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1.
Sci Rep ; 10(1): 19995, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33203955

RESUMO

GPR37L1 is an orphan G protein-coupled receptor expressed exclusively in the brain and linked to seizures, neuroprotection and cardiovascular disease. Based upon the observation that fragments of the GPR37L1 N-terminus are found in human cerebrospinal fluid, we hypothesized that GPR37L1 was subject to post-translational modification. Heterologous expression of GPR37L1-eYFP in either HEK293 or U87 glioblastoma cells yielded two cell surface species of approximately equivalent abundance, the larger of which is N-glycosylated at Asn105. The smaller species is produced by matrix metalloprotease/ADAM-mediated proteolysis (shown by the use of pharmacological inhibitors) and has a molecular weight identical to that of a mutant lacking the entire N-terminus, Δ122 GPR37L1. Serial truncation of the N-terminus prevented GPR37L1 expression except when the entire N-terminus was removed, narrowing the predicted site of N-terminal proteolysis to residues 105-122. Using yeast expressing different G protein chimeras, we found that wild type GPR37L1, but not Δ122 GPR37L1, coupled constitutively to Gpa1/Gαs and Gpa1/Gα16 chimeras, in contrast to previous studies. We tested the peptides identified in cerebrospinal fluid as well as their putative newly-generated N-terminal 'tethered' counterparts in both wild type and Δ122 GPR37L1 Gpa1/Gαs strains but saw no effect, suggesting that GPR37L1 does not signal in a manner akin to the protease-activated receptor family. We also saw no evidence of receptor activation or regulation by the reported GPR37L1 ligand, prosaptide/TX14A. Finally, the proteolytically processed species predominated both in vivo and ex vivo in organotypic cerebellar slice preparations, suggesting that GPR37L1 is rapidly processed to a signaling-inactive form. Our data indicate that the function of GPR37L1 in vivo is tightly regulated by metalloprotease-dependent N-terminal cleavage.


Assuntos
Metaloproteinases da Matriz/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Linhagem Celular Tumoral , Cerebelo/metabolismo , Feminino , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia
2.
Sci Rep ; 10(1): 16656, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028854

RESUMO

Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is a cell adhesion molecule responsible for cell-to-cell interactions between immune cells and endothelial cells. In our previous paper, we have shown that Ninj1 plays an important role in the infiltration of neutrophils in the postischemic brain and that the dodecamer peptide harboring the Ninj1 N-terminal adhesion motif (N-NAM, Pro26-Asn37) inhibits infiltration of neutrophils in the postischemic brain and confers robust neuroprotective and anti-inflammatory effects. In the present study, we examinedt the pro-angiogenic effect of N-NAM using human umbilical vein endothelial cells (HUVECs) and rat MCAO (middle cerebral artery occlusion) model of stroke. We found that N-NAM promotes proliferation, migration, and tube formation of HUVECs and demonstrate that the suppression of endogenous Ninj1 is responsible for the N-NAM-mediated pro-angiogenic effects. Importantly, a pull-down assay revealed a direct binding between exogenously delivered N-NAM and endogenous Ninj1 and it is N-terminal adhesion motif dependent. In addition, N-NAM activated the Ang1-Tie2 and AKT signaling pathways in HUVECs, and blocking those signaling pathways with specific inhibitors suppressed N-NAM-induced tube formation, indicating critical roles of those signaling pathways in N-NAM-induced angiogenesis. Moreover, in a rat MCAO model, intranasal administration of N-NAM beginning 4 days post-MCAO (1.5 µg daily for 3 days) augmented angiogenesis in the penumbra of the ipsilateral hemisphere of the brain and significantly enhanced total vessel lengths, vessel densities, and pro-angiogenic marker expression. These results demonstrate that the 12-amino acid Ninj1 peptide, which contains the N-terminal adhesion motif of Ninj1, confers pro-angiogenic effects and suggest that those effects might contribute to its neuroprotective effects in the postischemic brain.


Assuntos
Indutores da Angiogênese/farmacologia , Moléculas de Adesão Celular Neuronais/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Fármacos Neuroprotetores/farmacologia , Indutores da Angiogênese/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fatores de Crescimento Neural/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos
3.
Sci Rep ; 10(1): 13526, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782285

RESUMO

Many neurons concurrently and/or differentially release multiple neurotransmitter substances to selectively modulate the activity of distinct postsynaptic targets within a network. However, the molecular mechanisms that produce synaptic heterogeneity by regulating the cotransmitter release characteristics of individual presynaptic terminals remain poorly defined. In particular, we know little about the regulation of neuropeptide corelease, despite the fact that they mediate synaptic transmission, plasticity and neuromodulation. Here, we report that an identified Lymnaea neuron selectively releases its classical small molecule and peptide neurotransmitters, acetylcholine and FMRFamide-derived neuropeptides, to differentially influence the activity of distinct postsynaptic targets that coordinate cardiorespiratory behaviour. Using a combination of electrophysiological, molecular, and pharmacological approaches, we found that neuropeptide cotransmitter release was regulated by cross-talk between extrinsic neurotrophic factor signaling and target-specific retrograde arachidonic acid signaling, which converged on modulation of glycogen synthase kinase 3. In this context, we identified a novel role for the Lymnaea synaptophysin homologue as a specific and synapse-delimited inhibitory regulator of peptide neurotransmitter release. This study is among the first to define the cellular and molecular mechanisms underlying the differential release of cotransmitter substances from individual presynaptic terminals, which allow for context-dependent tuning and plasticity of the synaptic networks underlying patterned motor behaviour.


Assuntos
Lymnaea/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Sinapses/fisiologia , Transmissão Sináptica , Animais , Células Cultivadas , Lymnaea/genética , Fatores de Crescimento Neural/genética , Terminações Pré-Sinápticas/fisiologia , Receptores Nicotínicos/metabolismo
4.
Nat Commun ; 11(1): 3942, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770063

RESUMO

Though discovered over 100 years ago, the molecular foundation of sporadic Alzheimer's disease (AD) remains elusive. To better characterize the complex nature of AD, we constructed multiscale causal networks on a large human AD multi-omics dataset, integrating clinical features of AD, DNA variation, and gene- and protein-expression. These probabilistic causal models enabled detection, prioritization and replication of high-confidence master regulators of AD-associated networks, including the top predicted regulator, VGF. Overexpression of neuropeptide precursor VGF in 5xFAD mice partially rescued beta-amyloid-mediated memory impairment and neuropathology. Molecular validation of network predictions downstream of VGF was also achieved in this AD model, with significant enrichment for homologous genes identified as differentially expressed in 5xFAD brains overexpressing VGF. Our findings support a causal role for VGF in protecting against AD pathogenesis and progression.


Assuntos
Doença de Alzheimer/etiologia , Encéfalo/patologia , Fatores de Crescimento Neural/metabolismo , Mapas de Interação de Proteínas , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Crescimento Neural/genética , Mapeamento de Interação de Proteínas , Proteômica
5.
PLoS One ; 15(8): e0237933, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822407

RESUMO

Defining the relationship between maternal care, sensory development and brain gene expression in neonates is important to understand the impact of environmental challenges during sensitive periods in early life. In this study, we used a selection approach to test the hypothesis that variation in maternal licking and grooming (LG) during the first week of life influences sensory development in Wistar rat pups. We tracked the onset of the auditory brainstem response (ABR), the timing of eye opening (EO), middle ear development with micro-CT X-ray tomography, and used qRT-PCR to monitor changes in gene expression of the hypoxia-sensitive pathway and neurotrophin signaling in pups reared by low-LG or high-LG dams. The results show the first evidence that the transcription of genes involved in the hypoxia-sensitive pathway and neurotrophin signaling is regulated during separate sensitive periods that occur before and after hearing onset, respectively. Although the timing of ABR onset, EO, and the relative mRNA levels of genes involved in the hypoxia-sensitive pathway did not differ between pups from different LG groups, we found statistically significant increases in the relative mRNA levels of four genes involved in neurotrophin signaling in auditory brain regions from pups of different LG backgrounds. These results suggest that sensitivity to hypoxic challenge might be widespread in the auditory system of neonate rats before hearing onset, and that maternal LG may affect the transcription of genes involved in experience-dependent neuroplasticity.


Assuntos
Comportamento Animal/fisiologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Asseio Animal/fisiologia , Comportamento Materno/fisiologia , Fatores de Crescimento Neural/metabolismo , Animais , Animais Recém-Nascidos , Olho/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Audição , Hipóxia/genética , Hipóxia/metabolismo , Fatores de Crescimento Neural/genética , Plasticidade Neuronal/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Microtomografia por Raio-X
6.
J Neurosci ; 40(32): 6146-6164, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32631936

RESUMO

Cerebral dopamine neurotrophic factor (CDNF) protects dopaminergic neurons against toxic damage in the rodent brain and is in clinical trials to treat Parkinson's disease patients. Yet the underlying mechanism is poorly understood. To examine its significance for neural circuits and behavior, we examined the development of neurotransmitter systems from larval to male adult mutant zebrafish lacking cdnf Although a lack of cdnf did not affect overall brain dopamine levels, dopaminergic neuronal clusters showed significant abnormalities. The number of histamine neurons that surround the dopaminergic neurons was significantly reduced. Expression of tyrosine hydroxylase 2 in the brain was elevated in cdnf mutants throughout their lifespan. There were abnormally few GABA neurons in the hypothalamus in the mutant larvae, and expression of glutamate decarboxylase was reduced throughout the brain. cdnf mutant adults showed a range of behavioral phenotypes, including increased sensitivity to pentylenetetrazole-induced seizures. Shoaling behavior of mutant adults was abnormal, and they did not display social attraction to conspecifics. CDNF plays a profound role in shaping the neurotransmitter circuit structure, seizure susceptibility, and complex behaviors in zebrafish. These findings are informative for dissecting the diverse functions of this poorly understood factor in human conditions related to Parkinson's disease and complex behaviors.SIGNIFICANCE STATEMENT A zebrafish lacking cdnf grows normally and shows no overt morphologic phenotype throughout the life span. Remarkably, impaired social cohesion and increased seizure susceptibility were found in adult cdnf KO fish conceivably associated with significant changes of dopaminergic, GABAergic, and histaminergic systems in selective brain areas. These findings suggest that cdnf has broad effects on regulating neurogenesis and maturation of transmitter-specific neuronal types during development and throughout adulthood, rather than ones restricted to the dopaminergic systems.


Assuntos
Comportamento Animal , Neurônios Dopaminérgicos/metabolismo , Neurônios GABAérgicos/metabolismo , Fatores de Crescimento Neural/metabolismo , Doença de Parkinson/metabolismo , Convulsões/genética , Proteínas de Peixe-Zebra/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Deleção de Genes , Histamina/metabolismo , Masculino , Fatores de Crescimento Neural/genética , Doença de Parkinson/genética , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
7.
Mem Inst Oswaldo Cruz ; 115: e200075, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32696914

RESUMO

BACKGROUND: Although Mycobacterium leprae (ML) is well characterised as the causative agent of leprosy, the pathophysiological mechanisms underlying peripheral nerve damage still need further understanding. In vitro and in vivo studies have yielded insights into molecular mechanisms of ML interaction with Schwann cells (SC), indicating the regulation of genes and proteins crucial to neural plasticity. OBJECTIVES: We aimed to investigate the effect of ML on neurotrophins expression in human SC (hSC) and mice sciatic nerves to better understand their role in leprosy neuropathy, and aiming to contribute to future therapeutic approaches. METHODS: We evaluated mRNA and protein expression of BDNF, NGF, NT-3, NT-4 in hSC from amputation nerve fragments, as well as in athymic nude mice, infected by ML for eight months. FINDINGS AND MAIN CONCLUSIONS: Our in vitro results showed a trend to decline in NGF and BDNF mRNA in ML-treated hSC, compared to controls. The immunodetection of BDNF and NT-4 was significantly downregulated in ML-treated hSC. Conversely, ML-infected mice demonstrated upregulation of NT-3, compared to non-infected animals. Our findings indicate that ML may be involved in neurotrophins regulation, suggesting that a pathogen-related imbalance of these growth factors may have a role in the neural impairment of leprosy.


Assuntos
Mycobacterium leprae , Fatores de Crescimento Neural/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Animais , Humanos , Camundongos , Camundongos Nus
8.
J Neurosci ; 40(30): 5709-5723, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32554548

RESUMO

The paranodal junctions flank mature nodes of Ranvier and provide a barrier between ion channels at the nodes and juxtaparanodes. These junctions also promote node assembly and maintenance by mechanisms that are poorly understood. Here, we examine their role in the accumulation of NF186, a key adhesion molecule of PNS and CNS nodes. We previously showed that NF186 is initially targeted/accumulates via its ectodomain to forming PNS (hemi)nodes by diffusion trapping, whereas it is later targeted to mature nodes by a transport-dependent mechanism mediated by its cytoplasmic segment. To address the role of the paranodes in this switch, we compared accumulation of NF186 ectodomain and cytoplasmic domain constructs in WT versus paranode defective (i.e., Caspr-null) mice. Both pathways are affected in the paranodal mutants. In the PNS of Caspr-null mice, diffusion trapping mediated by the NF186 ectodomain aberrantly persists into adulthood, whereas the cytoplasmic domain/transport-dependent targeting is impaired. In contrast, accumulation of NF186 at CNS nodes does not undergo a switch; it is predominantly targeted to both forming and mature CNS nodes via its cytoplasmic domain and requires intact paranodes. Fluorescence recovery after photobleaching analysis indicates that the paranodes provide a membrane diffusion barrier that normally precludes diffusion of NF186 to nodes. Linkage of paranodal proteins to the underlying cytoskeleton likely contributes to this diffusion barrier based on 4.1B and ßII spectrin expression in Caspr-null mice. Together, these results implicate the paranodes as membrane diffusion barriers that regulate targeting to nodes and highlight differences in the assembly of PNS and CNS nodes.SIGNIFICANCE STATEMENT Nodes of Ranvier are essential for effective saltatory conduction along myelinated axons. A major question is how the various axonal proteins that comprise the multimeric nodal complex accumulate at this site. Here we examine how targeting of NF186, a key nodal adhesion molecule, is regulated by the flanking paranodal junctions. We show that the transition from diffusion-trapping to transport-dependent accumulation of NF186 requires the paranodal junctions. We also demonstrate that these junctions are a barrier to diffusion of axonal proteins into the node and highlight differences in PNS and CNS node assembly. These results provide new insights into the mechanism of node assembly and the pathophysiology of neurologic disorders in which impaired paranodal function contributes to clinical disability.


Assuntos
Moléculas de Adesão Celular/metabolismo , Gânglios Espinais/metabolismo , Fatores de Crescimento Neural/metabolismo , Nós Neurofibrosos/metabolismo , Animais , Moléculas de Adesão Celular/análise , Células Cultivadas , Feminino , Gânglios Espinais/química , Gânglios Espinais/citologia , Junções Intercelulares/química , Junções Intercelulares/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores de Crescimento Neural/análise , Nós Neurofibrosos/química
9.
Biochim Biophys Acta Mol Basis Dis ; 1866(10): 165858, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32531260

RESUMO

As the population ages, obesity and metabolic complications as well as neurological disorders are becoming more prevalent, with huge economic burdens on both societies and families. New therapeutics are urgently needed. Nerve growth factor (NGF), first discovered in 1950s, is a neurotrophic factor involved in regulating cell proliferation, growth, survival, and apoptosis in both central and peripheral nervous systems. NGF and its precursor, proNGF, bind to TrkA and p75 receptors and initiate protein phosphorylation cascades, resulting in changes of cellular functions, and are associated with obesity, diabetes and its complications, and Alzheimer's disease. In this article, we summarize changes in NGF levels in metabolic and neuronal disorders, the signal transduction initiated by NGF and proNGF, the physiological and pathophysiological relevance, and therapeutic potential in treating chronic metabolic diseases and cognitive decline.


Assuntos
Doença de Alzheimer/patologia , Neuropatias Diabéticas/patologia , Retinopatia Diabética/patologia , Fator de Crescimento Neural/metabolismo , Obesidade/complicações , Precursores de Proteínas/metabolismo , Adipocinas/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/terapia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Cognição/efeitos dos fármacos , Cognição/fisiologia , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/metabolismo , Neuropatias Diabéticas/terapia , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/terapia , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Camundongos , Fator de Crescimento Neural/administração & dosagem , Fator de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Células-Tronco Neurais/transplante , Obesidade/metabolismo , Obesidade/patologia , Obesidade/terapia , Soluções Oftálmicas/administração & dosagem , Parvovirinae/genética , Precursores de Proteínas/administração & dosagem , Ratos , Receptor trkA/metabolismo , Transdução de Sinais
10.
FASEB J ; 34(6): 8702-8720, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32385864

RESUMO

Disruption of colonic homeostasis caused by aberrant M1/M2 macrophage polarization and dysbiosis contributes to inflammatory bowel disease (IBD) pathogenesis. However, the molecular factors mediating colonic homeostasis are not well characterized. Here, we found that Ninjurin1 (Ninj1) limits colon inflammation by regulating macrophage polarization and microbiota composition under homeostatic conditions and during colitis development. Ninj1 deletion in mice induced hypersusceptibility to colitis, with increased prevalence of colitogenic Prevotellaceae strains and decreased immunoregulatory Lachnospiraceae strains. Upon co-housing (CoH) with WT mice, Ninj1-/- mice showed increased Lachnospiraceae and decreased Prevotellaceae abundance, with subsequent improvement of colitis. Under homeostatic conditions, M1 macrophage frequency was higher in the Ninj1-/- mouse colons than wild-type (WT) mouse colons, which may contribute to increased basal colonic inflammation and microbial imbalance. Following colitis induction, Ninj1 expression was increased in macrophages; meanwhile Ninj1-/- mice showed severe colitis development and impaired recovery, associated with decreased M2 macrophages and escalated microbial imbalance. In vitro, Ninj1 knockdown in mouse and human macrophages activated M1 polarization and restricted M2 polarization. Finally, the transfer of WT macrophages ameliorated severe colitis in Ninj1-/- mice. These findings suggest that Ninj1 mediates colonic homeostasis by modulating M1/M2 macrophage balance and preventing extensive dysbiosis, with implications for IBD prevention and therapy.


Assuntos
Moléculas de Adesão Celular Neuronais/deficiência , Colite/metabolismo , Colite/patologia , Microbioma Gastrointestinal/fisiologia , Macrófagos/metabolismo , Macrófagos/patologia , Fatores de Crescimento Neural/deficiência , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Colite/microbiologia , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Modelos Animais de Doenças , Homeostase/fisiologia , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/patologia , Ativação de Macrófagos/fisiologia , Masculino , Camundongos , Fatores de Crescimento Neural/metabolismo , Células THP-1/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(21): 11450-11458, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385162

RESUMO

Dynamic remodeling of the extracellular matrix affects many cellular processes, either directly or indirectly, through the regulation of soluble ligands; however, the mechanistic details of this process remain largely unknown. Here we propose that type I collagen remodeling regulates the receptor-binding activity of pigment epithelium-derived factor (PEDF), a widely expressed secreted glycoprotein that has multiple important biological functions in tissue and organ homeostasis. We determined the crystal structure of PEDF in complex with a disulfide cross-linked heterotrimeric collagen peptide, in which the α(I) chain segments-each containing the respective PEDF-binding region (residues 930 to 938)-are assembled with an α2α1α1 staggered configuration. The complex structure revealed that PEDF specifically interacts with a unique amphiphilic sequence, KGHRGFSGL, of the type I collagen α1 chain, with its proposed receptor-binding sites buried extensively. Molecular docking demonstrated that the PEDF-binding surface of type I collagen contains the cross-link-susceptible Lys930 residue of the α1 chain and provides a good foothold for stable docking with the α1(I) N-telopeptide of an adjacent triple helix in the fibril. Therefore, the binding surface is completely inaccessible if intermolecular crosslinking between two crosslink-susceptible lysyl residues, Lys9 in the N-telopeptide and Lys930, is present. These structural analyses demonstrate that PEDF molecules, once sequestered around newly synthesized pericellular collagen fibrils, are gradually liberated as collagen crosslinking increases, making them accessible for interaction with their target cell surface receptors in a spatiotemporally regulated manner.


Assuntos
Colágeno Tipo I/metabolismo , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Serpinas/química , Serpinas/metabolismo , Sítios de Ligação , Dicroísmo Circular , Colágeno Tipo I/química , Cristalografia por Raios X , Dissulfetos/química , Lisina/química , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Transdução de Sinais , Análise Espaço-Temporal
12.
J Biol Chem ; 295(22): 7566-7583, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32327487

RESUMO

We have previously demonstrated that ischemia/reperfusion (I/R) impairs endoplasmic reticulum (ER)-based protein folding in the heart and thereby activates an unfolded protein response sensor and effector, activated transcription factor 6α (ATF6). ATF6 then induces mesencephalic astrocyte-derived neurotrophic factor (MANF), an ER-resident protein with no known structural homologs and unclear ER function. To determine MANF's function in the heart in vivo, here we developed a cardiomyocyte-specific MANF-knockdown mouse model. MANF knockdown increased cardiac damage after I/R, which was reversed by AAV9-mediated ectopic MANF expression. Mechanistically, MANF knockdown in cultured neonatal rat ventricular myocytes (NRVMs) impaired protein folding in the ER and cardiomyocyte viability during simulated I/R. However, this was not due to MANF-mediated protection from reactive oxygen species generated during reperfusion. Because I/R impairs oxygen-dependent ER protein disulfide formation and such impairment can be caused by reductive stress in the ER, we examined the effects of the reductive ER stressor DTT. MANF knockdown in NRVMs increased cell death from DTT-mediated reductive ER stress, but not from nonreductive ER stresses caused by thapsigargin-mediated ER Ca2+ depletion or tunicamycin-mediated inhibition of ER protein glycosylation. In vitro, recombinant MANF exhibited chaperone activity that depended on its conserved cysteine residues. Moreover, in cells, MANF bound to a model ER protein exhibiting improper disulfide bond formation during reductive ER stress but did not bind to this protein during nonreductive ER stress. We conclude that MANF is an ER chaperone that enhances protein folding and myocyte viability during reductive ER stress.


Assuntos
Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Sobrevivência Celular , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Glicosilação , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Fatores de Crescimento Neural/genética , Espécies Reativas de Oxigênio
13.
Exp Neurol ; 329: 113288, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32229226

RESUMO

The peri-infarct region after ischemic stroke is the anatomical location for many of the endogenous recovery processes; however, -the molecular events in the peri-infarct region remain poorly characterized. In this study, we examine the molecular profile of the peri-infarct region on post-stroke day four, a time when reparative processes are ongoing. We used a multiomics approach, involving RNA sequencing, and mass spectrometry-based proteomics and metabolomics to characterize molecular changes in the peri-infarct region. We also took advantage of our previously developed method to express transgenes in the peri-infarct region where self-complementary adeno-associated virus (AAV) vectors were injected into the brain parenchyma on post-stroke day 2. We have previously used this method to show that mesencephalic astrocyte-derived neurotrophic factor (MANF) enhances functional recovery from stroke and recruits phagocytic cells to the peri-infarct region. Here, we first analyzed the effects of stroke to the peri-infarct region on post-stroke day 4 in comparison to sham-operated animals, finding that strokeinduced changes in 3345 transcripts, 341 proteins, and 88 metabolites. We found that after stroke, genes related to inflammation, proliferation, apoptosis, and regeneration were upregulated, whereas genes encoding neuroactive ligand receptors and calcium-binding proteins were downregulated. In proteomics, we detected upregulation of proteins related to protein synthesis and downregulation of neuronal proteins. Metabolomic studies indicated that in after stroke tissue there is an increase in saccharides, sugar phosphates, ceramides and free fatty acids and a decrease of adenine, hypoxantine, adenosine and guanosine. We then compared the effects of post-stroke delivery of AAV1-MANF to AAV1-eGFP (enhanced green fluorescent protein). MANF administration increased the expression of 77 genes, most of which were related to immune response. In proteomics, MANF administration reduced S100A8 and S100A9 protein levels. In metabolomics, no significant differences between MANF and eGFP treatment were detected, but relative to sham surgery group, most of the changes in lipids were significant in the AAV-eGFP group only. This work describes the molecular profile of the peri-infarct region during recovery from ischemic stroke, and establishes a resource for further stroke studies. These results provide further support for parenchymal MANF as a modulator of phagocytic function.


Assuntos
Infarto Cerebral/genética , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Proteômica/métodos , Acidente Vascular Cerebral/genética , Transcriptoma/genética , Animais , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Infarto Cerebral/metabolismo , Infarto Cerebral/patologia , Técnicas de Transferência de Genes , Masculino , Metabolômica/métodos , Fatores de Crescimento Neural/administração & dosagem , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Fatores de Tempo
14.
Exp Eye Res ; 195: 108030, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32272114

RESUMO

Retinopathy of prematurity (ROP) is a growing cause of lifelong blindness and visual defects as improved neonatal care worldwide increases survival in very-low-birthweight preterm newborns. Advancing ROP is managed by laser surgery or a single intravitreal injection of anti-VEGF, typically at 33-36 weeks gestational age. While newer methods of scanning and telemedicine improve monitoring ROP, the above interventions are more difficult to deliver in developing countries. There is also concern as to laser-induced detachment and adverse developmental effects in newborns of anti-VEGF treatment, spurring a search for alternative means of mitigating ROP. Pigment epithelium-derived factor (PEDF), a potent angiogenesis inhibitor appears late in gestation, is undetected in 25-28 week vitreous, but present at full term. Its absence may contribute to ROP upon transition from high-to-ambient oxygen environment or with intermittent hypoxia. We recently described antiangiogenic PEDF-derived small peptides which inhibit choroidal neovascularization, and suggested that their target may be laminin receptor, 67LR. The latter has been implicated in oxygen-induced ischemic retinopathy (OIR). Here we examined the effect of a nonapeptide, PEDF 336, in a newborn mouse OIR model. Neovascularization was significantly decreased in a dose-responsive manner by single intravitreal (IVT) injections of 1.25-7.5 µg/eye (1.0-6.0 nmol/eye). By contrast, anti-mouse VEGFA164 was only effective at 25 ng/eye, with limited dose-response. Combination of anti-VEGFA164 with PEDF 336 gave only the poorer anti-VEGF response while abrogating the robust inhibition seen with peptide-alone, suggesting a need for VEGF in sensitizing the endothelium to the peptide. VEGF stimulated 67LR presentation on endothelial cells, which was decreased in the presence of PEDF 336. Mouse and rabbit eyes showed no histopathology or inflammation after IVT peptide injection. Thus, PEDF 336 is a potential ROP therapeutic, but is not expected to be beneficial in combination with anti-VEGF.


Assuntos
Animais Recém-Nascidos , Bevacizumab/administração & dosagem , Proteínas do Olho/metabolismo , Isquemia/tratamento farmacológico , Fatores de Crescimento Neural/metabolismo , Neovascularização Retiniana/tratamento farmacológico , Serpinas/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Injeções Intravítreas , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/toxicidade , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores
15.
Biomed Res Int ; 2020: 6301697, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32280693

RESUMO

Objective: To investigate the therapeutic effect of combined application of Wuweizi (Schisandrae Chinensis Fructus) and dexamethasone in rats with idiopathic pulmonary fibrosis (IPF) and the possible protective effect of Wuweizi against dexamethasone-induced glucocorticoid osteoporosis (GIOP). Methods: There were five groups in this study, including the sham operation group, model group, Wuweizi group, dexamethasone group, and the combination group. A rat IPF model was made by the endotracheal injection of bleomycin. After modeling, rats were given drug interventions for 7 and 28 days. Rats were sacrificed for pathological morphology examination of the bone and lung and quantitative determination of biochemical markers of bone metabolism and angiogenesis-related cytokine to observe therapeutic efficacy on the 7th and 28th day. ELISA was used for the quantitative determination of tartrate-resistant acid phosphatase (TRACP), bone alkaline phosphatase (BALP), hypoxia-inducible factor (HIF-1α), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), and endostatin in serum. The concentrations of calcium (Ca) and phosphorus (P) were detected with the automatic biochemical analyzer. Results: After drug interventions for 7 and 28 days, alveolitis and pulmonary fibrosis in treatment groups showed significant improvement compared with those in the model group (P < 0.05). Bone histopathological figures showed severely damaged trabecular bone and bone marrow cavity in the dexamethasone group, but it was significantly alleviated in the combination group. The concentrations of BALP and Ca in the combination group were significantly higher than those in the dexamethasone group after treatment, while the concentrations of TRACP and P were lower than those in the dexamethasone group (P < 0.05). Bone histopathological figures showed severely damaged trabecular bone and bone marrow cavity in the dexamethasone group, but it was significantly alleviated in the combination group. The concentrations of BALP and Ca in the combination group were significantly higher than those in the dexamethasone group after treatment, while the concentrations of TRACP and P were lower than those in the dexamethasone group (α), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), and endostatin in serum. The concentrations of calcium (Ca) and phosphorus (P) were detected with the automatic biochemical analyzer. P < 0.05). Bone histopathological figures showed severely damaged trabecular bone and bone marrow cavity in the dexamethasone group, but it was significantly alleviated in the combination group. The concentrations of BALP and Ca in the combination group were significantly higher than those in the dexamethasone group after treatment, while the concentrations of TRACP and P were lower than those in the dexamethasone group (P < 0.05). Bone histopathological figures showed severely damaged trabecular bone and bone marrow cavity in the dexamethasone group, but it was significantly alleviated in the combination group. The concentrations of BALP and Ca in the combination group were significantly higher than those in the dexamethasone group after treatment, while the concentrations of TRACP and P were lower than those in the dexamethasone group (α), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), and endostatin in serum. The concentrations of calcium (Ca) and phosphorus (P) were detected with the automatic biochemical analyzer. Conclusions: The combination therapy of Wuweizi and dexamethasone effectively treated IPF rats by regulating angiogenesis, meanwhile distinctly alleviating dexamethasone-induced GIOP.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Glucocorticoides/uso terapêutico , Fibrose Pulmonar Idiopática/complicações , Osteoporose/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Schisandra/química , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bleomicina/efeitos adversos , Medula Óssea/metabolismo , Medula Óssea/patologia , Osso e Ossos/patologia , Osso Esponjoso/patologia , Dexametasona , Modelos Animais de Doenças , Endostatinas/metabolismo , Proteínas do Olho/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Fatores de Crescimento Neural/metabolismo , Osteoporose/induzido quimicamente , Osteoporose/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ratos , Ratos Wistar , Serpinas/metabolismo , Fosfatase Ácida Resistente a Tartarato
16.
Life Sci ; 252: 117642, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32259600

RESUMO

AIMS: To determine whether ginsenoside Rg1 is involved in scratch wound healing through altered expression of related molecules in astrocytes and improved functional recovery after spinal cord injury (SCI). MATERIALS AND METHODS: Astrocytes were isolated from rats, followed by Rg1 treatment. The wound healing test was performed to observe the scratch wound healing in different groups. The expression of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and components of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway were detected by western blot. Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the altered expression of laminin (LN) and fibronectin (FN). A revised Allen's method for the SCI model was performed, followed by Rg1 treatment. Then, functional scoring was conducted to evaluate the functional recovery. Hematoxylin-eosin (HE) staining showed changes in the void area. Finally, western blot assessed the expression of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs). KEY FINDINGS: Rg1 mediated scratch wound healing through inducing an increased release of LN, FN, NGF, GDNF, and bFGF in vitro. Additionally, Rg1 activated the PI3K/Akt signaling pathway and promoted the functional recovery of hindlimb movement in rats. Furthermore, Rg1 significantly reduced the void area and downregulated the expression of GFAP and CSPGs. SIGNIFICANCE: Rg1 not only enhanced the scratch wound repair in vitro through the release of astroglial neurotrophic factors, adhesion factors, and inhibitory factors, but it also improved the functional recovery in vivo following SCI.


Assuntos
Astrócitos/efeitos dos fármacos , Ginsenosídeos/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Feminino , Masculino , Fatores de Crescimento Neural/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Transdução de Sinais/efeitos dos fármacos , Traumatismos da Medula Espinal/fisiopatologia
17.
Diabetes ; 69(6): 1264-1278, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32312869

RESUMO

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neurotrophic factor widely expressed in mammalian tissues, and it exerts critical protective effects on neurons and other cell types in various disease models, such as those for diabetes. However, to date, the expression and roles of MANF in the cornea, with or without diabetic keratopathy (DK), remain unclear. Here, we demonstrate that MANF is abundantly expressed in normal corneal epithelial cells; however, MANF expression was significantly reduced in both unwounded and wounded corneal epithelium in streptozotocin-induced type 1 diabetic C57BL/6 mice. Recombinant human MANF significantly promoted normal and diabetic corneal epithelial wound healing and nerve regeneration. Furthermore, MANF inhibited hyperglycemia-induced endoplasmic reticulum (ER) stress and ER stress-mediated apoptosis. Attenuation of ER stress with 4-phenylbutyric acid (4-PBA) also ameliorated corneal epithelial closure and nerve regeneration. However, the beneficial effects of MANF and 4-PBA were abolished by an Akt inhibitor and Akt-specific small interfering RNA (siRNA). Finally, we reveal that the subconjunctival injection of MANF-specific siRNA prevents corneal epithelial wound healing and nerve regeneration. Our results provide important evidence that hyperglycemia-suppressed MANF expression may contribute to delayed corneal epithelial wound healing and impaired nerve regeneration by increasing ER stress, and MANF may be a useful therapeutic modality for treating DK.


Assuntos
Lesões da Córnea/patologia , Diabetes Mellitus Experimental/complicações , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Regeneração Nervosa/fisiologia , Cicatrização/fisiologia , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Hiperglicemia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/administração & dosagem , Fatores de Crescimento Neural/genética , RNA Bacteriano , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia
18.
Int J Med Sci ; 17(4): 480-489, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32174778

RESUMO

Amyotrophic lateral sclerosis (ALS) is a serious disease characterized by the degeneration of motor neurons resulting in muscle weakness and paralysis. The neuroendocrine polypeptide VGF is localized in the central nervous system and peripheral endocrine neurons and is cleaved into several polypeptides with multiple functions. Previous studies revealed that VGF was decreased in the cerebrospinal fluid of ALS model mice and sporadic ALS patients. However, it is unknown which cells supply VGF in the spinal cord and a detailed localization is lacking. In this study, we evaluated the VGF-producing cells and protein localization using in situ hybridization and immunostaining in the spinal cords of ALS and control patients. VGF mRNA was localized both in the dorsal and anterior horns of the spinal cords. Moreover, in the anterior horn, VGF mRNA co-localized with a neurofilament heavy chain, which is a motor neuron marker, and VGF mRNA-positive motor neurons were decreased in the spinal cords of ALS patients. We revealed that VGF protein level was decreased in the anterior horn of ALS patients; however, the expression level of VGF protein was not changed in the posterior horn or white matter. Furthermore, the expression level of VGF protein was conserved in ALS patients with long-term survival. These results reveal that VGF is mainly supplied by human motor neurons, and suggest that VGF expression changes may be involved in ALS pathology.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Neurônios Motores/metabolismo , Fatores de Crescimento Neural/metabolismo , Medula Espinal/metabolismo , Idoso , Esclerose Amiotrófica Lateral/patologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Fatores de Crescimento Neural/genética , RNA Mensageiro/metabolismo
19.
Sci Rep ; 10(1): 5380, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32214122

RESUMO

Dogs share many chronic morbidities with humans and thus represent a powerful model for translational research. In comparison to rodents, the canine ganglioside metabolism more closely resembles the human one. Gangliosides are components of the cell plasma membrane playing a role in neuronal development, intercellular communication and cellular differentiation. The present in vitro study aimed to characterize structural and functional changes induced by GM1 ganglioside (GM1) in canine dorsal root ganglia (DRG) neurons and interactions of GM1 with nerve growth factor (NGF) and fibroblast growth factor (FGF2) using immunofluorescence for several cellular proteins including neurofilaments, synaptophysin, and cleaved caspase 3, transmission electron microscopy, and electrophysiology. GM1 supplementation resulted in increased neurite outgrowth and neuronal survival. This was also observed in DRG neurons challenged with hypoxia mimicking neurodegenerative conditions due to disruptions of energy homeostasis. Immunofluorescence indicated an impact of GM1 on neurofilament phosphorylation, axonal transport, and synaptogenesis. An increased number of multivesicular bodies in GM1 treated neurons suggested metabolic changes. Electrophysiological changes induced by GM1 indicated an increased neuronal excitability. Summarized, GM1 has neurotrophic and neuroprotective effects on canine DRG neurons and induces functional changes. However, further studies are needed to clarify the therapeutic value of gangliosides in neurodegenerative diseases.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Gangliosídeo G(M1)/metabolismo , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cães , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Gangliosídeos/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia
20.
ACS Appl Mater Interfaces ; 12(14): 16168-16177, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32182427

RESUMO

Peripheral nerve injury (PNI) was the leading cause of permanent dysfunction in movement and sensation. Synthesized nerve guide conduits (NGCs) with Schwann Cells (SCs) can help peripheral nerve regeneration. However, poor accessibility of SCs and lack of full coverage of seeded cells on NGCs can lead to failure of nerve regeneration across long gaps and full functional recovery. To overcome these limitations, bone marrow stromal cells (BMSCs) and a novel culture method were proposed in the current study. BMSCs were harvested and seeded on a never growth factor (NGF)-loaded PCL nanofibrous NGCs and cultured with a rotary cell culture system (RCCS) before implantation. The NGCs were tested in vitro with PC-12 cells to validate the bioactivity of released NGF and to access its ability to promote neurite extension. Also, the NGCs were tested in vivo with rat sciatic nerve model to exam its potential in bridging the long gap (15 mm segmental defect). The efficacy of the NGCs was investigated based on the results of the functional test, electrophysiology test, muscle atrophy, and histological analysis. The results of in vitro PC-12 cell study confirmed the bioactivity of released NGF and showed a significant increase in the neurite extension with the help of PEG-diamine and BSA. These results showed that the novel loading method could preserve the bioactivity of growth factors and achieve a sustained release in vitro. Besides, the results of the in vivo study exhibited a significant increase with the combination of all additives. These results showed that with the help of NGF and RCCS, the NGCs with the seeded BMSCs could enhance peripheral nerve regeneration across long nerve injury gaps.


Assuntos
Nanofibras/química , Regeneração Nervosa/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/terapia , Nervo Isquiático/efeitos dos fármacos , Animais , Reatores Biológicos , Técnicas de Cultura de Células , Modelos Animais de Doenças , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanofibras/uso terapêutico , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Células PC12 , Traumatismos dos Nervos Periféricos/patologia , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/crescimento & desenvolvimento , Nervos Periféricos/patologia , Ratos , Células de Schwann/efeitos dos fármacos , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/patologia
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