RESUMO
BACKGROUND: The glial cell line-derived neurotrophic factor has a potent neuroprotective action on mesencephalic dopamine neurons, which are progressively lost in Parkinson's disease. Intrastriatal administration of this factor is a promising therapy for Parkinson's disease. Glial cell line-derived neurotrophic factor is naturally produced in restricted cerebral regions, such as the striatum, septum, and thalamus; however, its effects in the adult brain remain under debate. OBJECTIVES: We sought to clarify the physiologic role of endogenous glial cell line-derived neurotrophic factor in the survival of catecholaminergic neurons of the substantia nigra pars compacta and the locus coeruleus in adult mice. METHODS: We used 2 new Cre recombinase-based mouse models to delete a floxed-glial cell line-derived neurotrophic factor gene. The first model had Cre expression in the parvalbumin expressing interneurons, as these cells represent the major source of striatal glial cell line-derived neurotrophic factor. The second model was an estrogen receptor 2-based inducible Cre triggered by tamoxifen at 2 months of age. RESULTS: We found that the floxed-glial cell line-derived neurotrophic factor gene was resilient to ablation by Cre-induced recombination and that parvalbumin-driven Cre was particularly inefficient to do so. The inducible-Cre model allowed an average 70% to 80% reduction in glial cell line-derived neurotrophic factor messenger ribonucleic acid and protein in striatum and septum with moderate significant loss of catecholamine neurons in the nigrostriatal pathway and, more markedly, in the locus coeruleus. This was accompanied with mild locomotor decline. CONCLUSIONS: Our data support qualitatively the view that brain glial cell line-derived neurotrophic factor is needed for the maintenance of adult central catecholaminergic neurons. © 2020 International Parkinson and Movement Disorder Society.
Assuntos
Fármacos Neuroprotetores , Doença de Parkinson , Animais , Corpo Estriado , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Camundongos , Neostriado , Neurônios , Substância NegraRESUMO
Reproduction is controlled in the brain by a neural network that drives the secretion of gonadotropin-releasing hormone (GnRH). Various permissive homeostatic signals must be integrated to achieve ovulation in mammals. However, the neural events controlling the timely activation of GnRH neurons are not completely understood. Here we show that kisspeptin, a potent activator of GnRH neuronal activity, directly communicates with neurons that synthesize the gaseous transmitter nitric oxide (NO) in the preoptic region to coordinate the progression of the ovarian cycle. Using a transgenic Gpr54-null IRES-LacZ knock-in mouse model, we demonstrate that neurons containing neuronal NO synthase (nNOS), which are morphologically associated with kisspeptin fibers, express the kisspeptin receptor GPR54 in the preoptic region, but not in the tuberal region of the hypothalamus. The activation of kisspeptin signaling in preoptic neurons promotes the activation of nNOS through its phosphorylation on serine 1412 via the AKT pathway and mimics the positive feedback effects of estrogens. Finally, we show that while NO release restrains the reproductive axis at stages of the ovarian cycle during which estrogens exert their inhibitory feedback, it is required for the kisspeptin-dependent preovulatory activation of GnRH neurons. Thus, interactions between kisspeptin and nNOS neurons may play a central role in regulating the hypothalamic-pituitary-gonadal axis in vivo.
Assuntos
Hipotálamo/citologia , Kisspeptinas/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Ovulação/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Ciclo Estral/efeitos dos fármacos , Ciclo Estral/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hipotálamo/efeitos dos fármacos , Kisspeptinas/deficiência , Kisspeptinas/farmacologia , Hormônio Luteinizante/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/deficiência , Ovulação/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Receptores Acoplados a Proteínas G/deficiência , Receptores de Kisspeptina-1 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esteroides/farmacologiaRESUMO
The anteroventral periventricular nucleus (AVPV) is thought to play a key role in regulating the excitability of gonadotropin-releasing hormone (GnRH) neurons that control fertility. Using an angled, parahorizontal brain slice preparation we have undertaken a series of electrophysiological experiments to examine how the AVPV controls GnRH neurons in adult male and female mice. More than half (59%) of GnRH neurons located in the rostral preoptic area were found to receive monosynaptic inputs from the AVPV in a sex-dependent manner. AVPV stimulation frequencies <1 Hz generated short-latency action potentials in GnRH neurons with GABA and glutamate mediating >90% of the evoked fast synaptic currents. The AVPV GABA input was dominant and found to excite or inhibit GnRH neurons in a cell-dependent manner. Increasing the AVPV stimulation frequency to 5-10 Hz resulted in the appearance of additional poststimulus inhibitory as well as delayed excitatory responses in GnRH neurons that were independent of ionotropic amino acid receptors. The inhibition observed immediately following the end of the stimulation period was mediated partly by GABA(B) receptors, while the delayed activation was mediated by the neuropeptide kisspeptin. The latter response was essentially absent in Gpr54 knock-out mice and abolished by a Gpr54 antagonist. Together, these studies show that AVPV neurons provide direct amino acid and neuropeptidergic inputs to GnRH neurons. Low-frequency activation generates predominant GABA/glutamate release with higher frequency activation recruiting release of kisspeptin. This frequency-dependent release of amino acid and neuropeptide neurotransmitters greatly expands the range of AVPV control of GnRH neuron excitability.
Assuntos
Aminoácidos/metabolismo , Núcleos Anteriores do Tálamo/citologia , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Biofísica , Estimulação Elétrica/métodos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Ácido Glutâmico/farmacologia , Hormônio Liberador de Gonadotropina/genética , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurotransmissores/metabolismo , Técnicas de Patch-Clamp , Tempo de Reação/fisiologia , Receptores Acoplados a Proteínas G/deficiência , Receptores de Kisspeptina-1 , Estatísticas não Paramétricas , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Valina/análogos & derivados , Valina/farmacologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
NMDA and kisspeptins can stimulate gonadotropin-releasing hormone (GnRH) release after peripheral or central administration in mice. To determine whether these agonists act independently or through a common pathway, we have examined their ability to stimulate GnRH/luteinizing hormone (LH) release after peripheral or central administration in Kiss1- or Gpr54 (Kiss1r)-null mutant mice. Peripheral injection of NMDA failed to stimulate GnRH/LH release in prepubertal or gonadally intact mutant male mice. Dual-labeling experiments indicated a direct activation of Kiss1-expressing neurons in the arcuate nucleus. In contrast, central injection of NMDA into the lateral ventricle increased plasma LH levels in both Kiss1 and Gpr54 mutant male mice similar to the responses in wild-type mice. Central injection of NMDA stimulated c-Fos expression throughout the hypothalamus but not in GnRH neurons, suggesting an action at the nerve terminals only. In contrast, kisspeptin-10 stimulated LH release after both central and peripheral injection but induced c-Fos expression in GnRH neurons only after central administration. Finally, central injection of NMDA induces c-Fos expression in catecholamine- and nitric oxide-producing neurons in the hypothalamus of mutant mice, indicating a possible kisspeptin-independent GnRH/LH release by NMDA through activation of these neurons. Thus, NMDA may act at both GnRH cell bodies (kisspeptin-independent) and nerve terminals (kisspeptin-dependent) in a dual way to participate in the GnRH/LH secretion in the male mouse.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , N-Metilaspartato/administração & dosagem , Neurônios/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Análise de Variância , Animais , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Imuno-Histoquímica , Injeções Intraperitoneais , Injeções Intraventriculares , Kisspeptinas , Hormônio Luteinizante/sangue , Masculino , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1 , Proteínas Supressoras de Tumor/genéticaRESUMO
Kisspeptins are a group of peptides that stimulate GnRH release and are required for puberty and maintenance of normal reproductive function. This review focuses on our understanding of the way in which kisspeptin signaling regulates mammalian fertility and how they act as central integrators of different hormonal and physiological signals.
Assuntos
Reprodução/genética , Reprodução/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Animais , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/fisiologia , Humanos , Kisspeptinas , Neurônios/fisiologia , Proteínas/genética , Ratos , Proteínas Supressoras de Tumor/agonistas , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
BACKGROUND: Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods. RESULTS: We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot. CONCLUSIONS: Taken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.
Assuntos
Redes Reguladoras de Genes , Hipotálamo/metabolismo , Proteínas/genética , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Testosterona/metabolismo , Animais , Genótipo , Kisspeptinas , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Kisspeptina-1 , Transcrição GênicaRESUMO
Nitric oxide (NO) is a peculiar chemical transmitter that freely diffuses through aqueous and lipid environments and plays a role in major aspects of brain function. Within the hypothalamus, NO exerts critical effects upon the gonadotropin-releasing hormone (GnRH) network to maintain fertility. Here, we review recent evidence that NO regulates major aspects of the GnRH neuron physiology. Far more active than once thought, NO powerfully controls GnRH neuronal activity, GnRH release and structural plasticity at the neurohemal junction. In the preoptic region, neuronal nitric oxide synthase (nNOS) activity is tightly regulated by estrogens and is found to be maximal at the proestrus stage. Natural fluctuations of estrogens control both the differential coupling of this Ca²+-activated enzyme to glutamate N-methyl-D-aspartic acid receptor channels and phosphorylation-mediated nNOS activation. Furthermore, NO endogenously produced by neurons expressing nNOS acutely and directly suppresses spontaneous firing in GnRH neurons, which suggests that neuronal NO may serve as a synchronizing switch within the preoptic region. At the median eminence, NO is spontaneously released from an endothelial source and follows a pulsatile and cyclic pattern of secretion. Importantly, GnRH release appears to be causally related to endothelial NO release. NO is also highly involved in mediating the dialogue set in motion between vascular endothelial cells and tanycytes that control the direct access of GnRH neurons to the pituitary portal blood during the estrous cycle. Altogether, these data raise the intriguing possibility that the neuroendocrine brain uses NO to coordinate both GnRH neuronal activity and GnRH release at key stages of reproductive physiology.
Assuntos
Encéfalo/fisiologia , Células Endoteliais/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Sistemas Neurossecretores/fisiologia , Óxido Nítrico/fisiologia , Reprodução/fisiologia , Transdução de Sinais/fisiologia , Animais , Hormônio Liberador de Gonadotropina/fisiologia , Modelos Biológicos , Óxido Nítrico/biossínteseRESUMO
Administration of recombinant glial cell line-derived neurotrophic factor into the putamen has been tested in preclinical and clinical studies to evaluate its neuroprotective effects on the progressive dopaminergic neuronal degeneration that characterizes Parkinson's disease. However, intracerebral glial cell line-derived neurotrophic factor infusion is a challenging therapeutic strategy, with numerous potential technical and medical limitations. Most of these limitations could be avoided if the production of endogenous glial cell line-derived neurotrophic factor could be increased. Glial cell line-derived neurotrophic factor is naturally produced in the striatum from where it exerts a trophic action on the nigrostriatal dopaminergic pathway. Most of striatal glial cell line-derived neurotrophic factor is synthesized by a subset of GABAergic interneurons characterized by the expression of parvalbumin. We sought to identify molecular targets specific to those neurons and which are putatively associated with glial cell line-derived neurotrophic factor synthesis. To this end, the transcriptomic differences between glial cell line-derived neurotrophic factor-positive parvalbumin neurons in the striatum and parvalbumin neurons located in the nearby cortex, which do not express glial cell line-derived neurotrophic factor, were analysed. Using mouse reporter models, we have defined the genomic signature of striatal parvalbumin interneurons obtained by fluorescence-activated cell sorting followed by microarray comparison. Short-listed genes were validated by additional histological and molecular analyses. These genes code for membrane receptors (Kit, Gpr83, Tacr1, Tacr3, Mc3r), cytosolic proteins (Pde3a, Crabp1, Rarres2, Moxd1) and a transcription factor (Lhx8). We also found the proto-oncogene cKit to be highly specific of parvalbumin interneurons in the non-human primate striatum, thus highlighting a conserved expression between species and suggesting that specific genes identified in mouse parvalbumin neurons could be putative targets in the human brain. Pharmacological stimulation of four G-protein-coupled receptors enriched in the striatal parvalbumin interneurons inhibited Gdnf expression presumably by decreasing cyclic adenosine monophosphate formation. Additional experiments with pharmacological modulators of adenylyl cyclase and protein kinase A indicated that this pathway is a relevant intracellular route to induce Gdnf gene activation. This preclinical study is an important step in the ongoing development of a specific pro-endo-glial cell line-derived neurotrophic factor pharmacological strategy to treat Parkinson's disease.
RESUMO
Kisspeptin and its receptor GPR54 have recently been identified as key signaling partners in the neural control of fertility in animal models and humans. The gonadotropin-releasing hormone (GnRH) neurons represent the final output neurons of the neural network controlling fertility and are suspected to be the primary locus of kisspeptin-GPR54 signaling. Using mouse models, the present study addressed whether kisspeptin and GPR54 have a key role in the activation of GnRH neurons to generate the luteinizing hormone (LH) surge responsible for ovulation. Dual-label immunocytochemistry experiments showed that 40-60% of kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) expressed estrogen receptor alpha and progesterone receptors. Using an ovariectomized, gonadal steroid-replacement regimen, which reliably generates an LH surge, approximately 30% of RP3V kisspeptin neurons were found to express c-FOS in surging mice compared with 0% in nonsurging controls. A strong correlation was found between the percentage of c-FOS-positive kisspeptin neurons and the percentage of c-FOS-positive GnRH neurons. To evaluate whether kisspeptin and/or GPR54 were essential for GnRH neuron activation and the LH surge, Gpr54- and Kiss1-null mice were examined. Whereas wild-type littermates all exhibited LH surges and c-FOS in approximately 50% of their GnRH neurons, none of the mutant mice from either line showed an LH surge or any GnRH neurons with c-FOS. These observations provide the first evidence that kisspeptin-GPR54 signaling is essential for GnRH neuron activation that initiates ovulation. This broadens considerably the potential roles and therapeutic possibilities for kisspeptin and GPR54 in fertility regulation.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Luteinizante/metabolismo , Neurônios/metabolismo , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Encéfalo/citologia , Estradiol/farmacologia , Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Kisspeptinas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Ovariectomia/métodos , Progesterona/farmacologia , Progestinas/farmacologia , Proteínas/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Receptores de Kisspeptina-1 , Transdução de Sinais/efeitos dos fármacosRESUMO
Estrogens and nitric oxide (NO) exert wide-ranging effects on brain function. Recent evidence suggested that one important mechanism for the regulation of NO production may reside in the differential coupling of the calcium-activated neuronal NO synthase (nNOS) to glutamate NMDA receptor channels harboring NR2B subunits by the scaffolding protein post-synaptic density-95 (PSD-95), and that estrogens promote the formation of this ternary complex. Here, we demonstrate that 30-min estradiol-treatment triggers the production of NO by physically and functionally coupling NMDA receptors to nNOS in primary neurons of the rat preoptic region in vitro. The ability of estradiol to activate neuronal NO signaling in preoptic neurons and to promote changes in protein-protein interactions is blocked by ICI 182,780, an estrogen receptor antagonist. In addition, blockade of NMDA receptor NR2B subunit activity with ifenprodil or disruption of PSD-95 synthesis in preoptic neurons by treatment with an anti-sense oligodeoxynucleotide inhibited the estradiol-promoted stimulation of NO release in cultured preoptic neurons. Thus, estrogen receptor-mediated stimulation of the nNOS/PSD-95/NMDA receptor complex assembly is likely to be a critical component of the signaling process by which estradiol facilitates coupling of glutamatergic fluxes for NO production in neurons.
Assuntos
Estradiol/fisiologia , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/biossíntese , Receptores de Estrogênio/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Neurônios/citologia , Neurônios/enzimologia , Óxido Nítrico/química , Óxido Nítrico Sintase Tipo I/química , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/química , Receptores de N-Metil-D-Aspartato/químicaRESUMO
Considerable research has been devoted to the understanding of how nitric oxide (NO) influences brain function. Few studies, however, have addressed how its production is physiologically regulated. Here, we report that protein-protein interactions between neuronal NO synthase (nNOS) and glutamate NMDA receptors via the scaffolding protein postsynaptic density-95 (PSD-95) in the hypothalamic preoptic region of adult female rats is sensitive to cyclic estrogen fluctuation. Coimmunoprecipitation experiments were used to assess the physical association between nNOS and NMDA receptor NR2B subunit in the preoptic region of the hypothalamus. We found that nNOS strongly interacts with NR2B at the onset of the preovulatory surge at proestrus (when estrogen levels are highest) compared with basal-stage diestrous rats. Consistently, estrogen treatment of gonadectomized female rats also increases nNOS/NR2B complex formation. Moreover, endogenous fluctuations in estrogen levels during the estrous cycle coincide with changes in the physical association of nNOS to PSD-95 and the magnitude of NO release in the preoptic region. Finally, temporary and local in vivo suppression of PSD-95 synthesis by using antisense oligodeoxynucleotides leads to inhibition of nNOS activity in the preoptic region and disrupted estrous cyclicity, a process requiring coordinated activation of neurons containing gonadotropin-releasing hormone (the neuropeptide controlling reproductive function). In conclusion, our findings identify a novel steroid-mediated molecular mechanism that enables the adult mammalian brain to control NO release under physiological conditions.
Assuntos
Estrogênios/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Reprodução/fisiologia , Fatores Etários , Animais , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Ciclo Estral/metabolismo , Feminino , RatosRESUMO
The G protein-coupled receptor GPR54, and its peptide ligand kisspeptin (Kp), are crucial for the induction and maintenance of mammalian reproductive function. GPR54 is expressed by GnRH neurons and is directly activated by Kp to stimulate GnRH release. We hypothesized that Kp may be able to act at the GnRH nerve terminals located in the mediobasal hypothalamus (MBH) region. To test this hypothesis, we used organotypic culture of MBH explants challenged with Kp, followed by RIA to detect GnRH released into the cultured medium. Kp stimulation for 1 h induced GnRH release from wild-type male MBH in a dose-dependent manner, whereas this did not occur in MBH explants isolated from Gpr54 null mice. Continuous Kp stimulation caused a sustained GnRH release for 4 h, followed by a decrease of GnRH release, suggesting a desensitization of GPR54 activity. Tetrodotoxin did not alter the Kp-induced GnRH release, indicating that Kp can act directly at the GnRH nerve terminals. To localize Gpr54 expression within the MBH, we used transgenic mice, in which Gpr54 expression is tagged with an IRES-LacZ reporter gene and can be visualized by beta-galactosidase staining. Gpr54 expression was detected outside of the median eminence, in the pars tuberalis. In conclusion, our results provide evidence for a potent stimulating effect of Kp at GnRH nerve terminals in the MBH of the mouse. This study suggests a new point at which Kp can act on GnRH neurons.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Oligopeptídeos/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Animais , Células Cultivadas , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Kisspeptinas , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Kisspeptina-1 , Tetrodotoxina/farmacologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0176821.].
RESUMO
There is emerging evidence that glial-derived neurotrophic factor (GDNF) is a potent inducer of restrictive barrier function in tight junction-forming microvascular endothelium and epithelium, including the human blood-nerve barrier (BNB) in vitro. We sought to determine the role of GDNF in restoring BNB function in vivo by evaluating sciatic nerve horseradish peroxidase (HRP) permeability in tamoxifen-inducible GDNF conditional knockout (CKO) adult mice following non-transecting crush injury via electron microscopy, with appropriate wildtype (WT) and heterozygous (HET) littermate controls. A total of 24 age-, genotype- and sex-matched mice >12 weeks of age were injected with 30 mg/kg HRP via tail vein injection 7 or 14 days following unilateral sciatic nerve crush, and both sciatic nerves were harvested 30 minutes later for morphometric assessment by light and electron microscopy. The number and percentage of HRP-permeable endoneurial microvessels were ascertained to determine the effect of GDNF in restoring barrier function in vivo. Following sciatic nerve crush, there was significant upregulation in GDNF protein expression in WT and HET mice that was abrogated in CKO mice. GDNF significantly restored sciatic nerve BNB HRP impermeability to near normal levels by day 7, with complete restoration seen by day 14 in WT and HET mice. A significant recovery lag was observed in CKO mice. This effect was independent on VE-Cadherin or claudin-5 expression on endoneurial microvessels. These results imply an important role of GDNF in restoring restrictive BNB function in vivo, suggesting a potential strategy to re-establish the restrictive endoneurial microenvironment following traumatic peripheral neuropathies.
Assuntos
Barreira Hematoneural/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Compressão Nervosa , Regeneração Nervosa/fisiologia , Doenças do Sistema Nervoso Periférico/metabolismo , Permeabilidade , Recuperação de Função Fisiológica/fisiologiaRESUMO
Kisspeptins regulate the mammalian reproductive axis by stimulating release of gonadotrophin releasing hormone (GnRH). Different length kisspeptins (KP) are found of 54, 14, 13 or 10 amino-acids which share a common C-terminal 10-amino acid sequence. KP-54 and KP-10 have been widely used to stimulate the reproductive axis but data suggest that KP-54 and KP-10 are not equally effective at eliciting reproductive hormone secretion after peripheral delivery. To confirm this, we analysed the effect of systemic administration of KP-54 or KP-10 on luteinizing hormone (LH) secretion into the bloodstream of male mice. Plasma LH measurements 10 min or 2 hours after kisspeptin injection showed that KP-54 can sustain LH release far longer than KP-10, suggesting a differential mode of action of the two peptides. To investigate the mechanism for this, we evaluated the pharmacokinetics of the two peptides in vivo and their potential to cross the blood brain barrier (BBB). We found that KP-54 has a half-life of ~32 min in the bloodstream, while KP-10 has a half-life of ~4 min. To compensate for this difference in half-life, we repeated injections of KP-10 every 10 min over 1 hr but failed to reproduce the sustained rise in LH observed after a single KP-54 injection, suggesting that the failure of KP-10 to sustain LH release may not just be related to peptide clearance. We tested the ability of peripherally administered KP-54 and KP-10 to activate c-FOS in GnRH neurons behind the blood brain barrier (BBB) and found that only KP-54 could do this. These data are consistent with KP-54 being able to cross the BBB and suggest that KP10 may be less able to do so.
Assuntos
Fármacos do Sistema Nervoso Central/farmacologia , Kisspeptinas/farmacologia , Análise de Variância , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Fármacos do Sistema Nervoso Central/farmacocinética , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Imuno-Histoquímica , Kisspeptinas/farmacocinética , Hormônio Luteinizante/sangue , Hormônio Luteinizante/metabolismo , Masculino , Camundongos da Linhagem 129 , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
Gender difference in Parkinson's disease (PD) suggests that female sex steroids may promote dopaminergic neuron survival and protect them from degeneration. The glial cell line-derived neurotrophic factor (GDNF) is believed to be dopaminotrophic; thus it is considered as a potential therapeutic target in PD. Additionally, GDNF is endogenously synthetized in the caudate/putamen of humans and striatum in rodents. A neuroprotective role of estrogens on the nigrostriatal pathway via the stimulation of GDNF has been proposed. Since the GDNF-producing parvalbumin (Parv) interneurons express the estrogen receptor alpha in the mouse striatum, we sought to determine whether ectopic estrogenic compound modulates the GDNF synthesis in mice. Using an ovariectomized-estradiol (E2) replacement regimen, which reliably generates a rise of plasma estradiol, we assessed the effects of different levels of E2 on the activation of striatal neuronal populations, and GDNF production. A strong correlation was found between plasma E2 and the expression of the immediate early gene cFos in the striatum, as well as in other cortical regions. However, moderate and high E2 treatments failed to induce any striatal GDNF mRNA and protein synthesis. High E2 only stimulates cFos induction in a low percentage of striatal Parv neurons whereas the majority of cFos-positive cells are medium spiny neurons. Activation of these projecting neurons by E2 suggests a role of circulating sex steroids in the modulation of striatal neural pathways.
Assuntos
Estradiol/sangue , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Imunoensaio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/metabolismoRESUMO
The glial cell line-derived neurotrophic factor (GDNF) is a well-established trophic agent for dopaminergic (DA) neurons in vitro and in vivo. GDNF is necessary for maintenance of neuronal morphological and neurochemical phenotype and protects DA neurons from toxic damage. Numerous studies on animal models of Parkinson's disease (PD) have reported beneficial effects of GDNF on nigrostriatal DA neuron survival. However, translation of these observations to the clinical setting has been hampered so far by side effects associated with the chronic continuous intra-striatal infusion of recombinant GDNF. In addition, double blind and placebo-controlled clinical trials have not reported any clinically relevant effect of GDNF on PD patients. In the past few years, experiments with conditional Gdnf knockout mice have suggested that GDNF is necessary for maintenance of DA neurons in adulthood. In parallel, new methodologies for exogenous GDNF delivery have been developed. Recently, it has been shown that a small population of scattered, electrically interconnected, parvalbumin positive (PV+) GABAergic interneurons is responsible for most of the GDNF produced in the rodent striatum. In addition, cholinergic striatal interneurons appear to be also involved in the modulation of striatal GDNF. In this review, we summarize current knowledge on brain GDNF delivery, homeostasis, and its effects on nigrostriatal DA neurons. Special attention is paid to the therapeutic potential of endogenous GDNF stimulation in PD.
RESUMO
Chronic hypoxemia, as evidenced in de-acclimatized high-altitude residents or in patients with chronic obstructive respiratory disorders, is a common medical condition that can produce serious neurological alterations. However, the pathogenesis of this phenomenon is unknown. We have found that adult rodents exposed for several days/weeks to hypoxia, with an arterial oxygen tension similar to that of chronically hypoxemic patients, manifest a partially irreversible structural disarrangement of the subventricular neurogenic niche (subventricular zone) characterized by displacement of neurons and myelinated axons, flattening of the ependymal cell layer, and thinning of capillary walls. Despite these abnormalities, the number of neuronal and oligodendrocyte progenitors, neuroblasts, and neurosphere-forming cells as well as the proliferative activity in subventricular zone was unchanged. These results suggest that neural stem cells and their undifferentiated progeny are resistant to hypoxia. However, in vivo and in vitro experiments indicate that severe chronic hypoxia decreases the survival of newly generated neurons and oligodendrocytes, with damage of myelin sheaths. These findings help explain the effects of hypoxia on adult neurogenesis and provide new perspectives on brain responsiveness to persistent hypoxemia.
RESUMO
The transition to puberty and adult fertility both require a minimum level of energy availability. The adipocyte-derived hormone leptin signals the long-term status of peripheral energy stores and serves as a key metabolic messenger to the neuroendocrine reproductive axis. Humans and mice lacking leptin or its receptor fail to complete puberty and are infertile. Restoration of leptin levels in these individuals promotes sexual maturation, which requires the pulsatile, coordinated delivery of gonadotropin-releasing hormone to the pituitary and the resulting surge of luteinizing hormone (LH); however, the neural circuits that control the leptin-mediated induction of the reproductive axis are not fully understood. Here, we found that leptin coordinated fertility by acting on neurons in the preoptic region of the hypothalamus and inducing the synthesis of the freely diffusible volume-based transmitter NO, through the activation of neuronal NO synthase (nNOS) in these neurons. The deletion of the gene encoding nNOS or its pharmacological inhibition in the preoptic region blunted the stimulatory action of exogenous leptin on LH secretion and prevented the restoration of fertility in leptin-deficient female mice by leptin treatment. Together, these data indicate that leptin plays a central role in regulating the hypothalamo-pituitary-gonadal axis in vivo through the activation of nNOS in neurons of the preoptic region.