RESUMO
Coronavirus disease 2019 (COVID-19) is a worldwide pandemic. It has a high transmission rate among humans, and is a threat to global public health. However, there are no effective prophylactics or therapeutics available. It is necessary to identify vulnerable and susceptible groups for adequate protection and care against this disease. Recent studies have reported that COVID-19 has angiotensin-converting enzyme 2 (ACE2) as a functional receptor, which may lead to the development of severe cerebrovascular diseases (CVD), including strokes, in patients with risk factors for CVD such as diabetes and smoking. Thus, the World Health Organization (WHO) advised caution against COVID-19 for smokers and patients with underlying clinical symptoms, including cardiovascular diseases. Here, we observed ACE2 expression in the brain of rat middle cerebral artery occlusion (MCAO) model and evaluated the effects of cigarette smoke extract (CSE) and diabetes on ACE2 expression in vessels. We showed that the levels of ACE2 expression was increased in the cortex penumbra after ischemic injuries. CSE treatment significantly elevated ACE2 expression in human brain vessels. We found that ACE2 expression was upregulated in primary cultured human blood vessels with diabetes compared to healthy controls. This study demonstrates that ACE2 expression is increased in ischemic brains and vessels exposed to diabetes or smoking, makes them vulnerable to COVID-19 infection.
Assuntos
Betacoronavirus/metabolismo , Isquemia Encefálica/virologia , Encéfalo/irrigação sanguínea , Diabetes Mellitus , Peptidil Dipeptidase A/biossíntese , Receptores Virais/biossíntese , Fumantes , Acidente Vascular Cerebral/virologia , Regulação para Cima , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus/patogenicidade , Encéfalo/efeitos dos fármacos , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , COVID-19 , Infecções por Coronavirus/genética , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Infarto da Artéria Cerebral Média/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pandemias , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Ratos , Ratos Sprague-Dawley , Receptores Virais/genética , SARS-CoV-2 , Fumaça/efeitos adversos , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Proper communication among neurons depends on an appropriately formed dendritic arbor, and thus, aberrant changes to the arbor are implicated in many pathologies, ranging from cognitive disorders to neurodegenerative diseases. Due to the importance of dendritic shape to neuronal network function, the morphology of dendrites is tightly controlled and is influenced by both intrinsic and extrinsic factors. In this work, we examine how brain-derived neurotrophic factor (BDNF), one of the most well-studied extrinsic regulators of dendritic branching, affects the arbor when it is applied locally via microbeads to cultures of hippocampal neurons. We found that local application of BDNF increases both proximal and distal branching in a time-dependent manner and that local BDNF application attenuates pruning of dendrites that occurs with neuronal maturation. Additionally, we examined whether cytosolic PSD-95 interactor (cypin), an intrinsic regulator of dendritic branching, plays a role in these changes and found strong evidence for the involvement of cypin in BDNF-promoted increases in dendrites after 24 but not 48 h of application. This current study extends our previous work in which we found that bath application of BDNF for 72 h, but not shorter times, increases proximal dendrite branching and that this increase occurs through transcriptional regulation of cypin. Moreover, this current work illustrates how dendritic branching is regulated differently by the same growth factor depending on its spatial localization, suggesting a novel pathway for modulation of dendritic branching locally.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Dendritos/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Plasticidade Neuronal/efeitos dos fármacos , Animais , Dendritos/metabolismo , Dendritos/ultraestrutura , Proteína 4 Homóloga a Disks-Large , Embrião de Mamíferos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Microesferas , Plasticidade Neuronal/genética , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
More than 31,000 protein-coding sequences (CCDS) have been identified in the human genome. Here, we analyzed codon usage in all human CCDS and found that there is a preferential usage of minor codons for Ala (CGC), Pro (CCG), Ser (UCG), and Thr (ACG) in the initial 50-codon sequences of the CCDS. These codons, with consensus XCG sequences, are most infrequently used among their synonymous codons. Thus, the tRNA concentrations per codon are considered to be highest for the minor codons for Ala, Pro, Ser and Thr in comparison with other synonymous codons for each of them to enhance the translation efficiency. This suggests that human genes are regulated at the level of translation by preferentially using minor codons within the first 50 codons of the CCDS. This hypothesis was experimentally confirmed by comparing the expression of the luciferase gene encoded by minor codons with that encoded by major codons.
Assuntos
Códon , Genoma Humano , Iniciação Traducional da Cadeia Peptídica/genética , Regulação da Expressão Gênica , Humanos , Fases de Leitura AbertaRESUMO
Parkinson's disease is a degenerative brain disorder characterized by dopamine neuronal degeneration and dopamine transporter loss. In this study, we generated an induced pluripotent stem cell (iPSC) line, KNIHi001-A, from the peripheral blood mononuclear cells (PBMCs) of a 76-year-old man with Parkinson's disease. The non-integrating Sendai virus was used to reprogram iPSCs. iPSCs exhibit pluripotent markers, a normal karyotype, viral clearance, and the ability to differentiate into the three germ layers.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Masculino , Humanos , Idoso , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/metabolismo , Leucócitos Mononucleares/metabolismo , Camadas Germinativas/metabolismo , Vírus Sendai/genética , Reprogramação Celular , Diferenciação Celular/fisiologiaRESUMO
Alterations in dendrite branching and morphology are present in many neurodegenerative diseases. These variations disrupt postsynaptic transmission and affect neuronal communication. Thus, it is important to understand the molecular mechanisms that regulate dendritogenesis and how they go awry during disease states. Previously, our laboratory showed that cypin, a mammalian guanine deaminase, increases dendrite number when overexpressed and decreases dendrite number when knocked down in cultured hippocampal neurons. Here, we report that exposure to brain-derived neurotrophic factor (BDNF), an important mediator of dendrite arborization, for 72 h but not for 24 h or less increases cypin mRNA and protein levels in rat hippocampal neurons. BDNF signals through cypin to regulate dendrite number, since knocking down cypin blocks the effects of BDNF. Furthermore, BDNF increases cypin levels via mitogen-activated protein kinase and transcription-dependent signaling pathways. Moreover, the cypin promoter region contains putative conserved cAMP response element (CRE) regions, which we found can be recognized and activated by CRE-binding protein (CREB). In addition, exposure of the neurons to BDNF increased CREB binding to the cypin promoter and, in line with these data, expression of a dominant negative form of CREB blocked BDNF-promoted increases in cypin protein levels and proximal dendrite branches. Together, these studies suggest that BDNF increases neuronal cypin expression by the activation of CREB, increasing cypin transcription leading to increased protein expression, thus identifying a novel pathway by which BDNF shapes the dendrite network.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Proteínas de Transporte/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dendritos/efeitos dos fármacos , Guanina Desaminase/genética , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dendritos/genética , Dendritos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Guanina Desaminase/metabolismo , Hipocampo/metabolismo , Imuno-Histoquímica , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transcrição GênicaRESUMO
Little is known about how the neuronal cytoskeleton is regulated when a dendrite decides whether to branch or not. Previously, we reported that postsynaptic density protein 95 (PSD-95) acts as a stop signal for dendrite branching. It is yet to be elucidated how PSD-95 affects the cytoskeleton and how this regulation relates to the dendritic arbor. Here, we show that the SH3 (src homology 3) domain of PSD-95 interacts with a proline-rich region within the microtubule end-binding protein EB3. Overexpression of PSD-95 or mutant EB3 results in a decreased lifetime of EB3 comets in dendrites. In line with these data, transfected rat neurons show that overexpression of PSD-95 results in less organized microtubules at dendritic branch points and decreased dendritogensis. The interaction between PSD-95 and EB3 elucidates a function for a novel region of EB3 and provides a new and important mechanism for the regulation of microtubules in determining dendritic morphology.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Animais , Células Cultivadas , Dendritos/metabolismo , Proteína 4 Homóloga a Disks-Large , Hipocampo/citologia , Hipocampo/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Microscopia Eletrônica , Neurônios/citologia , Ligação Proteica , Ratos , TransfecçãoRESUMO
Cytosolic PSD-95 interactor (cypin) regulates many aspects of neuronal development and function, ranging from dendritogenesis to synaptic protein localization. While it is known that removal of postsynaptic density protein-95 (PSD-95) from the postsynaptic density decreases synaptic N-methyl-D-aspartate (NMDA) receptors and that cypin overexpression protects neurons from NMDA-induced toxicity, little is known about cypin's role in AMPA receptor clustering and function. Experimental work shows that cypin overexpression decreases PSD-95 levels in synaptosomes and the PSD, decreases PSD-95 clusters/µm2, and increases mEPSC frequency. Analysis of microelectrode array (MEA) data demonstrates that cypin or cypinΔPDZ overexpression increases sensitivity to CNQX (cyanquixaline) and AMPA receptor-mediated decreases in spike waveform properties. Network-level analysis of MEA data reveals that cypinΔPDZ overexpression causes networks to be resilient to CNQX-induced changes in local efficiency. Incorporating these findings into a computational model of a neural circuit demonstrates a role for AMPA receptors in cypin-promoted changes to networks and shows that cypin increases firing rate while changing network functional organization, suggesting cypin overexpression facilitates information relay but modifies how information is encoded among brain regions. Our data show that cypin promotes changes to AMPA receptor signaling independent of PSD-95 binding, shaping neural circuits and output to regions beyond the hippocampus.
RESUMO
During neuronal development, neurons form elaborate dendritic arbors that receive signals from axons. Additional studies are needed to elucidate the factors regulating the establishment of dendritic patterns. Our work explored possible roles played by nitric oxide synthase 1 adaptor protein (NOS1AP; also known as C-terminal PDZ ligand of neuronal nitric oxide synthase or CAPON) in dendritic patterning of cultured hippocampal neurons. Here we report that the long isoform of NOS1AP (NOS1AP-L) plays a novel role in regulating dendrite outgrowth and branching. NOS1AP-L decreases dendrite number when overexpressed at any interval between day in vitro (DIV) 0 and DIV 12, and knockdown of NOS1AP-L results in increased dendrite number. In contrast, the short isoform of NOS1AP (NOS1AP-S) decreases dendrite number only when overexpressed during DIV 5-7. Using mutants of NOS1AP-L, we show that neither the PDZ-binding domain nor the PTB domain is necessary for the effects of NOS1AP-L. We have functionally narrowed the region of NOS1AP-L that mediates this effect to the middle amino acids 181-307, a region that is not present in NOS1AP-S. Furthermore, we performed a yeast two-hybrid screen and identified carboxypeptidase E (CPE) as a binding partner for the middle region of NOS1AP-L. Biochemical and cellular studies reveal that CPE mediates the effects of NOS1AP on dendrite morphology. Together, our results suggest that NOS1AP-L plays an important role in the initiation, outgrowth, and maintenance of dendrites during development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Padronização Corporal/fisiologia , Carboxipeptidase H/metabolismo , Dendritos/fisiologia , Hipocampo/citologia , Neurônios/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Padronização Corporal/genética , Células COS , Carboxipeptidase H/genética , Técnicas de Cultura de Células , Chlorocebus aethiops , DNA Complementar , Dendritos/metabolismo , Regulação para Baixo/fisiologia , Vetores Genéticos , Proteínas de Fluorescência Verde/química , Humanos , Imuno-Histoquímica , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Neurônios/metabolismo , Plasmídeos , RNA Interferente Pequeno , Ratos , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologiaRESUMO
The guanine deaminase cypin (cytosolic PSD-95 interactor) binds to PSD-95 (postsynaptic density protein 95) and regulates dendrite branching by promoting microtubule polymerization. Here, we identify a novel short isoform of cypin, termed cypinS, which is expressed in mouse and human, but not rat, tissues. Cypin and cypinS mRNA and protein levels peak at P7 and P14 in the mouse brain, suggesting a role for these isoforms during development. Interestingly, although cypinS lacks guanine deaminase activity, overexpression of cypinS increases dendrite branching. This increase occurs further away from soma than do increases resulting from overexpression of cypin. In contrast, overexpression of cypin, but not cypinS, decreases dendritic spine density and maturity. This suggests that changes to spines, but not to dendrites, may be dependent on guanine deaminase activity. Furthermore, overexpression of either cypin or cypinS increases miniature excitatory postsynaptic current (mEPSC) frequency, pointing to a presynaptic role for both isoforms. Interestingly, overexpression of cypinS results in a significantly greater increase in frequency than does overexpression of cypin. Thus, cypin and cypinS play distinct roles in neuronal development.
Assuntos
Proteína 4 Homóloga a Disks-Large/metabolismo , Guanina Desaminase/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Dendritos/metabolismo , Proteína 4 Homóloga a Disks-Large/genética , Potenciais Pós-Sinápticos Excitadores , Guanina Desaminase/genética , Células HEK293 , Hipocampo/metabolismo , Humanos , Camundongos , Especificidade de Órgãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Receptor tyrosine kinase Mer (MerTK) has been shown to be highly expressed in Glioblastoma multiforme (GBM) in comparison to its healthy counterpart and is implicated in brain tumorigenesis. Clarifying the underlying mechanism of MerTK induced invasiveness would result in novel strategies to improve patient's response to chemotherapeutics. In vitro and in vivo assays were performed to examine the functional role of cancer stem sell (CSC) maintenance in MerTK associated invasiveness. In this article, we demonstrate that apart from GBM cells, MerTK is also upregulated in GBM stem-like cells and associated with an increased infiltrative potential of brain tumors in vivo. Silencing of MerTK suppressed the self-renewal of patient-derived GBM stem-like cells. The signaling mechanisms by which MerTK contributes to CSC maintenance have largely been obscure. Molecular analyses revealed that high expression of the signal transducer and activator of transcription 3 (STAT3)- Kirsten rat sarcoma viral oncogene homolog (KRAS) and proto-oncogene tyrosine-protein kinase SRC axis supports MerTK-induced CSC maintenance in GBM spheroids. Furthermore, a short-hairpin RNA-mediated MerTK knockdown effectively blocked invasiveness and N-cadherin expression in mouse xenografts. Collectively, our results uncover a critical function of MerTK in CSC maintenance. Considering the low basal level of MerTK expression in healthy brain cells, evaluation of MerTK as a therapeutic target should advance the research into better therapeutics for GBM.
Assuntos
Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , c-Mer Tirosina Quinase/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Invasividade Neoplásica , Prognóstico , Proto-Oncogene Mas , c-Mer Tirosina Quinase/deficiência , c-Mer Tirosina Quinase/genéticaRESUMO
BACKGROUND: Where a neuron is positioned in the brain during development determines neuronal circuitry and information processing needed for normal brain function. When aberrations in this process occur, cognitive disorders may result. Patients diagnosed with schizophrenia have been reported to show altered neuronal connectivity and heterotopias. To elucidate pathways by which this process occurs and become aberrant, we have chosen to study the long isoform of nitric oxide synthase 1 adaptor protein (NOS1AP), a protein encoded by a susceptibility gene for schizophrenia. METHODS: To determine whether NOS1AP plays a role in cortical patterning, we knocked down or co-overexpressed NOS1AP and a green fluorescent protein or red fluorescent protein (TagRFP) reporter in neuronal progenitor cells of the embryonic rat neocortex using in utero electroporation. We analyzed sections of cortex (ventricular zone, intermediate zone, and cortical plate [CP]) containing green fluorescent protein or red fluorescent protein TagRFP positive cells and counted the percentage of positive cells that migrated to each region from at least three rats for each condition. RESULTS: NOS1AP overexpression disrupts neuronal migration, resulting in increased cells in intermediate zone and less cells in CP, and decreases dendritogenesis. Knockdown results in increased migration, with more cells reaching the CP. The phosphotyrosine binding region, but not the PDZ-binding motif, is necessary for NOS1AP function. Amino acids 181 to 307, which are sufficient for NOS1AP-mediated decreases in dendrite number, have no effect on migration. CONCLUSIONS: Our studies show for the first time a critical role for the schizophrenia-associated gene NOS1AP in cortical patterning, which may contribute to underlying pathophysiology seen in schizophrenia.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Movimento Celular/genética , Neocórtex/citologia , Células-Tronco Neurais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Recém-Nascidos , Células COS , Chlorocebus aethiops , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Mutação/genética , Células-Tronco Neurais/metabolismo , Domínios PDZ/genética , Gravidez , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Transgênicos , Transfecção , Proteína Vermelha FluorescenteRESUMO
Determining the shape of cell-specific dendritic arbors is a tightly regulated process that occurs during development. When this regulation is aberrant, which occurs during disease or injury, alterations in dendritic shape result in changes to neural circuitry. There has been significant progress on characterizing extracellular and intrinsic factors that regulate dendrite number by our laboratory and others. Generally, changes to the dendritic arbor are assessed by Sholl analysis or simple dendrite counting. However, we have found that this general method often overlooks local changes to the arbor. Previously, we developed a program (titled Bonfire) to facilitate digitization of neurite morphology and subsequent Sholl analysis and to assess changes to root, intermediate, and terminal neurites. Here, we apply these different Sholl analyses, and a novel Sholl analysis, to uncover previously unknown changes to the dendritic arbor when we overexpress an important regulator of dendrite branching, cytosolic PSD-95 interactor (cypin), at two developmental time points. Our results suggest that standard Sholl analysis and simple dendrite counting are not sufficient for uncovering local changes to the dendritic arbor.
RESUMO
The calcium phosphate transfection is a widely used method for introducing foreign DNA plasmids into cells. Mechanisms underlying this transfection method are not yet defined; however, DNA-calcium phosphate precipitates are internalized by the cells and DNA is efficiently expressed in almost all cell types. The cost-efficiency and simplicity of this method allows for use in primary neuronal cultures, despite issues of neurotoxicity. Here, we describe an optimized calcium phosphate transfection method for the delivery of DNA plasmid into primary dissociated neuronal cultures.
Assuntos
Fosfatos de Cálcio/farmacologia , DNA/metabolismo , Transfecção/métodos , AnimaisRESUMO
Cullin-RING ligases (CRLs) regulate diverse cellular functions such as cell cycle progression and cytokine signaling by ubiquitinating key regulatory proteins. The activity of CRLs is controlled by Nedd8 modification of the cullin subunits. Recent reports have suggested that CAND1, which specifically binds to unmodified CUL1 but not to neddylated one, is required for the in vivo function of SCFs, the CUL1-containing CRLs. We show here that CAND1 and COP9 signalosome (CSN), the major deneddylase of cullins, bind to unneddylated CUL1 in a mutually exclusive way. The suppression of CAND1 expression by small inhibitory RNA enhanced the interaction between CUL1 and CSN, suggesting that CAND1 inhibited the binding of CSN to CUL1. We found that the binding of CSN to CUL1 required the four helix bundle in CUL1 C-terminal domain, which was wrapped around by CAND1 in the CAND1-CUL1-Rbx1 complex. CAND1 greatly facilitated CSN-mediated deneddylation of CUL1 in vitro, which was dependent on its binding to CUL1. Our data suggest that enhancement of CSN-mediated deneddylation by CAND1 may contribute to its function as a positive regulator of SCFs in vivo.