
Assuntos
Movimento Celular , Polaridade Celular , Proteínas rac1 de Ligação ao GTP/genética , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Polaridade Celular/genética , Humanos , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We mutated the focal adhesion kinase (FAK) catalytic domain to inhibit binding of the chaperone Cdc37 and ATP, mimicking the actions of a FAK kinase inhibitor. We reexpressed mutant and wild-type FAK in squamous cell carcinoma (SCC) cells from which endogenous FAK had been deleted, genetically fixing one axis of a FAK inhibitor combination high-content phenotypic screen to discover drugs that may synergize with FAK inhibitors. Histone deacetylase (HDAC) inhibitors represented the major class of compounds that potently induced multiparametric phenotypic changes when FAK was rendered kinase-defective or inhibited pharmacologically in SCC cells. Combined FAK and HDAC inhibitors arrest proliferation and induce apoptosis in a subset of cancer cell lines in vitro and efficiently inhibit their growth as tumors in vivo Mechanistically, HDAC inhibitors potentiate inhibitor-induced FAK inactivation and impair FAK-associated nuclear YAP in sensitive cancer cell lines. Here, we report the discovery of a new, clinically actionable, synergistic combination between FAK and HDAC inhibitors.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Proliferação de Células , Sinergismo Farmacológico , Humanos , Camundongos , Transdução de SinaisRESUMO
Molecular chaperones promote the folding and macromolecular assembly of a diverse set of 'client' proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that primary ciliary dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease.
Assuntos
Axonema/metabolismo , Cílios/metabolismo , Proteínas de Ligação a DNA/genética , Dineínas/química , Proteínas de Choque Térmico HSP90/genética , Chaperonas Moleculares/genética , Proteínas de Ligação a Tacrolimo/genética , Animais , Animais Recém-Nascidos , Axonema/ultraestrutura , Sequência de Bases , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular , Cílios/ultraestrutura , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/metabolismo , Dineínas/genética , Dineínas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Cultura Primária de Células , Proteínas de Ligação a Tacrolimo/metabolismo , Traqueia/citologia , Traqueia/metabolismoRESUMO
Here, using mouse squamous cell carcinoma cells, we report a completely new function for the autophagy protein Ambra1 as the first described 'spatial rheostat' controlling the Src/FAK pathway. Ambra1 regulates the targeting of active phospho-Src away from focal adhesions into autophagic structures that cancer cells use to survive adhesion stress. Ambra1 binds to both FAK and Src in cancer cells. When FAK is present, Ambra1 is recruited to focal adhesions, promoting FAK-regulated cancer cell direction-sensing and invasion. However, when Ambra1 cannot bind to FAK, abnormally high levels of phospho-Src and phospho-FAK accumulate at focal adhesions, positively regulating adhesion and invasive migration. Spatial control of active Src requires the trafficking proteins Dynactin one and IFITM3, which we identified as Ambra1 binding partners by interaction proteomics. We conclude that Ambra1 is a core component of an intracellular trafficking network linked to tight spatial control of active Src and FAK levels, and so crucially regulates their cancer-associated biological outputs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma de Células Escamosas/fisiopatologia , Adesão Celular , Movimento Celular , Quinase 1 de Adesão Focal/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular Tumoral , Complexo Dinactina/metabolismo , Proteínas de Membrana/metabolismo , CamundongosRESUMO
Amino acid hydroxylation is a post-translational modification that regulates intra- and inter-molecular protein-protein interactions. The modifications are regulated by a family of 2-oxoglutarate- (2OG) dependent enzymes and, although the biochemistry is well understood, until now only a few substrates have been described for these enzymes. Using quantitative interaction proteomics, we screened for substrates of the proline hydroxylase PHD3 and the asparagine hydroxylase FIH, which regulate the HIF-mediated hypoxic response. We were able to identify hundreds of potential substrates. Enrichment analysis revealed that the potential substrates of both hydroxylases cluster in the same pathways but frequently modify different nodes of signaling networks. We confirm that two proteins identified in our screen, MAPK6 (Erk3) and RIPK4, are indeed hydroxylated in a FIH- or PHD3-dependent mechanism. We further determined that FIH-dependent hydroxylation regulates RIPK4-dependent Wnt signaling, and that PHD3-dependent hydroxylation of MAPK6 protects the protein from proteasomal degradation.
Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Aminoácidos Dicarboxílicos/química , Cromatografia Líquida de Alta Pressão , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Immunoblotting , Imunoprecipitação , Proteína Quinase 6 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Peptídeos/análise , Peptídeos/química , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , UbiquitinaçãoRESUMO
BACKGROUND: Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and is required for the derivation of induced pluripotent stem cells (iPSCs). However, the role of autophagy during iPSC maintenance remains undefined. METHODS: Human iPSCs were investigated by microscopy, immunofluorescence, and immunoblotting to detect autophagy machinery. Cells were treated with rapamycin to activate autophagy and with bafilomycin to block autophagy during iPSC maintenance. High concentrations of rapamycin treatment unexpectedly resulted in spontaneous formation of round floating spheres of uniform size, which were analyzed for differentiation into three germ layers. Mass spectrometry was deployed to reveal altered protein expression and pathways associated with rapamycin treatment. RESULTS: We demonstrate that human iPSCs express high basal levels of autophagy, including key components of APMKα, ULK1/2, BECLIN-1, ATG13, ATG101, ATG12, ATG3, ATG5, and LC3B. Block of autophagy by bafilomycin induces iPSC death and rapamycin attenuates the bafilomycin effect. Rapamycin treatment upregulates autophagy in iPSCs in a dose/time-dependent manner. High concentration of rapamycin reduces NANOG expression and induces spontaneous formation of round and uniformly sized embryoid bodies (EBs) with accelerated differentiation into three germ layers. Mass spectrometry analysis identifies actin cytoskeleton and adherens junctions as the major targets of rapamycin in mediating iPSC detachment and differentiation. CONCLUSIONS: High levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of end cell products. This research therefore highlights the potential of rapamycin in producing uniform EBs and in shortening iPSC differentiation duration.
Assuntos
Autofagia/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Sirolimo/farmacologia , Autofagia/fisiologia , Adesão Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/fisiologia , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/fisiologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/fisiologia , Humanos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
With the advent of the "-omics" era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches.
RESUMO
In certain Ras mutant cell lines, the inhibition of extracellular signal-regulated kinase (ERK) signaling increases RhoA activity and inhibits cell motility, which was attributed to a decrease in Fra-1 levels. Here we report a Fra-1-independent augmentation of RhoA signaling during short-term inhibition of ERK signaling. Using mass spectrometry-based proteomics, we identified guanine exchange factor H1 (GEF-H1) as mediating this effect. ERK binds to the Rho exchange factor GEF-H1 and phosphorylates it on S959, causing inhibition of GEF-H1 activity and a consequent decrease in RhoA activity. Knockdown experiments and expression of a nonphosphorylatable S959A GEF-H1 mutant showed that this site is crucial in regulating cell motility and invasiveness. Thus, we identified GEF-H1 as a critical ERK effector that regulates motility, cell morphology, and invasiveness.
Assuntos
Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Movimento Celular , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutação , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-fos/metabolismo , Interferência de RNA , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/química , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Transdução de SinaisRESUMO
Alzheimer's disease constitutes a rising threat to public health. Despite extensive research in cellular and animal models, identifying the pathogenic agent present in the human brain and showing that it confers key features of Alzheimer's disease has not been achieved. We extracted soluble amyloid-beta protein (Abeta) oligomers directly from the cerebral cortex of subjects with Alzheimer's disease. The oligomers potently inhibited long-term potentiation (LTP), enhanced long-term depression (LTD) and reduced dendritic spine density in normal rodent hippocampus. Soluble Abeta from Alzheimer's disease brain also disrupted the memory of a learned behavior in normal rats. These various effects were specifically attributable to Abeta dimers. Mechanistically, metabotropic glutamate receptors were required for the LTD enhancement, and N-methyl D-aspartate receptors were required for the spine loss. Co-administering antibodies to the Abeta N-terminus prevented the LTP and LTD deficits, whereas antibodies to the midregion or C-terminus were less effective. Insoluble amyloid plaque cores from Alzheimer's disease cortex did not impair LTP unless they were first solubilized to release Abeta dimers, suggesting that plaque cores are largely inactive but sequester Abeta dimers that are synaptotoxic. We conclude that soluble Abeta oligomers extracted from Alzheimer's disease brains potently impair synapse structure and function and that dimers are the smallest synaptotoxic species.