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1.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669795

RESUMO

AdipoRon, an adiponectin receptor agonist, elicits similar antidiabetic, anti-atherogenic, and anti-inflammatory effects on mouse models as adiponectin does. Since AdipoRon can cross the blood-brain barrier, its chronic effects on regulating hippocampal function are yet to be examined. This study investigated whether AdipoRon treatment promotes hippocampal neurogenesis and spatial recognition memory in a dose-dependent manner. Adolescent male C57BL/6J mice received continuous treatment of either 20 mg/kg (low dose) or 50 mg/kg (high dose) AdipoRon or vehicle intraperitoneally for 14 days, followed by the open field test to examine anxiety and locomotor activity, and the Y maze test to examine hippocampal-dependent spatial recognition memory. Immunopositive cell markers of neural progenitor cells, immature neurons, and newborn cells in the hippocampal dentate gyrus were quantified. Immunosorbent assays were used to measure the serum levels of factors that can regulate hippocampal neurogenesis, including adiponectin, brain-derived neurotrophic factor (BDNF), and corticosterone. Our results showed that 20 mg/kg AdipoRon treatment significantly promoted hippocampal cell proliferation and increased serum levels of adiponectin and BDNF, though there were no effects on spatial recognition memory and locomotor activity. On the contrary, 50 mg/kg AdipoRon treatment impaired spatial recognition memory, suppressed cell proliferation, neuronal differentiation, and cell survival associated with reduced serum levels of BDNF and adiponectin. The results suggest that a low-dose AdipoRon treatment promotes hippocampal cell proliferation, while a high-dose AdipoRon treatment is detrimental to the hippocampus function.


Assuntos
Envelhecimento/fisiologia , Hipocampo/fisiologia , Neurogênese/efeitos dos fármacos , Piperidinas/farmacologia , Adiponectina/sangue , Animais , Glicemia/metabolismo , Fator Neurotrófico Derivado do Encéfalo/sangue , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Corticosterona/sangue , Giro Denteado/efeitos dos fármacos , Giro Denteado/fisiologia , Hipocampo/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Memória Espacial/efeitos dos fármacos
2.
Int J Biol Macromol ; 171: 366-381, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33422514

RESUMO

Despite the poor regenerative capacity of the adult central nervous system (CNS) in mammals, two distinct regions, subventricular zone (SVZ) and the subgranular zone (SGZ), continue to generate new functional neurons throughout life which integrate into the pre-existing neuronal circuitry. This process is not fixed but highly modulated, revealing many intrinsic and extrinsic mechanisms by which this performance can be optimized for a given environment. The capacity for self-renewal, proliferation, migration, and multi-lineage potency of neural stem cells (NSCs) underlines the necessity of controlling stem cell fate. In this context, the native and local microenvironment plays a critical role, and the application of this highly organized architecture in the CNS has been considered as a fundamental concept in the generation of new effective therapeutic strategies in tissue engineering approaches. The brain extracellular matrix (ECM) is composed of biomacromolecules, including glycosaminoglycans, proteoglycans, and glycoproteins that provide various biological actions through biophysical and biochemical signaling pathways. Herein, we review predominantly the structure and function of the mentioned ECM composition and their regulatory impact on multiple and diversity of biological functions, including neural regeneration, survival, migration, differentiation, and final destiny of NSCs.


Assuntos
Proteínas da Matriz Extracelular/farmacologia , Matriz Extracelular/química , Glicosaminoglicanos/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Proteoglicanas/farmacologia , Regeneração/efeitos dos fármacos , Materiais Biomiméticos/isolamento & purificação , Materiais Biomiméticos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proteínas da Matriz Extracelular/isolamento & purificação , Glicosaminoglicanos/isolamento & purificação , Humanos , Células-Tronco Neurais/citologia , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Proteoglicanas/isolamento & purificação
3.
Neurosci Lett ; 746: 135648, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33444672

RESUMO

Vitronectin, an extracellular matrix protein, controls the differentiation of cerebellar granule cell precursors (CGCPs) via αvß5 integrin, particularly in the initial stage of differentiation to granule cells. In this study, we determined whether vitronectin regulates axon specification in this initial differentiation stage of CGCPs. First, we analyzed whether vitronectin deficiency, ß5 integrin knockdown (KD), and ß5 integrin overexpression affect axon specification of primary cultured CGCPs. Vitronectin deficiency and ß5 integrin KD inhibited axon formation, while vitronectin administrated- and ß5 integrin overexpressed-neurons formed multiple axons. Moreover, KD of ß5 integrin suppressed vitronectin-induced multiple axon formation. These findings indicate that vitronectin contributes to regulating axon specification via αvß5 integrin in CGCPs. Next, we determined the signaling pathway involved in regulating vitronectin-induced axon specification. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited vitronectin-induced multiple axon specification, and lithium chloride, an inhibitor of glyocogen synthase kinase 3 beta (GSK3ß), attenuated the inhibitory effect of vitronectin-KO and ß5 integrin KD on the specification of CGCPs. In addition, vitronectin induced the phosphorylation of protein kinase B (Akt) and GSK3ß in neuroblastoma Neuro2a cells. Taken together, our results indicate that vitronectin plays an important factor in axon formation process in CGCPs via a ß5 integrin/PI3K/GSK3ß pathway.


Assuntos
Axônios/metabolismo , Diferenciação Celular/fisiologia , Cerebelo/metabolismo , Células-Tronco Neurais/metabolismo , Receptores de Vitronectina/metabolismo , Vitronectina/metabolismo , Animais , Axônios/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/efeitos dos fármacos , Gravidez , Vitronectina/farmacologia
4.
Int J Mol Sci ; 22(2)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445804

RESUMO

Neural progenitor cells (NPCs) are self-renewing and multipotent cells that persist in the postnatal and adult brain in the subventricular zone and the hippocampus. NPCs can be expanded in vitro to be used in cell therapy. However, expansion is limited, since the survival and proliferation of adult NPCs decrease with serial passages. Many signaling pathways control NPC survival and renewal. Among these, purinergic receptor activation exerts differential effects on the biology of adult NPCs depending on the cellular context. In this study, we sought to analyze the effect of a general blockade of purinergic receptors with suramin on the proliferation and survival of NPCs isolated from the subventricular zone of postnatal rats, which are cultured as neurospheres. Treatment of neurospheres with suramin induced a significant increase in neurosphere diameter and in NPC number attributed to a decrease in apoptosis. Proliferation and multipotency were not affected. Suramin also induced an increase in the gap junction protein connexin43 and in vascular endothelial growth factor, which might be involved in the anti-apoptotic effect. Our results offer a valuable tool for increasing NPC survival before implantation in the lesioned brain and open the possibility of using this drug as adjunctive therapy to NPC transplantation.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Antagonistas Purinérgicos/farmacologia , Receptores Purinérgicos/metabolismo , Células-Tronco/efeitos dos fármacos , Suramina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ventrículos Laterais/efeitos dos fármacos , Ventrículos Laterais/metabolismo , Masculino , Células-Tronco Neurais/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Nat Commun ; 12(1): 662, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510165

RESUMO

Dynamic assembly and disassembly of primary cilia controls embryonic development and tissue homeostasis. Dysregulation of ciliogenesis causes human developmental diseases termed ciliopathies. Cell-intrinsic regulatory mechanisms of cilia disassembly have been well-studied. The extracellular cues controlling cilia disassembly remain elusive, however. Here, we show that lysophosphatidic acid (LPA), a multifunctional bioactive phospholipid, acts as a physiological extracellular factor to initiate cilia disassembly and promote neurogenesis. Through systematic analysis of serum components, we identify a small molecular-LPA as the major driver of cilia disassembly. Genetic inactivation and pharmacological inhibition of LPA receptor 1 (LPAR1) abrogate cilia disassembly triggered by serum. The LPA-LPAR-G-protein pathway promotes the transcription and phosphorylation of cilia disassembly factors-Aurora A, through activating the transcription coactivators YAP/TAZ and calcium/CaM pathway, respectively. Deletion of Lpar1 in mice causes abnormally elongated cilia and decreased proliferation in neural progenitor cells, thereby resulting in defective neurogenesis. Collectively, our findings establish LPA as a physiological initiator of cilia disassembly and suggest targeting the metabolism of LPA and the LPA pathway as potential therapies for diseases with dysfunctional ciliogenesis.


Assuntos
Cílios/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Neurogênese/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Transdução de Sinais , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cílios/genética , Cílios/metabolismo , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Ligação Proteica , Interferência de RNA , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
6.
Nat Commun ; 12(1): 640, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510167

RESUMO

Neural stem/progenitor cells (NSPCs) persist over the lifespan while encountering constant challenges from age or injury related brain environmental changes like elevated oxidative stress. But how oxidative stress regulates NSPC and its neurogenic differentiation is less clear. Here we report that acutely elevated cellular oxidative stress in NSPCs modulates neurogenic differentiation through induction of Forkhead box protein O3 (FOXO3)-mediated cGAS/STING and type I interferon (IFN-I) responses. We show that oxidative stress activates FOXO3 and its transcriptional target glycine-N-methyltransferase (GNMT) whose upregulation triggers depletion of s-adenosylmethionine (SAM), a key co-substrate involved in methyl group transfer reactions. Mechanistically, we demonstrate that reduced intracellular SAM availability disrupts carboxymethylation and maturation of nuclear lamin, which induce cytosolic release of chromatin fragments and subsequent activation of the cGAS/STING-IFN-I cascade to suppress neurogenic differentiation. Together, our findings suggest the FOXO3-GNMT/SAM-lamin-cGAS/STING-IFN-I signaling cascade as a critical stress response program that regulates long-term regenerative potential.


Assuntos
Proteína Forkhead Box O3/metabolismo , Interferon Tipo I/metabolismo , Laminas/metabolismo , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Acetilcisteína/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Depuradores de Radicais Livres/farmacologia , Glicina N-Metiltransferase/metabolismo , Células HEK293 , Herbicidas/farmacologia , Humanos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Paraquat/farmacologia , S-Adenosilmetionina/metabolismo , Transdução de Sinais
7.
Methods Mol Biol ; 2258: 151-169, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340360

RESUMO

Self-assembling brain spheroids derived from human stem cells closely emulate the tangled connectivity of the human brain, recapitulate aspects of organized tissue structure, and are relatively easy to manipulate compared to other existing three-dimensional (3D) cellular models. However, current platforms generate heterogeneously sized and short-lived spheroids, which do not robustly and reproducibly model human brain development and diseases. Here, we present a method to generate large-scale arrays of homogeneously sized 3D brain spheroids derived from human-induced pluripotent stem cells (hiPSCs) or immortalized neural progenitor cells to recapitulate Alzheimer's disease (AD) pathology in vitro. When embedded in extracellular matrix, these brain spheroids develop extensive outward projection of neurites and form networks, which are mediated by thick bundles of dendrites. This array facilitates cost-effective, high-throughput drug screening and mechanistic studies to better understand human brain development and neurodegenerative conditions, such as AD .


Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese , Neurônios/fisiologia , Engenharia Tecidual , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Técnicas de Cultura de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Microscopia Eletrônica de Varredura , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/ultraestrutura , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Organoides
8.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348868

RESUMO

Fish are a convenient model for the study of reparative and post-traumatic processes of central nervous system (CNS) recovery, because the formation of new cells in their CNS continues throughout life. After a traumatic injury to the cerebellum of juvenile masu salmon, Oncorhynchus masou, the cell composition of the neurogenic zones containing neural stem cells (NSCs)/neural progenitor cells (NPCs) in the acute period (two days post-injury) changes. The presence of neuroepithelial (NE) and radial glial (RG) neuronal precursors located in the dorsal, lateral, and basal zones of the cerebellar body was shown by the immunohistochemical (IHC) labeling of glutamine synthetase (GS). Progenitors of both types are sources of neurons in the cerebellum of juvenile O. masou during constitutive growth, thus, playing an important role in CNS homeostasis and neuronal plasticity during ontogenesis. Precursors with the RG phenotype were found in the same regions of the molecular layer as part of heterogeneous constitutive neurogenic niches. The presence of neuroepithelial and radial glia GS+ cells indicates a certain proportion of embryonic and adult progenitors and, obviously, different contributions of these cells to constitutive and reparative neurogenesis in the acute post-traumatic period. Expression of nestin and vimentin was revealed in neuroepithelial cerebellar progenitors of juvenile O. masou. Patterns of granular expression of these markers were found in neurogenic niches and adjacent areas, which probably indicates the neurotrophic and proneurogenic effects of vimentin and nestin in constitutive and post-traumatic neurogenesis and a high level of constructive metabolism. No expression of vimentin and nestin was detected in the cerebellar RG of juvenile O. masou. Thus, the molecular markers of NSCs/NPCs in the cerebellum of juvenile O. masou are as follows: vimentin, nestin, and glutamine synthetase label NE cells in intact animals and in the post-traumatic period, while GS expression is present in the RG of intact animals and decreases in the acute post-traumatic period. A study of distribution of cystathionine ß-synthase (CBS) in the cerebellum of intact young O. masou showed the expression of the marker mainly in type 1 cells, corresponding to NSCs/NCPs for other molecular markers. In the post-traumatic period, the number of CBS+ cells sharply increased, which indicates the involvement of H2S in the post-traumatic response. Induction of CBS in type 3 cells indicates the involvement of H2S in the metabolism of extracellular glutamate in the cerebellum, a decrease in the production of reactive oxygen species, and also arrest of the oxidative stress development, a weakening of the toxic effects of glutamate, and a reduction in excitotoxicity. The obtained results allow us to consider H2S as a biologically active substance, the numerous known effects of which can be supplemented by participation in the processes of constitutive neurogenesis and neuronal regeneration.


Assuntos
Poluentes Atmosféricos/farmacologia , Cerebelo/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Células-Tronco Neurais/citologia , Neurogênese , Oncorhynchus/crescimento & desenvolvimento , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Oncorhynchus/metabolismo
9.
Cell Death Dis ; 11(12): 1059, 2020 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-33311479

RESUMO

Recent studies on developing three-dimensional (3D) brain organoids from stem cells have allowed the generation of in vitro models of neural disease and have enabled the screening of drugs because these organoids mimic the complexity of neural tissue. Niemann-Pick disease, type C (NPC) is a neurodegenerative lysosomal storage disorder caused by mutations in the NPC1 or NPC2. The pathological features underlying NPC are characterized by the abnormal accumulation of cholesterol in acidic compartments, including late endosomes and lysosomes. Due to the inaccessibility of brain tissues from human NPC patients, we developed NPC brain organoids with induced neural stem cells from NPC patient-derived fibroblasts. NPC organoids exhibit significantly reduced size and proliferative ability, which are accompanied by accumulation of cholesterol, impairment in neuronal differentiation, and autophagic flux and dysfunction of lysosomes; therefore, NPC organoids can recapitulate the main phenotypes of NPC patients. Furthermore, these pathological phenotypes observed in NPC organoids were reversed by treatment with valproic acid and HPBCD, which are known to be an effective treatment for several neurodegenerative diseases. Our data present patient-specific phenotypes in 3D organoid-based models of NPC and highlight the application of this model to drug screening in vitro.


Assuntos
Encéfalo/patologia , Modelos Biológicos , Células-Tronco Neurais/patologia , Doença de Niemann-Pick Tipo C/patologia , Organoides/patologia , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Niemann-Pick Tipo C/genética , Organoides/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácido Valproico/farmacologia
10.
J Toxicol Sci ; 45(10): 639-650, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33012732

RESUMO

Bisphenol A (BPA), an endocrine disruptor with estrogenic effects, is widely used as a raw material for manufacturing polycarbonate plastic and epoxy resins. Prenatal and postnatal exposure to BPA affects brain morphogenesis. However, the effects of prenatal and postnatal BPA exposure on postnatal neurogenesis in mice are poorly understood. In this study, we developed a mouse model of prenatal and postnatal BPA exposure and analyzed its effects on hippocampal neurogenesis. The hippocampal dentate gyrus is vulnerable to chemical exposure, as neurogenesis continues in this region even after birth. Our results showed that in mice, prenatal and postnatal BPA exposure decreased the number of type-1, 2a, 2b, and 3 neural progenitor cells, as well as in granule cells, in the hippocampal dentate gyrus on postnatal days 16 and 70. The effect of prenatal and postnatal BPA exposure on neural progenitors were affected at all differentiation stages. In addition, prenatal and postnatal BPA exposure affects the maintenance of long-term memory on postnatal day 70. Our results suggest that neurodevelopmental toxicity due to prenatal and postnatal BPA exposure might affect postnatal morphogenesis and functional development of the hippocampal dentate gyrus.


Assuntos
Animais Recém-Nascidos , Compostos Benzidrílicos/toxicidade , Giro Denteado/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Hipocampo/efeitos dos fármacos , Exposição Materna/efeitos adversos , Troca Materno-Fetal/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Fenóis/toxicidade , Animais , Diferenciação Celular/efeitos dos fármacos , Feminino , Masculino , Camundongos , Modelos Animais , Gravidez
11.
Stroke ; 51(9): 2844-2853, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32772683

RESUMO

BACKGROUND AND PURPOSE: Although VEGF165 (vascular endothelial growth factor-165) is able to enhance both angiogenesis and neurogenesis, it also increases vascular permeability through the blood-brain barrier. Heparan sulfate (HS) sugars play important roles in regulating VEGF bioactivity in the pericellular compartment. Here we asked whether an affinity-purified VEGF165-binding HS (HS7) could augment endogenous VEGF activity during stroke recovery without affecting blood-brain barrier function. METHODS: Both rat brain endothelial cell line 4 and primary rat neural progenitor cells were used to evaluate the potential angiogenic and neurogenic effects of HS7 in vitro. For in vivo experiments, male Sprague-Dawley rats were subjected to 100 minutes of transient focal cerebral ischemia, then treated after 4 days with either PBS or HS7. One week later, infarct volume, behavioral sequelae, immunohistochemical markers of angiogenesis and neural stem cell proliferation were assessed. RESULTS: HS7 significantly enhanced VEGF165-mediated angiogenesis in rat brain endothelial cell line 4 brain endothelial cells, and increased the proliferation and differentiation of primary neural progenitor cells, both via the VEGFR2 (vascular endothelial growth factor receptor 2) pathway. Intracerebroventricular injection of HS7 improved neurological outcome in ischemic rats without changing infarct volumes. Immunostaining of the compromised cerebrum demonstrated increases in collagen IV/Ki67 and nestin/Ki67 after HS7 exposure, consistent with its ability to promote angiogenesis and neurogenesis, without compromising blood-brain barrier integrity. CONCLUSIONS: A VEGF-activating glycosaminoglycan sugar, by itself, is able to enhance endogenous VEGF165 activity during the post-ischemic recovery phase of stroke.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Heparitina Sulfato/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Heparitina Sulfato/administração & dosagem , Infarto da Artéria Cerebral Média/prevenção & controle , Injeções Intraventriculares , Ataque Isquêmico Transitório/tratamento farmacológico , Ataque Isquêmico Transitório/fisiopatologia , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
12.
PLoS One ; 15(8): e0237025, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32797057

RESUMO

Troxerutin (TRX) is a water-soluble flavonoid which occurs commonly in the edible plants. Recent studies state that TRX improves the functionality of the nervous system and neutralizes Amyloid-ß induced neuronal toxicity. In this study, an in vitro assay based upon Neural stem cell (NSCs) isolated from the subventricular zone of the postnatal balb/c mice was established to explore the impact of TRX on individual neurogenesis processes in general and neuroprotective effect against ß-amyloid 1-42 (Aß42) induced inhibition in differentiation in particular. NSCs were identified exploiting immunostaining of the NSCs markers. Neurosphere clonogenic assay and BrdU/Ki67 immunostaining were employed to unravel the impact of TRX on proliferation. Differentiation experiments were carried out for a time span lasting from 48 h to 7 days utilizing ß-tubulin III and GFAP as neuronal and astrocyte marker respectively. Protective effects of TRX on Aß42 induced depression of NSCs differentiation were determined after 48 h of application. A neurosphere migration assay was carried out for 24 h in the presence and absence of TRX. Interestingly, TRX enhanced neuronal differentiation of NSCs in a dose-dependent manner after 48 h and 7 days of incubation and significantly enhanced neurite growth. A higher concentration of TRX also neutralized the inhibitory effects of Aß42 on neurite outgrowth and length after 48 h of incubation. TRX significantly stimulated cell migration. Overall, TRX not only promoted NSCs differentiation and migration but also neutralized the inhibitory effects of Aß42 on NSCs. TRX, therefore, offers an interesting lead structure from the perspective of drug design especially to promote neurogenesis in neurological disorders i.e. Alzheimer's disease.


Assuntos
Hidroxietilrutosídeo/análogos & derivados , Neuritos/efeitos dos fármacos , Crescimento Neuronal/fisiologia , Precursor de Proteína beta-Amiloide/farmacologia , Animais , Astrócitos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Flavonoides/farmacologia , Hidroxietilrutosídeo/metabolismo , Hidroxietilrutosídeo/farmacologia , Ventrículos Laterais/efeitos dos fármacos , Ventrículos Laterais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/metabolismo , Neuroproteção , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia
13.
J Vis Exp ; (162)2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32831302

RESUMO

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect. Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model. The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology."


Assuntos
Bioensaio/métodos , Células-Tronco Neurais/patologia , Crescimento Neuronal , Neurônios/patologia , Neurotoxinas/toxicidade , Animais , Diferenciação Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Epigênese Genética/efeitos dos fármacos , Fluorescência , Congelamento , Humanos , Células-Tronco Neurais/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Software , Coloração e Rotulagem
14.
Proc Natl Acad Sci U S A ; 117(32): 19578-19589, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32727894

RESUMO

The CreER/LoxP system is widely accepted to track neural lineages and study gene functions upon tamoxifen (TAM) administration. We have observed that prenatal TAM treatment caused high rates of delayed delivery and fetal mortality. This substance could produce undesired results, leading to data misinterpretation. Here, we report that administration of TAM during early stages of cortical neurogenesis promoted precocious neural differentiation, while it inhibited neural progenitor cell (NPC) proliferation. The TAM-induced inhibition of NPC proliferation led to deficits in cortical neurogenesis, dendritic morphogenesis, synaptic formation, and cortical patterning in neonatal and postnatal offspring. Mechanistically, by employing single-cell RNA-sequencing (scRNA-seq) analysis combined with in vivo and in vitro assays, we show TAM could exert these drastic effects mainly through dysregulating the Wnt-Dmrta2 signaling pathway. In adult mice, administration of TAM significantly attenuated NPC proliferation in both the subventricular zone and the dentate gyrus. This study revealed the cellular and molecular mechanisms for the adverse effects of TAM on corticogenesis, suggesting that care must be taken when using the TAM-induced CreER/LoxP system for neural lineage tracing and genetic manipulation studies in both embryonic and adult brains.


Assuntos
Encéfalo/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/patologia , Tamoxifeno/efeitos adversos , Animais , Encéfalo/embriologia , Encéfalo/patologia , Diferenciação Celular , Proliferação de Células , Giro Denteado/efeitos dos fármacos , Giro Denteado/patologia , Feminino , Ventrículos Laterais/efeitos dos fármacos , Ventrículos Laterais/patologia , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , RNA-Seq , Análise de Célula Única , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos
15.
Life Sci ; 256: 118031, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32615186

RESUMO

AIMS: We had previously reported that addition of putrescine to the culture medium was reported to reduce methylmercury toxicity in C17.2 neural stem cells. Here, we have examined the inhibition of methylmercury-induced cytotoxicity by putrescine using ODC1-overexpressing C17.2 cells. MATERIALS AND METHODS: We established stable ODC1-overexpressing C17.2 cells and evaluated methylmercury-induced apoptosis by examining the TUNEL assay and cleaved caspase-3 levels. Mitochondria-mediated apoptosis was also evaluated by reduction of mitochondrial membrane potential and recruitment of Bax and Bak to the mitochondria. KEY FINDINGS: ODC is encoded by ODC1 gene, and putrescine levels in ODC1-overexpressing cells were significantly higher than in control cells. Overexpression of ODC1 and addition of putrescine to the culture medium suppressed DNA fragmentation and caspase-3 activation, which are observed when apoptosis is induced by methylmercury. Moreover, mitochondrial dysfunction and reactive oxygen species (ROS) generation, caused by methylmercury, were also inhibited by the overexpression of ODC1 and putrescine; pretreatment with ODC inhibitor, however, promoted both ROS generation and apoptosis by methylmercury. Finally, we found that Bax and Bak, the apoptosis-promoting factors, to be increased in mitochondria, following methylmercury treatment, and the same was inhibited by overexpression of ODC1. These results suggest that overexpression of ODC1 may prevent mitochondria-mediated apoptosis by methylmercury via increase of putrescine levels. SIGNIFICANCE: Our findings provide important clues to clarify mechanisms involved in the defense against methylmercury toxicity and suggest novel biological functions of putrescine.


Assuntos
Compostos de Metilmercúrio/toxicidade , Mitocôndrias/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Ornitina Descarboxilase/genética , Putrescina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Marcação In Situ das Extremidades Cortadas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/patologia , Células-Tronco Neurais/patologia
16.
Nat Commun ; 11(1): 3258, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591533

RESUMO

Tauopathies are neurodegenerative diseases associated with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacologically targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-molecule screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compounds are more potent than rapamycin, and robustly downregulate phosphorylated and insoluble tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged reduction of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.


Assuntos
Autofagia , Neurônios/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estresse Fisiológico , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fenótipo , Ratos Wistar , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Tauopatias/patologia , Fatores de Tempo
17.
J Neurosci ; 40(21): 4116-4129, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32385093

RESUMO

The cellular and molecular basis of metaplasia and declining neurogenesis in the aging olfactory epithelium (OE) remains unknown. The horizontal basal cell (HBC) is a dormant tissue-specific stem cell presumed to only be forced into self-renewal and differentiation by injury. Here we analyze male and female mice and show that HBCs also are activated with increasing age as well as non-cell-autonomously by increased expression of the retinoic acid-degrading enzyme CYP26B1. Activating stimuli induce HBCs throughout OE to acquire a rounded morphology and express IP3R3, which is an inositol-1,4,5-trisphosphate receptor constitutively expressed in stem cells of the adjacent respiratory epithelium. Odor/air stimulates CYP26B1 expression in olfactory sensory neurons mainly located in the dorsomedial OE, which is spatially inverse to ventrolateral constitutive expression of the retinoic acid-synthesizing enzyme (RALDH1) in supporting cells. In ventrolateral OE, HBCs express low p63 levels and preferentially differentiate instead of self-renewing when activated. When activated by chronic CYP26B1 expression, repeated injury, or old age, ventrolateral HBCs diminish in number and generate a novel type of metaplastic respiratory cell that is RALDH- and secretes a mucin-like mucus barrier protein (FcγBP). Conversely, in the dorsomedial OE, CYP26B1 inhibits injury-induced and age-related replacement of RALDH- supporting cells with RALDH1+ ciliated respiratory cells. Collectively, these results support the concept that inositol-1,4,5-trisphosphate type 3 receptor signaling in HBCs, together with altered retinoic acid metabolism within the niche, promote HBC lineage commitment toward two types of respiratory cells that will maintain epithelial barrier function once the capacity to regenerate OE cells ceases.SIGNIFICANCE STATEMENT Little is known about signals that activate dormant stem cells to self-renew and regenerate odor-detecting neurons and other olfactory cell types after loss due to injury, infection, or toxin exposure in the nose. It is also unknown why the stem cells do not prevent age-dependent decline of odor-detecting neurons. We show that (1) stem cells are kept inactive by the vitamin A derivative retinoic acid, which is synthesized and degraded locally by olfactory cells; (2) old age as well as repeated injuries activate the stem cells and exhaust their potential to produce olfactory cells; and (3) exhausted stem cells alter the local retinoic acid metabolism and maintain the epithelial tissue barrier by generating airway cells instead of olfactory cells.


Assuntos
Envelhecimento/metabolismo , Isotretinoína/farmacologia , Células-Tronco Neurais/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Ácido Retinoico 4 Hidroxilase/metabolismo , Animais , Feminino , Masculino , Metaplasia/metabolismo , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/efeitos dos fármacos
18.
Ecotoxicol Environ Saf ; 200: 110733, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32450442

RESUMO

Paraquat (1,1'-dimethyl-4,4'-bipyridium dichloride, PQ), a non-selective and efficient herbicide, causes neuroinflammation, neurodegeneration and memory dysfunction. However, adverse effects of PQ on the neuroimmune interactions have rarely been investigated. Female adult C57/BL6 mice were divided into 3 groups and treated with PQ (intraperitoneal injection, 1 mg/kg or 5 mg/kg) or the vehicle (an equivalent volume of 0.9% saline) every two days, at day 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, for a total of 14 doses. We evaluated blood-brain barrier (BBB) integrity and PQ concentrations during the course of PQ exposure and tested interleukin-1ß (IL-1ß) concentrations in dentate gyrus (DG) after 28 days PQ exposure. In addition, memory function, neural stem cells (NSCs) proliferation, neurogenesis and microglia polarization were analyzed after PQ exposure. Furthermore, mice were intraperitoneal injections of anti-IL-1ß during 5 mg/kg PQ exposure to test the rule of IL-1ß. Blood-brain barrier (BBB) permeability and PQ concentrations increased gradually during PQ exposure (n = 6). Moreover, memory function, NSCs proliferation and neurogenesis were impaired after 5 mg/kg PQ exposure (n = 6). Further analyses revealed that 'classically' activated (M1) microglia and IL-1ß concentrations in DG were increased after 5 mg/kg PQ treatment (n = 6). Moreover, we found that neutralization of IL-1ß partly restored PQ-induced NSCs impairments and memory dysfunction (n = 6). In conclusion, our results revealed that PQ induced NSCs impairments and memory dysfunction in adult mice, which was related to the release of IL-1ß by M1-polarized microglia in DG. These findings may help understand the neurotoxic effect of PQ.


Assuntos
Giro Denteado/efeitos dos fármacos , Herbicidas/toxicidade , Interleucina-1beta/metabolismo , Neurogênese/efeitos dos fármacos , Paraquat/toxicidade , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Giro Denteado/citologia , Feminino , Memória/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos
19.
Am J Obstet Gynecol ; 223(5): 753.e1-753.e14, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32416155

RESUMO

BACKGROUND: Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. OBJECTIVE: We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. STUDY DESIGN: Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For in vitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. RESULTS: Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes in vivo and high glucose in vitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. CONCLUSION: The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.


Assuntos
Autofagia/genética , Estresse do Retículo Endoplasmático/genética , Doenças Fetais/genética , Células-Tronco Neurais/metabolismo , Defeitos do Tubo Neural/genética , Gravidez em Diabéticas/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Resposta a Proteínas não Dobradas/genética , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Glicemia/metabolismo , Óxidos N-Cíclicos/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Doenças Fetais/etiologia , Doenças Fetais/metabolismo , Glucose/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/metabolismo , Células Neuroepiteliais/efeitos dos fármacos , Células Neuroepiteliais/metabolismo , Neurulação/genética , Estresse Oxidativo , Gravidez , Gravidez em Diabéticas/metabolismo , Marcadores de Spin , Resposta a Proteínas não Dobradas/efeitos dos fármacos
20.
PLoS Comput Biol ; 16(4): e1007780, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32298259

RESUMO

Metabolism plays an essential role in cell fate decisions. However, the methods used for metabolic characterization and for finding potential metabolic regulators are still based on characterizing cellular metabolic steady-state which is dependent on the extracellular environment. In this work, we hypothesized that the response dynamics of intracellular metabolic pools to extracellular stimuli is controlled in a cell type-specific manner. We applied principles of process dynamics and control to human induced pluripotent stem cells (hiPSC) and human neural stem cells (hNSC) subjected to a sudden extracellular glutamine step. The fold-changes of steady-states and the transient profiles of metabolic pools revealed that dynamic responses were reproducible and cell type-specific. Importantly, many amino acids had conserved dynamics and readjusted their steady state concentration in response to the increased glutamine influx. Overall, we propose a novel methodology for systematic metabolic characterization and identification of potential metabolic regulators.


Assuntos
Células-Tronco Pluripotentes Induzidas , Redes e Vias Metabólicas/fisiologia , Células-Tronco Neurais , Reatores Biológicos , Células Cultivadas , Biologia Computacional , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Glutamina/metabolismo , Glutamina/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo
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