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Insulin resistance is a major public health burden that often results in other comorbidities including type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), and cardiovascular disease. An insulin sensitizer has the potential to become a disease-modifying therapy. It remains an unmet medical need to identify therapeutics that target the insulin signaling pathway to treat insulin resistance. Low-molecular-weight protein tyrosine phosphatase (LMPTP) negatively regulates insulin signaling and has emerged as a potential therapeutic target for insulin sensitization. Genetic studies have demonstrated that LMPTP is positively associated with obesity in humans and promotes insulin resistance in rodents. A recent study showed that pharmacological inhibition or genetic deletion of LMPTP protects mice from high-fat diet-induced insulin resistance and diabetes. Here, we show that loss of LMPTP by genetic deletion has no significant effects on improving glucose tolerance in lean or diet-induced obese mice. Furthermore, our data demonstrate that LMPTP deficiency potentiates cardiac hypertrophy that leads to mild cardiac dysfunction. Our findings suggest that the development of LMPTP inhibitors for the treatment of insulin resistance and type 2 diabetes should be reevaluated, and further studies are needed to characterize the molecular and pathophysiological role of LMPTP.NEW & NOTEWORTHY Inhibition of LMPTP with a small-molecule inhibitor, Cmpd23, improves glucose tolerance in mice as reported earlier. However, genetic deficiency of the LMPTP-encoding gene, Acp1, has limited effects on glucose metabolism but leads to mild cardiac hypertrophy in mice. The findings suggest the potential off-target effects of Cmpd23 and call for reevaluation of LMPTP as a therapeutic target for the treatment of insulin resistance and type 2 diabetes.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Animales , Cardiomegalia/genética , Cardiomegalia/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/genética , Dieta Alta en Grasa , Glucosa/metabolismo , Insulina/metabolismo , Resistencia a la Insulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas/uso terapéutico , DelgadezRESUMEN
Cytotoxic CD8+ T cells are the primary effector cells mediating anti-tumor responses. In vivo monitoring of CD8+ T cells has broad implications for the development of novel cancer therapies. Here we describe the development of a genetically engineered mouse model (GEMM) in which CD8+ T cells are labeled with an optical reporter, enabling in vivo, longitudinal monitoring using bioluminescence imaging (BLI). Firefly luciferase (Luc2), human diphtheria toxin receptor (DTR), and enhanced green fluorescence protein (eGFP) cDNAs are engineered under the CD8α promoter to generate a transgenic mouse line. Luciferase mRNA and CD8α mRNA were generally correlated in various tissues from these mice. Sorted splenic CD8+ T cells, CD4+ T cells and CD3- non-T cells verified that the luciferase signal is specific to CD8+ T cells. In vivo imaging showed that luciferase signal was detected in various immune organs, such as lymph nodes, thymus, and spleen, and the detection was confirmed by ex vivo examination. Administration of diphtheria toxin markedly reduced luciferase signal systemically, confirming the function of the DTR. In the MC38 mouse syngeneic model, we observed significant increases in CD8+ T cells with mDX400 treatment, an anti PD-1 mouse monoclonal antibody that correlated with tumor growth inhibition. This novel reporter GEMM is a valuable drug discovery tool for profiling compounds and understanding mechanisms of action in immunotherapy of cancer.
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Linfocitos T CD8-positivos , Luciferasas , Animales , Anticuerpos Monoclonales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Genes Reporteros/genética , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismoRESUMEN
Programmed cell death-1 (PD-1) blockade has a profound effect on the ability of the immune system to eliminate tumors, but many questions remain about the cell types involved and the underlying mechanisms of immune activation. To shed some light on this, the cellular and molecular events following inhibition of PD-1 signaling was investigated in the MC-38 colon carcinoma model using constitutive (PD-1 KO) and conditional (PD1cKO) mice and in wild-type mice treated with PD-1 antibody. The impact on both tumor growth and the development of tumor immunity was assessed. In the PD-1cKO mice, a complete deletion of Pdcd1 in tumor-infiltrating T cells (TILs) after tamoxifen treatment led to the inhibition of tumor growth of both small and large tumors. Extensive immune phenotypic analysis of the TILs by flow and mass cytometry identified 20-different T cell subsets of which specifically 5-CD8 positive ones expanded in all three models after PD-1 blockade. All five subsets expressed granzyme B and interferon gamma (IFNγ). Gene expression analysis of the tumor further supported the phenotypic analysis in both PD-1cKO- and PD-1 Ab-treated mice and showed an upregulation of pathways related to CD4 and CD8 T-cell activation, enhanced signaling through costimulatory molecules and IFNγ, and non-T-cell processes. Altogether, using PD-1cKO mice, we define the intrinsic nature of PD-1 suppression of CD8 T-cell responses in tumor immunity.
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Linfocitos T CD8-positivos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Experimentales/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Femenino , Inhibidores de Puntos de Control Inmunológico/farmacología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Muerte Celular Programada 1/deficienciaRESUMEN
3D imaging in animal models, during development or in adults, facilitates the identification of structural morphological changes that cannot be achieved with traditional 2D histological staining. Through the reconstruction of whole embryos or a region-of-interest, specific changes are better delimited and can be easily quantified. We focused here on high-resolution episcopic microscopy (HREM), and its potential for visualizing and quantifying the organ systems of normal and genetically altered embryos and adult organisms. Although the technique is based on episcopic images, these are of high resolution and are close to histological quality. The images reflect the tissue structure and densities revealed by histology, albeit in a grayscale color map. HREM technology permits researchers to take advantage of serial 2D aligned stacks of images to perform 3D reconstructions. Three-dimensional visualization allows for an appreciation of topology and morphology that is difficult to achieve with classical histological studies. The nature of the data lends itself to novel forms of computational analysis that permit the accurate quantitation and comparison of individual embryos in a manner that is impossible with histology. Here, we have developed a new HREM prototype consisting of the assembly of a Leica Biosystems Nanocut rotary microtome with optics and a camera. We describe some examples of applications in the prenatal and adult lifestage of the mouse to show the added value of HREM for phenotyping experimental cohorts to compare and quantify structure volumes. At prenatal stages, segmentations and 3D reconstructions allowed the quantification of neural tissue and ventricular system volumes of normal brains at E14.5 and E16.5 stages. 3D representations of normal cranial and peripheric nerves at E15.5 and of the normal urogenital system from stages E11.5 to E14.5 were also performed. We also present a methodology to quantify the volume of the atherosclerotic plaques of ApoEtm1Unc/tm1Unc mutant mice and illustrate a 3D reconstruction of knee ligaments in adult mice.
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Gene targeting and additive (random) transgenesis have proven to be powerful technologies with which to decipher the mammalian genome. With the advent of CRISPR/Cas9 genome editing, the ability to inactivate or modify the function of a gene has become even more accessible. However, the impact of each generated modification may be different from what was initially desired. Minimal validation of mutant alleles from genetically altered (GA) rodents remains essential to guarantee the interpretation of experimental results. The protocol described here combines design strategies for genomic and functional validation of genetically modified alleles with droplet digital PCR (ddPCR) or quantitative PCR (qPCR) for target DNA or mRNA quantification. In-depth analysis of the results obtained with GA models through the analysis of target DNA and mRNA quantification is also provided, to evaluate which pitfalls can be detected using these two methods, and we propose recommendations for the characterization of different type of mutant allele (knock-out, knock-in, conditional knock-out, FLEx, IKMC model or transgenic). Our results also highlight the possibility that mRNA expression of any mutated allele can be different from what might be expected in theory or according to common assumptions. For example, mRNA analyses on knock-out lines showed that nonsense-mediated mRNA decay is generally not achieved with a critical-exon approach. Likewise, comparison of multiple conditional lines crossed with the same CreERT2 deleter showed that the inactivation outcome was very different for each conditional model. DNA quantification by ddPCR of G0 to G2 generations of transgenic rodents generated by pronuclear injection showed an unexpected variability, demonstrating that G1 generation rodents cannot be considered as established lines.
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Sistemas CRISPR-Cas , Alelos , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas/genética , ADN , Genómica , ARN Mensajero , Reacción en Cadena en Tiempo Real de la Polimerasa , Roedores/genéticaRESUMEN
[This corrects the article DOI: 10.3389/fimmu.2020.573405.].
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The molecule "T cell immunoreceptor with immunoglobulin and ITIM domain," or TIGIT, has recently received much attention as a promising target in the treatment of various malignancies. In spite of the quick progression of anti-TIGIT antibodies into clinical testing both as monotherapy and in combination with programmed cell death-1 (PD-1)-directed immune checkpoint blockade, the molecular mechanism behind the observed therapeutic benefits remains poorly understood. Here we demonstrate, using mouse tumor models, that TIGIT blocking antibodies with functional Fc binding potential induce effective anti-tumor response. Our observations reveal that the anti-TIGIT therapeutic effect is not achieved by depletion of intratumoral regulatory T cells (Treg) or any cell population expressing TIGIT, but instead is mediated by possible "reverse activating signals" through FcγRs on myeloid cells, inducing expression of various mediators such as cytokines and chemokines. Furthermore, we discovered an induction of a robust and persistent granzyme B and perforin response, distinct from a predominantly interferon-γ (IFN-γ)-driven anti-PD-1 blockade. Our observations, for the first time, provide the basis for a mechanistic hypothesis involving the requirement of a functional Fc domain of anti-TIGIT monoclonal antibodies, of which various isotypes are currently under intense clinical investigation.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/farmacología , Células Mieloides/efectos de los fármacos , Receptores de IgG/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Femenino , Granzimas/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/inmunología , Células Mieloides/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Microambiente TumoralRESUMEN
Analgesic properties of orthosteric agonists of the muscarinic M4 receptor subtype have been documented in literature reports, with evidence from pharmacological and in vivo receptor knock out (KO) studies. Constitutive M4 receptor KO mice demonstrated an increased response in the formalin pain model, supporting this hypothesis. Two novel positive allosteric modulators (PAM) of the M4 receptor, Compounds 1 and 2, were characterized in rodent models of acute nociception. Results indicated decreased time spent on nociceptive behaviors in the mouse formalin model, and efficacy in the mouse tail flick assay. The analgesic-like effects of Compounds 1 and 2 were shown to be on target, as the compounds lacked any activity in constitutive M4 KO mice, while retaining activity in wild type control littermates. The analgesic-like effects of Compounds 1 and 2 were significantly diminished in KO mice that have selective deletion of the M4 receptor in neurons that co-express the dopaminergic D1 receptor subtype, suggesting a centrally-mediated effect on nociception. The opioid antagonist naloxone did not diminish the effect of Compound 1, indicating the effects of Compound 1 are not secondarily linked to opioid pathways. Compound 1 was evaluated in the rat, where it demonstrated analgesic-like effects in tail flick and a subpopulation of spinal nociceptive sensitive neurons, suggesting some involvement of spinal mechanisms of nociceptive modulation. These studies indicate that M4 PAMs may be a tractable target for pain management assuming an appropriate safety profile, and it appears likely that both spinal and supraspinal pathways may mediate the antinociceptive-like effects.
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Regulación Alostérica/efectos de los fármacos , Nocicepción/efectos de los fármacos , Receptor Muscarínico M4/agonistas , Regulación Alostérica/fisiología , Analgésicos/farmacología , Analgésicos Opioides/farmacología , Animales , Colinérgicos/farmacología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antagonistas de Narcóticos/farmacología , Nocicepción/fisiología , Dolor/metabolismo , Dolor/fisiopatología , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M4/efectos de los fármacos , Receptor Muscarínico M4/metabolismoRESUMEN
BACE1 is the first enzyme involved in APP processing, thus it is a strong therapeutic target candidate for Alzheimer's disease. The observation of deleterious phenotypes in BACE1 Knock-out (KO) mouse models (germline and conditional) raised some concerns on the safety and tolerability of BACE1 inhibition. Here, we have employed a tamoxifen inducible BACE1 conditional Knock-out (cKO) mouse model to achieve a controlled partial depletion of BACE1 in adult mice. Biochemical and behavioural characterization was performed at two time points: 4-5 months (young mice) and 12-13 months (aged mice). A ~50% to ~70% BACE1 protein reduction in hippocampus and cortex, respectively, induced a significant reduction of BACE1 substrates processing and decrease of Aßx-40 levels at both ages. Hippocampal axonal guidance and peripheral nerve myelination were not affected. Aged mice displayed a CA1 long-term potentiation (LTP) deficit that was not associated with memory impairment. Our findings indicate that numerous phenotypes observed in germline BACE1 KO reflect a fundamental role of BACE1 during development while other phenotypes, observed in adult cKO, may be absent when partially rather than completely deleting BACE1. However, we demonstrated that partial depletion of BACE1 still induces CA1 LTP impairment, supporting a role of BACE1 in synaptic plasticity in adulthood.
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Secretasas de la Proteína Precursora del Amiloide/deficiencia , Ácido Aspártico Endopeptidasas/deficiencia , Orientación del Axón/genética , Región CA1 Hipocampal , Corteza Cerebral , Eliminación de Gen , Plasticidad Neuronal/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Región CA1 Hipocampal/enzimología , Región CA1 Hipocampal/patología , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Ratones , Ratones NoqueadosRESUMEN
Fructose consumption in humans and animals has been linked to enhanced de novo lipogenesis, dyslipidemia, and insulin resistance. Hereditary deficiency of ketohexokinase (KHK), the first enzymatic step in fructose metabolism, leads to essential fructosuria in humans, characterized by elevated levels of blood and urinary fructose following fructose ingestion but is otherwise clinically benign. To address whether KHK deficiency is associated with altered glucose and lipid metabolism, a Khk knockout (KO) mouse line was generated and characterized. NMR spectroscopic analysis of plasma following ingestion of [6-13C] fructose revealed striking differences in biomarkers of fructose metabolism. Significantly elevated urine and plasma 13C-fructose levels were observed in Khk KO vs. wild-type (WT) control mice, as was reduced conversion of 13C-fructose into plasma 13C-glucose and 13C-lactate. In addition, the observation of significant levels of fructose-6-phosphate in skeletal muscle tissue of Khk KO, but not WT, mice suggests a potential mechanism, whereby fructose is metabolized via muscle hexokinase in the absence of KHK. Khk KO mice on a standard chow diet displayed no metabolic abnormalities with respect to ambient glucose, glucose tolerance, body weight, food intake, and circulating trigylcerides, ß-hydroxybutyrate, and lactate. When placed on a high-fat and high-fructose (HF/HFruc) diet, Khk KO mice had markedly reduced liver weight, triglyceride levels, and insulin levels. Together, these results suggest that Khk KO mice may serve as a good model for essential fructosuria in humans and that inhibition of KHK offers the potential to protect from diet-induced hepatic steatosis and insulin resistance.
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Dieta , Fructoquinasas/deficiencia , Errores Innatos del Metabolismo de la Fructosa/genética , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal/genética , Dieta Alta en Grasa , Ingestión de Alimentos/genética , Fructoquinasas/genética , Fructoquinasas/metabolismo , Errores Innatos del Metabolismo de la Fructosa/metabolismo , Fructosafosfatos/sangre , Intolerancia a la Glucosa/genética , Resistencia a la Insulina , Metabolismo de los Lípidos/genética , Ratones NoqueadosRESUMEN
Although tau pathology, behavioral deficits, and neuronal loss are observed in patients with tauopathies, the relationship between these endpoints has not been clearly established. Here we found that rTg4510 mice, which overexpress human mutant tau in the forebrain, develop progressive age-dependent increases in locomotor activity (LMA), which correlates with neurofibrillary tangle (NFT) pathology, hyperphosphorylated tau levels, and brain atrophy. To further clarify the relationship between these endpoints, we treated the rTg4510 mice with either doxycycline to reduce mutant tau expression or an O-GlcNAcase inhibitor Thiamet G, which has been shown to ameliorate tau pathology in animal models. We found that both doxycycline and Thiamet G treatments starting at 2 months of age prevented the progression of hyperactivity, slowed brain atrophy, and reduced brain hyperphosphorylated tau. In contrast, initiating doxycycline treatment at 4 months reduced neither brain hyperphosphorylated tau nor hyperactivity, further confirming the relationship between these measures. Collectively, our results demonstrate a unique behavioral phenotype in the rTg4510 mouse model of tauopathy that strongly correlates with disease progression, and that early interventions which reduce tau pathology ameliorate the progression of the locomotor dysfunction. These findings suggest that better understanding the relationship between locomotor deficits and tau pathology in the rTg4510 model may improve our understanding of the mechanisms underlying behavioral disturbances in patients with tauopathies.
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Tauopatías/tratamiento farmacológico , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Doxiciclina/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Actividad Motora/genética , Actividad Motora/fisiología , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ovillos Neurofibrilares/patología , Fosforilación , Piranos/uso terapéutico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tauopatías/patología , Tauopatías/fisiopatología , Tiazoles/uso terapéutico , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , Proteínas tau/genéticaRESUMEN
Histone Deacetylase 11 (HDAC11) is highly expressed in the central nervous system where it has been reported to have roles in neural differentiation. In contrast with previous studies showing nuclear and cytoplasmic localisation, we observed synaptic enrichment of HDAC11. Knockout mouse models for HDACs 1-9 have been important for guiding the development of isoform specific HDAC inhibitors as effective therapeutics. Given the close relationship between HDAC11 and neural cells in vitro, we examined neural tissue in a previously uncharacterised Hdac11 knockout mouse (Hdac11 KO/KO). Loss of HDAC11 had no obvious impact on brain morphology and neural stem/precursor cells isolated from Hdac11 KO/KO mice had comparable proliferation and differentiation characteristics. However, in differentiating neural cells we observed decreased expression of schizophrenia-associated gene Fez1 (fasciculation and elongation protein zeta 1), a gene previously reported to be regulated by HDAC11 activity. FEZ1 has been associated with the dendritic growth of neurons and risk of schizophrenia via its interaction with DISC1 (disrupted in schizophrenia 1). Examination of cortical, cerebellar and hippocampal tissue reveal decreased Fez1 expression specifically in the hippocampus of adult mice. The results of this study demonstrate that loss of HDAC11 has age dependent and brain-region specific consequences.
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Proteínas Adaptadoras Transductoras de Señales/genética , Regulación de la Expresión Génica , Hipocampo/metabolismo , Histona Desacetilasas/genética , Proteínas del Tejido Nervioso/genética , Esquizofrenia/genética , Envejecimiento , Animales , Línea Celular , Hipocampo/ultraestructura , Ratones , Ratones Noqueados , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , NeurogénesisRESUMEN
ATP6AP2 codes for the (pro)renin receptor and is an essential component of vacuolar H+ ATPase. Activating (pro)renin for conversion of Angiotensinogen to Angiotensin makes ATP6AP2 attractive for drug intervention. Tissue-specific ATP6AP2 inactivation in mouse suggested a strong impact on various organs. Consistent with this, we found that embryonic ablation of Atp6ap2 resulted in both male hemizygous lethality and female haploinsufficiency. Next, we examined the phenotype of an induced inactivation in the adult animal, most akin to detect potential effect of functional interference of ATP6AP2 through drug therapy. Induced ablation of Atp6ap2, even without equal efficiency in all tissues (aorta, brain and kidney), resulted in rapid lethality marked by weight loss, changes in nutritional as well as blood parameters, leukocyte depletion, and bone marrow hypoplasia. Upon Atp6ap2 ablation, the colon demonstrated a rapid disruption of crypt morphology, aberrant proliferation, cell-death activation, as well as generation of microadenomas. Consequently, disruption of ATP6AP2 is extremely poorly tolerated in the adult, and severely affects various organ systems demonstrating that ATP6AP2 is an essential gene implicated in basic cellular mechanisms and necessary for multiple organ function. Accordingly, any potential drug targeting of this gene product must be strictly assessed for safety.
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Insuficiencia Multiorgánica/mortalidad , Insuficiencia Multiorgánica/patología , ATPasas de Translocación de Protón/deficiencia , Receptores de Superficie Celular/deficiencia , Animales , Técnicas de Inactivación de Genes , Ratones , Análisis de SupervivenciaRESUMEN
BACKGROUND: Hyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies. METHODS: Genetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A "click" chemistry labeling method is developed for the detection of O-GlcNAcylated tau. RESULTS: Substantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity. CONCLUSION: The present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer's disease and other neurodegenerative tauopathies.
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Tauopatías/metabolismo , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , Proteínas tau/metabolismo , Animales , Ratones , Ratones Transgénicos , Procesamiento Proteico-Postraduccional , Piranos/farmacología , Tiazoles/farmacologíaRESUMEN
Previous work has suggested that activation of mGlu5 receptor augments NMDA receptor function and thereby may constitute a rational approach addressing glutamate hypofunction in schizophrenia and a target for novel antipsychotic drug development. Here, we report the in vitro activity, in vivo efficacy and safety profile of 5PAM523 (4-Fluorophenyl){(2R,5S)-5-[5-(5-fluoropyridin-2-yl)-1,2,4-oxadiazol-3-yl]-2-methylpiperidin-1-yl}methanone), a structurally novel positive allosteric modulator selective of mGlu5. In cells expressing human mGlu5 receptor, 5PAM523 potentiated threshold responses to glutamate in fluorometric calcium assays, but does not have any intrinsic agonist activity. 5PAM523 acts as an allosteric modulator as suggested by the binding studies showing that 5PAM523 did not displace the binding of the orthosteric ligand quisqualic acid, but did partially compete with the negative allosteric modulator, MPyEP. In vivo, 5PAM523 reversed amphetamine-induced locomotor activity in rats. Therefore, both the in vitro and in vivo data demonstrate that 5PAM523 acts as a selective mGlu5 PAM and exhibits anti-psychotic like activity. To study the potential for adverse effects and particularly neurotoxicity, brain histopathological exams were performed in rats treated for 4 days with 5PAM523 or vehicle. The brain exam revealed moderate to severe neuronal necrosis in the rats treated with the doses of 30 and 50 mg/kg, particularly in the auditory cortex and hippocampus. To investigate whether this neurotoxicity is mechanism specific to 5PAM523, similar safety studies were carried out with three other structurally distinct selective mGlu5 PAMs. Results revealed a comparable pattern of neuronal cell death. Finally, 5PAM523 was tested in mGlu5 knock-out (KO) and wild type (WT) mice. mGlu5 WT mice treated with 5PAM523 for 4 days at 100 mg/kg presented significant neuronal death in the auditory cortex and hippocampus. Conversely, mGlu5 KO mice did not show any neuronal loss by histopathology, suggesting that enhancement of mGlu5 function is responsible for the toxicity of 5PAM523. This study reveals for the first time that augmentation of mGlu5 function with selective allosteric modulators results in neurotoxicity.
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Antipsicóticos/toxicidad , Benzamidas/toxicidad , Encéfalo/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Fármacos actuantes sobre Aminoácidos Excitadores/toxicidad , Oxadiazoles/toxicidad , Receptor del Glutamato Metabotropico 5/metabolismo , Regulación Alostérica , Animales , Antipsicóticos/química , Antipsicóticos/farmacocinética , Benzamidas/química , Benzamidas/farmacocinética , Encéfalo/patología , Encéfalo/fisiopatología , Células CHO , Muerte Celular/fisiología , Células Cultivadas , Cricetulus , Fármacos actuantes sobre Aminoácidos Excitadores/química , Fármacos actuantes sobre Aminoácidos Excitadores/farmacocinética , Femenino , Humanos , Masculino , Ratones de la Cepa 129 , Ratones Noqueados , Necrosis/patología , Necrosis/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/fisiología , Síndromes de Neurotoxicidad/patología , Síndromes de Neurotoxicidad/fisiopatología , Oxadiazoles/química , Oxadiazoles/farmacocinética , Ratas Sprague-Dawley , Ratas Wistar , Receptor del Glutamato Metabotropico 5/genéticaRESUMEN
Modulation of TASK-3 (Kcnk9) potassium channels affect neurotransmitter release in thalamocortical centers and other sleep-related nuclei having the capacity to regulate arousal cycles and REM sleep changes associated with mood disorders and antidepressant action. Circumstantial evidence from this and previous studies suggest the potential for TASK-3 to be a novel antidepressant therapeutic target; TASK-3 knock-out mice display augmented circadian amplitude and exhibit sleep architecture characterized by suppressed REM activity. Detailed analysis of locomotor activity indicates that the amplitudes of activity bout duration and bout number are augmented in TASK-3 mutants well beyond that seen in wildtypes, findings substantiated by amplitude increases in body temperature and EEG recordings of sleep stage bouts. Polysomnographic analysis of TASK-3 mutants reveals increases in nocturnal active wake and suppressed REM sleep time while increased slow wave sleep typifies the inactive phase, findings that have implications for the cognitive impact of reduced TASK-3 activity. In direct measures of their resistance to despair behavior, TASK-3 knock-outs displayed significant decreases in immobility relative to wildtype controls in both tail suspension and forced swim tests. Treatment of wildtype animals with the antidepressant Fluoxetine markedly reduced REM sleep, while leaving active wake and slow wave sleep relatively intact. Remarkably, these effects were absent in TASK-3 mutants indicating that TASK-3 is either directly involved in the mechanism of this drug's action, or participates in parallel pathways that achieve the same effect. Together, these results support the TASK-3 channel to act as a therapeutic target for antidepressant action.
Asunto(s)
Antidepresivos de Segunda Generación/farmacología , Nivel de Alerta/fisiología , Ritmo Circadiano/fisiología , Fluoxetina/farmacología , Canales de Potasio/metabolismo , Sueño REM/fisiología , Animales , Conducta Animal/fisiología , Depresión/tratamiento farmacológico , Depresión/metabolismo , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Masculino , Análisis por Apareamiento , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes Neurológicos , Fenotipo , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genéticaRESUMEN
Chondroitin sulphate proteoglycans (CSPGs) upregulated in the glial scar inhibit axon regeneration via their sulphated glycosaminoglycans (GAGs). Chondroitin 6-sulphotransferase-1 (C6ST-1) is upregulated after injury leading to an increase in 6-sulphated GAG. In this study, we ask if this increase in 6-sulphated GAG is responsible for the increased inhibition within the glial scar, or whether it represents a partial reversion to the permissive embryonic state dominated by 6-sulphated glycosaminoglycans (GAGs). Using C6ST-1 knockout mice (KO), we studied post-injury changes in chondroitin sulphotransferase (CSST) expression and the effect of chondroitin 6-sulphates on both central and peripheral axon regeneration. After CNS injury, wild-type animals (WT) showed an increase in mRNA for C6ST-1, C6ST-2 and C4ST-1, but KO did not upregulate any CSSTs. After PNS injury, while WT upregulated C6ST-1, KO showed an upregulation of C6ST-2. We examined regeneration of nigrostriatal axons, which demonstrate mild spontaneous axon regeneration in the WT. KO showed many fewer regenerating axons and more axonal retraction than WT. However, in the PNS, repair of the median and ulnar nerves led to similar and normal levels of axon regeneration in both WT and KO. Functional tests on plasticity after the repair also showed no evidence of enhanced plasticity in the KO. Our results suggest that the upregulation of 6-sulphated GAG after injury makes the extracellular matrix more permissive for axon regeneration, and that the balance of different CSs in the microenvironment around the lesion site is an important factor in determining the outcome of nervous system injury.
Asunto(s)
Axones/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Regeneración , Sulfatos/metabolismo , Animales , Conducta Animal/fisiología , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Fuerza de la Mano/fisiología , Ratones , Plasticidad Neuronal/fisiología , Sistema Nervioso Periférico/citología , Sistema Nervioso Periférico/lesiones , Sistema Nervioso Periférico/metabolismo , Sistema Nervioso Periférico/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismo , Médula Espinal/fisiología , Sustancia Negra/citología , Sustancia Negra/metabolismo , Sustancia Negra/fisiología , Sulfotransferasas/deficiencia , Sulfotransferasas/genética , Regulación hacia Arriba , Carbohidrato SulfotransferasasRESUMEN
Synaptic transmission and long-term potentiation (LTP) in the CA1 region of hippocampal slices have been studied during ageing of a double transgenic mouse strain relevant to early-onset familial Alzheimer's disease (AD). This strain, which over-expresses both the 695 amino acid isoform of human amyloid precursor protein (APP) with K670N and M671L mutations and presenilin 1 with the A246E mutation, has accelerated amyloidosis and plaque formation. There was a decrease in synaptic transmission in both wildtype and transgenic mice between 2 and 9 months of age. However, preparing slices from 14 month old animals in kynurenic acid (1 mM) counteracted this age-related deficit. Basal transmission and paired-pulse facilitation was similar between the two groups at all ages (2, 6, 9 and 14 months) tested. Similarly, at all ages LTP, induced either by theta burst stimulation or by multiple tetani, was normal. These data show that a prolonged, substantially elevated level of Abeta are not sufficient to cause deficits in the induction or expression of LTP in the CA1 hippocampal region.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Potenciación a Largo Plazo/fisiología , Proteínas Mutantes/metabolismo , Presenilina-1/metabolismo , Envejecimiento/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Región CA1 Hipocampal/fisiopatología , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/fisiología , Humanos , Ratones , Ratones Transgénicos , Proteínas Mutantes/genética , Placa Amiloide/metabolismo , Placa Amiloide/patología , Presenilina-1/genética , Transmisión Sináptica/fisiología , Tetania/fisiopatologíaRESUMEN
Because GABA(A) receptors containing alpha2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine's ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha2-GABA(A) receptors (alpha2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.
Asunto(s)
Trastornos Relacionados con Cocaína/metabolismo , Trastornos Relacionados con Cocaína/psicología , Cocaína/farmacología , Aprendizaje/efectos de los fármacos , Aprendizaje/fisiología , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-A/fisiología , Adulto , Animales , Azidas/farmacología , Benzodiazepinas/farmacología , Sitios de Unión/genética , Estudios de Casos y Controles , Trastornos Relacionados con Cocaína/genética , Condicionamiento Psicológico , Dopamina/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Mutación Puntual , Polimorfismo de Nucleótido Simple , Receptores de GABA-A/deficiencia , Recompensa , Adulto JovenRESUMEN
The mechanism by which aggregates of the beta-amyloid peptide (Abeta) mediate their toxicity is uncertain. We show here that the expression of the 42-amino-acid isoform of Abeta (Abeta(1-42)) changes the expression of genes involved in oxidative stress in a Drosophila model of Alzheimer's disease. A subsequent genetic screen confirmed the importance of oxidative stress and a molecular dissection of the steps in the cellular metabolism of reactive oxygen species revealed that the iron-binding protein ferritin and the H(2)O(2) scavenger catalase are the most potent suppressors of the toxicity of wild-type and Arctic (E22G) Abeta(1-42). Likewise, treatment with the iron-binding compound clioquinol increased the lifespan of flies expressing Arctic Abeta(1-42). The effect of iron appears to be mediated by oxidative stress as ferritin heavy chain co-expression reduced carbonyl levels in Abeta(1-42) flies by 65% and restored the survival and locomotion function to normal. This was achieved despite the presence of elevated levels of the Abeta(1-42). Taken together, our data show that oxidative stress, probably mediated by the hydroxyl radical and generated by the Fenton reaction, is essential for Abeta(1-42) toxicity in vivo and provide strong support for Alzheimer's disease therapies based on metal chelation.