RESUMO
Peripheral nerve injuries have common clinical problems that are often accompanied by sensory and motor dysfunction and failure of axonal regeneration. Although various therapeutic approaches have been attempted, full functional recovery and axonal regeneration are rarely achieved in patients. In this study, we investigated the effects of recombinant adeno-associated virus (AAV) of mesencephalic astrocyte-derived neurotrophic factor (AAV-MANF) or placental growth factor (AAV-PlGF) transduced into mesenchymal stem cells (hMSC-MANF and hMSC-PlGF), which were then transplanted using human decellularized nerves (HDN) into sciatic nerve injury model. Our results showed that both AAV-MANF and AAV-PlGF were expressed in MSCs transplanted into the injury site. Behavioral measurements performed 2, 4, 6, 8, and 12 weeks after injury indicated that MANF facilitated the rapid and improved recovery of sensory and motor functions than PlGF. In addition, immunohistochemical analysis was used to quantitatively analyze the myelination of neurofilaments, Schwann cells, and regrowth axons. Both hMSC-MANF and hMSC-PlGF increased axon numbers and immunoreactive areas of axons and Schwann cells compared with the hMSC-GFP group. However, hMSC-MANF significantly improved the thickness of axons and Schwann cells compared with hMSC-PlGF. G-ratio analysis also showed a marked increase in axon myelination in axons thicker than 2.0 µm treated with MANF than that treated with PlGF. Our study suggests that transplantation of hMSC transduced with AAV-MANF has a potential to provide a novel and efficient strategy for promoting functional recovery and axonal regeneration in peripheral nerve injury.
Assuntos
Traumatismos dos Nervos Periféricos , Neuropatia Ciática , Humanos , Feminino , Traumatismos dos Nervos Periféricos/metabolismo , Recuperação de Função Fisiológica/fisiologia , Astrócitos/metabolismo , Regeneração Nervosa/fisiologia , Fator de Crescimento Placentário/metabolismo , Neuropatia Ciática/metabolismo , Axônios/metabolismo , Fatores de Crescimento Neural/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismoRESUMO
NANOG plays a key role in cellular plasticity and the acquisition of the stem cell state during reprogramming, but its role in the regenerative process remains unclear. Here, we show that the induction of NANOG in neuronal cells is necessary for the physiological initiation of neuronal regeneration in response to ischemic stress. Specifically, we found that NANOG was preferentially expressed in undifferentiated neuronal cells, and forced expression of Nanog in neural progenitor cells (NPCs) promoted their self-renewing expansion both in ex-vivo slice cultures and in vitro limiting dilution analysis. Notably, the upstream region of the Nanog gene contains sequence motifs for hypoxia-inducible factor-1 alpha (HIF-1α). Therefore, cerebral neurons exposed to hypoxia significantly upregulated NANOG expression selectively in primitive (CD133+) cells, but not in mature cells, leading to the expansion of NPCs. Notably, up to 80% of the neuronal expansion induced by hypoxia was attributed to NANOG-expressing neuronal cells, whereas knockdown during hypoxia abolished this expansion and was accompanied by the downregulation of other pluripotency-related genes. Moreover, the number of NANOG-expressing neuronal cells were transiently increased in response to ischemic insult, predominantly in the infarct area of brain regions undergoing neurogenesis, but not in non-neurogenic loci. Together, these findings reveal a functional effect of NANOG-induction for the initiation of adaptive neuronal regeneration among heterogeneous NPC subsets, pointing to cellular plasticity as a potential link between regeneration and reprogramming processes.
Assuntos
Proteína Homeobox Nanog , Células-Tronco Neurais , Encéfalo/metabolismo , Hipóxia/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/metabolismo , AnimaisRESUMO
The sources of mesenchymal stromal cells (MSCs) for cell therapy trials are expanding, increasing the need for their characterization. Here, we characterized multi-donor, turbinate-derived MSCs (TB-MSCs) that develop from the neural crest, and compared them to bone marrow-derived MSCs (BM-MSCs). TB-MSCs had higher proliferation potential and higher self-renewal of colony forming cells, but lower potential for multi-lineage differentiation than BM-MSCs. TB-MSCs expressed higher levels of neural crest markers and lower levels of pericyte-specific markers. These neural crest-like properties of TB-MSCs were reflected by their propensity to differentiate into neuronal cells and proliferative response to nerve growth factors. Proteomics (LC-MS/MS) analysis revealed a distinct secretome profile of TB-MSCs compared to BM and adipose tissue-derived MSCs, exhibiting enrichments of factors for cell-extracellular matrix interaction and neurogenic signaling. However, TB-MSCs and BM-MSCs exhibited comparable suppressive effects on the allo-immune response and comparable stimulatory effects on hematopoietic stem cell self-renewal. In contrast, TB-MSCs stimulated growth and metastasis of breast cancer cells more than BM-MSCs. Altogether, our multi-donor characterization of TB-MSCs reveals distinct cell autonomous and paracrine properties, reflecting their unique developmental origin. These findings support using TB-MSCs as an alternative source of MSCs with distinct biological characteristics for optimal applications in cell therapy.
RESUMO
Guanine nucleotide binding protein (G protein) gamma 8 (Gng8) is a subunit of G proteins and expressed in the medial habenula (MHb) and interpeduncular nucleus (IPN). Recent studies have demonstrated that Gng8 is involved in brain development; however, the roles of Gng8 on cognitive function have not yet been addressed. In the present study, we investigated the expression of Gng8 in the brain and found that Gng8 was predominantly expressed in the MHb-IPN circuit of the mouse brain. We generated Gng8 knockout (KO) mice by CRISPR/Cas9 system in order to assess the role of Gng8 on cognitive function. Gng8 KO mice exhibited deficiency in learning and memory in passive avoidance and Morris water maze tests. In addition, Gng8 KO mice significantly reduced long-term potentiation (LTP) in the hippocampus compared to that of wild-type (WT) mice. Furthermore, we observed that levels of acetylcholine (ACh) and choline acetyltransferase (ChAT) in the MHb and IPN of Gng8 KO mice were significantly decreased, compared to WT mice. The administration of nAChR α4ß2 agonist A85380 rescued memory impairment in the Gng8 KO mice, suggesting that Gng8 regulates cognitive function via modulation of cholinergic activity. Taken together, Gng8 is a potential therapeutic target for memory-related diseases and/or neurodevelopmental diseases.
Assuntos
Habenula , Acetilcolina , Animais , Aprendizagem , Aprendizagem em Labirinto , Camundongos , Camundongos Knockout , Agonistas NicotínicosRESUMO
Neuronal loss caused by neurodegenerative diseases, traumatic brain injury and stroke results in cognitive dysfunctioning. Implantation of neural stem/precursor cells (NPCs) can improve the brain function by replacing lost neurons. Proper synaptic integration following neuronal differentiation of implanted cells is believed to be a prerequisite for the functional recovery. In the present study, we characterized the functional properties of immortalized neural progenitor HiB5 cells implanted into the rat hippocampus with chemically induced lesion. The implanted HiB5 cells migrated toward CA1 pyramidal layer and differentiated into vGluT1-positive glutamatergic neurons with morphological and electrophysiological properties of endogenous CA1 pyramidal cells. Functional synaptic integration of HiB5 cell-derived neurons was also evidenced by immunohistochemical and electrophysiological data. Lesion-caused memory deficit was significantly recovered after the implantation when assessed by inhibitory avoidance (IA) learning. Remarkably, IA learning preferentially produced long-term potentiation (LTP) at the synapses onto HiB5 cell-derived neurons, which occluded paring protocol-induced LTP ex vivo. We conclude that the implanted HiB5 cell-derived neurons actively participate in learning process through LTP formation, thereby counteracting lesion-mediated memory impairment.
Assuntos
Diferenciação Celular , Transtornos Cognitivos/terapia , Células-Tronco Neurais/transplante , Neurônios/metabolismo , Animais , Região CA1 Hipocampal/crescimento & desenvolvimento , Região CA1 Hipocampal/fisiopatologia , Transtornos Cognitivos/fisiopatologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/fisiopatologia , Humanos , Aprendizagem/fisiologia , Células-Tronco Neurais/fisiologia , Neurônios/patologia , Ratos , Sinapses/fisiologiaRESUMO
At high concentrations, manganese (Mn) promotes cellular neurodevelopment but causes toxicity. Here, we report that Mn ion at high concentrations can be delivered to pheochromocytoma 12 (PC12) cells using gold nanoparticles (AuNPs) to enhance cellular neurodevelopment without toxicity. Mn(2+) release from AuNPs was designed to be pH-responsive so that low pH condition of the cell endosomes can trigger in situ release of Mn(2+) from AuNPs after cellular uptake of Mn-incorporated AuNPs (MnAuNPs). Due to the differences in reduction potentials of Mn and Au, only Mn ionized and released while Au remained intact when MnAuNPs were uptaken by cells. Compared to PC12 cells treated with a high concentration of free Mn(2+), PC12 cells treated with an equal concentration of MnAuNPs resulted in significantly enhanced cellular neurodevelopment with decreased apoptosis and necrosis. Treatment with a high concentration of free Mn(2+) led to an abrupt consumption of a large amount of ATP for the intracellular transport of Mn(2+) through the ion channel of the cell membrane and to mitochondrial damage caused by the high intracellular concentration of Mn(2+), both of which resulted in cell necrosis and apoptosis. In contrast, MnAuNP-treated cells consumed much smaller amount of ATP for the intracellular transport of MnAuNPs by endocytosis and showed pH-triggered in situ release of Mn(2+) from the MnAuNPs in the endosomes of the cells, both of which prevented the cell death caused by ATP depletion and mitochondrial damage. To our knowledge, this is the first report on the use of AuNPs as a vehicle for pH-responsive, intracellular delivery of metal ion, which may open a new window for drug delivery and clinical therapy.
Assuntos
Diferenciação Celular , Sistemas de Liberação de Medicamentos , Ouro/química , Manganês/química , Nanopartículas Metálicas/química , Neurônios/citologia , Trifosfato de Adenosina/química , Animais , Apoptose , Membrana Celular/metabolismo , Endocitose , Concentração de Íons de Hidrogênio , Íons , Ácido Láctico/química , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Necrose , Neurônios/efeitos dos fármacos , Células PC12/citologia , Células PC12/efeitos dos fármacos , RatosRESUMO
Soy (Glycine Max Merr, family Leguminosae) has been reported to possess anti-cancer, anti-lipidemic, estrogen-like, and memory-enhancing effects. We investigated the memory-enhancing effects and the underlying mechanisms of soyasaponin I (soya-I), a major constituent of soy. Impaired learning and memory were induced by injecting ibotenic acid into the entorhinal cortex of adult rat brains. The effects of soya-I were evaluated by measuring behavioral tasks and neuronal regeneration of memory-deficient rats. Oral administration of soya-I exhibited significant memory-enhancing effects in the passive avoidance, Y-maze, and Morris water maze tests. Soya-Ι also increased BrdU incorporation into the dentate gyrus and the number of cell types (GAD67, ChAT, and VGluT1) in the hippocampal region of memory-deficient rats, whereas the number of reactive microglia (OX42) decreased. The mechanism underlying memory improvement was assessed by detecting the differentiation and proliferation of neural precursor cells (NPCs) prepared from the embryonic hippocampus (E16) of timed-pregnant Sprague-Dawley rats using immunocytochemical staining and immunoblotting analysis. Addition of soya-Ι in the cultured NPCs significantly elevated the markers for cell proliferation (Ki-67) and neuronal differentiation (NeuN, TUJ1, and MAP2). Finally, soya-I increased neurite lengthening and the number of neurites during the differentiation of NPCs. Soya-Ι may improve hippocampal learning and memory impairment by promoting proliferation and differentiation of NPCs in the hippocampus through facilitation of neuronal regeneration and minimization of neuro-inflammation.
Assuntos
Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/fisiopatologia , Memória/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Saponinas/uso terapêutico , Animais , Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Hipocampo/patologia , Ácido Ibotênico , Masculino , Microglia/efeitos dos fármacos , Microglia/patologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Ácido Oleanólico/administração & dosagem , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Ácido Oleanólico/uso terapêutico , Gravidez , Ratos , Ratos Sprague-Dawley , Saponinas/administração & dosagem , Saponinas/químicaRESUMO
WIP1 (wild-type p53-induced phosphatase 1) functions as a homeostatic regulator of the ataxia telangiectasia mutated (ATM)-mediated signaling pathway in response to ionizing radiation (IR). Here we identify homeodomain-interacting protein kinase 2 (HIPK2) as a protein kinase that targets WIP1 for phosphorylation and proteasomal degradation. In unstressed cells, WIP1 is constitutively phosphorylated by HIPK2 and maintained at a low level by proteasomal degradation. In response to IR, ATM-dependent AMPKα2-mediated HIPK2 phosphorylation promotes inhibition of WIP1 phosphorylation through dissociation of WIP1 from HIPK2, followed by stabilization of WIP1 for termination of the ATM-mediated double-strand break (DSB) signaling cascade. Notably, HIPK2 depletion impairs IR-induced γ-H2AX foci formation, cell-cycle checkpoint activation, and DNA repair signaling, and the survival rate of hipk2+/- mice upon γ-irradiation is markedly reduced compared to wild-type mice. Taken together, HIPK2 plays a critical role in the initiation of DSB repair signaling by controlling WIP1 levels in response to IR.
Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA/efeitos da radiação , Reparo do DNA , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Dano ao DNA/genética , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Fosforilação , Proteína Fosfatase 2C , Radiação Ionizante , Transdução de Sinais , UbiquitinaçãoRESUMO
Mesenchymal stem cells (MSCs) are effective vectors in delivering a gene of interest into degenerating brain. In ex vivo gene therapy, viability of transplanted MSCs is correlated with the extent of functional recovery. It has been reported that BDNF facilitates survival of MSCs but dividing MSCs do not express the BDNF receptor, TrkB. In this study, we found that the expression of TrkB is upregulated in human MSCs by the addition of forskolin (Fsk), an activator of adenylyl cyclase. To increase survival rate of MSCs and their secretion of tropic factors that enhance regeneration of endogenous cells, we pre-exposed hMSCs with Fsk and transduced with BDNF-adenovirus before transplantation into the brain of memory deficient rats, a degenerating brain disease model induced by ibotenic acid injection. Viability of MSCs and expression of a GABA synthesizing enzyme were increased. The pre-treatment improved learning and memory, as detected by the behavioral tests including Y-maze task and passive avoidance test. These results suggest that TrkB expression of hMSCs elevates the neuronal regeneration and efficiency of BDNF delivery for treating degenerative neurological diseases accompanying memory loss.
Assuntos
Colforsina/farmacologia , Terapia Genética/métodos , Transtornos da Memória/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Receptor trkB/biossíntese , Regeneração/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Transdução Genética , Regulação para Cima , Ácido gama-Aminobutírico/metabolismoRESUMO
Berberine, an isoquinoline alkaloid component of Coptidis Rhizoma (goldenthread) extract, has been reported to have therapeutic potential for central nervous system disorders such as Alzheimer's disease, cerebral ischemia, and schizophrenia. We have previously shown that berberine promotes the survival and differentiation of hippocampal precursor cells. In a memory-impaired rat model induced by ibotenic acid injection, the survival of pyramidal and granular cells was greatly increased in the hippocampus by berberine administration. In the present study, we investigated the effects of berberine on neurite outgrowth in the SH-SY5Y neuronal cell line and axonal regeneration in the rat peripheral nervous system (PNS). Berberine enhanced neurite extension in differentiating SH-SY5Y cells at concentrations of 0.25-3 µg/mL. In an injury model of the rat sciatic nerve, we examined the neuroregenerative effects of berberine on axonal remyelination by using immunohistochemical analysis. Four weeks after berberine administration (20 mg/kg i.p. once per day for 1 week), the thickness of remyelinated axons improved approximately 1.4-fold in the distal stump of the injury site. Taken together, these results indicate that berberine promotes neurite extension and axonal regeneration in injured nerves of the PNS.
Assuntos
Berberina/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Animais , Axônios/metabolismo , Axônios/patologia , Linhagem Celular , Humanos , Masculino , Neurônios/citologia , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/patologiaRESUMO
Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways, respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.
Assuntos
Actinas/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Papillomavirus Humano 18/genética , Proteínas Oncogênicas Virais/genética , Ativação Transcricional , Neoplasias do Colo do Útero/virologia , Actinas/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Feminino , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas Oncogênicas Virais/antagonistas & inibidores , Regiões Promotoras Genéticas , Interferência de RNA , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismoRESUMO
BACKGROUND AND AIMS: Various techniques have been investigated to enhance peripheral nerve regeneration including the application of low-intensity electrical stimulation (ES) and the administration of growth factors, especially brain-derived neurotrophic factor (BDNF). The purpose of this study was to investigate the effects of combining short-term (ES) and recombinant adenoviral vector-mediated BDNF (BDNF-Ad) transfer, in comparison to each sole modality, on peripheral nerve regeneration in a rat model with crush-injured sciatic nerve. METHODS: Sixty male Sprague-Dawley rats (250-300 g) were equally distributed into four groups; the control group, the ES group, the BDNF-Ad group, and the combination group (n = 15 each). A standard crush injury was introduced to the sciatic nerve. The control group received no treatment after injury, the ES group received 30 minutes of low-intensity ES, the BDNF-Ad group received an injection of recombinant BDNF-Ad (concentration = 10(11) pfu/µl, 3 µl/rat) after injury, and the combination group received both ES and BDNF-Ad. The rats were followed-up for 3 weeks. RESULTS: At the end of the follow-up period, the sciatic function index (ES =-39, BDNF-Ad =-38) and number of the retrogradely labeled sensory neurons were significantly increased in the ES group and the BDNF-Ad group (ES = 326, BDNF-Ad = 264), but not in the combined treatment group, compared to the control group (SFI = -53, retrogradely labeled neurons = 229). Axonal counts were highest in the ES group (7,208 axons), axonal densities in the BDNF group (10,598 axons/mm(2)), and the myelin thickness was greater in both groups as compared to the control group. The combined treatment group showed no signs of superior recovery compared to the other groups. CONCLUSIONS: Both the ES and the BDNF-Ad treatments were effective techniques enhancing the sciatic nerve regeneration following a crush injury in rats. Nevertheless, the combined treatment with ES and BDNF-Ad produces neither a synergistic effect nor an improvement in this injury model.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Terapia por Estimulação Elétrica/métodos , Técnicas de Transferência de Genes , Regeneração Nervosa/genética , Neuropatia Ciática/genética , Neuropatia Ciática/terapia , Adenoviridae/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Terapia Combinada/métodos , Modelos Animais de Doenças , Técnicas de Transferência de Genes/tendências , Vetores Genéticos/administração & dosagem , Vetores Genéticos/uso terapêutico , Masculino , Compressão Nervosa/métodos , Regeneração Nervosa/fisiologia , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/fisiopatologiaRESUMO
Wogonin is a flavonoid isolated from Scutellaria baicalensis root, and has multiple pharmacological effects, including anti-inflammatory, anti-oxidant, and anti-cancer effects. It is also neuroprotective in the brain under many stress conditions, but wogonin does not elevate neuronal cell survival. Thus, the mechanisms controlling the neuroprotective effect of wogonin are not clear. Neural precursor cells (NPCs), present in the hippocampus and subventricular zone of adult brains, replace damaged cells. In this study we investigated the biological functions underlying the neuroprotective effect of wogonin on NPCs. We initially examined survival of NPCs but found it was slightly reduced at concentrations higher than 2µg/ml. When we explored differentiation of NPCs into neuronal cells, the number of differentiated cells expressing neurofilaments was increased remarkably (fourfold) in the hippocampal NPCs treated with wogonin. Wogonin maximally elevated the expressions of presynaptic protein, synapsin I and postsynaptic protein (PSD95) at a concentration of 0.7µg/ml. Differentiated cells containing longer neurites were significantly increased in cortical NPCs, primarily cultured from rat E14 embryonic brain. Wogonin also promoted differentiation of NPCs into mature neurons in vivo. When transplanted into the adult rat hippocampus, NPCs differentiated into cells expressing NeuN, the mature neuron marker, by 4weeks after transplantation. These data indicate that wogonin induces differentiation of NPCs both in culture and in vivo, and suggest that facilitation of NPC differentiation is a biological activity by which wogonin protects neurons in damaged brain.
Assuntos
Flavanonas/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Neurogênese , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Masculino , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/transplante , Neuritos/fisiologia , Ratos , Ratos Endogâmicos , Transplante de Células-TroncoRESUMO
Neuregulin 1 (NRG1) and epidermal growth factor receptor (ErbB) signaling pathways control Schwann cells during axonal regeneration in an injured peripheral nervous system. We investigated whether a persistent supply of recombinant NRG1 to the injury site could improve axonal growth and recovery of sensory and motor functions in rats during nerve regeneration. We generated a recombinant adenovirus expressing a secreted form of EGF-like domain from Heregulinß (sHRGßE-Ad). This virus, sHRGßE-Ad allowed for the secretion of 30-50 ng of small sHRGßE peptides per 10(7-8) virus particle outside cells and was able to phosphorylate ErbB receptors. Transduction of the concentrated sHRGßE-Ad into an axotomy model of sciatic nerve damage caused an effective promotion of nerve regeneration, as shown by histological features of the axons and Schwann cells, as well as increased expression of neurofilaments, GAP43 and S100 in the distal stump of the injury site. This result is consistent with longer axon lengths and thicker calibers observed in the sHRGßE-Ad treated animals. Furthermore, sensory and motor functions were significantly improved in sHRGßE-Ad treated animals when evaluated by a behavioral test. These results suggest a therapeutic potential for sHRGßE-Ad in treatment of peripheral nerve injury.
Assuntos
Axônios/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Neuregulina-1/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Adenoviridae/genética , Animais , Axônios/metabolismo , Axônios/fisiologia , Axotomia , Linhagem Celular , DNA Recombinante/genética , Fator de Crescimento Epidérmico/metabolismo , Proteína GAP-43/genética , Humanos , Masculino , Neuregulina-1/genética , Neuregulina-1/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptor ErbB-3/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas S100/genética , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Nervo Isquiático/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Although donepezil, a potent acetylcholinesterase (AChE) inhibitor, has been used to treat Alzheimer's disease (AD) due to its neuroprotective effects, its mode of action to inhibit the growth of cancer cells is poorly understood. In the present study, we investigated the pro-apoptotic activities of donepezil in HL-60 human promyelocytic leukemia cells and the underlying molecular mechanism involved. It was found that donepezil induced the apoptosis of HL-60 and U937 cells in a dose- and time-dependent manner, as evidenced by the formation of DNA fragmentation and the accumulation of positive cells for Annexin V. In addition, the activations of caspase-8, -9, and -3 were significantly increased 36 h after donepezil treatment. Furthermore, the broad caspase inhibitor (z-VAD-fmk) blocked donepezil-induced apoptosis. In addition, donepezil was found to cause the loss of mitochondrial membrane potential (DeltaPsi(m)), to increase the release of cytochrome c to the cytosol, and to alter the expressions of Bcl-2 family proteins. Taken together, these results demonstrate for the first time that donepezil displayed an induction of apoptosis in HL-60 cells via a mitochondria-mediated caspase-dependent pathway.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Inibidores da Colinesterase/farmacologia , Indanos/farmacologia , Leucemia Promielocítica Aguda/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Piperidinas/farmacologia , Anexina A5/metabolismo , Citocromos c/metabolismo , Citosol , Fragmentação do DNA , Donepezila , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células HL-60 , Células HeLa , Humanos , Leucemia Promielocítica Aguda/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células U937RESUMO
The p62 protein has been identified as a major component of the protein aggregations associated with neurodegenerative disease. Oxidative insult has also been identified as a principal cause of neurodegenerative disease. Thus, in the present study, we investigated the potential role of p62 in oxidative stress-induced cell death in SH-SY5Y human neuroblastoma cells. The results indicated that H(2)O(2) treatment induced p62 expression in SH-SY5Y cells. In addition, p62 showed neuroprotective effects against H(2)O(2)-induced cell death in differentiated SH-SY5Y cells. p62 expression prolonged Akt phosphorylation during the later stages of H(2)O(2)-induced cell death. Furthermore, coexpression of p62 and wild-type PDK1, the upstream kinase of Akt, further increased Akt phosphorylation and cell viability, whereas the expression of kinase-defective PDK1 reversed the cytoprotective effects of p62 under oxidative stress. Overexpression of p62 led to the dissociation of PDK1 from the 14-3-3theta protein, which is thought to be a negative regulator of PDK1 kinase activity. These findings suggest a mechanism that involves the p62-mediated modulation of the interaction between signaling molecules and results in cell survival.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peróxido de Hidrogênio/toxicidade , Neurônios/fisiologia , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Morfolinas/farmacologia , Neurônios/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteína Quinase C/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Proteína Sequestossoma-1 , Transdução de Sinais , TransfecçãoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria baicalensis Georgi (Labiatae) extracts have been used as traditional Korean medicine, to treat cerebral ischemia in addition to bacterial infection and inflammatory diseases. AIM OF THE STUDY: The improvement effect on learning and memory by the administration of Scutellaria baicalensis extracts was evaluated and the underlying mechanisms were investigated. MATERIALS AND METHODS: Memory behavior was tested by the passive avoidance test and Y-maze test. We also investigated the cells expressing neuronal markers related to memory processes by immunofluorescence staining analysis in memory deficient animal model (Ibo model) rats and in hippocampal progenitor cells. RESULTS: We found neuronal cells immunoreactive to choline acetyltransferase (ChAT), a marker for cholinergic neurons were increased in the hippocampus, while cells producing GABA and glutamate were not after 30 mg/kg Scutellaria baicalensis administration. Futhermore, Scutellaria baicalensis extracts enhanced the survival of a hippocampal progenitor cell line, HiB5 and its differentiation to ChAT immunoreactive cells. The increased expression of memory related neurotransmitter, NMDA receptor and a reduction of activated microglia in the hippocampus were also observed in the Ibo model when administrated Scutellaria baicalensis extracts. CONCLUSIONS: These results imply that Scutellaria baicalensis has significant neuroprotective effects in the Ibo model.
Assuntos
Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fitoterapia , Extratos Vegetais/farmacologia , Scutellaria baicalensis , Animais , Sobrevivência Celular , Agonistas de Aminoácidos Excitatórios/efeitos adversos , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Ácido Ibotênico/efeitos adversos , Masculino , Aprendizagem em Labirinto , Microglia/efeitos dos fármacos , Modelos Animais , Fármacos Neuroprotetores/uso terapêutico , Extratos Vegetais/uso terapêutico , Raízes de Plantas , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Neurogenesis persists in the adult mammalian brain and can be a target for modulation for therapeutic purposes. This study investigated the effect of a Polygala tenuifolia root extract on the proliferation of a stem cell population in the rat hippocampus. The root extract of P. tenuifolia (2 mg/kg/day, 14 times intraperitoneal injections) increased the incorporation of bromodeoxyuridine (BrdU) into cells in the hippocampal CA1 region. This activity was enriched in the saponin-containing fraction. The majority of cells labelled with BrdU were immunoreactive to nestin or Tuj1 and the percentages of nestin/BrdU- and Tuj1/BrdU-double positive cells were increased by the P. tenuifolia root extract, suggesting that the P. tenuifolia root extract promotes the proliferation of neural stem cells. In addition, this extract promoted the neurite outgrowth of rat neuronal precursor cells, HiB5. These activities of P. tenuifolia root extract may contribute to the therapeutic benefits of herbal medicines containing P. tenuifolia root for the treatment of patients with insomnia, neurosis and dementia.
Assuntos
Proliferação de Células/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Sistema Nervoso/efeitos dos fármacos , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Polygala/química , Células-Tronco/efeitos dos fármacos , Animais , Linhagem Celular , Hipocampo/citologia , Imuno-Histoquímica , Masculino , Sistema Nervoso/citologia , Ratos , Ratos Sprague-DawleyRESUMO
PDGFRB is located on chromosome 5q31-q32, a chromosomal region identified by linkage analyses to contain a susceptibility gene for schizophrenia (SCZ). Recent research has focused on the role of the N-methyl-d-aspartate (NMDA) receptor in the pathogenesis of SCZ. D4 dopamine receptor-mediated transactivation of the gene encoding platelet-derived growth factor receptor beta (PDGFRB) has immediate effects on synaptic neurotransmission via calcium-dependent inactivation of NMDA receptors. In this study, we investigate the association between the PDGFRB gene and SCZ in a Korean population. We screened 6 single-nucleotide polymorphisms (SNPs) in the 5'-upstream region of PDGFRB and conducted a case-control study of 381 SCZ patients and 752 controls. The genotype and haplotype frequencies of 3 of the 6 SNPs [SNP1 (g.-1924T>C, rs3756314), SNP3 (g.-1772A>G, rs3756312) and SNP4 (rs3756311, g.-1658G>A)] were significantly associated with SCZ [SNP1, corrected p=0.012 (co-dominant model), 0.002 (Dominant model), and 0.506 (Recessive model); SNP3 and 4, corrected p=0.003, 0.009, and 0.049]. Haplotype analysis also revealed that ht1 (CGG) and ht2 (TAA) were significantly associated with SCZ (ht1, corrected p=0.018, 0.340, and 0.010; ht2, corrected p=0.002, 0.009, and 0.016). Transient transfection in neuronal cells revealed that ht1 had higher luciferase activity than the vector alone. Furthermore, Pdgfrb expression was increased in the frontal cortex and hippocampus in a mouse model of SCZ induced by MK801. We conclude that SNPs of the 5'-upstream region of PDGFRB are associated with SCZ in a Korean population. These are weak positives that require future studies to confirm these results.
Assuntos
Polimorfismo de Nucleotídeo Único/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Esquizofrenia/genética , Adulto , Animais , Estudos de Casos e Controles , Cromossomos Humanos Par 5/genética , Modelos Animais de Doenças , Feminino , Lobo Frontal/patologia , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Genótipo , Haplótipos , Hipocampo/patologia , Humanos , Coreia (Geográfico) , Masculino , Camundongos , Camundongos Endogâmicos ICR , Pessoa de Meia-IdadeRESUMO
To further clarify schizophrenia (SCZ), disrupted in schizophrenia 1 (DISC1) is a promising candidate gene expressed predominantly within the hippocampus. Several lines of evidence suggest that DISC1 may be involved in susceptibility to SCZ. In this study, we investigated whether genetic polymorphisms in the coding region of DISC1 were associated with several SCZ clinical phenotypes in a Korean population. To examine any association between DISC1 and SCZ, we genotyped three clinical single nucleotide polymorphisms (SNPs) (rs3738401, R264Q; rs3738402, L465L; rs821616, S704C) in the coding region of the DISC1 gene using the Illumina Sentrix Array Matrix chip and direct sequencing in 303 patients with SCZ and 300 healthy controls. Our case-control analysis showed that none of these SNPs was associated with SCZ. In further endophenotype stratification, however, we found a significant association between rs821616 and the poor concentration subgroup of SCZ, determined using the Operational Criteria Checklist (codominant model, p=0.015). Our results suggest that DISC1 may be a susceptibility gene for poor concentration among Korean patients with SCZ.