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1.
Mol Cell ; 73(2): 364-376.e8, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30581142

RESUMO

Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Mitofagia , Trifosfato de Adenosina/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Dinaminas , Metabolismo Energético , GTP Fosfo-Hidrolases/genética , Células HEK293 , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Mutação , Neurônios/metabolismo , Neurônios/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos Sprague-Dawley , Transdução de Sinais , Fatores de Tempo , Zinco/metabolismo
2.
Nat Chem Biol ; 20(3): 353-364, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37973890

RESUMO

Proteases function as pivotal molecular switches, initiating numerous biological events. Notably, potyviral protease, derived from plant viruses, has emerged as a trusted proteolytic switch in synthetic biological circuits. To harness their capabilities, we have developed a single-component photocleavable switch, termed LAUNCHER (Light-Assisted UNcaging switCH for Endoproteolytic Release), by employing a circularly permutated tobacco etch virus protease and a blue-light-gated substrate, which are connected by fine-tuned intermodular linkers. As a single-component system, LAUNCHER exhibits a superior signal-to-noise ratio compared with multi-component systems, enabling precise and user-controllable release of payloads. This characteristic renders LAUNCHER highly suitable for diverse cellular applications, including transgene expression, tailored subcellular translocation and optochemogenetics. Additionally, the plug-and-play integration of LAUNCHER into existing synthetic circuits facilitates the enhancement of circuit performance. The demonstrated efficacy of LAUNCHER in improving existing circuitry underscores its significant potential for expanding its utilization in various applications.


Assuntos
Peptídeo Hidrolases , Potyvirus , Luz Azul , Proteólise , Razão Sinal-Ruído
3.
Mol Ther ; 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38845196

RESUMO

Systemic administration of adeno-associated virus (AAV) vectors for spinal cord gene therapy has challenges including toxicity at high doses and pre-existing immunity that reduces efficacy. Intrathecal (IT) delivery of AAV vectors into cerebral spinal fluid can avoid many issues, although distribution of the vector throughout the spinal cord is limited, and vector entry to the periphery sometimes initiates hepatotoxicity. Here we performed biopanning in non-human primates (NHPs) with an IT injected AAV9 peptide display library. We identified top candidates by sequencing inserts of AAV DNA isolated from whole tissue, nuclei, or nuclei from transgene-expressing cells. These barcoded candidates were pooled with AAV9 and compared for biodistribution and transgene expression in spinal cord and liver of IT injected NHPs. Most candidates displayed increased retention in spinal cord compared with AAV9. Greater spread from the lumbar to the thoracic and cervical regions was observed for several capsids. Furthermore, several capsids displayed decreased biodistribution to the liver compared with AAV9, providing a high on-target/low off-target biodistribution. Finally, we tested top candidates in human spinal cord organoids and found them to outperform AAV9 in efficiency of transgene expression in neurons and astrocytes. These capsids have potential to serve as leading-edge delivery vehicles for spinal cord-directed gene therapies.

4.
J Neurochem ; 162(2): 190-206, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35567753

RESUMO

The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2 × Myc and CYFIP2-3 × Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2 × Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Astrócitos , Adesões Focais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Astrócitos/metabolismo , Proteínas de Transporte/genética , Adesões Focais/metabolismo , Camundongos
5.
J Biol Chem ; 291(48): 25088-25095, 2016 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-27687730

RESUMO

Proper positioning of neurons is fundamental for brain functions. However, little is known on how adult-born neurons generated in the hilar side of hippocampal dentate gyrus migrate into the granular cell layer. Because class 3 Semaphorins (Sema3) are involved in dendritic growth of these newborn neurons, we examined whether they are essential for cell positioning. We disrupted Sema3 signaling by silencing neuropilin 1 (NRP1) or 2 (NRP2), the main receptors for Sema3A and Sema3F, in neural progenitors of adult mouse dentate gyrus. Silencing of NRP2, but not NRP1, affected cell positioning of adult newborn neurons. Glycogen synthase kinase-3ß (GSK3ß) knockdown phenocopied this NRP2 silencing-mediated cell positioning defect, but did not affect dendritic growth. Furthermore, GSK3ß is activated upon stimulation with Sema3F, and GSK3ß overexpression rescued the cell positioning phenotypes seen in NRP2-deficient neurons. These results point to a new role for NRP2 in the positioning of neurons during adult hippocampal neurogenesis, acting via the GSK3ß signaling pathway.


Assuntos
Giro Denteado/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Neuropilina-2/metabolismo , Transdução de Sinais/fisiologia , Animais , Giro Denteado/citologia , Feminino , Glicogênio Sintase Quinase 3 beta/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Semaforina-3A/genética , Semaforina-3A/metabolismo
6.
Biochem Biophys Res Commun ; 494(3-4): 581-586, 2017 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-29111324

RESUMO

Variants of the SHANK3 gene, which encodes a core scaffold protein of the postsynaptic density of excitatory synapses, have been causally associated with numerous brain disorders. Shank3 proteins directly bind zinc ions through their C-terminal sterile α motif domain, which enhances the multimerization and synaptic localization of Shank3, to regulate excitatory synaptic strength. However, no studies have explored whether zinc affects the protein interactions of Shank3, which might contribute to the synaptic changes observed after zinc application. To examine this, we first purified Shank3 protein complexes from mouse brain synaptosomal lysates that were incubated with different concentrations of ZnCl2, and analyzed them with mass spectrometry. We used strict criteria to identify 71 proteins that specifically interacted with Shank3 when extra ZnCl2 was added to the lysate. To characterize the zinc-induced Shank3 interactome, we performed various bioinformatic analyses that revealed significant associations of the interactome with subcellular compartments, including mitochondria, and brain disorders, such as bipolar disorder and schizophrenia. Together, our results showing that zinc affected the Shank3 protein interactions of in vitro mouse synaptosomes provided an additional link between zinc and core synaptic proteins that have been implicated in multiple brain disorders.


Assuntos
Encefalopatias/metabolismo , Cloretos/administração & dosagem , Doenças Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Mapas de Interação de Proteínas/fisiologia , Proteoma/metabolismo , Sinaptossomos/metabolismo , Compostos de Zinco/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Humanos , Metaboloma/efeitos dos fármacos , Metaboloma/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso/genética , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos
7.
Histochem Cell Biol ; 148(1): 13-20, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28314909

RESUMO

Dynamin-related protein1 (Drp1) plays an essential role in mitochondrial fission: Cytosolic Drp1 is translocated to the mitochondria upon stimulus, and oligomerized Drp1 constricts mitochondria by aid of actin filaments. Drp1 completes the fission process with GTP hydrolysis by its own GTPase activity. The importance of actin filament and its interaction with Drp1 in the mitochondrial fission process have been demonstrated. In this study, we found that Drp1 is enriched in the actin-rich leading edge of lamellipodia of mouse embryonic fibroblasts (MEFs) wherein mitochondria or peroxisomes are absent. Mff-binding mutant (A395D) of Drp1, which cannot be recruited to mitochondria, was also localized in lamellipodia, indicating that Drp1 in lamellipodia is not related to mitochondria. When lamellipodia formation was induced by platelet-derived growth factor (PDGF) in MEFs, S616 phosphorylated form of Drp1 was accumulated to the lamellipodia. Inhibition of Drp1 with Mdivi-1 or a specific shRNA significantly decreased PDGF-induced lamellipodia formation or initial cell spreading during re-plating of the cells, respectively. Interestingly, defective lamellipodia formation and cell adhesion caused by Drp1 inhibition were not rescued by supplementing L-carnitine, although it restored mitochondrial energy loss caused by Drp1 inhibition. Collectively, these results favor the idea that Drp1 might play a significant role in lamellipodia formation and cell spreading through a different mechanism from that used for regulating mitochondrial dynamics/function.


Assuntos
Dinaminas/metabolismo , Pseudópodes/metabolismo , Actinas/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez
9.
IBRO Neurosci Rep ; 17: 22-31, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38881849

RESUMO

Symmetry breaking leading to axis formation and spatial patterning is crucial for achieving more accurate recapitulation of human development in organoids. While these processes can occur spontaneously by self-organizing capabilities of pluripotent stem cells, they can often result in variation in structure and composition of cell types within organoids. To address this limitation, bioengineering techniques that utilize geometric, topological and stiffness factors are increasingly employed to enhance control and consistency. Here, we review how spontaneous manners and engineering tools such as micropattern, microfluidics, biomaterials, etc. can facilitate the process of symmetry breaking leading to germ layer patterning and the formation of anteroposterior and dorsoventral axes in blastoids, gastruloids, neuruloids and neural organoids. Furthermore, brain assembloids, which are composed of multiple brain regions through fusion processes are discussed. The overview of organoid polarization in terms of patterning tools can offer valuable insights for enhancing the physiological relevance of organoid system.

10.
Stem Cells Transl Med ; 13(3): 268-277, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38103168

RESUMO

Fetal spinal cord ischemia is a serious medical condition that can result in significant neurological damage and adverse outcomes for the fetus. However, the lack of an appropriate experimental model has hindered the understanding of the pathology and the development of effective treatments. In our study, we established a system for screening drugs that affect fetal spinal cord ischemia using spinal cord organoids. Importantly, we produced necrotic core-free human spinal cord organoids (nf-hSCOs) by reducing the organoid size to avoid potential complications of spontaneous necrosis in large organoids. Exposing nf-hSCOs to CoCl2 as a hypoxia mimetic and hypoglycemic conditions resulted in significant neuronal damage, as assessed by multiple assay batteries. By utilizing this model, we tested chemicals that have been reported to exhibit beneficial effects in brain organoid-based ischemia models. Surprisingly, these chemicals did not provide sufficient benefit, and we discovered that rapamycin is a mild neuroprotective reagent for both axon degeneration and neuronal survival. We propose that nf-hSCO is suitable for large-scale screening of fetal neural ischemia due to its scalability, ease of ischemic induction, implementation of quantifiable assay batteries, and the absence of spontaneous necrosis.


Assuntos
Isquemia , Isquemia do Cordão Espinal , Humanos , Isquemia/patologia , Isquemia do Cordão Espinal/etiologia , Isquemia do Cordão Espinal/patologia , Isquemia do Cordão Espinal/prevenção & controle , Medula Espinal/patologia , Necrose/complicações , Necrose/patologia , Feto/patologia , Organoides/patologia
11.
Nat Commun ; 15(1): 2000, 2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38448437

RESUMO

Bioresorbable neural implants based on emerging classes of biodegradable materials offer a promising solution to the challenges of secondary surgeries for removal of implanted devices required for existing neural implants. In this study, we introduce a fully bioresorbable flexible hybrid opto-electronic system for simultaneous electrophysiological recording and optogenetic stimulation. The flexible and soft device, composed of biodegradable materials, has a direct optical and electrical interface with the curved cerebral cortex surface while exhibiting excellent biocompatibility. Optimized to minimize light transmission losses and photoelectric artifact interference, the device was chronically implanted in the brain of transgenic mice and performed to photo-stimulate the somatosensory area while recording local field potentials. Thus, the presented hybrid neural implant system, comprising biodegradable materials, promises to provide monitoring and therapy modalities for versatile applications in biomedicine.


Assuntos
Implantes Absorvíveis , Depressores do Sistema Nervoso Central , Animais , Camundongos , Optogenética , Artefatos , Encéfalo , Eletrônica , Camundongos Transgênicos
12.
Immunol Rev ; 228(1): 170-83, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19290927

RESUMO

Stimulation of the T-cell antigen receptor (TCR) leads to the activation of signaling pathways that are essential for T-cell development and the response of mature T cells to antigens. The TCR has no intrinsic catalytic activity, but TCR engagement results in tyrosine phosphorylation of downstream targets by non-receptor tyrosine kinases. Three families of tyrosine kinases have long been recognized to play critical roles in TCR-dependent signaling. They are the Src, zeta-associated protein of 70 kDa, and Tec families of kinases. More recently, the Abelson (Abl) tyrosine kinases have been shown to be activated by TCR engagement and to be required for maximal TCR signaling. Using T-cell conditional knockout mice deficient for Abl family kinases, Abl (Abl1) and Abl-related gene (Arg) (Abl2), it was recently shown that loss of Abl kinases results in defective T-cell development and a partial block in the transition to the CD4(+)CD8(+) stage. Abl/Arg double null T cells exhibit impaired TCR-induced signaling, proliferation, and cytokine production. Moreover, conditional knockout mice lacking Abl and Arg in T cells exhibit impaired CD8(+) T-cell expansion in vivo upon Listeria monocytogenes infection. Thus, Abl kinase signaling is required for both T-cell development and mature T-cell function.


Assuntos
Proteínas Proto-Oncogênicas c-abl/imunologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Animais , Humanos , Linfócitos T/citologia , Linfócitos T/imunologia
13.
Chem Commun (Camb) ; 59(67): 10109-10112, 2023 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-37528768

RESUMO

Despite the apparent copious fluorescent probes targeting mitochondria, the development of low cytotoxic probes is still needed for improving validation of mitochondrial function assessment. Herein, we report a novel cyanine-based NIR fluorescent probe, T2, which selectively targets mitochondria with significantly low toxicity by modulating the intracellular redox status. Additionally, T2 inhibits oxidative stress-induced cell death in cortical neurons. This study provides new insight into developing low-toxic mitochondrial imaging agents by regulating redox homeostasis.


Assuntos
Diagnóstico por Imagem , Estresse Oxidativo , Morte Celular , Oxirredução , Corantes Fluorescentes/toxicidade , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo
14.
Adv Sci (Weinh) ; 10(20): e2301787, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37170679

RESUMO

Axis formation and related spatial patterning are initiated by symmetry breaking during development. A geometrically confined culture of human pluripotent stem cells (hPSCs) mimics symmetry breaking and cell patterning. Using this, polarized spinal cord organoids (pSCOs) with a self-organized dorsoventral (DV) organization are generated. The application of caudalization signals promoted regionalized cell differentiation along the radial axis and protrusion morphogenesis in confined hPSC colonies. These detached colonies grew into extended spinal cord-like organoids, which established self-ordered DV patterning along the long axis through the spontaneous expression of polarized DV patterning morphogens. The proportions of dorsal/ventral domains in the pSCOs can be controlled by the changes in the initial size of micropatterns, which altered the ratio of center-edge cells in 2D. In mature pSCOs, highly synchronized neural activity is separately detected in the dorsal and ventral side, indicating functional as well as structural patterning established in the organoids. This study provides a simple and precisely controllable method to generate spatially ordered organoids for the understanding of the biological principles of cell patterning and axis formation during neural development.


Assuntos
Padronização Corporal , Células-Tronco Pluripotentes , Humanos , Medula Espinal , Morfogênese , Organoides
15.
Stem Cell Reports ; 17(1): 68-81, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-34919810

RESUMO

Human pluripotent stem cells (hPSCs) grow as colonies with epithelial-like features including cell polarity and position-dependent features that contribute to symmetry breaking during development. Our study provides evidence that hPSC colonies exhibit position-dependent differences in apical structures and functions. With this apical difference, edge cells were preferentially labeled with amphipathic dyes, which enabled separation of edge and center cells by fluorescence-activated cell sorting. Transcriptome comparison between center and edge cells showed differential expression of genes related to apicobasal polarization, cell migration, and endocytosis. Accordingly, different kinematics and mechanical dynamics were found between center and edge cells, and perturbed actin dynamics disrupted the position-dependent apical polarity. In addition, our dye-labeling approach could be utilized to sort out a certain cell population in differentiated micropatterned colonies. In summary, hPSC colonies have position-dependent differences in apical structures and properties, and actin dynamics appear to play an important role in the establishment of this position-dependent cell polarity.


Assuntos
Citoesqueleto de Actina/metabolismo , Diferenciação Celular , Polaridade Celular , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Citoesqueleto de Actina/genética , Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imunofenotipagem
16.
Front Neuroanat ; 15: 746057, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34744642

RESUMO

The function of a neural circuit can be determined by the following: (1) characteristics of individual neurons composing the circuit, (2) their distinct connection structure, and (3) their neural circuit activity. However, prior research on correlations between these three factors revealed many limitations. In particular, profiling and modeling of the connectivity of complex neural circuits at the cellular level are highly challenging. To reduce the burden of the analysis, we suggest a new approach with simplification of the neural connection in an array of honeycomb patterns on 2D, using a microcontact printing technique. Through a series of guided neuronal growths in defined honeycomb patterns, a simplified neuronal circuit was achieved. Our approach allowed us to obtain the whole network connectivity at cellular resolution using a combination of stochastic multicolor labeling via viral transfection. Therefore, we were able to identify several types of hub neurons with distinct connectivity features. We also compared the structural differences between different circuits using three-node motif analysis. This new model system, iCANN, is the first experimental model of neural computation at the cellular level, providing neuronal circuit structures for the study of the relationship between anatomical structure and function of the neuronal network.

17.
Nitric Oxide ; 22(1): 18-29, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19887113

RESUMO

The role of protease-activated receptor (PARs) in the regulation of microglial activation process is increasingly evident. In the present study, we have investigated the role of PAR-2, which can be activated by trypsin-like proteases, in microglial activation and neuronal cell death. In cultured rat primary microglia, activation of PAR-2 induced nitrite production by PKC- and MAPKs-dependent mechanism. Among the three members of MAPK pathway, ERK and JNK but not p38 mediated PAR-2-induced microglial activation. The down-stream regulator of PAR-2-PKC-MAPK pathway-induced microglial activation was NF-kappaB pathway. Besides nitrite, PAR-2 activation increased production of a variety of inflammatory mediators such as ROS and pro-inflammatory cytokines including TNF-alpha and IL-1beta. The addition of culture spent media from PAR-2 activated microglia induced neuronal cell death in primary rat cortical neuron cultures with apoptotic features such as increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive neurons, dissipation of mitochondrial membrane potential, increased expression of pro-apoptotic Bax, decreased expression of anti-apoptotic Bcl-2, Bcl-X(L), and activation of caspase-3 in neurons. Interestingly, the increased production of cytoactive molecules as well as the neuronal cell death was normalized by PAR-2 or trypsin inhibitor or an NO synthase inhibitor, N(G)-nitro-l-arginine-methyl ester. Taken together, these results suggest that overt PAR-2 activation may induce microglial activation, which contributes to neuronal cell death.


Assuntos
Microglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Receptor PAR-2/metabolismo , Animais , Morte Celular , Células Cultivadas , Citocinas/biossíntese , Microglia/citologia , Microglia/enzimologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptor PAR-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tripsina/metabolismo
18.
Cell Death Dis ; 11(10): 888, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087694

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of motor neurons (MNs) and subsequent muscle weakness. These pathological features are associated with numerous cellular changes, including alteration in mitochondrial morphology and function. However, the molecular mechanisms associating mitochondrial structure with ALS pathology are poorly understood. In this study, we found that Dynamin-related protein 1 (Drp1) was dephosphorylated in several ALS models, including those with SOD1 and TDP-43 mutations, and the dephosphorylation was mediated by the pathological induction of protein phosphatase 1 (PP1) activity in these models. Suppression of the PP1-Drp1 cascade effectively prevented ALS-related symptoms, including mitochondrial fragmentation, mitochondrial complex I impairment, axonal degeneration, and cell death, in primary neuronal culture models, iPSC-derived human MNs, and zebrafish models in vivo. These results suggest that modulation of PP1-Drp1 activity may be a therapeutic target for multiple pathological features of ALS.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/metabolismo , Proteína Fosfatase 1/metabolismo , Animais , Morte Celular/genética , Morte Celular/fisiologia , Modelos Animais de Doenças , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Mutação/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Peixe-Zebra
19.
Front Mol Neurosci ; 13: 614435, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33505245

RESUMO

Variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3), which encodes postsynaptic scaffolds, are associated with brain disorders. The targeted alleles in a few Shank3 knock-out (KO) lines contain a neomycin resistance (Neo) cassette, which may perturb the normal expression of neighboring genes; however, this has not been investigated in detail. We previously reported an unexpected increase in the mRNA expression of Shank3 exons 1-12 in the brains of Shank3B KO mice generated by replacing Shank3 exons 13-16 with the Neo cassette. In this study, we confirmed that the increased Shank3 mRNA in Shank3B KO brains produced an unusual ∼60 kDa Shank3 isoform (Shank3-N), which did not properly localize to the synaptic compartment. Functionally, Shank3-N overexpression altered the dendritic spine morphology in cultured neurons. Importantly, Shank3-N expression in Shank3B KO mice was not a compensatory response to a reduction of full-length Shank3 because expression was still detected in the brain after normalizing the level of full-length Shank3. Moreover, in another Shank3 KO line (Shank3 gKO) with a similar Shank3 exonal deletion as that in Shank3B KO mice but without a Neo cassette, the mRNA expression levels of Shank3 exons 1-12 were lower than those of wild-type mice and Shank3-N was not detected in the brain. In addition, the expression levels of genes neighboring Shank3 on chromosome 15 were altered in the striatum of Shank3B KO but not Shank3 gKO mice. These results suggest that the Neo cassette has potential off-target effects in Shank3B KO mice.

20.
Nitric Oxide ; 21(2): 110-9, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19527794

RESUMO

Protease-activated receptors (PARs) play important roles in the regulation of brain function such as neuroinflammation by transmitting the signal from proteolytic enzymes such as thrombin and trypsin. We and others have reported that a member of the family, PAR-2 is activated by trypsin, whose involvement in the neurophysiological process is increasingly evident, and is involved in the neuroinflammatory processes including morphological changes of astrocytes. In this study, we investigated the role of PAR-2 in the production of nitric oxide (NO) in rat primary astrocytes. Treatment of PAR-2 agonist trypsin increased NO production in a dose-dependent manner, which was mediated by the induction of inducible nitric-oxide synthase. The trypsin-mediated production of NO was mimicked by PAR-2 agonist peptide and reduced by either pharmacological PAR-2 antagonist peptide or by siRNA-mediated inhibition of PAR-2 expression, which suggests the critical role of PAR-2 in this process. NO production by PAR-2 was mimicked by PMA, a PKC activator, and was attenuated by Go6976, a protein kinase C (PKC) inhibitor. PAR-2 stimulation activated three subtypes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. NO production by PAR-2 was blocked by inhibition of ERK, p38, and JNK pathways. PAR-2 stimulation also activated nuclear factor-kappaB (NF-kappaB) DNA binding and transcriptional activity as well as IkappaBalpha phosphorylation. Inhibitors of NF-kappaB pathway inhibited PAR-2-mediated NO production. In addition, inhibitors of MAPK pathways prevented transcriptional activation of NF-kappaB reporter constructs. These results suggest that PAR-2 activation-mediated NO production in astrocytes is transduced by the activation of MAPKs followed by NF-kappaB pathways.


Assuntos
Astrócitos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico/biossíntese , Receptor PAR-2/metabolismo , Análise de Variância , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Sobrevivência Celular/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Tripsina/farmacologia
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