RESUMEN
AMPA receptors (AMPARs) play a critical role in synaptic plasticity and learning and memory, and dysfunction or dysregulation of AMPARs could lead to various neurological and psychiatric disorders, such as Alzheimer's disease (AD). However, the dynamics and/or longitudinal changes of AMPARs in vivo during AD pathogenesis remain elusive. Here, employing 5xFAD SEP-GluA1 KI mice, we investigated endogenous AMPA receptor dynamics in a whisker deflection-associated Go/No-go learning paradigm. We found a significant increase in synaptosomal AMPA receptor subunits GluA1 in WT mice after learning, while no such changes were detected in 7-mo-old 5xFAD mice. Daily training led to an increase in endogenous spine surface GluA1 in Control mice, while this increase was absent in 5xFAD-KI mice which correlates with its learning defects in Go/No-go paradigm. Furthermore, we demonstrated that the onset of abnormal AMPAR dynamics corresponds temporally with microglia and astrocyte overactivation. Our results have shown that impairments in endogenous AMPA receptor dynamics play an important role in learning deficits in 5xFAD mice and AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer , Receptores AMPA , Humanos , Animales , Ratones , Aprendizaje , Astrocitos , MicroglíaRESUMEN
BACKGROUND: Working memory capacity impairment is an early sign of Alzheimer's disease, but the underlying mechanisms remain unclear. Clarifying how working memory capacity is affected will help us better understand the pathological mechanism of Alzheimer's disease. We used the olfactory working memory capacity paradigm to evaluate memory capacity in 3-month-old 5XFAD (an animal model of Alzheimer's disease) mice. Immunofluorescence staining of the prefrontal cortex was performed to detect the number of FOS-positive neurons, calmodulin-dependent protein kinase II-positive neurons, and glutamate decarboxylase-positive neurons in the prelimbic cortex and infralimbic cortex. A chemogenetic method was then used to modulate the inhibition and activation of excitatory neurons in the prelimbic cortex of wild-type and 5XFAD mice and to measure the memory capacity of mice. RESULTS: Working memory capacity was significantly diminished in 5XFAD mice compared to littermate wild-type mice. Neuronal activation of the prelimbic cortex, but not the infralimbic cortex, was attenuated in 5XFAD mice performing the olfactory working memory capacity task. Subsequently, the FOS-positive neurons were co-localized with both calmodulin-dependent protein kinase II-positive neurons and glutamate decarboxylase-positive neurons. The results showed that the activation of excitatory neurons in the prelimbic cortex was correlated with working memory capacity in mice. Our results further demonstrate that the chemogenetic inhibition of prelimbic cortex excitatory neurons resulted in reduced working memory capacity in wild-type mice, while the chemogenetic activation of prelimbic cortex excitatory neurons improved the working memory capacity of 5XFAD mice. CONCLUSION: The diminished activation of prelimbic cortex excitatory neurons in 5XFAD mice during task performance is associated with reduced working memory capacity, and activation modulation of excitatory neurons by chemogenetic methods can improve memory capacity impairment in 5XFAD mice. These findings may provide a new direction for exploring Alzheimer's disease therapeutic approaches.
Asunto(s)
Enfermedad de Alzheimer , Memoria a Corto Plazo , Ratones , Animales , Memoria a Corto Plazo/fisiología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Glutamato Descarboxilasa/metabolismo , Neuronas/metabolismo , Modelos Animales de Enfermedad , Ratones TransgénicosRESUMEN
The DNA damage response (DDR) is essential for the maintenance of genomic stability. Protein posttranslational modifications play pivotal roles in regulating the DDR process. Here, we found that SUMOylated RNF168 undergoes liquid-liquid phase separation (LLPS), which restricts the recruitment of RNF168 to DNA damage sites, reduces RNF168-catalyzed H2A ubiquitination, restrains 53BP1 in nuclear condensates, and ultimately impairs nonhomologous DNA end joining repair efficiency. Sentrin/SUMO-specific protease 1 (SENP1) was identified as a specific deSUMOylase of RNF168, and it was highly expressed in colorectal adenocarcinoma. In response to DNA damage, SENP1 decreased RNF168 SUMOylation and prevented RNF168 from forming nuclear condensates, thus promoting damage repair efficiency and cancer cell resistance to DNA damaging agents. Moreover, high SENP1 expression correlated with poor prognosis in patients with cancer, and SENP1 depletion sensitized cancer cells to chemotherapy. In summary, these findings reveal DDR is suppressed by SUMOylation-induced LLPS of RNF168 and suggest that SENP1 is a potential target for cancer therapy. SIGNIFICANCE: Sentrin/SUMO-specific protease 1 decreases RNF168 SUMOylation and liquid-liquid phase separation to promote DNA damage repair, safeguarding genomic integrity and driving chemotherapy resistance.
Asunto(s)
Neoplasias del Colon , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Péptido Hidrolasas/metabolismo , Reparación del ADN , Ubiquitinación , Daño del ADN , Endopeptidasas , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Resistencia a Medicamentos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismoRESUMEN
Proper microtubule dynamics are critical for neuronal morphogenesis and functions, and their dysregulation results in neurological disorders and regeneration failure. Superior cervical ganglion-10 (SCG10, also known as stathmin-2 or STMN2) is a well-known regulator of microtubule dynamics in neurons, but its functions in the peripheral nervous system remain largely unknown. Here, we show that Scg10 knockout mice exhibit severely progressive motor and sensory dysfunctions with significant sciatic nerve myelination deficits and neuromuscular degeneration. Additionally, increased microtubule stability, shown by a significant increase in tubulin acetylation and decrease in tubulin tyrosination, and decreased axonal transport were observed in Scg10 knockout dorsal root ganglion (DRG) neurons. Furthermore, SCG10 depletion impaired axon regeneration in both injured mouse sciatic nerve and cultured DRG neurons following replating, and the impaired axon regeneration was found to be induced by a lack of SCG10-mediated microtubule dynamics in the neurons. Thus, our results highlight the importance of SCG10 in peripheral axon maintenance and regeneration.
Asunto(s)
Axones , Tubulina (Proteína) , Animales , Ratones , Axones/fisiología , Ganglios Espinales , Regeneración Nerviosa/genética , Neuronas , Estatmina/genéticaRESUMEN
Transforming growth factor-ß (TGF-ß) is known to promote breast cancer cell migration, invasion, and dissemination; however, the underlying molecular mechanisms are not yet well characterized. Here, we report that TGF-ß induces pleckstrin-2 (PLEK2) expression by Smad3 and signal transducer and activator of transcription 3 (STAT3) activating PLEK2 promoter activity. Higher PLEK2 expression is associated with poor prognosis in breast cancer patients. Overexpression and knockout experiments in MDA-MB-231 and MCF-7 breast cancer cells revealed that PLEK2 promotes cell migration, invasion, and dissemination in 2D and 3D cell culture. Moreover, PLEK2 promotes metastasis of breast cancer cells in vivo. Pleckstrin-2 localizes to the cell membrane and cell protrusions following TGF-ß treatment. Furthermore, inhibition of PI3K phosphorylation abolishes TGF-ß- and PLEK2-induced cell invasion. The carboxyl-terminal PH domain of PLEK2 is critical for TGF-ß- and PLEK2-induced Akt activation and plays an important role in cell invasion. Pleckstrin-2 interacts with PPM1B and promotes its ubiquitin-dependent degradation. The PLEK2-PPM1B axis utilizes nuclear factor-κB signaling to promote cell migration and invasion. Our data implicate the TGF-ß-STAT3/Smad3-PLEK2-PPM1B signaling cascade in TGF-ß-induced breast cancer cell migration and invasion. These findings suggest that PLEK2/PPM1B could represent novel targets for the intervention of breast cancer metastasis.
Asunto(s)
Neoplasias de la Mama , Factor de Crecimiento Transformador beta , Femenino , Humanos , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Proteínas de la Membrana/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteína Fosfatasa 2C , Factor de Crecimiento Transformador beta/metabolismo , Factores de Crecimiento TransformadoresRESUMEN
The DNA damage response (DDR) is critical for maintaining cellular homeostasis and genome integrity. Mounting evidence has shown that posttranslational protein modifications play vital roles in the DDR. In this study, we showed that deubiquitinase OTUD6A is involved in the DDR and is important for maintaining genomic stability. Mechanistically, in response to DNA damage, the abundance of OTUD6A was increased; meanwhile, PP2A interacted with OTUD6A and dephosphorylated OTUD6A at sites S70/71/74, which promoted nuclear localization of OTUD6A. Subsequently, OTUD6A was recruited to the damage site, where it interacted with TopBP1 and blocked the interaction between TopBP1 and its ubiquitin E3 ligase UBR5, decreasing K48-linked polyubiquitination and increasing the stability of TopBP1. OTUD6A depletion impaired CHK1 S345 phosphorylation and blocked cell cycle progression under DNA replication stress. Consistently, knockout of OTUD6A rendered mice hypersensitive to irradiation, shortened survival, and inhibited tumor growth by regulating TopBP1 in xenografted nude mice. Moreover, OTUD6A is expressed at high levels in breast cancer, and OTUD6A overexpression promotes cell proliferation, migration and invasion, indicating that dysregulation of OTUD6A expression contributes to genomic instability and is associated with tumor development. In summary, this study demonstrates that OTUD6A plays a critical role in promoting tumor cell resistance to chemoradiotherapy by deubiquitinating and stabilizing TopBP1.
Asunto(s)
Proteínas de Unión al ADN , Enzimas Desubicuitinizantes , Neoplasias , Animales , Ratones , Proteínas Portadoras/metabolismo , Enzimas Desubicuitinizantes/metabolismo , ADN , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Ratones Desnudos , HumanosRESUMEN
Accurate and efficient non-rigid registration is important to investigate neural mechanisms in multi-session two-photon (2p) imaging across a few days. The 2p imaging recordings from different sessions usually possess certain complex misalignment or huge data variance due to relocation errors during experimental operations or brain recovery. Most of the reported neural image registration tools were able to solve the registration problem in the same session with small deformation. However, the registration of neural images across multi-sessions remains a challenge. In this study, we report the development of a non-rigid registration method for 2p imaging in mice based on image triangulation and piecewise affine transformation (TPAT) technologies. The TPAT method supported both automatic and semi-automatic operation types, and both showed great performance in the benchmark test of non-rigid neural image registration. The proposed method constitutes a step forward in promoting and accelerating discoveries from multi-session 2p imaging research.
Asunto(s)
Algoritmos , Encéfalo , Animales , Encéfalo/diagnóstico por imagen , Imagenología Tridimensional/métodos , Imagen por Resonancia Magnética/métodos , Ratones , FotonesRESUMEN
The centrosome composed of a pair of centrioles (mother and daughter) and pericentriolar material, and is mainly responsible for microtubule nucleation and anchorage in animal cells. The subdistal appendage (SDA) is a centriolar structure located at the mother centriole's subdistal region, and it functions in microtubule anchorage. However, the molecular composition and detailed structure of the SDA remain largely unknown. Here, we identified α-taxilin and γ-taxilin as new SDA components that form a complex via their coiled-coil domains and that serve as a new subgroup during SDA hierarchical assembly. The taxilins' SDA localization is dependent on ODF2, and α-taxilin recruits CEP170 to the SDA. Functional analyses suggest that α- and γ-taxilin are responsible for SDA structural integrity and centrosomal microtubule anchorage during interphase and for proper spindle orientation during metaphase. Our results shed light on the molecular components and functional understanding of the SDA hierarchical assembly and microtubule organization.
Asunto(s)
Centriolos/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Fenómenos Biológicos , Línea Celular , Línea Celular Tumoral , Humanos , Microtúbulos/metabolismo , Dominios ProteicosRESUMEN
Many people affected by fragile X syndrome (FXS) and autism spectrum disorders have sensory processing deficits, such as hypersensitivity to auditory, tactile, and visual stimuli. Like FXS in humans, loss of Fmr1 in rodents also cause sensory, behavioral, and cognitive deficits. However, the neural mechanisms underlying sensory impairment, especially vision impairment, remain unclear. It remains elusive whether the visual processing deficits originate from corrupted inputs, impaired perception in the primary sensory cortex, or altered integration in the higher cortex, and there is no effective treatment. In this study, we used a genetic knockout mouse model (Fmr1KO), in vivo imaging, and behavioral measurements to show that the loss of Fmr1 impaired signal processing in the primary visual cortex (V1). Specifically, Fmr1KO mice showed enhanced responses to low-intensity stimuli but normal responses to high-intensity stimuli. This abnormality was accompanied by enhancements in local network connectivity in V1 microcircuits and increased dendritic complexity of V1 neurons. These effects were ameliorated by the acute application of GABAA receptor activators, which enhanced the activity of inhibitory neurons, or by reintroducing Fmr1 gene expression in knockout V1 neurons in both juvenile and young-adult mice. Overall, V1 plays an important role in the visual abnormalities of Fmr1KO mice and it could be possible to rescue the sensory disturbances in developed FXS and autism patients.
Asunto(s)
Síndrome del Cromosoma X Frágil , Animales , Modelos Animales de Enfermedad , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/complicaciones , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Humanos , Ratones , Ratones Noqueados , Neuronas/metabolismoRESUMEN
Upregulation of programmed death ligand 1 (PD-L1) helps tumor cells escape from immune surveillance, and therapeutic antibodies targeting PD-1/PD-L1 have shown better patient outcomes only in several types of malignancies. Recent studies suggest that the clinical efficacy of anti-PD-1/PD-L1 treatments is associated with PD-L1 levels; however, the underlying mechanism of high PD-L1 protein levels in cancers is not well defined. Here, we report that the deubiquitinase OTUB1 positively regulates PD-L1 stability and mediates cancer immune responses through the PD-1/PD-L1 axis. Mechanistically, we demonstrate that OTUB1 interacts with and removes K48-linked ubiquitin chains from the PD-L1 intracellular domain in a manner dependent on its deubiquitinase activity to hinder the degradation of PD-L1 through the ERAD pathway. Functionally, depletion of OTUB1 markedly decreases PD-L1 abundance, reduces PD-1 protein binding to the tumor cell surface, and causes increased tumor cell sensitivity to human peripheral blood mononuclear cells (PBMCs)-mediated cytotoxicity. Meanwhile, OTUB1 ablation-induced PD-L1 destabilization facilitates more CD8+ T cells infiltration and increases the level of IFN-γ in serum to enhance antitumor immunity in mice, and the tumor growth suppression by OTUB1 silencing could be reversed by PD-L1 overexpression. Furthermore, we observe a significant correlation between PD-L1 abundance and OTUB1 expression in human breast carcinoma. Our study reveals OTUB1 as a deubiquitinating enzyme that influences cancer immunosuppression via regulation of PD-L1 stability and may be a potential therapeutic target for cancer immunotherapy.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Enzimas Desubicuitinizantes/metabolismo , Terapia de Inmunosupresión/métodos , Inmunoterapia/métodos , Receptor de Muerte Celular Programada 1/efectos de los fármacos , Adulto , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Voluntarios Sanos , Humanos , Ratones , Transfección , Regulación hacia Arriba , Adulto JovenRESUMEN
Studies demonstrated that pneumonia can decrease vitamin A productions and vitamin A reduction/deficiency may promote asthma development. Our previous study showed that neonatal Streptococcus pneumoniae (S. pneumoniae) infection promoted asthma development. Whether neonatal S. pneumoniae pneumonia induced asthma was associated with vitamin A levels remains unclear. The aim of this study was to investigate the effects of neonatal S. pneumoniae pneumonia on vitamin A expressions, to explore the effects of vitamin A supplement after neonatal S. pneumoniae pneumonia on adulthood asthma development. Non-lethal S. pneumoniae pneumonia was established by intranasal inoculation of neonatal (1-week-old) female BALB/c mice with D39. S. pneumoniae pneumonia mice were supplemented with or without all-trans retinoic acid 24 hours after infection. Vitamin A concentrations in lung, serum and liver were measured post pneumonia until early adulthood. Four weeks after pneumonia, mice were sensitized and challenged with OVA to induce allergic airway disease (AAD). Twenty-four hours after the final challenge, the lungs and bronchoalveolar lavage fluid (BALF) were collected to assess AAD. We stated that serum vitamin A levels in neonatal S. pneumoniae pneumonia mice were lower than 0.7µmol/L from day 2-7 post infection, while pulmonary vitamin A productions were significantly lower than those in the control mice from day 7-28 post infection. Vitamin A supplement after neonatal S. pneumoniae pneumonia significantly promoted Foxp3+Treg and Th1 productions, decreased Th2 and Th17 cells expressions, alleviated airway hyperresponsiveness (AHR) and inflammatory cells infiltration during AAD. Our data suggest that neonatal S. pneumoniae pneumonia induce serum vitamin A deficiency and long-time lung vitamin A reduction, vitamin A supplement after neonatal S. pneumoniae pneumonia inhibit the progression of asthma by altering CD4+T cell subsets.
Asunto(s)
Asma/prevención & control , Suplementos Dietéticos , Neumonía Neumocócica/complicaciones , Hipersensibilidad Respiratoria/prevención & control , Streptococcus pneumoniae/inmunología , Subgrupos de Linfocitos T/inmunología , Vitamina A/administración & dosificación , Animales , Animales Recién Nacidos , Asma/etiología , Asma/metabolismo , Asma/patología , Femenino , Ratones , Ratones Endogámicos BALB C , Neumonía Neumocócica/tratamiento farmacológico , Neumonía Neumocócica/metabolismo , Hipersensibilidad Respiratoria/etiología , Hipersensibilidad Respiratoria/metabolismo , Hipersensibilidad Respiratoria/patología , Streptococcus pneumoniae/aislamiento & purificación , Subgrupos de Linfocitos T/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Vitamina A/metabolismo , Vitaminas/administración & dosificación , Vitaminas/metabolismoRESUMEN
Acute myeloid leukemia (AML) is a genetically heterogeneous malignant disorder of the hematopoietic system, characterized by the accumulation of DNA-damaged immature myeloid precursors. Here, we find that hCINAP is involved in the repair of double-stranded DNA breaks (DSB) and that its expression correlates with AML prognosis. Following DSB, hCINAP is recruited to damage sites where it promotes SENP3-dependent deSUMOylation of NPM1. This in turn results in the dissociation of RAP80 from the damage site and CTIP-dependent DNA resection and homologous recombination. NPM1 SUMOylation is required for recruitment of DNA repair proteins at the early stage of DNA-damage response (DDR), and SUMOylated NPM1 impacts the assembly of the BRCA1 complex. Knockdown of hCINAP also sensitizes a patient-derived xenograft (PDX) mouse model to chemotherapy. In clinical AML samples, low hCINAP expression is associated with a higher overall survival rate in patients. These results provide mechanistic insight into the function of hCINAP during the DNA-damage response and its role in AML resistance to therapy.
Asunto(s)
Adenilato Quinasa/metabolismo , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/genética , Reparación del ADN por Recombinación , Adenilato Quinasa/genética , Adenilato Quinasa/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antineoplásicos/uso terapéutico , Proteína BRCA1/metabolismo , Cisteína Endopeptidasas/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Femenino , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Ratones , Persona de Mediana Edad , Proteínas Nucleares/metabolismo , Nucleofosmina , Sumoilación , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
Our previous study showed that neonatal S. pneumoniae infection aggravated airway inflammation and airway hyperresponsiveness (AHR) in an OVA-induced allergic asthma model. As airway smooth muscle (ASM) plays a pivotal role in AHR development, we aim to investigate the effects of neonatal S. pneumoniae pneumonia on ASM structure and AHR development. Non-lethal neonatal pneumonia was established by intranasally infecting 1-week-old BALB/C mice with the S. pneumoniae strain D39. Five weeks after infection, the lungs were collected to assess the levels of α-SMA and the contractile proteins of ASM. Our results indicate that neonatal S. pneumoniae pneumonia significantly increased adulthood lung α-SMA and SMMHC proteins production and aggravated airway inflammatory cells infiltration and cytokines release. In addition, the neonatal S. pneumoniae pneumonia group had significantly higher Penh values compared to the uninfected controls. These data suggest that neonatal S. pneumoniae pneumonia promoted an aberrant ASM phenotype and AHR development in mice model.
Asunto(s)
Pulmón/metabolismo , Músculo Liso/metabolismo , Neumonía/genética , Hipersensibilidad Respiratoria/genética , Actinas/genética , Animales , Animales Recién Nacidos , Líquido del Lavado Bronquioalveolar/microbiología , Modelos Animales de Enfermedad , Humanos , Pulmón/microbiología , Pulmón/patología , Ratones , Músculo Liso/microbiología , Músculo Liso/patología , Fenotipo , Neumonía/complicaciones , Neumonía/microbiología , Neumonía/patología , Hipersensibilidad Respiratoria/complicaciones , Hipersensibilidad Respiratoria/microbiología , Hipersensibilidad Respiratoria/patología , Streptococcus pneumoniae/patogenicidadRESUMEN
MOTIVATION: Functional imaging at single-neuron resolution offers a highly efficient tool for studying the functional connectomics in the brain. However, mainstream neuron-detection methods focus on either the morphologies or activities of neurons, which may lead to the extraction of incomplete information and which may heavily rely on the experience of the experimenters. RESULTS: We developed a convolutional neural networks and fluctuation method-based toolbox (ImageCN) to increase the processing power of calcium imaging data. To evaluate the performance of ImageCN, nine different imaging datasets were recorded from awake mouse brains. ImageCN demonstrated superior neuron-detection performance when compared with other algorithms. Furthermore, ImageCN does not require sophisticated training for users. AVAILABILITY AND IMPLEMENTATION: ImageCN is implemented in MATLAB. The source code and documentation are available at https://github.com/ZhangChenLab/ImageCN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Redes Neurales de la Computación , Programas Informáticos , Algoritmos , Animales , RatonesRESUMEN
BACKGROUND: Optical imaging is one of the most common, low-cost imaging tools used for investigating the tumor biological behavior in vivo. This study explores the feasibility and sensitivity of a near infrared fluorescent protein mKate2 for a long-term non-invasive tumor imaging in BALB/c nude mice, by using a low-power optical imaging system. METHODS: In this study, breast cancer cell line MDA-MB-435s expressing mKate2 and MDA-MB-231 expressing a dual reporter gene firefly luciferase (fLuc)-GFP were used as cell models. Tumor cells were implanted in different animal body compartments including subcutaneous, abdominal and deep tissue area and closely monitored in real-time. A simple and low-power optical imaging system was set up to image both fluorescence and bioluminescence in live animals. RESULTS: The presence of malignant tissue was further confirmed by histopathological assay. Considering its lower exposure time and no need of substrate injection, mKate2 is considered a superior choice for subcutaneous imaging compared with fLuc. On the contrary, fLuc has shown to be a better option when monitoring the tumor in a diffusive area such as abdominal cavity. Furthermore, both reporter genes have shown good stability and sensitivity for deep tissue imaging, i.e. tumor within the liver. In addition, fLuc has shown to be an excellent method for detecting tumor cells in the lung. CONCLUSIONS: The combination of mKate2 and fLuc offers a superior choice for long-term non-invasive real-time investigation of tumor biological behavior in vivo.
Asunto(s)
Proteínas Luminiscentes/metabolismo , Imagen Óptica/métodos , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Transformación Celular Neoplásica , Femenino , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
Short-term memory (STM) is crucial for animals to hold information for a small period of time. Persistent or recurrent neural activity, together with neural oscillations, is known to encode the STM at the cellular level. However, the coding mechanisms at the microcircuitry level remain a mystery. Here, we performed two-photon imaging on behaving mice to monitor the activity of neuronal microcircuitry. We discovered a neuronal subpopulation in the medial prefrontal cortex (mPFC) that exhibited emergent properties in a context-dependent manner underlying a STM-like behavior paradigm. These neuronal subpopulations exclusively comprise excitatory neurons and mainly represent a group of neurons with stronger functional connections. Microcircuitry plasticity was maintained for minutes and was absent in an animal model of Alzheimer's disease (AD). Thus, these results point to a functional coding mechanism that relies on the emergent behavior of a functionally defined neuronal assembly to encode STM.
Asunto(s)
Memoria a Corto Plazo/fisiología , Neuronas/fisiología , Corteza Prefrontal/fisiología , Animales , Conducta Animal , Extinción Psicológica , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Red Nerviosa/fisiología , Plasticidad Neuronal , Especificidad de Órganos , Dolor/fisiopatología , SonidoRESUMEN
Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene that inactivate expression of the gene product, the fragile X mental retardation 1 protein (FMRP). In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate Fmr1 knockout (KO) rats by disruption of the fourth exon of the Fmr1 gene. Western blotting analysis confirmed that the FMRP was absent from the brains of the Fmr1 KO rats (Fmr1exon4-KO ). Electrophysiological analysis revealed that the theta-burst stimulation (TBS)-induced long-term potentiation (LTP) and the low-frequency stimulus (LFS)-induced long-term depression (LTD) were decreased in the hippocampal Schaffer collateral pathway of the Fmr1exon4-KO rats. Short-term plasticity, measured as the paired-pulse ratio, remained normal in the KO rats. The synaptic strength mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) was also impaired. Consistent with previous reports, the Fmr1exon4-KO rats demonstrated an enhanced 3,5-dihydroxyphenylglycine (DHPG)-induced LTD in the present study, and this enhancement is insensitive to protein translation. In addition, the Fmr1exon4-KO rats showed deficits in the probe trial in the Morris water maze test. These results demonstrate that deletion of the Fmr1 gene in rats specifically impairs long-term synaptic plasticity and hippocampus-dependent learning in a manner resembling the key symptoms of FXS. Furthermore, the Fmr1exon4-KO rats displayed impaired social interaction and macroorchidism, the results consistent with those observed in patients with FXS. Thus, Fmr1exon4-KO rats constitute a novel rat model of FXS that complements existing mouse models.
RESUMEN
This corrects the article DOI: 10.1038/ncomms15308.
RESUMEN
Targeting the specific metabolic phenotypes of colorectal cancer stem cells (CRCSCs) is an innovative therapeutic strategy for colorectal cancer (CRC) patients with poor prognosis and relapse. However, the context-dependent metabolic traits of CRCSCs remain poorly elucidated. Here we report that adenylate kinase hCINAP is overexpressed in CRC tissues. Depletion of hCINAP inhibits invasion, self-renewal, tumorigenesis and chemoresistance of CRCSCs with a loss of mesenchymal signature. Mechanistically, hCINAP binds to the C-terminal domain of LDHA, the key regulator of glycolysis, and depends on its adenylate kinase activity to promote LDHA phosphorylation at tyrosine 10, resulting in the hyperactive Warburg effect and the lower cellular ROS level and conferring metabolic advantage to CRCSC invasion. Moreover, hCINAP expression is positively correlated with the level of Y10-phosphorylated LDHA in CRC patients. This study identifies hCINAP as a potent modulator of metabolic reprogramming in CRCSCs and a promising drug target for CRC invasion and metastasis.
Asunto(s)
Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , L-Lactato Deshidrogenasa/genética , Células Madre Neoplásicas/metabolismo , Proteínas Nucleares/genética , Procesamiento Proteico-Postraduccional , Animales , Antineoplásicos/farmacología , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN , Doxiciclina/farmacología , Resistencia a Antineoplásicos/genética , Glucólisis/efectos de los fármacos , Glucólisis/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lactato Deshidrogenasa 5 , Ratones , Ratones Desnudos , Invasividad Neoplásica , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fenotipo , Fosforilación , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tirosina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Herpes simplex virus type 1 strain 129 (H129) has represented a promising anterograde neuronal circuit tracing tool, which complements the existing retrograde tracers. However, the current H129 derived tracers are multisynaptic, neither bright enough to label the details of neurons nor capable of determining direct projection targets as monosynaptic tracer. METHODS: Based on the bacterial artificial chromosome of H129, we have generated a serial of recombinant viruses for neuronal circuit tracing. Among them, H129-G4 was obtained by inserting binary tandemly connected GFP cassettes into the H129 genome, and H129-ΔTK-tdT was obtained by deleting the thymidine kinase (TK) gene and adding tdTomato coding gene to the H129 genome. Then the obtained viral tracers were tested in vitro and in vivo for the tracing capacity. RESULTS: H129-G4 is capable of transmitting through multiple synapses, labeling the neurons by green florescent protein, and visualizing the morphological details of the labeled neurons. H129-ΔTK-tdT neither replicates nor spreads in neurons alone, but transmits to and labels the postsynaptic neurons with tdTomato in the presence of complementary expressed TK from a helper virus. H129-ΔTK-tdT is also capable to map the direct projectome of the specific neuron type in the given brain regions in Cre transgenic mice. In the tested brain regions where circuits are well known, the H129-ΔTK-tdT tracing patterns are consistent with the previous results. CONCLUSIONS: With the assistance of the helper virus complimentarily expressing TK, H129-ΔTK-tdT replicates in the initially infected neuron, transmits anterogradely through one synapse, and labeled the postsynaptic neurons with tdTomato. The H129-ΔTK-tdT anterograde monosynaptic tracing system offers a useful tool for mapping the direct output in neuronal circuitry. H129-G4 is an anterograde multisynaptic tracer with a labeling signal strong enough to display the details of neuron morphology.