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A 'genomically' humanized animal stably maintains and functionally expresses the genes on human chromosome fragment (hCF; <24 Mb) loaded onto mouse artificial chromosome (MAC); however, cloning of hCF onto the MAC (hCF-MAC) requires a complex process that involves multiple steps of chromosome engineering through various cells via chromosome transfer and Cre-loxP chromosome translocation. Here, we aimed to develop a strategy to rapidly construct the hCF-MAC by employing three alternative techniques: (i) application of human induced pluripotent stem cells (hiPSCs) as chromosome donors for microcell-mediated chromosome transfer (MMCT), (ii) combination of paclitaxel (PTX) and reversine (Rev) as micronucleation inducers and (iii) CRISPR/Cas9 genome editing for site-specific translocations. We achieved a direct transfer of human chromosome 6 or 21 as a model from hiPSCs as alternative human chromosome donors into CHO cells containing MAC. MMCT was performed with less toxicity through induction of micronucleation by PTX and Rev. Furthermore, chromosome translocation was induced by simultaneous cleavage between human chromosome and MAC by using CRISPR/Cas9, resulting in the generation of hCF-MAC containing CHO clones without Cre-loxP recombination and drug selection. Our strategy facilitates rapid chromosome cloning and also contributes to the functional genomic analyses of human chromosomes.
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Clonación Molecular , Animales , Cricetinae , Humanos , Ratones , Cromosomas Artificiales , Clonación Molecular/métodos , Cricetulus , Sistemas CRISPR-Cas , ADN , Edición Génica , Células Madre Pluripotentes Inducidas , Translocación GenéticaRESUMEN
Microcell-mediated chromosome transfer is an attractive technique for transferring chromosomes from donor cells to recipient cells and has enabled the generation of cell lines and humanized animal models that contain megabase-sized gene(s). However, improvements in chromosomal transfer efficiency are still needed to accelerate the production of these cells and animals. The chromosomal transfer protocol consists of micronucleation, microcell formation, and fusion of donor cells with recipient cells. We found that the combination of Taxol (paclitaxel) and reversine rather than the conventional reagent colcemid resulted in highly efficient micronucleation and substantially improved chromosomal transfer efficiency from Chinese hamster ovary donor cells to HT1080 and NIH3T3 recipient cells by up to 18.3- and 4.9-fold, respectively. Furthermore, chromosome transfer efficiency to human induced pluripotent stem cells, which rarely occurred with colcemid, was also clearly improved after Taxol and reversine treatment. These results might be related to Taxol increasing the number of spindle poles, leading to multinucleation and delaying mitosis, and reversine inducing mitotic slippage and decreasing the duration of mitosis. Here, we demonstrated that an alternative optimized protocol improved chromosome transfer efficiency into various cell lines. These data advance chromosomal engineering technology and the use of human artificial chromosomes in genetic and regenerative medical research.
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Radionuclide imaging and therapy are promising methods for controlling systemic cancers; however, their clinical application has been limited by excessive radionuclide accumulation in healthy tissues. To minimize radionuclide accumulation in non-cancerous tissues while ensuring sufficient build up in tumors, we aimed to develop a method that controlled the in vivo dynamics of radionuclides post-administration. To this end, we describe a novel strategy that combines liposomes, a potent carrier system for drug delivery, with unique radionuclide-ligand complexes based on 111In-ethylenedicysteine. Conventional 111In-ligand-complexes-carrying liposomes delivered substantial amounts of radionuclides to tumors; however, they also accumulated in the liver and spleen. In contrast, 111In-ethylenedicysteine-carrying liposomes greatly reduced non-specific accumulation, while being retained selectively at high doses within tumors. Liposomes were rapidly broken down in the liver, releasing encapsulated 111In-ligand complexes. Among the chelates used, only 111In-ethylenedicysteine could escape from the liver and be excreted in the urine. Instead, most liposomes remained intact in tumors, retaining the radionuclide-ligand complexes within them. Therefore, high tumor accumulation was obtained regardless of the type of 111In-ligand complexes in the liposomes. In vivo single photon emission computed tomography/computed tomography imaging with 111In-ethylenedicysteine-carrying liposomes accurately revealed tumor-selective radionuclide retention with little background. Hence, our new strategy could greatly enhance tumor-to-healthy tissue ratios, improve diagnostic imaging, boost therapeutic efficacy, reduce toxicity to healthy tissues, and facilitate radionuclide imaging and therapy.
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Liposomas , Neoplasias , Humanos , Ligandos , Radioisótopos , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológicoRESUMEN
We previously generated fully human antibody-producing TC-mAb mice for obtaining potential therapeutic monoclonal antibodies (mAbs). In this study, we investigated 377 clones of fully human mAbs against a tumor antigen, epithelial cell adhesion molecule (EpCAM), to determine their antigen binding properties. We revealed that a wide variety of mAbs against EpCAM can be obtained from TC-mAb mice by the combination of epitope mapping analysis of mAbs to EpCAM and native conformational recognition analysis. Analysis of 72 mAbs reacting with the native form of EpCAM indicated that the EpCL region (amino acids 24-80) is more antigenic than the EpRE region (81-265), consistent with numerous previous studies. To evaluate the potential of mAbs against antibody-drug conjugates, mAbs were directly labeled with DM1, a maytansine derivative, using an affinity peptide-based chemical conjugation (CCAP) method. The cytotoxicity of the conjugates against a human colon cancer cell line could be clearly detected with high-affinity as well as low-affinity mAbs by the CCAP method, suggesting the advantage of this method. Thus, this study demonstrated that TC-mAb mice can provide a wide variety of antibodies and revealed an effective way of identifying candidates for fully human ADC therapeutics.
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Neoplasias del Colon , Inmunoconjugados , Humanos , Ratones , Animales , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Molécula de Adhesión Celular Epitelial , Antígenos de Neoplasias , Neoplasias del Colon/patología , Anticuerpos MonoclonalesRESUMEN
Background: Cytoplasmic mislocalization of TAR-DNA binding protein of 43 kDa (TDP-43) is a major hallmark of amyotrophic lateral sclerosis (ALS). TDP-43 aggregation is detected in the cortical and spinal motor neurons in most ALS cases; however, pathological mechanism of this mislocalized TDP-43 remains unknown. Methods: We generated a tetracycline-inducible TDP-43 A315T system on a mouse artificial chromosome (MAC) vector to avoid transgene-insertional mutagenesis, established a mouse embryonic stem (ES) cell line holding this MAC vector system, and investigated whether overexpressed exogenous TDP-43 A315T was mislocalized in the cytoplasm of the ES cell-derived neurons and triggered the neurotoxic effects on these cells. Results: Inducible TDP-43 A315T system was successfully loaded onto the MAC and introduced into the mouse ES cells. These ES cells could differentiate into motor neurons and interneurons. Overexpression of TDP-43 A315T by addition of doxycycline in both neurons resulted in mislocalization to cytoplasm. Mislocalized TDP-43 caused cell death of motor neurons, but not interneurons. Conclusion: Vulnerability to cytoplasmic mislocalized TDP-43 is selective on neuronal types, whereas mislocalization of overexpressed TDP-43 occurs in even insusceptible neurons. This inducible gene expression system using MAC remains useful for providing critical insights into appearance of TDP-43 pathology.
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Homology-directed repair-mediated knock-in (HDR-KI) in combination with CRISPR-Cas9-mediated double strand break (DSB) leads to high frequency of site-specific HDR-KI. While this characteristic is advantageous for generating genetically modified cellular and animal models, HDR-KI efficiency in mammalian cells remains low. Since avian DT40 cells offer distinct advantage of high HDR-KI efficiency, we expanded this practicality to adapt to mammalian research through sequential insertion of target sequences into mouse/human artificial chromosome vector (MAC/HAC). Here, we developed the simultaneous insertion of multiple fragments by HDR method termed the simHDR wherein a target sequence and selection markers could be loaded onto MAC simultaneously. Additionally, preparing each HDR donor containing homology arm by PCR could bypass the cloning steps of target sequence and selection markers. To confirm the functionality of the loaded HDR donors, we constructed a MAC with human leukocyte antigen A (HLA-A) gene in the DT40 cells, and verified the expression of this genomic region by reverse transcription PCR (RT-PCR) and western blotting. Collectively, the simHDR offers a rapid and convenient approach to generate genetically modified models for investigating gene functions, as well as understanding disease mechanisms and therapeutic interventions.
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Sistemas CRISPR-Cas , Reparación del ADN por Recombinación , Ratones , Animales , Humanos , Sistemas CRISPR-Cas/genética , Cromosomas Artificiales , Genoma , Reacción en Cadena de la Polimerasa , Edición Génica , Mamíferos/genéticaRESUMEN
The fine-tuning of gene expression is critical for all cellular processes; aberrations in this activity can lead to pathology, and conversely, resilience. As their role in coordinating organismal responses to both internal and external factors have increasingly come into focus, small non-coding RNAs have emerged as an essential component to disease etiology. Using Systemic RNA interference Defective (SID) mutants of the nematode Caenorhabditis elegans, deficient in gene silencing, we examined the potential consequences of dysfunctional epigenomic regulation in the context of Parkinson's disease (PD). Specifically, the loss of either the sid-1 or sid-3 genes, which encode a dsRNA transporter and an endocytic regulatory non-receptor tyrosine kinase, respectively, conferred neuroprotection to dopaminergic (DA) neurons in an established transgenic C. elegans strain wherein overexpression of human α-synuclein (α-syn) from a chromosomally integrated multicopy transgene causes neurodegeneration. We further show that knockout of a specific microRNA, mir-2, attenuates α-syn neurotoxicity; suggesting that the native targets of mir-2-dependent gene silencing represent putative neuroprotective modulators. In support of this, we demonstrated that RNAi knockdown of multiple mir-2 targets enhanced α-syn-induced DA neurodegeneration. Moreover, we demonstrate that mir-2 overexpression originating in the intestine can induce neurodegeneration of DA neurons, an effect that was reversed by pharmacological inhibition of SID-3 activity. Interestingly, sid-1 mutants retained mir-2-induced enhancement of neurodegeneration. Transcriptomic analysis of α-syn animals with and without a sid-1 mutation revealed 27 differentially expressed genes with human orthologs related to a variety of diseases, including PD. Among these was pgp-8, encoding a P-glycoprotein-related ABC transporter. Notably, sid-1; pgp-8 double mutants abolished the neurodegeneration resulting from intestinal mir-2 overexpression. This research positions known regulators of small RNA-dependent gene silencing within a framework that facilitates mechanistic evaluation of epigenetic responses to exogenous and endogenous factors influencing DA neurodegeneration, revealing a path toward new targets for therapeutic intervention of PD.
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Proteínas de Caenorhabditis elegans , Enfermedad de Parkinson , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Humanos , Enfermedad de Parkinson/patología , Interferencia de ARN , ARN Bicatenario/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Mammalian artificial chromosomes derived from native chromosomes have been applied to biomedical research and development by generating cell sources and transchromosomic (Tc) animals. Human artificial chromosome (HAC) is a precedent chromosomal vector which achieved generation of valuable humanized animal models for fully human antibody production and human pharmacokinetics. While humanized Tc animals created by HAC vector have attained significant contributions, there was a potential issue to be addressed regarding stability in mouse tissues, especially highly proliferating hematopoietic cells. Mouse artificial chromosome (MAC) vectors derived from native mouse chromosome 11 demonstrated improved stability, and they were utilized for humanized Tc mouse production as a standard vector. In mouse, however, stability of MAC vector derived from native mouse chromosome other than mouse chromosome 11 remains to be evaluated. To clarify the potential of mouse centromeres in the additional chromosomes, we constructed a new MAC vector from native mouse chromosome 10 to evaluate the stability in Tc mice. The new MAC vector was transmitted through germline and stably maintained in the mouse tissues without any apparent abnormalities. Through this study, the potential of additional mouse centromere was demonstrated for Tc mouse production, and new MAC is expected to be used for various applications.
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Cromosomas Artificiales , Cromosomas/genética , Células Madre Embrionarias/metabolismo , Técnicas de Transferencia de Gen , Ingeniería Genética/métodos , Vectores Genéticos/genética , Recombinación Genética , Animales , Centrómero , Células Madre Embrionarias/citología , Femenino , Células Germinativas , Masculino , Ratones , Ratones Endogámicos ICRRESUMEN
Development of monoclonal antibody is critical for targeted drug delivery because its characteristics determine improved therapeutic efficacy and reduced side-effect. Antibody therapeutics target surface molecules; hence, internalization is desired for drug delivery. As an antibody-drug conjugate, a critical parameter is drug-to-antibody ratio wherein the quantity of drugs attached to the antibody influences the antibody structure, stability, and efficacy. Here, we established a cell-based immunotoxin screening system to facilitate the isolation of functional antibodies with internalization capacities, and discovered an anti-human CD71 monoclonal antibody. To overcome the limitation of drug-to-antibody ratio, we employed the encapsulation capacity of liposome, and developed anti-CD71 antibody-conjugated liposome that demonstrated antigen-antibody dependent cellular uptake when its synthesis was optimized. Furthermore, anti-CD71 antibody-conjugated liposome encapsulating doxorubicin demonstrated antigen-antibody dependent cytotoxicity. In summary, this study demonstrates the powerful pipeline to discover novel functional antibodies, and the optimal method to synthesize immunoliposomes. This versatile technology offers a rapid and direct approach to generate antibodies suitable for drug delivery modalities.
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Anticuerpos/inmunología , Liposomas , Animales , Complejo Antígeno-Anticuerpo , Antígenos CD/inmunología , Línea Celular Tumoral , Doxorrubicina/química , Humanos , Ratones , Ratones Endogámicos BALB C , Receptores de Transferrina/inmunologíaRESUMEN
Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional ITs are tested in the clinical trials or at the preclinical level. For next generation of ITs, as well as antibody-mediated drug delivery systems, specific targeting by monoclonal antibodies is critical to enhance efficacies and reduce side effects, and this methodological field remains open to discover potent therapeutic monoclonal antibodies. Here, we describe our application of engineered toxin termed a cell-based IT screening system. This unique screening strategy offers the following advantages: (1) identification of monoclonal antibodies that recognize cell-surface molecules, (2) selection of the antibodies that are internalized into the cells, (3) selection of the antibodies that induce cytotoxicity since they are linked with toxins, and (4) determination of state-specific activities of the antibodies by differential screening under multiple experimental conditions. Since the functional monoclonal antibodies with internalization capacities have been identified successfully, we have pursued their subsequent modifications beyond antibody drug conjugates, resulting in development of immunoliposomes. Collectively, this screening system by using engineered toxin is a versatile platform, which enables straight-forward and rapid selection for discovery of novel functional antibodies.
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Anticuerpos Monoclonales/farmacología , Membrana Celular/metabolismo , Ensayos Analíticos de Alto Rendimiento , Inmunoconjugados/farmacología , Inmunotoxinas/farmacología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Transporte Biológico , Membrana Celular/inmunología , Citotoxicidad Inmunológica , Toxina Diftérica/inmunología , Toxina Diftérica/metabolismo , Toxina Diftérica/farmacología , Exotoxinas/inmunología , Exotoxinas/metabolismo , Exotoxinas/farmacología , Humanos , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Inmunoconjugados/metabolismo , Inmunotoxinas/genética , Inmunotoxinas/inmunología , Inmunotoxinas/metabolismo , Interleucina-2/inmunología , Interleucina-2/metabolismo , Interleucina-2/farmacología , Liposomas , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
BACKGROUND/AIM: Tumor angiogenesis induced by vascular endothelial growth factor (VEGF) and/or fibroblast growth factor (FGF) plays an important role in tumor growth, metastasis, and drug resistance. However, the characteristics of tumor vessels derived from these angiogenic factors have not been fully explored. MATERIALS AND METHODS: To functionally examine tumor vessels, we developed in vivo VEGF- and FGF-induced tumor blood vessel models. We performed immunohistochemistry and Hoechst perfusion assay to elucidate histopathological differences between the derived tumor vessels. To kinetically understand tumor perfusion, we employed radiolabeled PEGylated liposomes. RESULTS: While tumor vessel density was substantially increased by enhanced expression levels of VEGF and FGF, permeability of VEGF-driven tumor vessels was significantly higher than that of FGF-driven ones, the latter demonstrating an increased number of pericyte-covered vessels. Accordingly, we observed an increased tumor retention of the PEGylated liposomes in the VEGF-driven tumor. CONCLUSION: Our in vivo models of tumor vessel demonstrate the frequency of pericyte coverage and tumor perfusion levels as major functional differences between VEGF- and FGF-driven tumor vessels.
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Factores de Crecimiento de Fibroblastos/metabolismo , Neoplasias/irrigación sanguínea , Neovascularización Patológica/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Factores de Crecimiento de Fibroblastos/genética , Humanos , Inmunohistoquímica , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neovascularización Patológica/metabolismo , Niacinamida/análogos & derivados , Niacinamida/farmacología , Compuestos de Fenilurea/farmacología , Sorafenib , Factor A de Crecimiento Endotelial Vascular/genética , GemcitabinaRESUMEN
We previously reported that eribulin mesylate (eribulin), a tubulin-binding drug (TBD), could remodel tumor vasculature (i.e. increase tumor vessels and perfusion) in human breast cancer xenograft models. However, the role of this vascular remodeling in antitumor effects is not fully understood. Here, we investigated the effects of eribulin-induced vascular remodeling on antitumor activities in multiple human cancer xenograft models. Microvessel densities (MVD) were evaluated by immunohistochemistry (CD31 staining), and antitumor effects were examined in 10 human cancer xenograft models. Eribulin significantly increased MVD compared to the controls in six out of 10 models with a correlation between enhanced MVD levels and antitumor effects (R2 = 0.54). Because of increased MVD, we next used radiolabeled liposomes to examine whether eribulin treatment would result in increased tumoral accumulation levels of these macromolecules and, indeed, we found that eribulin, unlike vinorelbine (another TBD) enhanced them. As eribulin increased accumulation of radiolabeled liposomes, we postulated that this treatment might enhance the antitumor effect of Doxil (a liposomal anticancer agent) and facilitate recruitment of immune cells into the tumor. As expected, eribulin enhanced antitumor activity of Doxil in a post-erlotinib treatment H1650 (PE-H1650) xenograft model. Furthermore, infiltrating CD11b-positive immune cells were significantly increased in multiple eribulin-treated xenografted tumors, and natural killer (NK) cell depletion reduced the antitumor effects of eribulin. These findings suggest a contribution of the immune cells for antitumor activities of eribulin. Taken together, our results suggest that vascular remodeling induced by eribulin acts as a microenvironment modulator and, consequently, this alteration enhanced the antitumor effects of eribulin.
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Furanos/administración & dosificación , Cetonas/administración & dosificación , Neoplasias/tratamiento farmacológico , Microambiente Tumoral/efectos de los fármacos , Remodelación Vascular/efectos de los fármacos , Animales , Doxorrubicina/administración & dosificación , Doxorrubicina/análogos & derivados , Femenino , Células HCT116 , Humanos , Ratones , Neoplasias/patología , Polietilenglicoles/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Liposomal anticancer agents can effectively deliver drugs to tumor lesions, but their therapeutic effects are enhanced in only limited number of patients. Appropriate biomarkers to identify responder patients to these liposomal agents will improve their treatment efficacies. We carried out pharmacological and histopathological analyses of mouse xenograft models bearing human ovarian cancers (Caov-3, SK-OV-3, KURAMOCHI, and TOV-112D) to correlate the therapeutic effects of doxorubicin-encapsulated liposome (Doxil(®) ) and histological characteristics linked to the enhanced permeability and retention effect. We next generated (111) In-encapsulated liposomes to examine their capacities to determine indications for Doxil(®) treatment by single-photon emission computed tomography (SPECT)/CT imaging. Antitumor activities of Doxil(®) were drastically enhanced in Caov-3, moderately in SK-OV-3, and minimally in KURAMOCHI and TOV-112D when compared to doxorubicin. Microvessel density and vascular perfusion were high in Caov-3 and SK-OV-3, indicating a close relation with the enhanced antitumor effects. Next, (111) In-encapsulated liposomes were given i.v. to the animals. Their tumor accumulation and area under the curve values over 72 h were high in Caov-3, relatively high in SK-OV-3, and low in two other tumors. Importantly, as both Doxil(®) effects and liposomal accumulation varied in the SK-OV-3 group, we individually obtained SPECT/CT images of SK-OV-3-bearing mouse (n = 11) before Doxil(®) treatment. Clear correlation between liposomal tumor accumulation and effects of Doxil(®) was confirmed (R(2) = 0.73). Taken together, our experiments definitely verified that enhanced therapeutic effects through liposomal formulations of anticancer agents depend on tumor accumulation of liposomes. Tumor accumulation of the radiolabeled liposomes evaluated by SPECT/CT imaging is applicable to appropriately determine indications for liposomal antitumor agents.
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Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/análogos & derivados , Neoplasias Ováricas/diagnóstico por imagen , Animales , Línea Celular Tumoral , Doxorrubicina/farmacocinética , Femenino , Humanos , Radioisótopos de Indio/farmacocinética , Liposomas , Ratones , Ratones Desnudos , Polietilenglicoles/farmacocinética , Tomografía Computarizada de Emisión de Fotón Único , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: We explored a recovery correction technique that can correct metabolite loss during perchloric acid (PCA) extraction and minimize inter-assay variance in quantitative (1)H nuclear magnetic resonance (NMR) spectroscopy of the brain and evaluated its efficacy in 5-fluorouracil (5-FU)- and saline-administered rats. METHODS: We measured the recovery of creatine and dl-valine-2,3-d2 from PCA extract containing both compounds (0.5 to 8 mM). We intravenously administered either 5-FU for 4 days (total, 100 mg/kg body weight) or saline into 2 groups of 11 rats each. We subsequently performed PCA extraction of the whole brain on Day 9, externally adding 7 µmol of dl-valine-2,3-d2. We estimated metabolite concentrations using an NMR spectrometer with recovery correction, correcting metabolite concentrations based on the recovery factor of dl-valine-2,3-d2. For each metabolite concentration, we calculated the coefficient of variation (CEV) and compared differences between the 2 groups using unpaired t-test. RESULTS: Equivalent recoveries of dl-valine-2,3-d2 (89.4 ± 3.9%) and creatine (89.7 ± 3.9%) in the PCA extract of the mixed solution indicated the suitability of dl-valine-2,3-d2 as an internal reference. In the rat study, recovery of dl-valine-2,3-d2 was 90.6 ± 9.2%. Nine major metabolite concentrations adjusted by recovery of dl-valine-2,3-d2 in saline-administered rats were comparable to data in the literature. CEVs of these metabolites were reduced from 10 to 17% before to 7 to 16% after correction. The significance of differences in alanine and taurine between the 5-FU- and saline-administered groups was determined only after recovery correction (0.75 ± 0.12 versus 0.86 ± 0.07 for alanine; 5.17 ± 0.59 versus 5.66 ± 0.42 for taurine [µmol/g brain tissue]; P < 0.05). CONCLUSION: A new recovery correction technique corrected metabolite loss during PCA extraction, minimized inter-assay variance in quantitative (1)H NMR spectroscopy of brain tissue, and effectively detected inter-group differences in concentrations of brain metabolites between 5-FU- and saline-administered rats.
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Encefalopatías/metabolismo , Encéfalo/metabolismo , Fluorouracilo/administración & dosificación , Espectroscopía de Resonancia Magnética/métodos , Percloratos , Valina/metabolismo , Animales , Creatina/metabolismo , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Cloruro de Sodio/administración & dosificaciónRESUMEN
Neurodegenerative diseases represent an increasing burden in our aging society, yet the underlying metabolic factors influencing onset and progression remain poorly defined. The relationship between impaired IGF-1/insulin-like signaling (IIS) and lifespan extension represents an opportunity to investigate the interface of metabolism with age-associated neurodegeneration. Using data sets of established DAF-2/IIS-signaling components in Caenorhabditis elegans, we conducted systematic RNAi screens in worms to select for daf-2-associated genetic modifiers of α-synuclein misfolding and dopaminergic neurodegeneration, two clinical hallmarks of Parkinson's disease. An outcome of this strategy was the identification of GPI-1/GPI, an enzyme in glucose metabolism, as a daf-2-regulated modifier that acts independent of the downstream cytoprotective transcription factor DAF-16/FOXO to modulate neuroprotection. Subsequent mechanistic analyses using Drosophila and mouse primary neuron cultures further validated the conserved nature of GPI neuroprotection from α-synuclein proteotoxicity. Collectively, these results support glucose metabolism as a conserved functional node at the intersection of proteostasis and neurodegeneration.
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Neuronas Dopaminérgicas/metabolismo , Glucosa-6-Fosfato Isomerasa/metabolismo , Envejecimiento , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Citocinas/antagonistas & inhibidores , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/citología , Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Transcripción Forkhead/metabolismo , Glucosa/metabolismo , Glucosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Glucosa-6-Fosfato Isomerasa/genética , Glucólisis , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor de Insulina/antagonistas & inhibidores , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
Alzheimer's disease (AD) is a common, progressive neurodegenerative disorder without effective disease-modifying therapies. The accumulation of amyloid-ß peptide (Aß) is associated with AD. However, identifying new compounds that antagonize the underlying cellular pathologies caused by Aß has been hindered by a lack of cellular models amenable to high-throughput chemical screening. To address this gap, we use a robust and scalable yeast model of Aß toxicity where the Aß peptide transits through the secretory and endocytic compartments as it does in neurons. The pathogenic Aß 1-42 peptide forms more oligomers and is more toxic than Aß 1-40 and genome-wide genetic screens identified genes that are known risk factors for AD. Here, we report an unbiased screen of â¼140,000 compounds for rescue of Aß toxicity. Of â¼30 hits, several were 8-hydroxyquinolines (8-OHQs). Clioquinol (CQ), an 8-OHQ previously reported to reduce Aß burden, restore metal homeostasis, and improve cognition in mouse AD models, was also effective and rescued the toxicity of Aß secreted from glutamatergic neurons in Caenorhabditis elegans. In yeast, CQ dramatically reduced Aß peptide levels in a copper-dependent manner by increasing degradation, ultimately restoring endocytic function. This mirrored its effects on copper-dependent oligomer formation in vitro, which was also reversed by CQ. This unbiased screen indicates that copper-dependent Aß oligomer formation contributes to Aß toxicity within the secretory/endosomal pathways where it can be targeted with selective metal binding compounds. Establishing the ability of the Aß yeast model to identify disease-relevant compounds supports its further exploitation as a validated early discovery platform.
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Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Clioquinol/farmacología , Endocitosis/fisiología , Proteolisis/efectos de los fármacos , Péptidos beta-Amiloides/toxicidad , Animales , Caenorhabditis elegans , Descubrimiento de Drogas/métodos , Endocitosis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , LevadurasRESUMEN
AIMS: Cells have developed quality control systems for protection against proteotoxicity. Misfolded and aggregation-prone proteins, which are behind the initiation and progression of many neurodegenerative diseases (ND), are known to challenge the proteostasis network of the cells. We aimed to explore the role of DNJ-27/ERdj5, an endoplasmic reticulum (ER)-resident thioredoxin protein required as a disulfide reductase for the degradation of misfolded proteins, in well-established Caenorhabditis elegans models of Alzheimer, Parkinson and Huntington diseases. RESULTS: We demonstrate that DNJ-27 is an ER luminal protein and that its expression is induced upon ER stress via IRE-1/XBP-1. When dnj-27 expression is downregulated by RNA interference we find an increase in the aggregation and associated pathological phenotypes (paralysis and motility impairment) caused by human ß-amyloid peptide (Aß), α-synuclein (α-syn) and polyglutamine (polyQ) proteins. In turn, DNJ-27 overexpression ameliorates these deleterious phenotypes. Surprisingly, despite being an ER-resident protein, we show that dnj-27 downregulation alters cytoplasmic protein homeostasis and causes mitochondrial fragmentation. We further demonstrate that DNJ-27 overexpression substantially protects against the mitochondrial fragmentation caused by human Aß and α-syn peptides in these worm models. INNOVATION: We identify C. elegans dnj-27 as a novel protective gene for the toxicity associated with the expression of human Aß, α-syn and polyQ proteins, implying a protective role of ERdj5 in Alzheimer, Parkinson and Huntington diseases. CONCLUSION: Our data support a scenario where the levels of DNJ-27/ERdj5 in the ER impact cytoplasmic protein homeostasis and the integrity of the mitochondrial network which might underlie its protective effects in models of proteotoxicity associated to human ND.
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Caenorhabditis elegans/genética , Proteínas del Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Enfermedades Neurodegenerativas/genética , Péptidos beta-Amiloides/metabolismo , Animales , Animales Modificados Genéticamente , Autofagia/genética , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animales de Enfermedad , Degradación Asociada con el Retículo Endoplásmico , Expresión Génica , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Mitocondrias/metabolismo , Chaperonas Moleculares/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Péptidos/metabolismo , Fenotipo , Complejo de la Endopetidasa Proteasomal , Proteolisis , Interferencia de ARN , alfa-Sinucleína/metabolismoRESUMEN
Aß (beta-amyloid peptide) is an important contributor to Alzheimer's disease (AD). We modeled Aß toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aß was previously unknown. The factors identified in yeast modified Aß toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aß impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aß, endocytosis, and human AD risk factors can be ascertained with yeast as a model system.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Endocitosis , Fragmentos de Péptidos/metabolismo , Saccharomyces cerevisiae , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Clatrina/metabolismo , Citoesqueleto/metabolismo , Susceptibilidad a Enfermedades , Estudios de Asociación Genética , Pruebas Genéticas , Glutamatos/metabolismo , Humanos , Proteínas de Ensamble de Clatrina Monoméricas/genética , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Neuronas/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Multimerización de Proteína , Transporte de Proteínas , Ratas , Factores de Riesgo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vías SecretorasRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinson's disease (PD) and have also been found in the more common sporadic form of PD, thus positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD.