RESUMO
Melanoma cells, deriving from neuroectodermal melanocytes, may exploit the nervous system's immune privilege for growth. Here we show that nerve growth factor (NGF) has both melanoma cell intrinsic and extrinsic immunosuppressive functions. Autocrine NGF engages tropomyosin receptor kinase A (TrkA) on melanoma cells to desensitize interferon γ signaling, leading to T and natural killer cell exclusion. In effector T cells that upregulate surface TrkA expression upon T cell receptor activation, paracrine NGF dampens T cell receptor signaling and effector function. Inhibiting NGF, either through genetic modification or with the tropomyosin receptor kinase inhibitor larotrectinib, renders melanomas susceptible to immune checkpoint blockade therapy and fosters long-term immunity by activating memory T cells with low affinity. These results identify the NGF-TrkA axis as an important suppressor of anti-tumor immunity and suggest larotrectinib might be repurposed for immune sensitization. Moreover, by enlisting low-affinity T cells, anti-NGF reduces acquired resistance to immune checkpoint blockade and prevents melanoma recurrence.
Assuntos
Melanoma , Receptor de Fator de Crescimento Neural , Humanos , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Tropomiosina , Melanoma/terapia , Receptor trkA/genética , Receptor trkA/metabolismo , Citoproteção , Inibidores de Checkpoint Imunológico , Células T de Memória , Terapia de Imunossupressão , Imunoterapia , Receptores de Antígenos de Linfócitos TRESUMO
Nerve growth factor (NGF) is the best-characterized neurotrophin and is primarily recognized for its key role in the embryonic development of the nervous system and neuronal cell survival/differentiation. Recently, unexpected actions of NGF in bone regeneration have emerged as NGF is able to enhance the osteogenic differentiation of mesenchymal stem cells. However, little is known regarding how NGF signaling regulates osteogenic differentiation through epigenetic mechanisms. In this study, using human dental mesenchymal stem cells (DMSCs), we demonstrated that NGF mediates osteogenic differentiation through p75NTR, a low-affinity NGF receptor. P75NTR-mediated NGF signaling activates the JNK cascade and the expression of KDM4B, an activating histone demethylase, by removing repressive H3K9me3 epigenetic marks. Mechanistically, NGF-activated c-Jun binds to the KDM4B promoter region and directly upregulates KDM4B expression. Subsequently, KDM4B directly and epigenetically activates DLX5, a master osteogenic gene, by demethylating H3K9me3 marks. Furthermore, we revealed that KDM4B and c-Jun from the JNK signaling pathway work in concert to regulate NGF-mediated osteogenic differentiation through simultaneous recruitment to the promoter region of DLX5. We identified KDM4B as a key epigenetic regulator during the NGF-mediated osteogenesis both in vitro and in vivo using the calvarial defect regeneration mouse model. In conclusion, our study thoroughly elucidated the molecular and epigenetic mechanisms during NGF-mediated osteogenesis.
Assuntos
Células-Tronco Mesenquimais , Osteogênese , Animais , Diferenciação Celular/genética , Epigênese Genética , Histona Desmetilases/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Osteogênese/genética , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismoRESUMO
The p75 neurotrophin (NT) receptor (p75NTR) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75NTR activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75NTR in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75NTR are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75NTR and mutated (mut) p75NTR differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75NTR , but not of mut p75NTR, to mediate immature NT (proNT)-induced apoptosis. Both p75NTR forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multifaceted activity of the p75NTR monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature.
Assuntos
Apoptose/fisiologia , Cones de Crescimento/fisiologia , Fatores de Crescimento Neural/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Sistema Nervoso/metabolismo , Fenômenos Fisiológicos do Sistema Nervoso/genética , Receptor de Fator de Crescimento Neural/genéticaRESUMO
There is increasing evidence that nerve growth factor (NGF) and its receptors, the neurotrophic receptor tyrosine kinase 1 (NTRK1/TrkA), the common neurotrophin receptor (NGFR/p75NTR) and the membrane receptor sortilin, participate in cancer growth. In melanoma, there have been some reports suggesting that NGF, TrkA and p75NTR are dysregulated, but the expression of the NGF precursor (proNGF) and its membrane receptor sortilin is unknown. In this study, we investigated the expression of NGF, proNGF, TrkA, p75NTR and sortilin by immunohistochemistry in a series of human tissue samples (n = 100), including non-cancerous nevi (n = 20), primary melanomas (n = 40), lymph node metastases (n = 20) and distant metastases (n = 20). Immunostaining was digitally quantified and revealed NGF and proNGF were expressed in all nevi and primary melanomas, and that the level of expression decreased from primary tumors to melanoma metastases (p = 0.0179 and p < 0.0001, respectively). Interestingly, TrkA protein expression was high in nevi and thin primary tumors but was strongly downregulated in thick primary tumors (p < 0.0001) and metastases (p < 0.0001). While p75NTR and sortilin were both expressed in most nevi and melanomas, there was no significant difference in expression between them. Together, these results pointed to a downregulation of NGF/ProNGF and TrkA in melanoma, and thus did not provide evidence to support the use of anti-proNGF/NGF or anti-TrkA therapies in advanced and metastatic forms of melanoma.
Assuntos
Melanoma , Nevo , Proteínas Adaptadoras de Transporte Vesicular , Humanos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Receptor de Fator de Crescimento Neural/genética , Receptor trkA/genética , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismoRESUMO
The objective of the study was to characterize the anatomical framework and sites of action of the nerve growth factor (NGF)-mediated ovulation-inducing system of llamas. The expression patterns of NGF and its receptors in the hypothalamus of llamas (n = 5) were examined using single and double immunohistochemistry/immunofluorescence. We also compare the expression pattern of the P75 receptor in the hypothalamus of llama and a spontaneous ovulator species (sheep, n = 5). Both NGF receptors (TrkA and P75) were highly expressed in the medial septum and diagonal band of Broca, and populations of TrkA cells were observed in the periventricular and dorsal hypothalamus. Unexpectedly, we found NGF immunoreactive cell bodies with widespread distribution in the hypothalamus but not in areas endowed with NGF receptors. The organum vasculosum of the lamina terminalis (OVLT) and the median eminence displayed immunoreactivity for P75. Double immunofluorescence using vimentin, a marker of tanycytes, confirmed that tanycytes were immunoreactive to P75 in the median eminence and in the OVLT. Additionally, tanycytes were in close association with GnRH and kisspeptin in the arcuate nucleus and median eminence of llamas. The choroid plexus of llamas contained TrkA and NGF immunoreactivity but no P75 immunoreactivity. Results of the present study demonstrate sites of action of NGF in the llama hypothalamus, providing support for the hypothesis of a central effect of NGF in the ovulation-inducing mechanism in llamas.
Assuntos
Hipotálamo/fisiologia , Fator de Crescimento Neural/metabolismo , Ovulação/fisiologia , Receptor de Fator de Crescimento Neural/metabolismo , Animais , Camelídeos Americanos , Plexo Corióideo , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Imuno-Histoquímica , Kisspeptinas/genética , Kisspeptinas/metabolismo , Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/genética , Receptor trkA/genética , Receptor trkA/metabolismo , Ovinos/fisiologia , Vimentina/genética , Vimentina/metabolismoRESUMO
Cardiac sympathetic innervation is critically involved in the regulation of circulatory dynamics. However, the molecular mechanism for the innervation patterning has remained elusive. Here, we demonstrate that nardilysin (NRDC, Nrdc), an enhancer of ectodomain shedding, regulates cardiac sympathetic innervation. Nardilysin-deficient (Nrdc-/- ) mice show hypoplastic hearts, hypotension, bradycardia, and abnormal sympathetic innervation patterning. While the innervation of left ventricle (LV) of wild-type mice is denser in the subepicardium than in the subendocardium, Nrdc-/- LV lacks such a polarity and is uniformly and more abundantly innervated. At the molecular level, the full-length form of p75 neurotrophin receptor (p75NTR , Ngfr) is increased in Nrdc-/- LV due to the reduced ectodomain shedding of p75NTR . Importantly, the reduction of p75NTR rescued the abnormal innervation phenotype of Nrdc-/- mice. Moreover, sympathetic neuron-specific, but not cardiomyocyte-specific deletion of Nrdc recapitulated the abnormal innervation patterning of Nrdc-/- mice. In conclusion, neuronal nardilysin critically regulates cardiac sympathetic innervation and circulatory dynamics via modulation of p75NTR .
Assuntos
Coração/inervação , Metaloendopeptidases/genética , Receptor de Fator de Crescimento Neural/genética , Sistema Nervoso Simpático/metabolismo , Animais , Pressão Sanguínea/genética , Pressão Sanguínea/fisiologia , Bradicardia/genética , Bradicardia/fisiopatologia , Células Cultivadas , Ecocardiografia , Coração/fisiopatologia , Frequência Cardíaca/genética , Frequência Cardíaca/fisiologia , Metaloendopeptidases/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Células PC12 , Ratos , Receptor de Fator de Crescimento Neural/deficiência , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/fisiopatologiaRESUMO
Hypertrophic scar (HS) results from abnormal wound healing, accompanied by excessive hypercellularity, migration, and extracellular matrix (ECM) deposition. Autophagy dysregulation plays crucial roles during HS formation. The overexpressed p75 neurotrophin receptor (p75NTR) in injured skin tissue after wound healing becomes a factor aggravating scar. This study was designed to investigate the role of p75NTR and p75NTR-mediated autophagy in the process of HS. The results revealed that p75NTR expression was significantly upregulated while that of autophagy proteins was downregulated in cicatrix at 3 and 6 months after a burn, which was recovered at 12 months. p75NTR silencing inhibited proliferation, migration, and ECM deposition of hypertrophic scar fibroblasts (HSF), whereas p75NTR overexpression presented the opposite results. Silencing of p75NTR reduced the expression of PI3K/Akt/mTOR signaling molecules while enhancing that of autophagy proteins. Importantly, PI3K agonist (IGF-1) intervention notably decreased the levels of LC3B II/I and Beclin-1 and restored the inhibitory effects of p75NTR silencing on proliferation, migration, and ECM deposition of HSF. Concurrently, autophagy inhibitor 3-methyladenine (3-MA) treatment exhibited the same variation trends with IGF-1. Taken together, these findings demonstrated that p75NTR silencing inhibits proliferation, migration, and ECM deposition of HSF by activating autophagy by inhibiting the PI3K/Akt/mTOR pathway.
Assuntos
Cicatriz Hipertrófica/genética , Cicatriz Hipertrófica/patologia , Matriz Extracelular/metabolismo , Fibroblastos/patologia , Inativação Gênica , Receptor de Fator de Crescimento Neural/genética , Transdução de Sinais/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Neurotrophins constitute a group of growth factor that exerts important functions in the nervous system of vertebrates. They act through two classes of transmembrane receptors: tyrosine-kinase receptors and the p75 neurotrophin receptor (p75NTR). The activation of p75NTR can favor cell survival or apoptosis depending on diverse factors. Several studies evidenced a link between p75NTR and the pathogenesis of prion diseases. In this study, we investigated the distribution of several neurotrophins and their receptors, including p75NTR, in the brain of naturally scrapie-affected sheep and experimentally infected ovinized transgenic mice and its correlation with other markers of prion disease. No evident changes in infected mice or sheep were observed regarding neurotrophins and their receptors except for the immunohistochemistry against p75NTR. Infected mice showed higher abundance of p75NTR immunostained cells than their non-infected counterparts. The astrocytic labeling correlated with other neuropathological alterations of prion disease. Confocal microscopy demonstrated the co-localization of p75NTR and the astrocytic marker GFAP, suggesting an involvement of astrocytes in p75NTR-mediated neurodegeneration. In contrast, p75NTR staining in sheep lacked astrocytic labeling. However, digital image analyses revealed increased labeling intensities in preclinical sheep compared with non-infected and terminal sheep in several brain nuclei. This suggests that this receptor is overexpressed in early stages of prion-related neurodegeneration in sheep. Our results confirm a role of p75NTR in the pathogenesis of classical ovine scrapie in both the natural host and in an experimental transgenic mouse model.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Scrapie/metabolismo , Ovinos/genética , Animais , Astrócitos/patologia , Encéfalo/patologia , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Camundongos Transgênicos , Receptor de Fator de Crescimento Neural/genética , Scrapie/genética , Ovinos/metabolismoRESUMO
BACE1 is a transmembrane aspartic protease that cleaves various substrates and it is required for normal brain function. BACE1 expression is high during early development, but it is reduced in adulthood. Under conditions of stress and injury, BACE1 levels are increased; however, the underlying mechanisms that drive BACE1 elevation are not well understood. One mechanism associated with brain injury is the activation of injurious p75 neurotrophin receptor (p75), which can trigger pathological signals. Here we report that within 72â¯h after controlled cortical impact (CCI) or laser injury, BACE1 and p75 are increased and tightly co-expressed in cortical neurons of mouse brain. Additionally, BACE1 is not up-regulated in p75 null mice in response to focal cortical injury, while p75 over-expression results in BACE1 augmentation in HEK-293 and SY5Y cell lines. A luciferase assay conducted in SY5Y cell line revealed that BACE1 expression is regulated at the transcriptional level in response to p75 transfection. Interestingly, this effect does not appear to be dependent upon p75 ligands including mature and pro-neurotrophins. In addition, BACE1 activity on amyloid precursor protein (APP) is enhanced in SY5Y-APP cells transfected with a p75 construct. Lastly, we found that the activation of c-jun n-terminal kinase (JNK) by p75 contributes to BACE1 up-regulation. This study explores how two injury-induced molecules are intimately connected and suggests a potential link between p75 signaling and the expression of BACE1 after brain injury.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Linhagem Celular Tumoral , Células Cultivadas , Córtex Cerebral/metabolismo , Células HEK293 , Humanos , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Receptor de Fator de Crescimento Neural/genética , Transdução de Sinais , Regulação para CimaRESUMO
The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aß, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy.
Assuntos
Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkB/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Substituição de Aminoácidos , Animais , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Membrana Celular/genética , Epilepsia/genética , Epilepsia/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Mutagênese , Mutação de Sentido Incorreto , Fosforilação , Domínios Proteicos , Ratos , Receptor de Fator de Crescimento Neural/genética , Receptor trkB/genética , Células Sf9 , SpodopteraRESUMO
This study focuses on the protective effect of epigenetic silencing of CyclinD1 against spinal cord injury (SCI) using bone marrow-derived mesenchymal stem cells (BMSCs) in rats. Eighty-eight adult female Wistar rats were randomly assigned into the sham group, the control group, the si-CyclinD1 + BMSCs group and the BMSCs group. CyclinD1 protein and mRNA expressions after siRNA transfection were detected by Western blotting and qRT-PCR. The siRNA-CyclinD1 BMSCs were transplanted into rats in the si-CyclinD1 + BMSCs group using stereotaxic method 6 hr after SCI. Hindlimb locomotor performance was determined using inclined plane test and Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Expressions of glial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) were detected by immunohistochemistry. Inclined plane and BBB scores in the control, si-CyclinD1 + BMSCs, and BMSCs groups were significantly lower than the sham group, but these scores were evidently decreased in the control group and increased in the si-CyclinD1 + BMSCs group compared with the BMSCs group. The repair degree of spinal cord tissues of rats in the si-CyclinD1 + BMSCs group was obvious than the BMSCs group. GFAP and NGF protein expressions were markedly decreased in the control, si-CyclinD1 + BMSCs and BMSCs groups when compared with the sham group. GFAP- and NGF-positive cells were significantly increased in the si-CyclinD1 + BMSCs group while decreased in the control group. Our study provides evidence that epigenetic silencing of CyclinD1 using BMSCs might accelerate the repair of SCI in rats.
Assuntos
Ciclina D1/genética , Epigênese Genética/genética , Transplante de Células-Tronco Mesenquimais , Traumatismos da Medula Espinal/genética , Animais , Células da Medula Óssea/citologia , Ciclina D1/antagonistas & inibidores , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/genética , Inativação Gênica , Proteína Glial Fibrilar Ácida , Humanos , Locomoção/genética , Locomoção/fisiologia , Células-Tronco Mesenquimais/citologia , RNA Interferente Pequeno/genética , Ratos , Receptor de Fator de Crescimento Neural/genética , Recuperação de Função Fisiológica/genética , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/terapiaRESUMO
Intravitreal injection (IVT) of antivascular endothelial growth factor (anti-VEGF) agents is widely used for the treatment of retinal vascular diseases. Recently, the injection of anti-VEGF agents in the ocular anterior chamber has been proposed for the treatment of neovascular glaucoma and potential side effects on the corneal structures have been investigated with contrasting results. Increasing evidence has demonstrated that VEGF inhibition is associated with cellular apoptotic changes and that this effect may be mediated by alterations in nerve growth factor (NGF) pathway. In this study, we demonstrated that anterior chamber injection (IC), but not IVT injection of two different anti-VEGF agents, aflibercept and ranibizumab, affects rabbit corneal endothelium in terms of survival and apoptosis and is associated with changes in endothelial expression of NGF precursor (proNGF) and p75 neurotrophin receptor (p75NTR) receptor. We observed an increase in corneal endothelial cell incorporation of trypan blue and expression of cleaved-caspase 3 (c-Casp3), p75NTR, and RhoA after IC injection of both anti-VEGF drugs when compared with the vehicle. Our results showed that apoptosis induction by aflibercept was more pronounced when compared with that of ranibizumab. Aflibercept also mediated a significant increase in endothelial expression of proNGF when compared with the vehicle. In line with these data, IC administration of both anti-VEGF agents induced the activation of apoptotic signals in endothelial cells, including an increase in c-Casp3, decrease in Bad Ser 112 phosphorylation, and unbalance of AKT phosphorylation. These results demonstrated that administration of anti-VEGF in the anterior chamber of rabbit affects endothelial cell survival by inducing apoptosis through alteration of NGF pathway.
Assuntos
Glaucoma Neovascular/tratamento farmacológico , Fator de Crescimento Neural/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/administração & dosagem , Animais , Câmara Anterior/efeitos dos fármacos , Câmara Anterior/patologia , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glaucoma Neovascular/genética , Glaucoma Neovascular/patologia , Humanos , Injeções Intravítreas , Coelhos , Ranibizumab/administração & dosagem , Receptor de Fator de Crescimento Neural/genética , Fator A de Crescimento do Endotélio Vascular/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Cholinergic basal forebrain neurons of the nucleus basalis of Meynert (nbM) regulate attentional and memory function and are exquisitely prone to tau pathology and neurofibrillary tangle (NFT) formation during the progression of Alzheimer's disease (AD). nbM neurons require the neurotrophin nerve growth factor (NGF), its cognate receptor TrkA, and the pan-neurotrophin receptor p75NTR for their maintenance and survival. Additionally, nbM neuronal activity and cholinergic tone are regulated by the expression of nicotinic (nAChR) and muscarinic (mAChR) acetylcholine receptors as well as receptors modulating glutamatergic and catecholaminergic afferent signaling. To date, the molecular and cellular relationships between the evolution of tau pathology and nbM neuronal survival remain unknown. To address this knowledge gap, we profiled cholinotrophic pathway genes within nbM neurons immunostained for pS422, a pretangle phosphorylation event preceding tau C-terminal truncation at D421, or dual-labeled for pS422 and TauC3, a later stage tau neo-epitope revealed by this same C-terminal truncation event, via single-population custom microarray analysis. nbM neurons were obtained from postmortem tissues from subjects who died with an antemortem clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or mild/moderate AD. Quantitative analysis revealed significant downregulation of mRNAs encoding TrkA as well as TrkB, TrkC, and the Trk-mediated downstream pro-survival kinase Akt in pS422+ compared to unlabeled, pS422-negative nbM neurons. In addition, pS422+ neurons displayed a downregulation of transcripts encoding NMDA receptor subunit 2B, metabotropic glutamate receptor 2, D2 dopamine receptor, and ß1 adrenoceptor. By contrast, transcripts encoding p75NTR were downregulated in dual-labeled pS422+/TauC3+ neurons. Appearance of the TauC3 epitope was also associated with an upregulation of the α7 nAChR subunit and differential downregulation of the ß2 nAChR subunit. Notably, we found that gene expression patterns for each cell phenotype did not differ with clinical diagnosis. However, linear regression revealed that global cognition and Braak stage were predictors of select transcript changes within both unlabeled and pS422+/TauC3- neurons. Taken together, these cell phenotype-specific gene expression profiling data suggest that dysregulation of neurotrophic and neurotransmitter signaling is an early pathogenic mechanism associated with NFT formation in vulnerable nbM neurons and cognitive decline in AD, which may be amenable to therapeutic intervention early in the disease process.
Assuntos
Doença de Alzheimer/patologia , Núcleo Basal de Meynert/patologia , Neurônios Colinérgicos/patologia , Progressão da Doença , Fatores de Crescimento Neural , Emaranhados Neurofibrilares/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Núcleo Basal de Meynert/metabolismo , Neurônios Colinérgicos/metabolismo , Feminino , Humanos , Masculino , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Emaranhados Neurofibrilares/genética , Emaranhados Neurofibrilares/metabolismo , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/metabolismoRESUMO
Itch, the unpleasant sensation that elicits a desire to scratch, is mediated by specific subtypes of cutaneous sensory neuron. Here, we identify a subpopulation of itch-sensing neurons based on their expression of the receptor tyrosine kinase Ret. We apply flow cytometry to isolate Ret-positive neurons from dorsal root ganglia and detected a distinct population marked by low levels of Ret and absence of isolectin B4 binding. We determine the transcriptional profile of these neurons and demonstrate that they express neuropeptides such as somatostatin (Sst), the NGF receptor TrkA, and multiple transcripts associated with itch. We validate the selective expression of Sst using an Sst-Cre driver line and ablated these neurons by generating mice in which the diphtheria toxin receptor is conditionally expressed from the sensory neuron-specific Avil locus. Sst-Cre::Avil(iDTR) mice display normal nociceptive responses to thermal and mechanical stimuli. However, scratching behavior evoked by interleukin-31 (IL-31) or agonist at the 5HT1F receptor is significantly reduced. Our data provide a molecular signature for a subpopulation of neurons activated by multiple pruritogens.
Assuntos
Gânglios Espinais/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Prurido/genética , Células Receptoras Sensoriais/metabolismo , Somatostatina/genética , Animais , Perfilação da Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Hibridização In Situ , Lectinas/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Neurônios Aferentes/metabolismo , Neuropeptídeos/metabolismo , Receptor de Fator de Crescimento Neural/genéticaRESUMO
Nogo-A restricts long-term potentiation (LTP) at the Schaffer collateral-CA1 pathway in the adult hippocampus via 2 extracellular domains: Nogo-A-Δ20 and Nogo-66. Nogo-66 signals via Nogo Receptor 1 (NgR1) to regulate synaptic function. Whether the NgR1 coreceptors Lingo1 and p75NTR are involved in the signaling in this context is still not known. Moreover, the intracellular cascade mediating the activity of Nogo-66 in restricting LTP is unexplored. We combine electrophysiology and biochemistry in acute hippocampal slices and demonstrate that a loss of function for Lingo1 results in a significant increase in LTP levels at the Schaffer collateral-CA1 pathway, and that Lingo1 is the NgR1 coreceptor mediating the role of Nogo-66 in restricting LTP. Our data show that p75NTR is not involved in mediating the Nogo-66 effect on LTP. Moreover, loss of function for p75NTR and NgR1 equally attenuate LTD, suggesting that p75NTR might mediate the NgR1-dependent regulation of LTD, independently of Nogo-66. Finally, our results indicate that Nogo-66 signaling limits LTP via the ROCK2-Cofilin pathway to control the dynamics of the actin cytoskeleton. The present results elucidate the signaling pathway activated by Nogo-66 to control LTP and contribute to the understanding of how Nogo-A stabilizes the neural circuits to limit activity-dependent plasticity events in the mature hippocampus.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Plasticidade Neuronal/fisiologia , Proteínas Nogo/metabolismo , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo , Fatores de Despolimerização de Actina/genética , Amidas/farmacologia , Animais , Biofísica , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Feminino , Hipocampo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Proteínas Nogo/antagonistas & inibidores , Proteínas Nogo/química , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Piridinas/farmacologia , Receptor de Fator de Crescimento Neural/deficiência , Receptor de Fator de Crescimento Neural/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genéticaRESUMO
This study reports the gene expression and immunolocalization of the low-affinity neurotrophin receptor, p75, in accessory glands (prostate and bulbourethral glands) and epididymis (caput and cauda) of male rabbits during sexual maturation. We showed that p75 was expressed in all tissues studied with similar mRNA levels during this period. However, it was differentially immunolocalized in bulbourethral glands and stereocilia of epididymis. These findings may be related with some remodeling processes in the accessory glands during sexual maturation, which could be associated with sperm maturation. The interaction of p75 with neurotrophins in rabbit male reproductive tract suggests the possible implication of this system in sexual maturation in rabbits.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coelhos/fisiologia , Receptor de Fator de Crescimento Neural/genética , Maturidade Sexual/genética , Maturação do Esperma/genética , Animais , Epididimo/fisiologia , Imuno-Histoquímica , Masculino , RNA Mensageiro/análiseRESUMO
Dimerization of single span transmembrane receptors underlies their mechanism of activation. p75 neurotrophin receptor plays an important role in the nervous system, but the understanding of p75 activation mechanism is still incomplete. The transmembrane (TM) domain of p75 stabilizes the receptor dimers through a disulfide bond, essential for the NGF signaling. Here we solved by NMR the three-dimensional structure of the p75-TM-WT and the functionally inactive p75-TM-C257A dimers. Upon reconstitution in lipid micelles, p75-TM-WT forms the disulfide-linked dimers spontaneously. Under reducing conditions, p75-TM-WT is in a monomer-dimer equilibrium with the Cys(257) residue located on the dimer interface. In contrast, p75-TM-C257A forms dimers through the AXXXG motif on the opposite face of the α-helix. Biochemical and cross-linking experiments indicate that AXXXG motif is not on the dimer interface of p75-TM-WT, suggesting that the conformation of p75-TM-C257A may be not functionally relevant. However, rather than mediating p75 homodimerization, mutagenesis of the AXXXG motif reveals its functional role in the regulated intramembrane proteolysis of p75 catalyzed by the γ-secretase complex. Our structural data provide an insight into the key role of the Cys(257) in stabilization of the weak transmembrane dimer in a conformation required for the NGF signaling.
Assuntos
Proteínas de Membrana/química , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Receptor de Fator de Crescimento Neural/química , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Western Blotting , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Células HeLa , Humanos , Lipídeos/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Micelas , Modelos Moleculares , Mutação , Oxirredução , Proteólise , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismoRESUMO
The neurotrophin receptor p75 (p75NTR) is a receptor for amyloid-beta (Aß) and mediates Aß-induced neurodegenerative signals. The ectodomain of p75NTR (p75ECD) is a physiological protective factor against Aß in Alzheimer's disease (AD). We have previously demonstrated that the shedding of p75ECD from the cell surface is down-regulated in AD brains and restoration of the p75ECD level in the brain, through intracranial administration of p75ECD by adeno-associated virus vectors, attenuates AD-like pathologies in an AD mouse model. In this study, we further investigated the feasibility and efficacy of peripheral administration of AAV-p75ECD on brain amyloid burden and associated pathogenesis. We found that intramuscular delivery of AAV-p75ECD increased the level of p75ECD in the blood, significantly improved the behavioral phenotype of amyloid precursor protein/PS1 transgenic mice, and reduced brain amyloid burden, attenuated Tau hyperphosphorylation, and neuroinflammation. Furthermore, intramuscular delivery of AAV-p75ECD was well tolerated. Our results indicate that peripheral delivery of p75ECD represents a safe and effective therapeutic strategy for AD. The ectodomain of p75NTR (p75ECD) is a physiological protective factor against amyloid-beta (Aß) in Alzheimer's disease (AD). Intramuscular delivery of AAV-p75ECD increased the p75ECD levels in the blood, reduced brain amyloid burden through a 'peripheral sink' mechanism and alleviates AD-type pathologies. Peripheral delivery of p75ECD represents a promising therapeutic strategy for AD.
Assuntos
Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Transtornos Cognitivos/terapia , Receptor de Fator de Crescimento Neural/química , Receptor de Fator de Crescimento Neural/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Transtornos Cognitivos/genética , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Injeções Intramusculares , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Presenilina-1/genética , Receptor de Fator de Crescimento Neural/genética , Transdução GenéticaRESUMO
Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Neoplasias Encefálicas/metabolismo , Encéfalo/patologia , Glioma/metabolismo , Células-Tronco Neoplásicas/patologia , Fatores de Crescimento Neural/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Glioma/genética , Glioma/patologia , Humanos , Mutação , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Receptor de Fator de Crescimento Neural/genéticaRESUMO
The ocular administration of nerve growth factor (NGF) as eye drops (oNGF) has been shown to exert protective effects in forebrain-injured animal models, including adult diabetes induced by a single injection of streptozotocin (STZ) (60 mg/kg body weight). This type 1 diabetes model was used in this study to investigate whether oNGF might extend its actions on neuronal precursors localised in the subventricular zone (SVZ). NGF or saline was administrated as eye drops twice daily for 2 weeks in rats with STZ-induced diabetes and healthy control rats. The expression of mature and precursor NGF and the NGF receptors, tropomyosin-related kinase A and neurotrophin receptor p75, and the levels of DNA fragmentation were analysed by ELISA and western blotting. Incorporation of bromodeoxyuridine was used to trace newly formed cells. Nestin, polysialylated neuronal cell adhesion molecule (PSA-NCAM), doublecortin (DCX) and glial fibrillary acidic protein antibodies were used to identify the SVZ cells by confocal microscopy. It was found that oNGF counteracts the STZ-induced cell death and the alteration of mature/pro-NGF expression in the SVZ. It also affects the survival and differentiation of SVZ progenitors. In particular, oNGF counteracts the reduction in the number of cells expressing PSA-NCAM/DCX (neuroblast type A cells) and the related reductions in the number and distribution of nestin/DCX-positive cells (C-type cells), or glia-committed cells (type B cells), observed in the SVZ of diabetic rats. These findings show that oNGF treatment counteracts the effect of type 1 diabetes on neuronal precursors in the SVZ, and further support the neuroprotective and reparative role of oNGF in the brain.