RESUMO
The growth and morphological differentiation of neurons are critical events in the establishment of proper neuronal connectivity and functioning. The developing nervous system is highly susceptible to damage caused by exposure to environmental contaminants. Glyphosate-containing herbicides are the most used agrochemicals in the world, particularly on genetically modified plants. Previous studies have demonstrated that glyphosate induces neurotoxicity in mammals. Therefore, its action mechanism on the nervous system needs to be determined. In this study, we report about impaired neuronal development caused by glyphosate exposure. Particularly, we observed that the initial axonal differentiation and growth of cultured neurons is affected by glyphosate since most treated cells remained undifferentiated after 1 day in culture. Although they polarized at 2 days in vitro, they elicited shorter and unbranched axons and they also developed less complex dendritic arbors compared to controls. To go further, we attempted to identify the cellular mechanism by which glyphosate affected neuronal morphology. Biochemical approaches revealed that glyphosate led to a decrease in Wnt5a level, a key factor for the initial neurite development and maturation, as well as inducing a down-regulation of CaMKII activity. This data suggests that the morphological defects would likely be a consequence of the decrease in both Wnt5a expression and CaMKII activity induced by glyphosate. Additionally, these changes might be reflected in a subsequent neuronal dysfunction. Therefore, our findings highlight the importance of establishing rigorous control on the use of glyphosate-based herbicides in order to protect mammals' health.
Assuntos
Axônios/efeitos dos fármacos , Glicina/análogos & derivados , Hipocampo/patologia , Neurogênese/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt-5a/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Glicina/toxicidade , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/patologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Células Piramidais/patologia , Ratos , Proteína Wnt-5a/biossíntese , GlifosatoRESUMO
Neurons are highly polarized cells, composed of one axon and several branching dendrites. One important issue in neurobiology is to understand the molecular factors that determine the neuron to develop polarized structures. A particularly early event, in neurons still lacking a discernible axon, is the segregation of IGF-1 (Insulin like Growth Factor-1) receptors in one neurite. This receptor can be activated by insulin in bulk, but, it is not known if changes of insulin organization as a monomolecular film may affect neuron polarization. To this end, in this work we developed solid-supported Langmuir-Blodgett films of insulin with different surface packing density. Hyppocampal pyramidal neurons, in early stage of differentiation, were cultured onto those substrates and polarization was studied after 24 h by confocal microscopy. Also we used surface reflection interference contrast microscopy and confocal microscopy to study attachment patterns and morphology of growth cones. We observed that insulin films packed at 14 mN/m induced polarization in a similar manner to high insulin concentration in bulk, but insulin packed at 44 mN/m did not induce polarization. Our results provide novel evidence that the neuron polarization through IGF-1 receptor activation can be selectively modulated by the lateral packing of insulin organized as a monomolecular surface for cell growth.
Assuntos
Polaridade Celular/efeitos dos fármacos , Insulina/farmacologia , Neurônios/citologia , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Cloretos/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Polilisina/farmacologia , Ratos , Receptor IGF Tipo 1/metabolismo , Propriedades de Superfície , Compostos de Zinco/farmacologiaRESUMO
Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for axonal outgrowth and is known to occur mainly at the nerve growth cone. We have demonstrated recently that plasmalemmal expansion is regulated at the growth cone by IGF-1, but not by BDNF, in a manner that is quasi independent of the neuron's perikaryon. To begin elucidating the signaling pathway by which exocytosis of the plasmalemmal precursor is regulated, we studied activation of the IRS/PI3K/Akt pathway in isolated growth cones and hippocampal neurons in culture stimulated with IGF-1 or BDNF. Our results show that IGF-1, but not BDNF, significantly and rapidly stimulates IRS/PI3K/Akt and membrane expansion. Inhibition of PI3K with Wortmannin or LY294002 blocked IGF-1-stimulated plasmalemmal expansion at the growth cones of cultured neurons. Finally, our results show that, upon stimulation with IGF-1, most active PI3K becomes associated with distal microtubules in the proximal or central domain of the growth cone. Taken together, our results suggest a critical role for IGF-1 and the IRS/PI3K/Akt pathway in the process of membrane assembly at the axonal growth cone.
Assuntos
Membrana Celular/metabolismo , Sistema Nervoso Central/embriologia , Exocitose/fisiologia , Cones de Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos , Exocitose/efeitos dos fármacos , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/ultraestrutura , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/crescimento & desenvolvimento , Fator de Crescimento Insulin-Like I/farmacologia , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/fisiologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Transdução de Sinais/fisiologia , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestruturaRESUMO
Based on evidence obtained during the past 50 years, the current hypothesis to explain the sexual dimorphism of structure and function in the brain of vertebrates maintains that these differences are produced by the epigenetic action of gonadal hormones. However, evidence has progressively accumulated suggesting that genetic mechanisms controlling sexual-specific neuronal characteristics precede, or occur in parallel with, hormonal effects. 1. In cultures of hypothalamic neurons taken from gestation day 16 (GD16) embryos, treatment of sexually segregated cultures with estradiol (E2) induces axon growth in neurons from male neurons, but not from female neurons. In these cultures treatment with E2 increased the levels of tyrosine kinase type B (TrkB) and insulin-like growth factor I (IGF-I) receptors in male but not in female neurons. This and other sex differences cannot be explained by differences in hormonal environment, because the donor embryos were obtained when gonadal secretion of steroids is just beginning, before the perinatal surge of testosterone that determines development of the male brain beginning at GD17/18. 2. The response to estrogen is contingent upon coculture with heterotopic glia (mostly astrocytes) from a target region (amygdala) harvested from same-sex fetuses at GD16, whereas in the presence of homotopic glia or in cultures without glia, E2 had no effect. It was concluded that the axogenic effect of E2 depends on interaction between neurons and glia from a target region and that neurons from fetal male donors appear to mature earlier than neurons from females, a differentiated response that takes place prior to divergent exposure to gonadal secretions. 3. The effects of target and nontarget glia-conditioned media (CM) on the E2-induced growth of neuronal processes of hypothalamic neurons obtained from sexually segregated fetal donors were also studied. Estrogen added to media conditioned by target glia modified the number of primary neurites and the growth of axons of hypothalamic neurons of males but not of females. 4. Neither the Type III steroidal receptor blocker tamoxifen nor Type I antiestrogen ICI 182,780 prevented the axogenic effects of the hormone. Estradiol made membrane-impermeable by conjugation to a protein of high molecular weight (E2-BSA) preserved its axogenic capacity, suggesting the possibility of a membrane effect responsible for the action of E2. 5. Western blot analysis of the tyrosine kinase type A (TrkA), type B (TrkB), type C (TrkC), and insulin-like growth factor (IGF-I R) receptors in extracts from homogenates of cultured hypothalamic neurons showed that in cultures of male-derived neurons grown with E2 and CM from target glia, the amounts of TrkB and IGF-I R increased notably. Densitometric quantification showed that these cultures had more TrkB than cultures with CM alone or E2 alone. On the contrary, in cultures of female-derived neurons, the presence of CM alone induced maximal levels of TrkB, which were not further increased by E2; female-derived neurons in all conditions did not contain IGF-I R. Levels of TrkC were not modified by any experimental condition in male- or female-derived cultures and Trk A was not found in the homogenates. These results are compared with similar data from other laboratories and integrated in a model for the confluent interaction of estrogen and neurotrophic factors released by glia that may contribute to the sexual differentiation of the brain.
Assuntos
Encéfalo/embriologia , Estrogênios/metabolismo , Cones de Crescimento/metabolismo , Fatores de Crescimento Neural/metabolismo , Processos de Determinação Sexual , Diferenciação Sexual/genética , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Comunicação Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Cones de Crescimento/efeitos dos fármacos , Humanos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Diferenciação Sexual/efeitos dos fármacosRESUMO
The purpose of the present work was to investigate the participation of estradiol receptors (ER) in estrogen-induced axon growth in vitro. Hypothalamic neurons from 16 day (E16) male rat fetuses were cultured with or without 17-beta-estradiol at 1 x 10(-7) M in basal medium or medium conditioned by astroglia derived from ventral mesencephalon (CM). After 48 hr in vitro, neurite outgrowth was quantified by morphometric analysis. An axogenic effect could be demonstrated for estradiol added to CM. With RT-PCR, the mRNA transcript for ERalpha was found in the donor tissues as well as in the neuron cultures. In this model two specific nuclear ER blockers (tamoxifen and ICI 182,780) were ineffective in blocking the neuritogenic effect, and a membrane-impermeable estrogen-albumin construct (E2-BSA) was as effective as estradiol. These results indicate that the axogenic effect of estradiol at E16 is not exerted through the classical intracellular receptor signal transduction system and suggest the possibility of a membrane-mediated mechanism. The data are discussed in light of our previous findings pointing to the interdependent activation of the estrogenic and the trophic factor signaling pathways that mediate stimulated axon growth.
Assuntos
Diferenciação Celular/fisiologia , Estradiol/metabolismo , Cones de Crescimento/metabolismo , Hipotálamo/embriologia , Fatores de Crescimento Neural/metabolismo , Receptores de Estrogênio/genética , Transdução de Sinais/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Feto , Genoma , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/ultraestrutura , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Masculino , Fatores de Crescimento Neural/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Receptores de Estrogênio/antagonistas & inibidores , Reprodução/efeitos dos fármacos , Reprodução/fisiologia , Soroalbumina Bovina/farmacologia , Fatores Sexuais , Transdução de Sinais/efeitos dos fármacosRESUMO
PC12 cell line is a cellular model to study neurite outgrowth and neurotransmitter release mechanisms. Molecular motors may be involved in these responses and myosin V could be a candidate to mediate these effects. Overlay experiments using [(125)I]-calmodulin showed that PC12 cells possess several calmodulin-binding proteins, some of them around 190-210 kDa. Western blots using affinity purified polyclonal antibodies raised against chicken brain myosin V revealed a component of 190 kDa, a molecular mass typical of myosin V. Furthermore, Northern blots using a myosin V probe also detected a transcript of around 12 kbp. Immunofluorescence cytochemistry demonstrated the localization of myosin V throughout the cytoplasm, in the neurites, growth cone tips, and with an intense asymmetrical perinuclear labeling. Western blot analyses of PC12 cellular extracts after FGF-2 and/or dibutyryl cAMP treatment revealed variations between myosin V and myosin II expression during neuronal differentiation. These results demonstrated the presence of myosin V in PC12 cells and also suggest a role for this motor molecule in the neuronal differentiation response in PC12 cells.