RESUMO
Nuclear Cyclin D1 (Ccnd1) is a main regulator of cell cycle progression and cell proliferation. Interestingly, Ccnd1 moves to the cytoplasm at the onset of differentiation in neuronal precursors. However, cytoplasmic functions and targets of Ccnd1 in post-mitotic neurons are unknown. Here we identify the α4 subunit of gamma-aminobutyric acid (GABA) type A receptors (GABAARs) as an interactor and target of Ccnd1-Cdk4. Ccnd1 binds to an intracellular loop in α4 and, together with Cdk4, phosphorylates the α4 subunit at threonine 423 and serine 431. These modifications upregulate α4 surface levels, increasing the response of α4-containing GABAARs, measured in whole-cell patch-clamp recordings. In agreement with this role of Ccnd1-Cdk4 in neuronal signalling, inhibition of Cdk4 or expression of the non-phosphorylatable α4 decreases synaptic and extra-synaptic currents in the hippocampus of newborn rats. Moreover, according to α4 functions in synaptic pruning, CCND1 knockout mice display an altered pattern of dendritic spines that is rescued by the phosphomimetic α4. Overall, our findings molecularly link Ccnd1-Cdk4 to GABAARs activity in the central nervous system and highlight a novel role for this G1 cyclin in neuronal signalling.
Assuntos
Ciclina D1 , Quinase 4 Dependente de Ciclina , Receptores de GABA-A , Animais , Camundongos , Ratos , Ciclina D1/genética , Ácido gama-Aminobutírico , Camundongos Knockout , Neurônios , Fosforilação , Receptores de GABA-A/genética , Quinase 4 Dependente de Ciclina/genéticaRESUMO
The Wolffian ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here, we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting of ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1+/- mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.
Assuntos
Organogênese , Ductos Mesonéfricos , Animais , Feminino , Masculino , Mamíferos , Camundongos , Camundongos Knockout , Mutação/genética , Transdução de Sinais , TirosinaRESUMO
Neuron migration is a hallmark of nervous system development that allows gathering of neurons from different origins for assembling of functional neuronal circuits. Cortical inhibitory interneurons arise in the ventral telencephalon and migrate tangentially forming three transient migratory streams in the cortex before reaching the final laminar destination. Although migration defects lead to the disruption of inhibitory circuits and are linked to aspects of psychiatric disorders such as autism and schizophrenia, the molecular mechanisms controlling cortical interneuron development and final layer positioning are incompletely understood. Here, we show that mouse embryos with a double deletion of FLRT2 and FLRT3 genes encoding cell adhesion molecules exhibit an abnormal distribution of interneurons within the streams during development, which in turn, affect the layering of somatostatin+ interneurons postnatally. Mechanistically, FLRT2 and FLRT3 proteins act in a noncell-autonomous manner, possibly through a repulsive mechanism. In support of such a conclusion, double knockouts deficient in the repulsive receptors for FLRTs, Unc5B and Unc5D, also display interneuron defects during development, similar to the FLRT2/FLRT3 mutants. Moreover, FLRT proteins are chemorepellent ligands for developing interneurons in vitro, an effect that is in part dependent on FLRT-Unc5 interaction. Together, we propose that FLRTs act through Unc5 receptors to control cortical interneuron distribution in a mechanism that involves cell repulsion.SIGNIFICANCE STATEMENT Disruption of inhibitory cortical circuits is responsible for some aspects of psychiatric disorders such as schizophrenia or autism. These defects include interneuron migration during development. A crucial step during this process is the formation of three transient migratory streams within the developing cortex that determine the timing of interneuron final positioning and the formation of functional cortical circuits in the adult. We report that FLRT proteins are required for the proper distribution of interneurons within the cortical migratory streams and for the final laminar allocation in the postnatal cortex. These results expand the multifunctional role of FLRTs during nervous system development in addition to the role of FLRTs in axon guidance and the migration of excitatory cortical neurons.
Assuntos
Córtex Cerebral/citologia , Interneurônios/citologia , Glicoproteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Adesão Celular , Movimento Celular/fisiologia , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Cruzamentos Genéticos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Receptores de Netrina/fisiologia , Organogênese , Mapeamento de Interação de Proteínas , Receptores de Superfície Celular/fisiologiaRESUMO
The forebrain includes the cerebral cortex, the thalamus, and the striatum and globus pallidus (GP) in the subpallium. The formation of these structures and their interconnections by specific axonal tracts take place in a precise and orchestrated time and spatial-dependent manner during development. However, the knowledge of the molecular and cellular mechanisms that are involved is rather limited. Moreover, while many extracellular cues and specific receptors have been shown to play a role in different aspects of nervous system development, including neuron migration and axon guidance, examples of intracellular signaling effectors involved in these processes are sparse. In the present work, we have shown that the atypical RhoGTPase, Rnd3, is expressed very early during brain development and keeps a dynamic expression in several brain regions including the cortex, the thalamus, and the subpallium. By using a gene-trap allele (Rnd3gt ) and immunological techniques, we have shown that Rnd3gt/gt embryos display severe defects in striatal and thalamocortical axonal projections (SAs and TCAs, respectively) and defects in GP formation already at early stages. Surprisingly, the corridor, an important intermediate target for TCAs is still present in these mutants. Mechanistically, a conditional genetic deletion approach revealed that Rnd3 is primarily required for the normal development of Medial Ganglionic Eminence-derived structures, such as the GP, and therefore acts non-cell autonomously in SAs and TCAs. In conclusion, we have demonstrated the important role of Rnd3 as an early regulator of subpallium development in vivo and revealed new insights about SAs and TCAs development.
Assuntos
Globo Pálido/anormalidades , Cápsula Interna/anormalidades , Proteínas rho de Ligação ao GTP/genética , Animais , Axônios/patologia , Encéfalo/crescimento & desenvolvimento , Química Encefálica/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Eminência Mediana/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neostriado/anormalidades , Vias Neurais/anormalidadesRESUMO
In recent decades, neurogenesis in the adult brain has been well demonstrated in a number of animal species, including humans. Interestingly, work with rodents has shown that adult neurogenesis in the dentate gyrus (DG) of the hippocampus is vital for some cognitive aspects, as increasing neurogenesis improves memory, while its disruption triggers the opposite effect. Adult neurogenesis declines with age and has been suggested to play a role in impaired progressive learning and memory loss seen in Alzheimer's disease (AD). Therefore, therapeutic strategies designed to boost adult hippocampal neurogenesis may be beneficial for the treatment of AD. The precursor forms of neurotrophins, such as pro-NGF, display remarkable increase during AD in the hippocampus and entorhinal cortex. In contrast to mature NGF, pro-NGF exerts adverse functions in survival, proliferation, and differentiation. Hence, we hypothesized that pro-NGF and its p75 neurotrophin receptor (p75NTR) contribute to disrupting adult hippocampal neurogenesis during AD. To test this hypothesis, in this study, we took advantage of the availability of mouse models of AD (APP/PS1), which display memory impairment, and AD human samples to address the role of pro-NGF/p75NTR signaling in different aspects of adult neurogenesis. First, we observed that DG doublecortin (DCX) + progenitors express p75NTR both, in healthy humans and control animals, although the percentage of DCX+ cells are significantly reduced in AD. Interestingly, the expression of p75NTR in these progenitors is significantly decreased in AD conditions compared to controls. In order to assess the contribution of the pro-NGF/p75NTR pathway to the memory deficits of APP/PS1 mice, we injected pro-NGF neutralizing antibodies (anti-proNGF) into the DG of control and APP/PS1 mice and animals are subjected to a Morris water maze test. Intriguingly, we observed that anti-pro-NGF significantly restored memory performance of APP/PS1 animals and significantly increase the percentage of DCX+ progenitors in the DG region of these animals. In summary, our results suggest that pro-NGF is involved in disrupting spatial memory in AD, at least in part by blocking adult neurogenesis. Moreover, we propose that adult neurogenesis alteration should be taken into consideration for better understanding of AD pathology. Additionally, we provide a new molecular entry point (pro-NGF/p75NTR signaling) as a promising therapeutic target in AD.
Assuntos
Doença de Alzheimer/complicações , Encéfalo/patologia , Transtornos da Memória/patologia , Fator de Crescimento Neural/metabolismo , Neurogênese , Neurônios/patologia , Precursores de Proteínas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Proteína Duplacortina , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Masculino , Transtornos da Memória/etiologia , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Fator de Crescimento Neural/genética , Neurônios/metabolismo , Precursores de Proteínas/genética , Memória Espacial , Adulto JovemRESUMO
BACKGROUND: Studies in mice suggest that perturbations of the GDNF-Ret signaling pathway are a major genetic cause of congenital anomalies of the kidney and urinary tract (CAKUT). Mutations in Sprouty1, an intracellular Ret inhibitor, results in supernumerary kidneys, megaureters, and hydronephrosis in mice. But the underlying molecular mechanisms involved and which structural domains are essential for Sprouty1 function are a matter of controversy, partly because studies have so far relied on ectopic overexpression of the gene in cell lines. A conserved N-terminal tyrosine has been frequently, but not always, identified as critical for the function of Sprouty1 in vitro. METHODS: We generated Sprouty1 knockin mice bearing a tyrosine-to-alanine substitution in position 53, corresponding to the conserved N-terminal tyrosine of Sprouty1. We characterized the development of the genitourinary systems in these mice via different methods, including the use of reporter mice expressing EGFP from the Ret locus, and whole-mount cytokeratin staining. RESULTS: Mice lacking this tyrosine grow ectopic ureteric buds that will ultimately form supernumerary kidneys, a phenotype indistinguishable to that of Sprouty1 knockout mice. Sprouty1 knockin mice also present megaureters and vesicoureteral reflux, caused by failure of ureters to separate from Wolffian ducts and migrate to their definitive position. CONCLUSIONS: Tyrosine 53 is absolutely necessary for Sprouty1 function during genitourinary development in mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Tirosina/genética , Sistema Urinário/embriologia , Alanina/genética , Animais , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Proteínas de Fluorescência Verde/metabolismo , Queratinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Fenótipo , Fosforilação , Domínios Proteicos , Proteínas Proto-Oncogênicas c-ret/genética , Ureter/anormalidades , Sistema Urinário/crescimento & desenvolvimento , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/genética , Ductos Mesonéfricos/metabolismoRESUMO
Netrin-1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A-Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin-1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine-rich transmembrane protein-3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5-positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2-expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ-derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5-positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.
Assuntos
Glicoproteínas de Membrana/fisiologia , Neurônios/metabolismo , Receptores de Superfície Celular/fisiologia , Animais , Axônios/metabolismo , Movimento Celular , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Immunoblotting , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Crescimento Neural/metabolismo , Receptores de Netrina , Netrina-1 , Neurônios/citologia , Ligação Proteica , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismoRESUMO
Genes of the Sprouty family (Spry1-4) restrain signaling by certain receptor tyrosine kinases. Consequently, these genes participate in several developmental processes and function as tumor suppressors in adult life. Despite these important roles, the biology of this family of genes still remains obscure. Here we show that Sprouty proteins are general mediators of cellular senescence. Induction of cellular senescence by several triggers in vitro correlates with upregulation of Sprouty protein levels. More importantly, overexpression of Sprouty genes is sufficient to cause premature cellular senescence, via a conserved N-terminal tyrosine (Tyrosine 53 of Sprouty1). Accordingly, fibroblasts from knockin animals lacking that tyrosine escape replicative senescence. In vivo, heterozygous knockin mice display delayed induction of cellular senescence during cutaneous wound healing and upon chemotherapy-induced cellular senescence. Unlike other functions of this family of genes, induction of cellular senescence appears to be independent of activation of the ERK1/2 pathway. Instead, we show that Sprouty proteins induce cellular senescence upstream of the p38 pathway in these in vitro and in vivo paradigms.
Assuntos
Senescência Celular , Fibroblastos , Proteínas de Membrana , Animais , Camundongos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Humanos , Fibroblastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases , CicatrizaçãoRESUMO
Self-renewal and differentiation of stem cell depend on a dynamic interplay of cell-extrinsic and -intrinsic regulators. However, how stem cells perceive the right amount of signal and at the right time to undergo a precise developmental program remains poorly understood. The cell surface proteins Glypicans act as gatekeepers of environmental signals to modulate their perception by target cells. Here, we show that one of these, Glypican4 (Gpc4), is specifically required to maintain the self-renewal potential of mouse embryonic stem cells (ESCs) and to fine tune cell lineage commitment. Notably, Gpc4-mutant ESCs contribute to all embryonic cell lineages when injected in blastocyts but lose their intrinsic tumorigenic properties after implantation into nude mice. Therefore, our molecular and functional studies reveal that Gpc4 maintains distinct stemness features. Moreover, we provide evidence that self-renewal and lineage commitment of different stem cell types is fine tuned by Gpc4 activity by showing that Gpc4 is required for the maintenance of adult neural stem cell fate in vivo. Mechanistically, Gpc4 regulates self-renewal of ESCs by modulating Wnt/ß-catenin signaling activities. Thus, our findings establish that Gpc4 acts at the interface of extrinsic and intrinsic signal regulation to fine tune stem cell fate. Moreover, the ability to uncouple pluripotent stem cell differentiation from tumorigenic potential makes Gpc4 as a promising target for cell-based regenerative therapies.
Assuntos
Transformação Celular Neoplásica/metabolismo , Células-Tronco Embrionárias/metabolismo , Glipicanas/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Processos de Crescimento Celular/fisiologia , Transformação Celular Neoplásica/patologia , Células Cultivadas , Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia , Transdução de SinaisRESUMO
Phosphatase and TENsin homolog (Pten) and p53 are two of the most frequently mutated tumor suppressor genes in endometrial cancer. However, the functional consequences and histopathological manifestation of concomitant p53 and Pten loss of function alterations in the development of endometrial cancer is still controversial. Here, it is demonstrated that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel intravaginal delivery method using HIV1 trans-activator of transcription cell penetrating peptide fused with a Cre recombinase protein (TAT-Cre), local ablation of both p53 and Pten is achieved specifically in the uterus. These mice developed high-grade endometrial carcinomas and a high percentage of uterine carcinosarcomas resembling those found in humans. To further demonstrate that carcinosarcomas arise from epithelium, double Pten/p53 deficient epithelial cells are mixed with wild type stromal and myometrial cells and subcutaneously transplanted to Scid mice. All xenotransplants resulted in the development of uterine carcinosarcomas displaying high nuclear pleomorphism and metastatic potential. Accordingly, in vivo CRISPR/Cas9 disruption of Pten and p53 also triggered the development of metastatic carcinosarcomas. The results unfadingly demonstrate that simultaneous deletion of p53 and Pten in endometrial epithelial cells is enough to trigger epithelial to mesenchymal transition that is consistently translated to the formation of uterine carcinosarcomas in vivo.
Assuntos
Carcinossarcoma , Neoplasias do Endométrio , Neoplasias Uterinas , Humanos , Feminino , Camundongos , Animais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Transição Epitelial-Mesenquimal , Sistemas CRISPR-Cas/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Carcinossarcoma/genética , Carcinossarcoma/patologiaRESUMO
Metformin is a widespread antidiabetic agent that is commonly used as a treatment against type 2 diabetes mellitus patients. Regarding its therapeutic potential, multiple studies have concluded that Metformin exhibits antineoplastic activity on several types of cancer, including endometrial carcinoma. Although Metformin's antineoplastic activity is well documented, its cellular and molecular anticancer mechanisms are still a matter of controversy because a plethora of anticancer mechanisms have been proposed for different cancer cell types. In this study, we addressed the cellular and molecular mechanisms of Metformin's antineoplastic activity by using both in vitro and in vivo studies of Pten-loss driven carcinoma mouse models. In vivo, Metformin reduced endometrial neoplasia initiated by Pten-deficiency. Our in vitro studies using Pten-deficient endometrial organoids focused on both cellular and molecular levels in Metformin's tumor suppressive action. At cellular level, we showed that Metformin is involved in not only the proliferation of endometrial epithelial cells but also their regulation via a variety of mechanisms of epithelial-to-mesenchymal transition (EMT) as well as TGF-ß-induced apoptosis. At the molecular level, Metformin was shown to affect the TGF-ß signalling., a widely known signal that plays a pivotal role in endometrial carcinogenesis. In this respect, Metformin restored TGF-ß-induced apoptosis of Pten-deficient endometrial organoids through a p38-dependent mechanism and inhibited TGF-ß-induced EMT on no-polarized endometrial epithelial cells by inhibiting ERK/MAPK signalling. These results provide new insights into the link between the cellular and molecular mechanism for Metformin's antineoplastic activity in Pten-deficient endometrial cancers.
Assuntos
Antineoplásicos , Diabetes Mellitus Tipo 2 , Neoplasias do Endométrio , Metformina , Humanos , Feminino , Animais , Camundongos , Metformina/farmacologia , Metformina/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fator de Crescimento Transformador beta/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias do Endométrio/patologia , Proliferação de CélulasRESUMO
The extracellular matrix and the correct establishment of epithelial cell polarity plays a critical role in epithelial cell homeostasis and cell polarity. In addition, loss of tissue structure is a hallmark of carcinogenesis. In this study, we have addressed the role of extracellular matrix in the cellular responses to TGF-ß. It is well known that TGF-ß is a double-edged sword: it acts as a tumor suppressor in normal epithelial cells, but conversely has tumor-promoting effects in tumoral cells. However, the factors that determine cellular outcome in response to TGF-ß remain controversial. Here, we have demonstrated that the lack of extracellular matrix and consequent loss of cell polarity inhibits TGF-ß-induced apoptosis, observed when endometrial epithelial cells are polarized in presence of extracellular matrix. Rather, in absence of extracellular matrix, TGF-ß-treated endometrial epithelial cells display features of epithelial-to-mesenchymal transition. We have also investigated the molecular mechanism of such a switch in cellular response. On the one hand, we found that the lack of Matrigel results in increased AKT signaling which is sufficient to inhibit TGF-ß-induced apoptosis. On the other hand, we demonstrate that TGF-ß-induced epithelial-to-mesenchymal transition requires ERK and SMAD2/3 activation. In summary, we demonstrate that loss of cell polarity changes the pro-apoptotic function of TGF-ß to tumor-associated phenotype such as epithelial-to-mesenchymal transition. These results may be important for understanding the dual role of TGF-ß in normal versus tumoral cells.
Assuntos
Transição Epitelial-Mesenquimal , Matriz Extracelular , Fator de Crescimento Transformador beta , Apoptose/efeitos dos fármacos , Carcinogênese/metabolismo , Endométrio/metabolismo , Células Epiteliais , Matriz Extracelular/metabolismo , Feminino , Humanos , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. We show that the Rac-specific GTPase-activating protein alpha2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. alpha-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. Our results reveal an alpha-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo.
Assuntos
Diferenciação Celular/genética , Sistema Nervoso Central/anormalidades , Sistema Nervoso Central/metabolismo , Quimerina 1/metabolismo , Cones de Crescimento/metabolismo , Receptor EphA4/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/anormalidades , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Comunicação Celular/genética , Células Cultivadas , Sistema Nervoso Central/citologia , Quimerina 1/genética , Regulação para Baixo/genética , Transtornos Neurológicos da Marcha/genética , Transtornos Neurológicos da Marcha/metabolismo , Transtornos Neurológicos da Marcha/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Cones de Crescimento/ultraestrutura , Camundongos , Camundongos Knockout , Vias Neurais/anormalidades , Vias Neurais/metabolismo , Vias Neurais/fisiopatologia , Ligação Proteica/genética , Tratos Piramidais/anormalidades , Tratos Piramidais/metabolismo , Tratos Piramidais/fisiopatologia , Transdução de Sinais/genética , Medula Espinal/anormalidades , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
TGF-ß has a dichotomous function, acting as tumor suppressor in premalignant cells but as a tumor promoter for cancerous cells. These contradictory functions of TGF-ß are caused by different cellular contexts, including both intracellular and environmental determinants. The TGF-ß/SMAD and the PI3K/PTEN/AKT signal transduction pathways have an important role in the regulation of epithelial cell homeostasis and perturbations in either of these two pathways' contributions to endometrial carcinogenesis. We have previously demonstrated that both PTEN and SMAD2/3 display tumor-suppressive functions in the endometrium, and genetic ablation of either gene results in sustained activation of PI3K/AKT signaling that suppresses TGF-ß-induced apoptosis and enhances cell proliferation of mouse endometrial cells. However, the molecular and cellular effects of PTEN deficiency on TGF-ß/SMAD2/3 signaling remain controversial. Here, using an in vitro and in vivo model of endometrial carcinogenesis, we have demonstrated that loss of PTEN leads to a constitutive SMAD2/3 nuclear translocation. To ascertain the function of nuclear SMAD2/3 downstream of PTEN deficiency, we analyzed the effects of double deletion PTEN and SMAD2/3 in mouse endometrial organoids. Double PTEN/SMAD2/3 ablation results in a further increase of cell proliferation and enlarged endometrial organoids compared to those harboring single PTEN, suggesting that nuclear translocation of SMAD2/3 constrains tumorigenesis induced by PTEN deficiency.
RESUMO
Signaling by receptor tyrosine kinases (RTKs) is mediated by their intrinsic kinase activity. Typically, kinase-activating mutations result in ligand-independent signaling and gain-of-function phenotypes. Like other RTKs, Ephs require kinase activity to signal, but signaling by Ephs in vitro also requires clustering by their membrane bound ephrin ligands. The relative importance of Eph kinase activity and clustering for in vivo functions is unknown. We find that knockin mice expressing a mutant form of EphA4 (EphA4(EE)), whose kinase is constitutively activated in the absence of ephrinB ligands, are deficient in the development of thalamocortical projections and some aspects of central pattern generator rhythmicity. Surprisingly, other functions of EphA4 were regulated normally by EphA4(EE), including midline axon guidance, hindlimb locomotion, in vitro growth cone collapse, and phosphorylation of ephexin1. These results suggest that signaling of Eph RTKs follows a multistep process of induced kinase activity and higher-order clustering different from RTKs responding to soluble ligands.
Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Efrinas/metabolismo , Cones de Crescimento/metabolismo , Vias Neurais/crescimento & desenvolvimento , Agregação de Receptores/fisiologia , Receptor EphA4/metabolismo , Animais , Animais Recém-Nascidos , Comunicação Celular/genética , Diferenciação Celular/genética , Células Cultivadas , Sistema Nervoso Central/metabolismo , Cones de Crescimento/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Mutação/fisiologia , Rede Nervosa/crescimento & desenvolvimento , Rede Nervosa/metabolismo , Vias Neurais/metabolismo , Fosforilação , Receptor EphA4/genética , Receptores da Família Eph/metabolismo , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismoRESUMO
EphA/ephrin signaling regulates axon growth and guidance of neurons, but whether this process occurs also independently of ephrins is unclear. We show that presenilin-1 (PS1)/γ-secretase is required for axon growth in the developing mouse brain. PS1/γ-secretase mediates axon growth by inhibiting RhoA signaling and cleaving EphA3 independently of ligand to generate an intracellular domain (ICD) fragment that reverses axon defects in PS1/γ-secretase- and EphA3-deficient hippocampal neurons. Proteomic analysis revealed that EphA3 ICD binds to non-muscle myosin IIA (NMIIA) and increases its phosphorylation (Ser1943), which promotes NMIIA filament disassembly and cytoskeleton rearrangement. PS1/γ-secretase-deficient neurons show decreased phosphorylated NMIIA and NMIIA/actin colocalization. Moreover, pharmacological NMII inhibition reverses axon retraction in PS-deficient neurons suggesting that NMIIA mediates PS/EphA3-dependent axon elongation. In conclusion, PS/γ-secretase-dependent EphA3 cleavage mediates axon growth by regulating filament assembly through RhoA signaling and NMIIA, suggesting opposite roles of EphA3 on inhibiting (ligand-dependent) and promoting (receptor processing) axon growth in developing neurons.
Assuntos
Axônios/fisiologia , Miosina não Muscular Tipo IIA/metabolismo , Presenilina-1/metabolismo , Receptor EphA3/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Alzheimer disease (AD) is a complex pathology related to multiple causes including oxidative stress. Brain-derived neurotrophic factor (BDNF) is a neutrotrophic factor essential for the survival and differentiation of neurons and is considered a key target in the pathophysiology of various neurodegenerative diseases, as for example AD. Contrarily to BDNF, the precursor form of BDNF (proBDNF) induces apoptosis through the specific interaction with p75 and its co-receptor, Sortilin.We used hippocampal tissue and cerebrospinal fluid from AD patients and controls. to study the localization and the levels of proBDNF, p75 and Sortilin as well as the post-traduccional modifications of proBDNF induced by Radical Oxygen Species, by immunofluorescence and Western blot. Differentiation and survival were assessed on differentiated mouse hippocampal neurons derived from postnatal neural stem cells from WT animals or from the transgenic AD animal model APP/PS1∆E9, based on mutations of familiar AD. In AD patients we observe a significative increase of proBDNF and Sortilin expression and a significative increase of the ratio proBDNF/BDNF in their cerebrospinal fluid compared to controls. In addition, the proBDNF of AD patients is modified by ROS-derived advanced glycation end products, which prevent the processing of the proBDNF to the mature BDNF, leading to an increase of pathogenicity and a decrease of trophic effects. The cerebrospinal fluid from AD patients, but not from controls, induces apoptosis in differentiated hippocampal neurons mainly by the action of AGE-modified proBDNF present in the cerebrospinal fluid of the patients. This effect is triggered by the activation and processing of p75 that stimulate the internalization of the intracellular domain (ICD) within the nucleus causing apoptosis. Induction of apoptosis and p75 ICD internalization by AD patients-derived proBDNF is further enhanced in neuron cultures from the AD model expressing the APP/PS1∆E9 transgene.Our results indicate the importance of proBDNF neurotoxic signaling in AD pathology essentially by three mechanisms: i) by an increase of proBDNF stability due to ROS-induced post-traductional modifications; ii) by the increase of expression of the p75 co-receptor, Sortilin and iii) by the increase of the basal levels of p75 processing found in AD.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Apoptose , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Neurônios/patologia , Processamento de Proteína Pós-Traducional , Receptor de Fator de Crescimento Neural/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/líquido cefalorraquidiano , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Hipocampo/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Aldeído Pirúvico/farmacologia , Adulto JovemRESUMO
The Fibronectin Leucine-Rich Transmembrane protein 2 (FLRT2) has been implicated in several hormone -and sex-dependent physiological and pathological processes (including chondrogenesis, menarche and breast cancer); is known to regulate developmental synapses formation, and is expressed in the hippocampus, a brain structure central for learning and memory. However, the role of FLRT2 in the adult hippocampus and its relevance in sex-dependent brain functions remains unknown. We here used adult single-allele FLRT2 knockout (FLRT2+/-) mice and behavioral, electrophysiological, and molecular/biological assays to examine the effects of FLRT2 haplodeficiency on synaptic plasticity and hippocampus-dependent learning and memory. Female and male FLRT2+/- mice presented morphological features (including body masses, brain shapes/weights, and brain macroscopic cytoarchitectonic organization), indistinguishable from their wild type counterparts. However, in vivo examinations unveiled enhanced hippocampus-dependent spatial memory recall in female FLRT2+/- animals, concomitant with augmented hippocampal synaptic plasticity and decreased levels of the glutamate transporter EAAT2 and beta estrogen receptors. In contrast, male FLRT2+/- animals exhibited deficient memory recall and decreased alpha estrogen receptor levels. These observations propose that FLRT2 can regulate memory functions in the adulthood in a sex-specific manner and might thus contribute to further research on the mechanisms linking sexual dimorphism and cognition.
Assuntos
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Plasticidade Neuronal/fisiologia , Memória Espacial/fisiologia , Animais , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Transportador 2 de Aminoácido Excitatório , Feminino , Proteínas de Transporte de Glutamato da Membrana Plasmática/genética , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Plasticidade Neuronal/genética , Fatores SexuaisRESUMO
Determining the time of stroke onset in order to apply recanalization therapies within the accepted therapeutic window and the correct diagnosis of transient ischemic attack (TIA) are two common clinical problems in acute cerebral ischemia management. Therefore, biomarkers helping in this conundrum could be very helpful. We developed mouse models of distal middle cerebral artery occlusion mimicking TIA and ischemic stroke (IS), respectively. Plasma samples were analyzed by metabolomics at 6, 12, 24, and 48 h post onset in order to find TIA- and time-related stroke biomarkers. The results were validated in a second experimental cohort. Plasma metabolomic profiles identified time after stroke events with a very high accuracy. Specific metabolites pointing to a recent event (< 6 h) were identified. A multivariate (partial least square discriminant analyses [PLS-DA]) model was also able to separate samples from TIA, IS, and sham events with high accuracy and to obtain specific metabolites for each time point. The combination of mice models of focal ischemia with plasma metabolomics allows the discovery of candidate biomarkers for the diagnosis and estimation of onset time of stroke and TIA diagnosis.