RESUMO
Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)-mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER-mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial- and NOX4-derived ROS. The TMX-knockdown-induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1-positive and NFAT1-negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial- and redox-related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox-controlled ER-mitochondria-NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease.
Assuntos
Melanoma/patologia , Proteínas de Membrana/metabolismo , Fatores de Transcrição NFATC/metabolismo , Oxirredução , Isomerases de Dissulfetos de Proteínas/metabolismo , Tiorredoxinas/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Progressão da Doença , Retículo Endoplasmático/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Melanoma/metabolismo , Proteínas de Membrana/genética , Camundongos , Mitocôndrias/metabolismo , NADPH Oxidase 4/metabolismo , Transplante de Neoplasias , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Análise de Sobrevida , Tiorredoxinas/genética , Regulação para CimaRESUMO
Synaptic transmission between neurons relies on the exact spatial organization of postsynaptic transmitter receptors, which are recruited and positioned by dedicated scaffolding and regulatory proteins. At GABAergic synapses, the regulatory protein collybistin (Cb, also known as ARHGEF9) interacts with small GTPases, cell adhesion proteins and phosphoinositides to recruit the scaffolding protein gephyrin and GABAA receptors to nascent synapses. We dissected the interaction of Cb with the small Rho-like GTPase TC10 (also known as RhoQ) and phospholipids. Our data define a protein-lipid interaction network that controls the clustering of gephyrin at synapses. Within this network, TC10 and monophosphorylated phosphoinositides, particulary phosphatidylinositol 3-phosphate (PI3P), provide a coincidence detection platform that allows the accumulation and activation of Cb in endomembranes. Upon activation, TC10 induces a phospholipid affinity switch in Cb, which allows Cb to specifically interact with phosphoinositide species present at the plasma membrane. We propose that this GTPase-based regulatory switch mechanism represents an important step in the process of tethering of Cb-dependent scaffolds and receptors at nascent postsynapses.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Transmissão Sináptica/genética , Análise por Conglomerados , Humanos , Sinapses/metabolismoRESUMO
Cerebral disease manifestation occurs in about two thirds of males with X-linked adrenoleukodystrophy (CALD) and is fatally progressive if left untreated. Early histopathologic studies categorized CALD as an inflammatory demyelinating disease, which led to repeated comparisons to multiple sclerosis (MS). The aim of this study was to revisit the relationship between axonal damage and myelin loss in CALD. We applied novel immunohistochemical tools to investigate axonal damage, myelin loss and myelin repair in autopsy brain tissue of eight CALD and 25 MS patients. We found extensive and severe acute axonal damage in CALD already in prelesional areas defined by microglia loss and relative myelin preservation. In contrast to MS, we did not observe selective phagocytosis of myelin, but a concomitant decay of the entire axon-myelin unit in all CALD lesion stages. Using a novel marker protein for actively remyelinating oligodendrocytes, breast carcinoma-amplified sequence (BCAS) 1, we show that repair pathways are activated in oligodendrocytes in CALD. Regenerating cells, however, were affected by the ongoing disease process. We provide evidence that-in contrast to MS-selective myelin phagocytosis is not characteristic of CALD. On the contrary, our data indicate that acute axonal injury and permanent axonal loss are thus far underestimated features of the disease that must come into focus in our search for biomarkers and novel therapeutic approaches.
Assuntos
Adrenoleucodistrofia , Esclerose Múltipla , Adrenoleucodistrofia/metabolismo , Axônios/metabolismo , Humanos , Masculino , Esclerose Múltipla/patologia , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismoRESUMO
A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.
Assuntos
Microscopia , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Myelination of axons facilitates the rapid propagation of electrical signals and the long-term integrity of axons. The ubiquitin-proteasome system is essential for proper protein homeostasis, which is particularly crucial for interactions of postmitotic cells. In our study, we examined how the E3 ubiquitin ligase FBXO7-SCF (SKP1, Cul1, F-box protein) expressed in myelinating cells affects the axon-myelin unit. Deletion of Fbxo7 in oligodendrocytes and Schwann cells in mice using the Cnp1-Cre driver line led to motor impairment due to hindlimb paresis. It did not result in apoptosis of myelinating cells, nor did it affect the proper myelination of axons or lead to demyelination. It however triggered axonal degeneration in the CNS and resulted in the severe degeneration of axons in the PNS, inducing a full-blown neuropathy. Both the CNS and PNS displayed inflammation, while the PNS was also characterized by fibrosis, massive infiltration of macrophages, and edema. Tamoxifen-induced deletion of Fbxo7, after myelination using the Plp1-CreERT2 line, led to a small number of degenerated axons and hence a very mild peripheral neuropathy. Interestingly, loss of Fbxo7 also resulted in reduced proteasome activity in Schwann cells but not in cerebellar granule neurons, indicating a specific sensitivity of the former cell type. Together, our results demonstrate an essential role for FBXO7 in myelinating cells to support associated axons, which is fundamental to the proper developmental establishment and the long-term integrity of the axon-myelin unit.SIGNIFICANCE STATEMENT The myelination of axons facilitates the fast propagation of electrical signals and the trophic support of the myelin-axon unit. Here, we report that deletion of Fbxo7 in myelinating cells in mice triggered motor impairment but had no effect on myelin biogenesis. Loss of Fbxo7 in myelinating glia, however, led to axonal degeneration in the CNS and peripheral neuropathy of the axonal type. In addition, we found that Schwann cells were particularly sensitive to Fbxo7 deficiency reflected by reduced proteasome activity. Based on these findings, we conclude that Fbxo7 is essential for the support of the axon-myelin unit and long-term axonal health.
Assuntos
Axônios/metabolismo , Proteínas F-Box/genética , Bainha de Mielina/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Animais , Apoptose , Axônios/patologia , Células Cultivadas , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Proteínas F-Box/metabolismo , Feminino , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/patologia , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologia , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Mutations in the FBXO7 (PARK15) gene have been implicated in a juvenile form of parkinsonism termed parkinsonian pyramidal syndrome (PPS), characterized by Parkinsonian symptoms and pyramidal tract signs. FBXO7 (F-box protein only 7) is a subunit of the SCF (SKP1/cullin-1/F-box protein) E3 ubiquitin ligase complex, but its relevance and function in neurons remain to be elucidated. Here, we report that the E3 ligase FBXO7-SCF binds to and ubiquitinates the proteasomal subunit PSMA2. In addition, we show that FBXO7 is a proteasome-associated protein involved in proteasome assembly. In FBXO7 knockout mice, we find reduced proteasome activity and early-onset motor deficits together with premature death. In addition, we demonstrate that NEX (neuronal helix-loop-helix protein-1)-Cre-induced deletion of the FBXO7 gene in forebrain neurons or the loss of FBXO7 in tyrosine hydroxylase (TH)-positive neurons results in motor defects, reminiscent of the phenotype in PARK15 patients. Taken together, our study establishes a vital role for FBXO7 in neurons, which is required for proper motor control and accentuates the importance of FBXO7 in proteasome function.
Assuntos
Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Técnicas de Inativação de Genes , Transtornos Parkinsonianos/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Camundongos Knockout , Processamento de Proteína Pós-Traducional , UbiquitinaçãoRESUMO
Pelizaeus-Merzbacher disease (PMD) is an untreatable and fatal leukodystrophy. In a model of PMD with perturbed blood-brain barrier integrity, cholesterol supplementation promotes myelin membrane growth. Here, we show that in contrast to the mouse model, dietary cholesterol in two PMD patients did not lead to a major advancement of hypomyelination, potentially because the intact blood-brain barrier precludes its entry into the CNS. We therefore turned to a PMD mouse model with preserved blood-brain barrier integrity and show that a high-fat/low-carbohydrate ketogenic diet restored oligodendrocyte integrity and increased CNS myelination. This dietary intervention also ameliorated axonal degeneration and normalized motor functions. Moreover, in a paradigm of adult remyelination, ketogenic diet facilitated repair and attenuated axon damage. We suggest that a therapy with lipids such as ketone bodies, that readily enter the brain, can circumvent the requirement of a disrupted blood-brain barrier in the treatment of myelin disease.
Assuntos
Doenças Desmielinizantes/patologia , Proteína Proteolipídica de Mielina/metabolismo , Oligodendroglia/fisiologia , Doença de Pelizaeus-Merzbacher/patologia , Animais , Dieta Cetogênica , Modelos Animais de Doenças , Camundongos , Oligodendroglia/metabolismo , Organogênese/fisiologiaRESUMO
The original article was published.
RESUMO
The cerebellum is crucial for sensorimotor coordination. The cerebellar architecture not only requires proper development but also long-term integrity to ensure accurate functioning. Developmental defects such as impaired neuronal migration or neurodegeneration are thus detrimental to the cerebellum and can result in movement disorders including ataxias. In this study, we identify FBXO41 as a novel CNS-specific F-box protein that localizes to the centrosome and the cytoplasm of neurons and demonstrate that cytoplasmic FBXO41 promotes neuronal migration. Interestingly, deletion of the FBXO41 gene results in a severely ataxic gait in mice, which show delayed neuronal migration of granule neurons in the developing cerebellum in addition to deformities and degeneration of the mature cerebellum. We show that FBXO41 is a critical factor, not only for neuronal migration in the cerebellum, but also for its long-term integrity.
Assuntos
Encéfalo/patologia , Movimento Celular/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/patologia , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia , Animais , Animais Recém-Nascidos , Sobrevivência Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Proteínas F-Box/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Fenótipo , Frações Subcelulares/metabolismoRESUMO
The ability of somatosensory neurons to perceive mechanical stimuli relies on specialized mechanotransducing proteins and their molecular environment. Only recently has the identity of a major transducer of mechanical forces in vertebrates been revealed by the discovery of Piezo2. Further work has established its pivotal role for innocuous touch in mice. Therefore, Piezo2 offers a unique platform for the molecular investigation of somatosensory mechanosensation. We performed a mass spectrometry-based interactomics screen on native Piezo2 in somatosensory neurons of mouse dorsal root ganglia (DRG). Stringent and quantitative data analysis yielded the identity of 36 novel binding partners of Piezo2. The biological significance of this data set is reflected by functional experiments demonstrating a role for Pericentrin in modulating Piezo2 activity and membrane expression in somatosensory neurons. Collectively, our findings provide a framework for understanding Piezo2 physiology and serve as a rich resource for the molecular dissection of mouse somatosensation.
Assuntos
Antígenos/metabolismo , Canais Iônicos/metabolismo , Córtex Somatossensorial/citologia , Animais , Antígenos/fisiologia , Gânglios Espinais/citologia , Mecanotransdução Celular , Camundongos , Ligação Proteica , Mapas de Interação de Proteínas , Córtex Somatossensorial/metabolismoRESUMO
KV10.1 potassium channels are implicated in a variety of cellular processes including cell proliferation and tumour progression. Their expression in over 70% of human tumours makes them an attractive diagnostic and therapeutic target. Although their physiological role in the central nervous system is not yet fully understood, advances in their precise cell localization will contribute to the understanding of their interactions and function. We have determined the plasma membrane (PM) distribution of the KV10.1 protein in an enriched mouse brain PM fraction and its association with cholesterol- and sphingolipid-rich domains. We show that the KV10.1 channel has two different populations in a 3:2 ratio, one associated to and another excluded from Detergent Resistant Membranes (DRMs). This distribution of KV10.1 in isolated PM is cholesterol- and cytoskeleton-dependent since alteration of those factors changes the relationship to 1:4. In transfected HEK-293 cells with a mutant unable to bind Ca(2+)/CaM to KV10.1 protein, Kv10.1 distribution in DRM/non-DRM is 1:4. Mean current density was doubled in the cholesterol-depleted cells, without any noticeable effects on other parameters. These results demonstrate that recruitment of the KV10.1 channel to the DRM fractions involves its functional regulation.
Assuntos
Membrana Celular/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Microdomínios da Membrana/metabolismo , Neurônios/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Western Blotting , Membrana Celular/química , Colesterol/metabolismo , Citoesqueleto/metabolismo , Detergentes/metabolismo , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/genética , Feminino , Células HEK293 , Humanos , Microdomínios da Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
l-3, 4-dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinson's disease but can induce debilitating abnormal involuntary movements (dyskinesia). Here we show that the development of L-DOPA-induced dyskinesia in the rat is accompanied by upregulation of an inflammatory cascade involving nitric oxide. Male Wistar rats sustained unilateral injections of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. After three weeks animals started to receive daily treatment with L-DOPA (30 mg/kg plus benserazide 7.5 mg/kg, for 21 days), combined with an inhibitor of neuronal NOS (7-nitroindazole, 7-NI, 30 mg/kg/day) or vehicle (saline-PEG 50%). All animals treated with L-DOPA and vehicle developed abnormal involuntary movements, and this effect was prevented by 7-NI. L-DOPA-treated dyskinetic animals exhibited an increased striatal and pallidal expression of glial fibrillary acidic protein (GFAP) in reactive astrocytes, an increased number of CD11b-positive microglial cells with activated morphology, and the rise of cells positive for inducible nitric oxide-synthase immunoreactivity (iNOS). All these indexes of glial activation were prevented by 7-NI co-administration. These findings provide evidence that the development of L-DOPA-induced dyskinesia in the rat is associated with activation of glial cells that promote inflammatory responses. The dramatic effect of 7-NI in preventing this glial response points to an involvement of nitric oxide. Moreover, the results suggest that the NOS inhibitor prevents dyskinesia at least in part via inhibition of glial cell activation and iNOS expression. Our observations indicate nitric oxide synthase inhibitors as a therapeutic strategy for preventing neuroinflammatory and glial components of dyskinesia pathogenesis in Parkinson's disease.
Assuntos
Antiparkinsonianos/efeitos adversos , Discinesia Induzida por Medicamentos/tratamento farmacológico , Discinesia Induzida por Medicamentos/metabolismo , Indazóis/farmacologia , Levodopa/efeitos adversos , Neuroglia/metabolismo , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico/metabolismo , Doença de Parkinson/tratamento farmacológico , Animais , Modelos Animais de Doenças , Inflamação/induzido quimicamente , Levodopa/administração & dosagem , Masculino , Ratos , Ratos Wistar , Regulação para CimaRESUMO
Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.
Assuntos
Astrócitos , Permeabilidade da Membrana Celular , Conexina 43 , Ubiquitina-Proteína Ligases Nedd4 , Canais de Potássio Corretores do Fluxo de Internalização , Animais , Humanos , Camundongos , Conexina 43/genética , Mutação de Sentido Incorreto , Proteostase , Canais de Potássio Corretores do Fluxo de Internalização/genética , Ubiquitina-Proteína Ligases Nedd4/genética , EpilepsiaRESUMO
Neuron-glia interactions play a key role in maintaining and regulating the central nervous system. Glial cells are implicated in the function of dopamine neurons and regulate their survival and resistance to injury. Parkinson's disease is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, decreased striatal dopamine levels and consequent onset of extrapyramidal motor dysfunction. Parkinson's disease is a common chronic, neurodegenerative disorder with no effective protective treatment. In the 6-OHDA mouse model of Parkinson's disease, doxycycline administered at a dose that both induces/represses conditional transgene expression in the tetracycline system, mitigates the loss of dopaminergic neurons in the substantia nigra compacta and nerve terminals in the striatum. This protective effect was associated with: (1) a reduction of microglia in normal mice as a result of doxycycline administration per se; (2) a decrease in the astrocyte and microglia response to the neurotoxin 6-OHDA in the globus pallidus and substantia nigra compacta, and (3) the astrocyte reaction in the striatum. Our results suggest that doxycycline blocks 6-OHDA neurotoxicity in vivo by inhibiting microglial and astrocyte expression. This action of doxycycline in nigrostriatal dopaminergic neuron protection is consistent with a role of glial cells in Parkinson's disease neurodegeneration. The neuroprotective effect of doxycycline may be useful in preventing or slowing the progression of Parkinson's disease and other neurodegenerative diseases linked to glia function.
Assuntos
Doxiciclina/uso terapêutico , Neuroglia/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Adrenérgicos/toxicidade , Análise de Variância , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Antígeno de Macrófago 1/metabolismo , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Oxidopamina/toxicidade , Doença de Parkinson/etiologia , Doença de Parkinson/fisiopatologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
SUMOylation is an evolutionarily conserved eukaryotic posttranslational protein modification with broad biological relevance. Differentiating between the major small ubiquitin-like modifier (SUMO) paralogs and uncovering paralog-specific functions in vivo has long been very difficult. To overcome this problem, we generated His6-HA-Sumo2 and HA-Sumo2 knockin mouse lines, expanding upon our existing His6-HA-Sumo1 mouse line, to establish a "toolbox" for Sumo1-Sumo2 comparisons in vivo. Leveraging the specificity of the HA epitope, we performed whole-brain imaging and uncovered regional differences between Sumo1 and Sumo2 expression. At the subcellular level, Sumo2 was specifically detected in extranuclear compartments, including synapses. Immunoprecipitation coupled with mass spectrometry identified shared and specific neuronal targets of Sumo1 and Sumo2. Target validation using proximity ligation assays provided further insight into the subcellular distribution of neuronal Sumo2-conjugates. The mouse models and associated datasets provide a powerful framework to determine the native SUMO "code" in cells of the central nervous system.
RESUMO
Pancreatic ductal adenocarcinoma (PDAC) progresses in an organ with a unique pH landscape, where the stroma acidifies after each meal. We hypothesized that disrupting this pH landscape during PDAC progression triggers pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs) to induce PDAC fibrosis. We revealed that alkaline environmental pH was sufficient to induce PSC differentiation to a myofibroblastic phenotype. We then mechanistically dissected this finding, focusing on the involvement of the Na+/H+ exchanger NHE1. Perturbing cellular pH homeostasis by inhibiting NHE1 with cariporide partially altered the myofibroblastic PSC phenotype. To show the relevance of this finding in vivo, we targeted NHE1 in murine PDAC (KPfC). Indeed, tumor fibrosis decreased when mice received the NHE1-inhibitor cariporide in addition to gemcitabine treatment. Moreover, the tumor immune infiltrate shifted from granulocyte rich to more lymphocytic. Taken together, our study provides mechanistic evidence on how the pancreatic pH landscape shapes pancreatic cancer through tuning PSC differentiation.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Camundongos , Animais , Células Estreladas do Pâncreas/patologia , Linhagem Celular Tumoral , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Fenótipo , Homeostase , Fibrose , Neoplasias PancreáticasRESUMO
Pyramidal neurons of the neocortex can be subdivided into two major groups: deep- (DL) and upper-layer (UL) neurons. Here we report that the expression of the AT-rich DNA-binding protein Satb2 defines two subclasses of UL neurons: UL1 (Satb2 positive) and UL2 (Satb2 negative). In the absence of Satb2, UL1 neurons lose their identity and activate DL- and UL2-specific genetic programs. UL1 neurons in Satb2 mutants fail to migrate to superficial layers and do not contribute to the corpus callosum but to the corticospinal tract, which is normally populated by DL axons. Ctip2, a gene required for the formation of the corticospinal tract, is ectopically expressed in all UL1 neurons in the absence of Satb2. Satb2 protein interacts with the Ctip2 genomic region and controls chromatin remodeling at this locus. Satb2 therefore is required for the initiation of the UL1-specific genetic program and for the inactivation of DL- and UL2-specific genes.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/fisiologia , Mitose/fisiologia , Neocórtex/citologia , Neurônios/fisiologia , Fatores de Transcrição/fisiologia , Animais , Carbocianinas/metabolismo , Diferenciação Celular , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Eletroporação/métodos , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Neocórtex/embriologia , Neocórtex/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
For a better understanding of the mode of action of duocarmycin and its analogs, the novel fluorescent duocarmycin derivatives 13-15 and 17b-19b were synthesized, and their bioactivity as well as their cellular uptake investigated using confocal laser scanning microscopy (CLSM) in live-cell imaging experiments.