RESUMO
Glial cell line-derived neurotrophic factor (GDNF) has neuroprotective effects and may be a promising candidate for regenerative strategies focusing on neurodegenerative diseases. As GDNF cannot cross the blood-brain barrier to potentially regenerate damaged brain areas, continuous in situ delivery with host cells is desired. Here, a non-viral Sleeping Beauty transposon was used to achieve continuous in vitro overexpression of GDNF in immune-privileged human adipose tissue-derived mesenchymal stromal cells (GDNF-tASCs). In addition, in vivo survival, tolerance, and effectiveness of transfected cells were tested in a very mild 6-hydroxydopamine (6-OHDA)-induced dopamine depletion rat model by means of intrastriatal injection on a sample basis up to 6 months after treatment. GDNF-tASCs showed vast in vitro gene overexpression up to 13 weeks post-transfection. In vivo, GDNF was detectable 4 days following transplantation, but no longer after 1 month, although adipose tissue-derived mesenchymal stromal cells (ASCs) could be visualized histologically even after 6 months. Despite successful long-term in vitro GDNF overexpression and its in vivo detection shortly after cell transplantation, the 6-OHDA model was too mild to enable sufficient evaluation of in vivo disease improvement. Still, in vivo immunocompatibility could be further examined. ASCs initially induced a pronounced microglial accumulation at transplantation site, particularly prominent in GDNF-tASCs. However, 6-OHDA-induced pro-inflammatory immune response was attenuated by ASCs, although delayed in the GDNF-tASCs group. To further test the therapeutic potential of the generated GDNF-overexpressing cells in a disease-related context, a follow-up study using a more appropriate 6-OHDA model is needed.
Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial , Células-Tronco Mesenquimais , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Modelos Animais de Doenças , Seguimentos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Oxidopamina/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
The first stage of the drug discovery process involves the identification of small compounds with biological activity. Iboga alkaloids are monoterpene indole alkaloids (MIAs) containing a fused isoquinuclidine-tetrahydroazepine ring. Both the natural products and the iboga-inspired synthetic analogs have shown a wide variety of biological activities. Herein, we describe the chemoenzymatic preparation of a small library of novel N-indolylethyl-substituted isoquinuclidines as iboga-inspired compounds, using toluene as a starting material and an imine Diels-Alder reaction as the key step in the synthesis. The new iboga series was investigated for its potential to promote the release of glial cell line-derived neurotrophic factor (GDNF) by C6 glioma cells, and to inhibit the growth of infective trypanosomes. GDNF is a neurotrophic factor widely recognized by its crucial role in development, survival, maintenance, and protection of dopaminergic neuronal circuitries affected in several neurological and psychiatric pathologies. Four compounds of the series showed promising activity as GDNF releasers, and a leading structure (compound 11) was identified for further studies. The same four compounds impaired the growth of bloodstream Trypanosoma brucei brucei (EC50 1-8 µM) and two of them (compounds 6 and 14) showed a good selectivity index.
Assuntos
Alcaloides , Antiprotozoários , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Tabernaemontana/química , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase Africana/tratamento farmacológico , Alcaloides/síntese química , Alcaloides/química , Alcaloides/farmacologia , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Antiprotozoários/farmacologia , Linhagem Celular Tumoral , Camundongos , Ratos , Tripanossomíase Africana/metabolismo , Tripanossomíase Africana/patologiaRESUMO
Glial cell line-derived neurotrophic factor (GDNF) is a powerful neuroprotective growth factor. However, systemic or intrathecal administration of GDNF is associated with side effects. Here, we aimed to avoid this by restricting the transgene expression to the skeletal muscle by gene therapy. To specifically target most skeletal muscles in the mouse model of amyotrophic lateral sclerosis (ALS), SOD1G93A transgenic mice were intravenously injected with adeno-associated vectors coding for GDNF under the control of the desmin promoter. Treated and control SOD1G93A mice were evaluated by rotarod and nerve conduction tests from 8 to 20 weeks of age, and then histological and molecular analyses were performed. Muscle-specific GDNF expression delayed the progression of the disease in SOD1G93A female and male mice by preserving the neuromuscular function; increasing the number of innervated neuromuscular junctions, the survival of spinal motoneurons; and reducing glial reactivity in treated SOD1G93A mice. These beneficial actions are attributed to a paracrine protective mechanism from the muscle to the motoneurons by GDNF. Importantly, no adverse secondary effects were detected. These results highlight the potential of muscle GDNF-targeted expression for ALS therapy.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Músculo Esquelético/metabolismo , Esclerose Lateral Amiotrófica/diagnóstico por imagem , Animais , Feminino , Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/diagnóstico por imagem , Superóxido Dismutase/genéticaRESUMO
Patients with isolated pulmonary embolism (PE) have a distinct clinical profile from those with deep vein thrombosis (DVT)-associated PE, with more pulmonary conditions and atherosclerosis. These findings suggest a distinct molecular pathophysiology and the potential involvement of alternative pathways in isolated PE. To test this hypothesis, data from 532 individuals from the Genotyping and Molecular Phenotyping of Venous ThromboEmbolism Project, a multicenter prospective cohort study with extensive biobanking, were analyzed. Targeted, high-throughput proteomics, machine learning, and bioinformatic methods were applied to contrast the acute-phase plasma proteomes of isolated PE patients (n = 96) against those of patients with DVT-associated PE (n = 276) or isolated DVT (n = 160). This resulted in the identification of shared molecular processes between PE phenotypes, as well as an isolated PE-specific protein signature. Shared processes included upregulation of inflammation, response to oxidative stress, and the loss of pulmonary surfactant. The isolated PE-specific signature consisted of 5 proteins: interferon-γ, glial cell line-derived neurotrophic growth factor, polypeptide N-acetylgalactosaminyltransferase 3, peptidyl arginine deiminase type-2, and interleukin-15 receptor subunit α. These proteins were orthogonally validated using cis protein quantitative trait loci. External replication in an independent population-based cohort (n = 5778) further validated the proteomic results and showed that they were prognostic for incident primary isolated PE in individuals without history of VTE (median time to event: 2.9 years; interquartile range: 1.6-4.2 years), supporting their possible involvement in the early pathogenesis. This study has identified molecular overlaps and differences between VTE phenotypes. In particular, the results implicate noncanonical pathways more commonly associated with respiratory and atherosclerotic disease in the acute pathophysiology of isolated PE.
Assuntos
Proteoma , Embolia Pulmonar/metabolismo , Transcriptoma , Proteínas de Fase Aguda/biossíntese , Adulto , Idoso , Aterosclerose/complicações , Comorbidade , Conjuntos de Dados como Assunto , Feminino , Seguimentos , Regulação da Expressão Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Interferon gama/biossíntese , Interferon gama/genética , Subunidade alfa de Receptor de Interleucina-15/biossíntese , Subunidade alfa de Receptor de Interleucina-15/genética , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , N-Acetilgalactosaminiltransferases/biossíntese , N-Acetilgalactosaminiltransferases/genética , Estresse Oxidativo , Estudos Prospectivos , Mapas de Interação de Proteínas , Proteína-Arginina Desiminase do Tipo 2/biossíntese , Proteína-Arginina Desiminase do Tipo 2/genética , Embolia Pulmonar/genética , Embolia Pulmonar/fisiopatologia , Surfactantes Pulmonares , Locos de Características Quantitativas , Tromboembolia Venosa/metabolismo , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disease, which is characterized by the progressive loss of dopaminergic neurons in the substantia nigra, leading to a decrease in striatal dopamine. There is no antiparkinsonian therapy that offers a true disease-modifying treatment till date and there is an urgent need for a safe and effective neuroprotective or neurorestorative therapy. Our previous study demonstrated that metformin upregulated dopamine in the mouse brain and provided significant neuroprotection in animal model of PD. Therefore, we designed this study to investigate the molecular mechanism underlying such pharmacological effect of metformin. Herein, we found that metformin enhanced the phosphorylation of tyrosine hydroxylase (TH) which was accompanied by increase in brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and activation of their downstream signaling pathways in the mouse brain and SH-SY5Y cells. We further investigated the role of the neurotrophic factors in the activation of TH and observed that both BDNF and GDNF-induction were essential for metformin-induced TH activation. We found that the AMPK/aPKCζ/CREB pathway was essential for metformin-induced GDNF upregulation and TH activation. Thus, this study reveals the TH-activating property of metformin in the brain via induction of neurotrophic factors along with the signaling mechanism. These results potentiate the candidacy of metformin not only as a neuroprotective agent, but also as restorative therapy for the treatment of PD.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dopamina/biossíntese , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Metformina/farmacologia , Proteína Quinase C/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Following nerve tissue damage, various events, such as inflammatory responses, microglial activation, endoplasmic reticulum stress, and apoptosis, can occur, which all lead to cell death, prevent axonal growth, and cause axonal circumvolution. So far, several researchers have tended to adopt strategies to reduce the harmful conditions associated with neurological disorders, and stemcellbased therapy is one of those promising strategies. Epidermal neural crest stem cells (EPINCSCs) are a type of stem cell that has widely been employed for the treatment of various neurological disorders. It has been suggested that these stem cells perform their supportive actions primarily through the release of different neurotrophic factors. Hence, in this study, the neuroprotective impacts of valproic acid (VPA) and crocin were evaluated on the mRNA expression levels of brainderived neurotrophic factor (BDNF) and glialcellderived neurotrophic factor (GDNF) in EPINCSCs. In this research, we isolated EPINCSCs from the hair follicles of the rat whisker pad. Then, the cells were treated with different concentrations of VPA and crocin for 72 h. Subsequently, an MTT assay was performed to define the suitable concentrations of drugs. Finally, realtime PCR was performed to evaluate the mRNA expression levels of BDNF and GDNF in these stem cells. The results of the MTT assay showed that the treatment of EPINCSCs with 1 mM VPA and 1.5 mM crocin, and the cotreatment with 1 mM VPA and 500 µM crocin, led to the survival and proliferation of these stem cells. Moreover, the realtime PCR results revealed that both VPA and crocin, both individually and in combination, can significantly increase the expression levels of BDNF and GDNF in EPINCSCs. According to the findings of this study, both VPA and crocin, alone and in combination, are potential candidates for enhancing the capacity of EPINCSCs to differentiate into neural lineages. Therefore, the cotreatment of EPINCSCs with these drugs can be employed for the treatment of various neurological disorders, such as spinal cord injury.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Carotenoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Crista Neural/efeitos dos fármacos , Ácido Valproico/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Células Cultivadas , Meios de Cultura/farmacologia , Sinergismo Farmacológico , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Folículo Piloso/citologia , Crista Neural/citologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RatosRESUMO
Glial cell line-derived neurotrophic factor (GDNF) is expressed at a high level in the human ovary and GDNF signaling is involved in the direct control of follicular activation and oocyte maturation. Transforming growth factor-ß1 (TGF-ß1) plays an important role in the regulation of various ovarian functions. Furin is an intracellular serine endopeptidase of the subtilisin family that is closely associated with the activation of multiple protein precursors. Despite the important roles of GDNF and TGF-ß1 in the regulation of follicular development, whether TGF-ß is able to regulate the expression and production of GDNF in human granulosa cells remains to be determined. The aim of this study was to investigate the effect of TGF-ß1 on the production of GDNF and its underlying mechanisms in human granulosa-lutein (hGL) cells. We used two types of hGL cells (primary hGL cells and an established immortalized hGL cell line, SVOG cells) as study models. Our results show that TGF-ß1 significantly induced the expression of GDNF and furin, which, in turn, increased the production of mature GDNF. Using a dual inhibition approach combining RNA interference and kinase inhibitors against cell signaling components, we showed that the TßRII type II receptor and ALK5 type I receptor are the principal receptors that mediated TGF-ß1-induced cellular activity in hGL cells. Additionally, Sma- and Mad-related protein (SMAD)3 and SMAD4 are the downstream signaling transducers that mediate the biological response induced by TGF-ß1. Furthermore, furin is the main proprotein convertase that induces the production of GDNF. These findings provide additional regulatory mechanisms by which an intrafollicular factor influences the production of another growth factor through a paracrine or autocrine interaction in hGL cells.
Assuntos
Furina/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Células da Granulosa/metabolismo , Células Lúteas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células Cultivadas , Feminino , Furina/metabolismo , Humanos , Regulação para CimaRESUMO
Multiple system atrophy (MSA) is a fatal disorder with no effective treatment. MSA pathology is characterized by α-synuclein (aSyn) accumulation in oligodendrocytes, the myelinating glial cells of the central nervous system (CNS). aSyn accumulation in oligodendrocytes forms the pathognomonic glial cytoplasmic inclusions (GCIs) of MSA. MSA aSyn pathology is also associated with motor and autonomic dysfunction, including an impaired ability to sweat. MSA patients have abnormal CNS expression of glial-cell-line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). Our prior studies using the parent compound FTY720, a food and drug administration (FDA) approved immunosuppressive for multiple sclerosis, reveal that FTY720 protects parkinsonian mice by increasing BDNF. Our FTY720-derivative, FTY720-Mitoxy, is known to increase expression of oligodendrocyte BDNF, GDNF, and nerve growth factor (NGF) but does not reduce levels of circulating lymphocytes as it is not phosphorylated so cannot modulate sphingosine 1 phosphate receptors (S1PRs). To preclinically assess FTY720-Mitoxy for MSA, we used mice expressing human aSyn in oligodendrocytes under a 2,' 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter. CNP-aSyn transgenic (Tg) mice develop motor dysfunction between 7 and 9 mo, and progressive GCI pathology. Using liquid chromatography-mass spectrometry (LC-MS/MS) and enzymatic assays, we confirmed that FTY720-Mitoxy was stable and active. Vehicle or FTY720-Mitoxy (1.1â¯mg/kg/day) was delivered to wild type (WT) or Tg littermates from 8.5-11.5 mo by osmotic pump. We behaviorally assessed their movement by rotarod and sweat production by starchiodine test. Postmortem tissues were evaluated by qPCR for BDNF, GDNF, NGF and GDNF-receptor RET mRNA and for aSyn, BDNF, GDNF, and Iba1 protein by immunoblot. MicroRNAs (miRNAs) were also assessed by qPCR. FTY720-Mitoxy normalized movement, sweat function and soleus muscle mass in 11.5 mo Tg MSA mice. FTY720-Mitoxy also increased levels of brain GDNF and reduced brain miR-96-5p, a miRNA that acts to decrease GDNF expression. Moreover, FTY720-Mitoxy blocked aSyn pathology measured by sequential protein extraction and immunoblot, and microglial activation assessed by immunohistochemistry and immunoblot. In the 3-nitropropionic acid (3NP) toxin model of MSA, FTY720-Mitoxy protected movement and mitochondria in WT and CNP-aSyn Tg littermates. Our data confirm potent in vivo protection by FTY720-Mitoxy, supporting its further evaluation as a potential therapy for MSA and related synucleinopathies.
Assuntos
Cloridrato de Fingolimode/análogos & derivados , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Atrofia de Múltiplos Sistemas/patologia , Fármacos Neuroprotetores/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Cloridrato de Fingolimode/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/efeitos dos fármacos , MicroRNAs/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , Proteínas Proto-Oncogênicas c-ret/biossíntese , Proteínas Proto-Oncogênicas c-ret/efeitos dos fármacos , alfa-Sinucleína/genéticaRESUMO
Adipose-derived mesenchymal stem cells (AMSCs) are a type of adult stem cell from the mesoderm with the capacity to migrate and differentiate into other cell lineages. As a morphogenetic state of stem cells, glial-derived neurotrophic factor (GDNF) has been found to promote cell proliferation and differentiation of stem cells. The aims of our study were to investigate the biological activity of AMSCs and whether the GDNF gene can enhance the anti-inflammatory properties of stem cells. In this study, stable proliferative GDNF-overexpressing AMSC lines were successfully established and the AMSCs/GDNF-AMSCs were cocultured with macrophages (Mφ) derived from THP-1 cells in a transwell system. The mRNA expression levels of tumor necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS), interleukin (IL)-10 and IL-4 were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, the expressions of CD163 and CD206, two markers of M2 macrophages, were detected with flow cytometric analysis. In animal experiments, AMSCs/GDNF-AMSCs (5 × 105) were administered to unilateral ureteral obstruction (UUO) nude mice for 3 or 7 days. The expression levels of cyclooxygenase-2 (COX-2), IL-6, transforming growth factor ß1 (TGF-ß1) and α-Smooth muscle actin (α-SMA) were determined by Western blotting. Renal pathological changes of all groups were observed by hematoxylin and eosin (HE) and Masson staining. In conclusion, in vitro cultured AMSCs induced a shift in macrophage phenotype from the inflammatory (M1) phenotype to the reparative (M2) phenotype. In the UUO model, AMSC treatment was conducive to the recovery of renal function and interstitial fibrosis. Therefore, we determined that AMSC therapy could promote the phenotypic transformation of macrophages and reduce the progression of renal fibrosis by suppressing inflammation. GDNF could enhance the anti-inflammatory effect of AMSCs.
Assuntos
Tecido Adiposo/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Nefropatias/terapia , Células-Tronco Mesenquimais/metabolismo , Tecido Adiposo/patologia , Animais , Fibrose , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Xenoenxertos , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/terapia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Nus , Células THP-1RESUMO
Different classes of antidepressants, such as tricyclic antidepressants, selective serotonin reuptake inhibitor (SSRI), and serotonin and norepinephrine reuptake inhibitor (SNRI), have been shown to increase GDNF production in astrocytes, which could be a key mechanism of the psychotropic effect of antidepressants. The antidepressant mirtazapine is a noradrenaline and specific serotonergic antidepressant (NaSSA) and does not block reuptake of catecholamines and serotonin. The present study examined the effect of mirtazapine on GDNF expression in rat C6 astroglial cells (C6 cells) and rat primary cultured cortical astrocytes (primary astrocytes). Mirtazapine treatment significantly increased GDNF mRNA expression and GDNF release in both C6 cells and primary astrocytes. In primary astrocytes, mirtazapine also increased the expressions of brain-derived neurotrophic factor mRNA. To mimic mirtazapine's putative mechanism of action, cells were treated with either a α2-adrenoceptor antagonist (yohimbine), 5-HT2 receptor antagonist (ketanserin), 5-HT3 receptor antagonist (ondansetron), or a mixture of these--no effect on GDNF mRNA expression was observed. Mirtazapine treatment increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and the mirtazapine-induced GDNF and BDNF expression were blocked by MAPK/ERK kinase (MEK) inhibitor (U0126). Furthermore, the effect of mirtazapine on ERK phosphorylation and expressions of GDNF and BDNF was antagonized by Gi/o inhibitor (pertussis toxin), lysophosphatidic acid-1 (LPA1) receptor antagonist (AM966), and LPA1/LPA3 receptors antagonist (Ki16425). The current findings demonstrate that the NaSSA mirtazapine, similar to other classes of antidepressants, increases GDNF expression through a Gi/o coupled LPA1 receptor-mediated ERK pathway. The current findings suggest a general mechanism underlying the psychotropic effect antidepressants.
Assuntos
Antidepressivos/farmacologia , Astrócitos/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mirtazapina/farmacologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologiaRESUMO
Sialylated milk oligosaccharides (SMOs) have a multifunctional health benefit, yet the molecular details underlying their potential role in modulating intestinal maturation remains unknown. To test the hypothesis that sialyllactose (SL) may mediate intestinal maturation and function through controlling neuronal function, studies were carried out where the diet of postnatal piglets was supplemented with a mixture of 3'- and 6'-sialyllactose from postnatal day 3 to 38. Gene transcription pathways regulating enteric nervous system function, polysialic acid (polySia) synthesis, and cell proliferation were quantified. Our new findings show that SL intervention: (1) upregulated the level of gene and protein expression of the glial-derived neurotrophic factor (GDNF) in the ileum; (2) upregulated phosphorylation of the cAMP responsive element-binding protein (CREB), the downstream target of GDNF signaling pathway; (3) promoted cell proliferation based on an increase in the number and density of Ki-67 positive cells in the crypts; (4) increased the crypt width in the ileum by 10%, while gene markers for the functional cells were not affected; (5) upregulated mRNA expression level of ST8Sia IV, a key polysialyltransferase responsible for synthesis of polySia-NCAM; (6) reduced the incidence and severity of diarrhea. These results show that SL promotes intestinal maturation in neonatal piglets by upregulating GDNF, synthesis of polySia and CREB-interactive pathway.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Diarreia/metabolismo , Sistema Nervoso Entérico/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Intestino Delgado/metabolismo , Lactose/análogos & derivados , Ácidos Siálicos/farmacologia , Animais , Animais Recém-Nascidos , Diarreia/prevenção & controle , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/crescimento & desenvolvimento , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/crescimento & desenvolvimento , Lactose/farmacologia , Masculino , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sus scrofa , Suínos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
OBJECTIVE: The present study aimed to investigate the effects of Mesenchymal stem cells/glial cell line derived neurotrophic factor (MSCs/GDNF) transplantation on nerve reconstruction in rats with intracerebral hemorrhage. METHODS: GDNF transduction to MSCs was using adenovirus vector pAdEasy-1-pAdTrack-CMV prepared. Intracerebral hemorrhage (ICH) was induced by injection of collagenase and heparin into the caudate putamen. At the third day after a collagenase-induced ICH, adult male SD rats were randomly divided into saline group, MSCs group and MSCs/GDNF group. Immunofluorescence and RT-PCR were performed to detect the differentiation of MSCs or MSCs with an adenovirus vector encoding GDNF gene in vivo and in vitro. RESULT: After 6 hours of induction, both MSCs and MSCs/GDNF expressed neuro or glial specific markers and synaptic-associated proteins (SYN, GAP-43, PSD-95); additionally, they secreted bioactive compounds (BDNF, NGF-ß). MSCs/GDNF transplantation, compared to MSCs and saline solution injection, significantly improved neurological functions after ICH. The grafted MSCs or MSCs/GDNF survived in the striatum after 2 weeks of transplantation and expressed the neural cell-specific biomarkers NSE, MAP2, and GFAP. CONCLUSION: These findings demonstrate that MSCs/GDNF transplantation contributes to improved neurological function in experimental ICH rats. The mechanisms are possibly due to neuronal replacement and enhanced neurotrophic factor secretion.
Assuntos
Encéfalo/fisiopatologia , Hemorragia Cerebral/cirurgia , Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Regeneração Nervosa , Células-Tronco Neurais/transplante , Transfecção , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Hemorragia Cerebral/genética , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/fisiopatologia , Modelos Animais de Doenças , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Masculino , Células-Tronco Neurais/metabolismo , Neurogênese , Fenótipo , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Fatores de TempoRESUMO
OBJECTIVE: Although glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-ß superfamily, its function in liver fibrosis has rarely been studied. Here, we investigated the role of GDNF in hepatic stellate cell (HSC) activation and liver fibrosis in humans and mice. DESIGN: GDNF expression was examined in liver biopsies and sera from patients with liver fibrosis. The functional role of GDNF in liver fibrosis was examined in mice with adenoviral delivery of the GDNF gene, GDNF sgRNA CRISPR/Cas9 and the administration of GDNF-blocking antibodies. GDNF was examined on HSC activation using human and mouse primary HSCs. The binding of activin receptor-like kinase 5 (ALK5) to GDNF was determined using surface plasmon resonance (SPR), molecular docking, mutagenesis and co-immunoprecipitation. RESULTS: GDNF mRNA and protein levels are significantly upregulated in patients with stage F4 fibrosis. Serum GDNF content correlates positively with α-smooth muscle actin (α-SMA) and Col1A1 mRNA in human fibrotic livers. Mice with overexpressed GDNF display aggravated liver fibrosis, while mice with silenced GDNF expression or signalling inhibition by GDNF-blocking antibodies have reduced fibrosis and HSC activation. GDNF is confined mainly to HSCs and contributes to HSC activation through ALK5 at His39 and Asp76 and through downstream signalling via Smad2/3, but not through GDNF family receptor alpha-1 (GFRα1). GDNF, ALK5 and α-SMA colocalise in human and mouse HSCs, as demonstrated by confocal microscopy. CONCLUSIONS: GDNF promotes HSC activation and liver fibrosis through ALK5/Smad signalling. Inhibition of GDNF could be a novel therapeutic strategy to combat liver fibrosis.
Assuntos
Regulação da Expressão Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Proteínas Smad/genética , Adulto , Animais , Biópsia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Seguimentos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Células Estreladas do Fígado/patologia , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , RNA/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/biossíntese , Estudos Retrospectivos , Transdução de Sinais , Proteínas Smad/biossíntese , Regulação para CimaRESUMO
Glial cell line-derived neurotrophic factor (GDNF) has potent neurotrophic effects and is known to promote the dopaminergic (DA) neuronal survival in cellular and animal models of Parkinson's disease (PD). However, long-term ectopic GDNF delivery is associated with long lasting adverse side effects in PD patients. Therefore, finding safer and effective ways to elevate endogenous GDNF levels is an active area of research. This study underlines the importance of sodium benzoate (NaB), a metabolite of commonly-used spice cinnamon, a food-additive and an FDA-approved drug against hyperammonemia, in stimulating GDNF in primary mouse and human astrocytes. Presence of cAMP response element (CRE) in the Gdnf gene promoter, recruitment of CREB to the Gdnf promoter by NaB and abrogation of NaB-mediated GDNF expression by siRNA knockdown of CREB suggest that NaB induces the transcription of Gdnf via CREB. Finally, oral administration of NaB and cinnamon itself increased the level of GDNF in vivo in the substantia nigra pars compacta (SNpc) of normal as well as MPTP-intoxicated mice. Accordingly, cinnamon and NaB treatment protected tyrosine hydroxylase positive neurons in the SNpc and fibers in the striatum, normalized striatal neurotransmitters, and improved locomotor activities in MPTP-intoxicated Gfapcre mice, but not GdnfΔastro mice lacking GDNF in astrocytes. These findings highlight the importance of astroglial GDNF in cinnamon- and NaB-mediated protection of the nigrostriatum in MPTP mouse model of PD and suggest possible therapeutic potential of cinnamon and NaB in PD patients. Graphical abstract Cinnamon metabolite sodium benzoate (NaB) activates cAMP-response element-binding (CREB) via protein kinase A (PKA) in astrocytes. Activated CREB then binds to cAMP-response element (CRE) present in GDNF gene promoter to stimulate the transcription of GDNF in astrocytes. This astrocytic GDNF leads to nigral trophism and protects dopaminergic neurons from MPTP insult.
Assuntos
Antiparkinsonianos/uso terapêutico , Astrócitos/metabolismo , Cinnamomum zeylanicum/metabolismo , Corpo Estriado/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Transtornos Parkinsonianos/tratamento farmacológico , Benzoato de Sódio/farmacologia , Substância Negra/efeitos dos fármacos , Animais , Antiparkinsonianos/farmacologia , Biotransformação , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Comportamento Exploratório , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Intoxicação por MPTP/tratamento farmacológico , Intoxicação por MPTP/patologia , Camundongos , Camundongos Endogâmicos C57BL , Transtornos Parkinsonianos/patologia , Parte Compacta da Substância Negra/efeitos dos fármacos , Parte Compacta da Substância Negra/metabolismo , Parte Compacta da Substância Negra/patologia , Casca de Planta , Regiões Promotoras Genéticas/genética , Teste de Desempenho do Rota-Rod , Substância Negra/metabolismo , Substância Negra/patologia , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The expression of glial cell line-derived neurothrophic factor (GDNF) transcript forms pre-(α)pro-gdnf, pre-(ß)pro-gdnf, and their common region m-gdnf in the pons as well as the inferior (IC) and superior colliculi in Krushinsky-Molodkina (KM) rats and in the strain "0" was analyzed by quantitative real-time polymerase chain reaction (PCR) in the control (unstimulated KM and "0" rats) and 1.5, 4.5, and 8â¯h after auditory stimulation. Such stimulation induced audiogenic seizures (AS) in KM rats. Audiogenic seizure was not observed in "0" rats, which was obtained by selection for the absence of AS in a population of F2 hybrids between KM and Wistar rats not predisposed to AS. A significant drop in the level of all transcripts was observed 1.5â¯h after auditory stimulation in both KM and "0" rats. In most cases, the average expression of α and ß isoforms and m-region 4.5â¯h after stimulation was greater than those after 1.5 and 8â¯h. At the same time, the expression of pre-(ß)pro-gdnf in the IC of KM rats 4.5â¯h after the stimulation was significantly lower than after 1.5 or 8â¯h. This work presents the first demonstration of different time courses of expression of the α and ß GDNF isoforms during physiological processes in genotype-specific pathology.
Assuntos
Estimulação Acústica/métodos , Encéfalo/metabolismo , Epilepsia Reflexa/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , RNA Mensageiro/biossíntese , Convulsões/metabolismo , Animais , Encéfalo/patologia , Epilepsia Reflexa/genética , Epilepsia Reflexa/patologia , Expressão Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Masculino , Neuroglia/metabolismo , Neuroglia/patologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Ratos , Ratos Wistar , Convulsões/genética , Convulsões/patologia , Fatores de TempoRESUMO
BACKGROUND/OBJECTIVE: Alzheimer's disease (AD) is a progressive incurable neurodegenerative disorder. Glial cell line-derived neurotrophic factor (GDNF) is a prominent regulator of brain tissue and has an impressive potential for use in AD therapy. While its metabolism is still not fully understood, delivering neuropeptides such as GDNF via umbilical cord blood mononuclear cells (UCBMCs) to the sites of neurodegeneration is a promising approach in the development of innovative therapeutic avenues. METHODS: UCBMCs were transduced with adenoviral vectors expressing GDNF and injected into AD transgenic mice. Various parameters including homing and survival of transplanted cells, expression of GDNF and synaptic proteins, as well as spatial memory were evaluated. RESULTS: UCBMCs were observed in the hippocampus and cortex several weeks after transplantation, and their long-term presence was associated with improved spatial memory. Post-synaptic density protein 95 (PSD-95) and synaptophysin levels in the hippocampus were also effectively restored following the procedure in AD mice. CONCLUSIONS: Our data indicate that gene-cell therapy with GDNF-overexpressing UCBMCs may produce long-lasting neuroprotection and stimulation of synaptogenesis. Such adenoviral constructs could potentially possess a high therapeutic potential for the treatment of AD.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/terapia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Modelos Animais de Doenças , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Hipocampo/metabolismo , Memória Espacial/fisiologia , Doença de Alzheimer/genética , Animais , Proteína 4 Homóloga a Disks-Large/biossíntese , Proteína 4 Homóloga a Disks-Large/genética , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Gravidez , Sinaptofisina/biossíntese , Sinaptofisina/genéticaRESUMO
Background and objectives: Paocai (pickled cabbage), which is fermented by lactic acid bacteria, is a traditional Chinese food. The microorganisms of Paocai were isolated and identified, and the constipation inhibition effect of one of the isolated Lactobacillus was investigated. Materials and Methods: The 16S rDNA technology was used for microbial identification. A mouse constipation model was established using activated carbon. After intragastric administration of Lactobacillus (108 CFU/mL), the mice were dissected to prepare pathological sections of the small intestine. Serum indicators were detected using kits, and the expression of small intestine-related mRNAs was detected by qPCR assay. Results: One strain of Lactobacillus was identified and named Lactobacillus fermentum CQPC03 (LF-CQPC03). Body weight and activated carbon propulsion rate were all higher in mice intragastrically administered with LF-CQPC03 compared with the control group, while the time to the first black stool in treated mice was lower than that in the control group. Serum assays showed that gastrin (Gas), endothelin (ET), and acetylcholinesterase (AchE) levels were significantly higher in the LF-CQPC03-treated mice than in the control group, while somatostatin (SS) levels were significantly lower than in the control mice. Mouse small intestine tissue showed that c-Kit, stem cell factor (SCF), and glial cell-derived neurotrophic factor (GDNF) mRNA expression levels were significantly higher in the LF-CQPC03 treated mice than in control mice, while transient receptor potential cation channel subfamily V member 1 (TRPV1) and inducible nitric oxide synthase (iNOS) expression levels were significantly lower in the LF-CQPC03 treated mice than in control mice. Conclusions: There is a better effect with high-dose LF-CQPC03, compared to the lower dose (LF-CQPC03-L), showing good probiotic potential, as well as development and application value.
Assuntos
Brassica/microbiologia , Constipação Intestinal/prevenção & controle , Alimentos Fermentados/microbiologia , Limosilactobacillus fermentum/isolamento & purificação , Limosilactobacillus fermentum/metabolismo , Probióticos/administração & dosagem , Acetilcolinesterase/sangue , Animais , Peso Corporal , Carbono/farmacologia , Constipação Intestinal/sangue , Constipação Intestinal/induzido quimicamente , Defecação , Modelos Animais de Doenças , Endotelinas/sangue , Fezes , Feminino , Fermentação , Gastrinas/sangue , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico Sintase Tipo II/biossíntese , Probióticos/isolamento & purificação , Probióticos/metabolismo , Somatostatina/sangue , Fator de Células-Tronco/biossíntese , Canais de Cátion TRPV/biossínteseRESUMO
Despite the success of tissue engineered nerve guidance conduits (NGCs) for the treatment of small peripheral nerve injuries, autografts remain the clinical gold standard for larger injuries. The delivery of neurotrophic factors from conduits might enhance repair for more effective treatment of larger injuries but the efficacy of such systems is dependent on a safe, effective platform for controlled and localised therapeutic delivery. Gene therapy might offer an innovative approach to control the timing, release and level of neurotrophic factor production by directing cells to transiently sustain therapeutic protein production in situ. In this study, a gene-activated NGC was developed by incorporating non-viral polyethyleneimine-plasmid DNA (PEI-pDNA) nanoparticles (N/P 7 ratio, 2⯵g dose) with the pDNA encoding for nerve growth factor (NGF), glial derived neurotrophic factor (GDNF) or the transcription factor c-Jun. The physicochemical properties of PEI-pDNA nanoparticles, morphology, size and charge, were shown to be suitable for gene delivery and demonstrated high Schwann cell transfection efficiency (60⯱â¯13%) in vitro. While all three genes showed therapeutic potential in terms of enhancing neurotrophic cytokine production while promoting neurite outgrowth, delivery of the gene encoding for c-Jun showed the greatest capacity to enhance regenerative cellular processes in vitro. Ultimately, this gene-activated NGC construct was shown to be capable of transfecting both Schwann cells (S42 cells) and neuronal cells (PC12 and dorsal root ganglia) in vitro, demonstrating potential for future therapeutic applications in vivo. STATEMENT OF SIGNIFICANCE: The basic requirements of biomaterial-based nerve guidance conduits have now been well established and include being able to bridge a nerve injury to support macroscopic guidance between nerve stumps, while being strong enough to withstand longitudinal tension and circumferential compression, in addition to being mechanically sound to facilitate surgical handling and implantation. While meeting these criteria, conduits are still limited to the treatment of small defects clinically and might benefit from additional biochemical stimuli to enhance repair for the effective treatment of larger injuries. In this study, a gene activated conduit was successfully developed by incorporating non-viral nanoparticles capable of efficient Schwann cell and neuronal cell transfection with therapeutic genes in vitro, which showed potential to enhance repair in future applications particularly when taking advantage of the transcription factor c-Jun. This innovative approach may provide an alternative to conduits used as platforms for the delivery neurotrophic factors or genetically modified cells (viral gene therapy), and a potential solution for the unmet clinical need to repair large peripheral nerve injury effectively.
Assuntos
DNA , Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Nanopartículas , Fator de Crescimento Neural , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-jun , Células de Schwann/metabolismo , Transfecção/métodos , Animais , DNA/química , DNA/genética , DNA/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Masculino , Nanopartículas/química , Nanopartículas/uso terapêutico , Fator de Crescimento Neural/biossíntese , Fator de Crescimento Neural/genética , Células PC12 , Polietilenoimina/química , Polietilenoimina/farmacologia , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-jun/genética , Ratos , Ratos WistarRESUMO
Traumatic, inherited, and age-related degenerative diseases of the retina, such as retinal detachment, glaucoma, retinitis pigmentosa, and age-related macular degeneration, are characterized by the irreversible loss of retinal neurons. Several growth factors, including glial cell-derived neurotrophic factor and pigment epithelium-derived factor, have been shown to rescue retinal neurons in animal models of retinal disease. Here we describe a scalable and robust system to study the growth factor induction in the retina: retinal organoids derived from the induced pluripotent stem cells. We have demonstrated that they secrete GDNF and PEDF at the levels tenfold above detection limit for ELISA. We also have shown that growth factor production in this system may be upregulated by specific trigger, demonstrating the feasibility of this approach for drug discovery.
Assuntos
Proteínas do Olho/biossíntese , Células-Tronco Pluripotentes Induzidas , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Retina/metabolismo , Animais , Descoberta de Drogas/métodos , Proteínas do Olho/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Camundongos , Morfogênese , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/metabolismo , Organoides/metabolismo , Retina/citologia , Serpinas/biossíntese , Serpinas/metabolismo , Engenharia Tecidual/métodosRESUMO
BACKGROUND: Several studies in recent years have confirmed that the direct application of pulsed radiofrequency (PRF) on peripheral nerve compression points can alleviate hyperalgesia in animal models of neuropathic pain (NP). However, the mechanism underlying the treatment of peripheral nerves by PRF is unclear. OBJECTIVES: We aim to observe changes in pain behavior after the application of PRF on the ligation site of the sciatic nerves (SNs) of rats with chronic constriction injury (CCI) and to investigate the effects of PRF on the transcription and translation levels of glial cell line-derived neurotrophic factor (GDNF) in nerve tissues at the treatment site. STUDY DESIGN: A randomized, experimental trial. SETTING: Experimental Animal Center, Beijing Tiantan Hospital. METHODS: Ninety-six adult male Sprague-Dawley rats were randomly divided into 4 groups: sham-sham (SS) group, sham-PRF (SP) group, CCI-sham (CS) group, and CCI-PRF (CP) group. The right SNs of rats in the CS and CP group were ligated to establish the CCI model. The right SNs in the SS and SP groups were isolated and exposed but without being ligated. On the fourteenth day after CCI/sham operation, PRF treatment was performed on the midpoint of the ligation sites of the SN in the CP group and the corresponding sites in the SP group. The electrode was only placed at the ligation sites of the SN in the CS group and the corresponding sites in the SS group without current being applied. The 50% paw withdrawal threshold (50%PWT) and thermal withdrawal latency (TWL) of rats in all of the groups were measured. The transcription and translation levels of GDNF of the PRF/sham treatment sites were measured before and after treatment by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assays (ELISAs). RESULTS: The 50%PWT value of the hind paws of rats in the CP group gradually increased on day 6 after the PRF treatment and was significantly higher than that in the CS group (6 days after treatment P < 0.05; 14 days after treatment P < 0.01). The TWL value in the CP group was higher than that in the CS group 2 days after treatment (P < 0.05) and was significantly higher (P < 0.01) from day 6 until the end of the experiment. On the day 6 and 14 after PRF treatment, the mRNA and protein expression levels of GDNF at the ligation sites of the SNs of rats in the CP group were higher than both the levels before treatment and those in the CS group (P < 0.01). LIMITATIONS: The efficacy of PRF treatment in the CCI model was only tested within 14 days, and the changes in GDNF levels were only tested at 3 time-points before and after treatment. CONCLUSIONS: The direct application of PRF on SN ligation sites in the CCI model can safely and effectively alleviate NP. One of the mechanisms of this effect could be the upregulation of the transcription and translation of GDNF in compressed SNs. KEY WORDS: Pulsed radiofrequency, chronic constriction injury, sciatic nerve, 50% paw withdrawal threshold, thermal withdrawal latency, glia cell line-derived neurotrophic factor, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay.