RESUMEN
Effective intracellular delivery of therapeutic proteins can potentially treat a wide array of diseases. However, efficient delivery of functional proteins across the cell membrane remains challenging. Exosomes are nanosized vesicles naturally secreted by various types of cells and may serve as promising nanocarriers for therapeutic biomolecules. Here, we engineered exosomes equipped with a photoinducible cargo protein release system, termed mMaple3-mediated protein loading into and release from exosome (MAPLEX), in which cargo proteins can be loaded into the exosomes by fusing them with photocleavable protein (mMaple3)-conjugated exosomal membrane markers and subsequently released from the exosomal membrane by inducing photocleavage with blue light illumination. Using this system, we first induced transcriptional regulation by delivering octamer-binding transcription factor 4 and SRY-box transcription factor 2 to fibroblasts in vitro. Second, we induced in vivo gene recombination in Cre reporter mice by delivering Cre recombinase. Last, we achieved targeted epigenome editing in the brains of 5xFAD and 3xTg-AD mice, two models of Alzheimer's disease. Administration of MAPLEXs loaded with ß-site amyloid precursor protein cleaving enzyme 1 (Bace1)-targeting single guide RNA-incorporated dCas9 ribonucleoprotein complexes, coupled with the catalytic domain of DNA methyltransferase 3A, resulted in successful methylation of the targeted CpG sites within the Bace1 promoter. This approach led to a significant reduction in Bace1 expression, improved recognition memory impairment, and reduced amyloid pathology in 5xFAD and 3xTg-AD mice. These results suggest that MAPLEX is an efficient intracellular protein delivery system that can deliver diverse therapeutic proteins for multiple diseases.
Asunto(s)
Enfermedad de Alzheimer , Sistemas CRISPR-Cas , Exosomas , Edición Génica , Exosomas/metabolismo , Animales , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/terapia , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Humanos , Ratones , Epigénesis Genética , Sistemas de Liberación de Medicamentos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Modelos Animales de Enfermedad , Integrasas/metabolismoRESUMEN
Systemic sclerosis is an autoimmune disease characterized by inflammatory reactions and fibrosis. Myofibroblasts are considered therapeutic targets for preventing and reversing the pathogenesis of fibrosis in systemic sclerosis. Although the mechanisms that differentiate into myofibroblasts are diverse, transforming growth factor ß (TGF-ß) is known to be a key mediator of fibrosis in systemic sclerosis. This study investigated the effects of extracellular vesicles derived from human adipose stem cells (ASC-EVs) in an in vivo systemic sclerosis model and in vitro TGF-ß1-induced dermal fibroblasts. The therapeutic effects of ASC-EVs on the in vivo systemic sclerosis model were evaluated based on dermal thickness and the number of α-smooth muscle actin (α-SMA)-expressing cells using hematoxylin and eosin staining and immunohistochemistry. Administration of ASC-EVs decreased both the dermal thickness and α-SMA expressing cell number as well as the mRNA levels of fibrotic genes, such as Acta2, Ccn2, Col1a1 and Comp. Additionally, we discovered that ASC-EVs can decrease the expression of α-SMA and CTGF and suppress the TGF-ß pathway by inhibiting the activation of SMAD2 in dermal fibroblasts induced by TGF-ß1. Finally, TGF-ß1-induced dermal fibroblasts underwent selective death through ASC-EVs treatment. These results indicate that ASC-EVs could provide a therapeutic approach for preventing and reversing systemic sclerosis.
RESUMEN
Intermittent fasting (IF) has been shown to reduce cardiovascular risk factors in both animals and humans, and can protect the heart against ischemic injury in models of myocardial infarction. However, the underlying molecular mechanisms behind these effects remain unclear. To shed light on the molecular and cellular adaptations of the heart to IF, we conducted comprehensive system-wide analyses of the proteome, phosphoproteome, and transcriptome, followed by functional analysis. Using advanced mass spectrometry, we profiled the proteome and phosphoproteome of heart tissues obtained from mice that were maintained on daily 12- or 16 hr fasting, every-other-day fasting, or ad libitum control feeding regimens for 6 months. We also performed RNA sequencing to evaluate whether the observed molecular responses to IF occur at the transcriptional or post-transcriptional levels. Our analyses revealed that IF significantly affected pathways that regulate cyclic GMP signaling, lipid and amino acid metabolism, cell adhesion, cell death, and inflammation. Furthermore, we found that the impact of IF on different metabolic processes varied depending on the length of the fasting regimen. Short IF regimens showed a higher correlation of pathway alteration, while longer IF regimens had an inverse correlation of metabolic processes such as fatty acid oxidation and immune processes. Additionally, functional echocardiographic analyses demonstrated that IF enhances stress-induced cardiac performance. Our systematic multi-omics study provides a molecular framework for understanding how IF impacts the heart's function and its vulnerability to injury and disease.
Asunto(s)
Ayuno Intermitente , Multiómica , Humanos , Ratones , Animales , Proteoma , Ayuno/fisiología , Metabolismo EnergéticoRESUMEN
Safety and efficacy are essential in the process of disease treatment. However, off-label medication use is inevitable because various medications do not contain regulatory labels for pediatric use. We aimed to examine off-label medication use and analyze the risk factors correlated with adverse drug reactions (ADRs). This study was performed retrospectively using electronic medical data from a pediatric intensive care unit (PICU) of a tertiary hospital in Korea from July 2019 to June 2020. A total 6,183 prescribed medications from 502 PICU patients were examined in the present study. A total of 80% were infants or children, and 96.0% of them were treated with off-label medications. It was discovered that 4,778 off-label cases (77.2%) of the top 100 drugs had prescriptions with dosage (67.8%). Drugs prescribed to patients admitted to the cardiothoracic department (odds ratio [OR], 3.248; p = 0.019), total number of medications (OR, 1.116; p = 0.001), and length of PICU stay of ≥ 7 days (OR, 4.981; p = 0.008) were significantly associated with ADRs. ADRs were noted to be more severe in off-label use (p = 0.0426). For appropriate medication use, evidence regarding the safety of off-label medications is required and ultimately reflected in the official regulation.
RESUMEN
Amyloid precursor protein (APP) is an evolutionarily conserved transmembrane protein and a well-characterized precursor protein of amyloid-beta (Aß) peptides, which accumulate in the brains of individuals with Alzheimer's disease (AD)-related pathologies. Aß has been extensively investigated since the amyloid hypothesis in AD was proposed. Besides Aß, previous studies on APP and its proteolytic cleavage products have suggested their diverse pathological and physiological functions. However, their roles still have not been thoroughly understood. In this review, we extensively discuss the evolutionarily-conserved biology of APP, including its structure and processing pathway, as well as recent findings on the physiological roles of APP and its fragments in the central nervous system and peripheral nervous system. We have also elaborated upon the current status of APP-targeted therapeutic approaches for AD treatment by discussing inhibitors of several proteases participating in APP processing, including α-, ß-, and γ-secretases. Finally, we have highlighted the future perspectives pertaining to further research and the potential clinical role of APP.
Asunto(s)
Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Amiloide , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , HumanosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is mediated by the interaction of the viral spike (S) protein with angiotensin-converting enzyme 2 (ACE2) on the host cell surface. Although a clinical trial testing soluble ACE2 (sACE2) for COVID-19 is currently ongoing, our understanding of the delivery of sACE2 via small extracellular vesicles (sEVs) is still rudimentary. With excellent biocompatibility allowing for the effective delivery of molecular cargos, sEVs are broadly studied as nanoscale protein carriers. In order to exploit the potential of sEVs, we design truncated CD9 scaffolds to display sACE2 on the sEV surface as a decoy receptor for the S protein of SARS-CoV-2. Moreover, to enhance the sACE2-S binding interaction, we employ sACE2 variants. sACE2-loaded sEVs exhibit typical sEVs characteristics and bind to the S protein. Furthermore, engineered sEVs inhibit the entry of wild-type (WT), the globally dominant D614G variant, Beta (K417N-E484K-N501Y) variant, and Delta (L452R-T478K-D614G) variant SARS-CoV-2 pseudovirus, and protect against authentic SARS-CoV-2 and Delta variant infection. Of note, sACE2 variants harbouring sEVs show superior antiviral efficacy than WT sACE2 loaded sEVs. Therapeutic efficacy of the engineered sEVs against SARS-CoV-2 challenge was confirmed using K18-hACE2 mice. The current findings provide opportunities for the development of new sEVs-based antiviral therapeutics.
Asunto(s)
Enzima Convertidora de Angiotensina 2/inmunología , COVID-19/inmunología , Vesículas Extracelulares/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Femenino , Células HEK293 , Humanos , Ratones , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
Mitochondrial dysfunction and oxidative stress are frequently observed in the early stages of Alzheimer's disease (AD). Studies have shown that presenilin-1 (PS1), the catalytic subunit of γ-secretase whose mutation is linked to familial AD (FAD), localizes to the mitochondrial membrane and regulates its homeostasis. Thus, we investigated how five PS1 mutations (A431E, E280A, H163R, M146V, and Δexon9) observed in FAD affect mitochondrial functions. Methods: We used H4 glioblastoma cell lines genetically engineered to inducibly express either the wild-type PS1 or one of the five PS1 mutants in order to examine mitochondrial morphology, dynamics, membrane potential, ATP production, mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), oxidative stress, and bioenergetics. Furthermore, we used brains of PS1M146V knock-in mice, 3xTg-AD mice, and human AD patients in order to investigate the role of PS1 in regulating MAMs formation. Results: Each PS1 mutant exhibited slightly different mitochondrial dysfunction. Δexon9 mutant induced mitochondrial fragmentation while A431E, E280A, H163R, and M146V mutants increased MAMs formation. A431E, E280A, M146V, and Δexon9 mutants also induced mitochondrial ROS production. A431E mutant impaired both complex I and peroxidase activity while M146V mutant only impaired peroxidase activity. All PS1 mutants compromised mitochondrial membrane potential and cellular ATP levels were reduced by A431E, M146V, and Δexon9 mutants. Through comparative profiling of hippocampal gene expression in PS1M146V knock-in mice, we found that PS1M146V upregulates Atlastin 2 (ATL2) expression level, which increases ER-mitochondria contacts. Down-regulation of ATL2 after PS1 mutant induction rescued abnormally elevated ER-mitochondria interactions back to the normal level. Moreover, ATL2 expression levels were significantly elevated in the brains of 3xTg-AD mice and AD patients. Conclusions: Overall, our findings suggest that each of the five FAD-linked PS1 mutations has a deleterious effect on mitochondrial functions in a variety of ways. The adverse effects of PS1 mutations on mitochondria may contribute to MAMs formation and oxidative stress resulting in an accelerated age of disease onset in people harboring mutant PS1.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Mitocondrias/fisiología , Presenilina-1/genética , Adenosina Trifosfato/metabolismo , Enfermedad de Alzheimer/genética , Animales , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Técnicas de Sustitución del Gen/métodos , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Estrés Oxidativo/fisiología , Presenilina-1/metabolismoRESUMEN
O-GlcNAcylation (O-linked ß-N-acetylglucosaminylation) is notably decreased in Alzheimer's disease (AD) brain. Necroptosis is activated in AD brain and is positively correlated with neuroinflammation and tau pathology. However, the links among altered O-GlcNAcylation, ß-amyloid (Aß) accumulation, and necroptosis are unclear. Here, we found that O-GlcNAcylation plays a protective role in AD by inhibiting necroptosis. Necroptosis was increased in AD patients and AD mouse model compared with controls; however, decreased necroptosis due to O-GlcNAcylation of RIPK3 (receptor-interacting serine/threonine protein kinase 3) was observed in 5xFAD mice with insufficient O-linked ß-N-acetylglucosaminase. O-GlcNAcylation of RIPK3 suppresses phosphorylation of RIPK3 and its interaction with RIPK1. Moreover, increased O-GlcNAcylation ameliorated AD pathology, including Aß burden, neuronal loss, neuroinflammation, and damaged mitochondria and recovered the M2 phenotype and phagocytic activity of microglia. Thus, our data establish the influence of O-GlcNAcylation on Aß accumulation and neurodegeneration, suggesting O-GlcNAcylation-based treatments as potential interventions for AD.
Asunto(s)
Enfermedad de Alzheimer , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Ratones , Necroptosis , FosforilaciónRESUMEN
Adult hippocampal neurogenesis supports the structural and functional plasticity of the brain, while its decline is associated with neurodegeneration common in Alzheimer's disease (AD). Although the dysregulation of certain microRNAs (miRNAs) in AD have been observed, the effects of miRNAs on hippocampal neurogenesis are largely unknown. In this study, we demonstrated miR-351-5p as a causative factor in hippocampal neural progenitor cell death through modulation of the mitochondrial guanosine triphosphatase (GTPase), Miro2. Downregulation of Miro2 by siMiro2 induced cell death, similar to miR-351-5p, whereas ectopic Miro2 expression using an adenovirus abolished these effects. Excessively fragmented mitochondria and dysfunctional mitochondria were indexed by decreased mitochondrial potential, and increased reactive oxygen species were identified in miR-351-5p-induced cell death. Moreover, subsequent induction of mitophagy via Pink1 and Parkin was observed in the presence of miR-351-5p and siMiro2. The suppression of mitochondrial fission by Mdivi-1 completely inhibited cell death by miR-351-5p. miR-351-5p expression increased whereas the level of Miro2 decreased in the hippocampus of AD model mice, emulating expression in AD patients. Collectively, the data indicate the mitochondrial fission and accompanying mitophagy by miR-351-5p/Miro2 axis as critical in hippocampal neural progenitor cell death, and a potential therapeutic target in AD.
RESUMEN
The accumulation of amyloid-ß (Aß) is a characteristic event in the pathogenesis of Alzheimer's disease (AD). Aquaporin 1 (AQP1) is a membrane water channel protein belonging to the AQP family. AQP1 levels are elevated in the cerebral cortex during the early stages of AD, but the role of AQP1 in AD pathogenesis is unclear. We first determined the expression and distribution of AQP1 in brain tissue samples of AD patients and two AD mouse models (3xTg-AD and 5xFAD). AQP1 accumulation was observed in vulnerable neurons in the cerebral cortex of AD patients, and in neurons affected by the Aß or tau pathology in the 3xTg-AD and 5xFAD mice. AQP1 levels increased in neurons as aging progressed in the AD mouse models. Stress stimuli increased AQP1 in primary cortical neurons. In response to cellular stress, AQP1 appeared to translocate to endocytic compartments of ß- and γ-secretase activities. Ectopic expression of AQP1 in human neuroblastoma cells overexpressing amyloid precussir protein (APP) with the Swedish mutations reduced ß-secretase (BACE1)-mediated cleavage of APP and reduced Aß production without altering the nonamyloidogenic pathway. Conversely, knockdown of AQP1 enhanced BACE1 activity and Aß production. Immunoprecipitation experiments showed that AQP1 decreased the association of BACE1 with APP. Analysis of a human database showed that the amount of Aß decreases as the expression of AQP1 increases. These results suggest that the upregulation of AQP1 is an adaptive response of neurons to stress that reduces Aß production by inhibiting the binding between BACE1 and APP.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/fisiología , Precursor de Proteína beta-Amiloide/fisiología , Amiloide/biosíntesis , Acuaporina 1/fisiología , Enfermedad de Alzheimer/metabolismo , Animales , Acuaporina 1/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Neuronas/metabolismoRESUMEN
Stem cell-derived extracellular vesicles (EVs) offer alternative approaches to stem cell-based therapy for regenerative medicine. In this study, stem cell EVs derived during differentiation are developed to use as cell-free therapeutic systems by inducing tissue-specific differentiation. EVs are isolated from human adipose-derived stem cells (HASCs) during white and beige adipogenic differentiation (D-EV and BD-EV, respectively) via tangential flow filtration. D-EV and BD-EV can successfully differentiate HASCs into white and beige adipocytes, respectively. D-EV are transplanted with collagen/methylcellulose hydrogels on the backs of BALB/c mice, and they produce numerous lipid droplets in injected sites. Treatments of BD-EV attenuate diet-induced obesity through browning of adipose tissue in mice. Furthermore, high-fat diet-induced hepatic steatosis and glucose tolerance are improved by BD-EV treatment. miRNAs are responsible for the observed effects of BD-EV. These results reveal that secreted EVs during stem cell differentiation into white adipocytes or beige adipocytes can promote cell reprogramming.
Asunto(s)
Adipocitos Beige/citología , Adipocitos Blancos/citología , Técnicas de Reprogramación Celular , Reprogramación Celular , Vesículas Extracelulares/metabolismo , Células Madre/citología , Células Madre/metabolismo , Adipocitos Beige/metabolismo , Adipocitos Blancos/metabolismo , Adipogénesis , Adipoquinas/metabolismo , Animales , Diferenciación Celular , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , MicroARNs/genéticaRESUMEN
Inhibition of Notch signalling has shown anti-inflammatory properties in vivo and in vitro models of rheumatoid arthritis (RA). The objective of this study was to determine whether Notch1 might play a role in regulating T-regulatory cells (Tregs) in animal models of RA. Methods: Collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) were induced in C57BL/6, Notch1 antisense transgenic (NAS) or DBA1/J mice. We examined whether pharmacological inhibitors of γ-secretase (an enzyme required for Notch1 activation) and antisense-mediated knockdown of Notch1 could attenuate the severity of inflammatory arthritis in CIA and CAIA mice. Proportions of CD4+CD25+Foxp3+ Treg cells were measured by flow cytometry. To assess the suppressive capacity of Treg toward responder cells, CFSE-based suppression assay of Treg was performed. Results: γ-secretase inhibitors and antisense-mediated knockdown of Notch1 reduced the severity of inflammatory arthritis in both CIA and CAIA mice. Pharmacological and genetic inhibition of Notch1 signalling induced significant elevation of Treg cell population in CIA and CAIA mice. We also demonstrated that inhibition of Notch signalling suppressed the progression of inflammatory arthritis through modulating the expansion and suppressive function of regulatory T (Treg) cells. Conclusion: Pharmacological and genetic inhibition of Notch1 signalling suppresses the progression of inflammatory arthritis through modulating the population and suppressive function of Treg cells in animal models of RA.
Asunto(s)
Artritis Reumatoide/patología , Artritis Reumatoide/fisiopatología , Receptor Notch1/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Artritis Reumatoide/inducido químicamente , Modelos Animales de Enfermedad , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/genética , Transducción de SeñalRESUMEN
Reactive oxygen species (ROS) are chemically reactive oxygen containing molecules. ROS consist of radical oxygen species including superoxide anion (O2 â¢-) and hydroxyl radical (â¢OH) and non-radical oxygen species such as hydrogen peroxide (H2O2), singlet oxygen (O2). ROS are generated by mitochondrial oxidative phosphorylation, environmental stresses including UV or heat exposure, and cellular responses to xenobiotics ( Ray et al., 2012 ). Excessive ROS production over cellular antioxidant capacity induces oxidative stress which results in harmful effects such as cell and tissue damage. Sufficient evidence suggests that oxidative stresses are involved in cancers, cardiovascular disease, and neurodegenerative diseases including Alzheimer's disease and Parkinson disease (Waris and Ahsan, 2006). Though excessive level of ROS triggers detrimental effects, ROS also have been implicated to regulate cellular processes. Since ROS function is context dependent, measurement of ROS level is important to understand cellular processes (Finkel, 2011). This protocol describes how to detect intracellular and mitochondrial ROS in live cells using popular chemical fluorescent dyes.
RESUMEN
Phytochemicals are reported to provide various biological functions leading to the promotion of health as well as the reduced risk of chronic diseases. Fat-soluble plant pigments, carotenoids, are extensively studied micronutrient phytochemicals for their potential health benefits. It is noteworthy that specific carotenoids may be responsible for different protective effects against certain diseases. In addition, each carotenoid can be obtained from different types of plant foods. Considering the fact that the phytochemical content in foods can vary according to, but not limited to, the varieties and culture conditions, it is important to establish a database of phytochemicals in locally produced plant foods. Currently, information on individual carotenoid content in plant foods commonly consumed in Korea is lacking. As the first step to support the production and consumption of sustainable local plant foods, carotenoids and total phenolic contents of plant foods commonly consumed in Korea are presented and their potential biological functions are discussed in this review.