RESUMEN
Mitochondria facilitate thousands of biochemical reactions, covering a broad spectrum of anabolic and catabolic processes. Here we demonstrate that the adipocyte mitochondrial proteome is markedly altered across multiple models of insulin resistance and reveal a consistent decrease in the level of the mitochondrial processing peptidase miPEP. OBJECTIVE: To determine the role of miPEP in insulin resistance. METHODS: To experimentally test this observation, we generated adipocyte-specific miPEP knockout mice to interrogate its role in the aetiology of insulin resistance. RESULTS: We observed a strong phenotype characterised by enhanced insulin sensitivity and reduced adiposity, despite normal food intake and physical activity. Strikingly, these phenotypes vanished when mice were housed at thermoneutrality, suggesting that metabolic protection conferred by miPEP deletion hinges upon a thermoregulatory process. Tissue specific analysis of miPEP deficient mice revealed an increment in muscle metabolism, and upregulation of the protein FBP2 that is involved in ATP hydrolysis in the gluconeogenic pathway. CONCLUSION: These findings suggest that miPEP deletion initiates a compensatory increase in skeletal muscle metabolism acting as a protective mechanism against diet-induced obesity and insulin resistance.
Asunto(s)
Adipocitos , Resistencia a la Insulina , Ratones Noqueados , Músculo Esquelético , Obesidad , Animales , Ratones , Obesidad/metabolismo , Obesidad/genética , Músculo Esquelético/metabolismo , Adipocitos/metabolismo , Masculino , Ratones Endogámicos C57BL , Mitocondrias/metabolismoRESUMEN
Snakebites affect about 1.8 million people annually. The current standard of care involves antibody-based antivenoms, which can be difficult to access and are generally not effective against local tissue injury, the primary cause of morbidity. Here, we used a pooled whole-genome CRISPR knockout screen to define human genes that, when targeted, modify cell responses to spitting cobra venoms. A large portion of modifying genes that conferred resistance to venom cytotoxicity was found to control proteoglycan biosynthesis, including EXT1, B4GALT7, EXT2, EXTL3, XYLT2, NDST1, and SLC35B2, which we validated independently. This finding suggested heparinoids as possible inhibitors. Heparinoids prevented venom cytotoxicity through binding to three-finger cytotoxins, and the US Food and Drug Administration-approved heparinoid tinzaparin was found to reduce tissue damage in mice when given via a medically relevant route and dose. Overall, our systematic molecular dissection of cobra venom cytotoxicity provides insight into how we can better treat cobra snakebite envenoming.
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Venenos Elapídicos , Mordeduras de Serpientes , Animales , Humanos , Mordeduras de Serpientes/tratamiento farmacológico , Ratones , Antídotos/farmacologíaRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) has become an integral part of the molecular biology toolkit. CRISPRa genetic screens are an exciting high-throughput means of identifying genes the upregulation of which is sufficient to elicit a given phenotype. Activation machinery is continually under development to achieve greater, more robust, and more consistent activation. In this review, we offer a succinct technological overview of available CRISPRa architectures and a comprehensive summary of pooled CRISPRa screens. Furthermore, we discuss contemporary applications of CRISPRa across broad fields of research, with the aim of presenting a view of exciting emerging applications for CRISPRa screening.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Sistemas CRISPR-Cas/genética , Humanos , Edición Génica/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Pruebas Genéticas/métodos , AnimalesRESUMEN
BACKGROUND: Widescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer's disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells. METHODS: iPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aß42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently-labelled fibrillar Aß42. RESULTS: AD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100ß and increased secretion and phagocytosis of Aß42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells showed exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia. CONCLUSION: Our study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a 'primed' phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aß42 production and phagocytosis.
Asunto(s)
Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Humanos , Astrocitos/metabolismo , Microglía/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Interleucina-8/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Citocinas/metabolismo , Fenotipo , Péptidos beta-Amiloides/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismoRESUMEN
Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , COVID-19/genética , Antivirales/farmacología , Enzima Convertidora de Angiotensina 2/metabolismo , Fibroblastos/metabolismo , Unión Proteica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
BACKGROUND: Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia, although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer's disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD. METHODS: We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling, insulin regulation of the major AD proteins Aß and/or tau, and/or calcium fluxes is altered by the PSEN2 N141I mutation. RESULTS: We report herein that wildtype, PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Aß42/40 ratio. Unexpectedly, we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux, which could be prevented by chronic exposure of BFCNs to insulin. CONCLUSIONS: Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Aß42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence, our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD.
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Enfermedad de Alzheimer/metabolismo , Neuronas Colinérgicas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Insulina/metabolismo , Enfermedad de Alzheimer/genética , Diabetes Mellitus Tipo 2/complicaciones , Femenino , Humanos , Resistencia a la Insulina/fisiología , Masculino , Mutación , Presenilina-2/genéticaRESUMEN
Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD, and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs, using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected, cell lines harboring the PSEN2 N141I mutation displayed an increase in the Aß42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit, restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased Aß42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in Aß42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Sistemas CRISPR-Cas , Neuronas Colinérgicas/fisiología , Edición Génica , Células Madre Pluripotentes Inducidas/fisiología , Presenilina-2/genética , Potenciales de Acción , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Proteínas Reguladoras de la Apoptosis , Prosencéfalo Basal/metabolismo , Muerte Celular , Línea Celular , Neuronas Colinérgicas/patología , Femenino , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Mutación , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Neurogénesis , Fragmentos de Péptidos/metabolismo , Presenilina-2/metabolismo , ARN Mensajero/metabolismoRESUMEN
Aging constitutes the central risk factor for major diseases including many forms of cancer, neurodegeneration, and cardiovascular diseases. The aging process is characterized by both global and tissue-specific changes in gene expression across taxonomically diverse species. While aging has historically been thought to entail cell-autonomous, even stochastic changes, recent evidence suggests that modulation of this process can be hierarchal, wherein manipulations of nutrient-sensing neurons (e.g., in the hypothalamus) produce peripheral effects that may modulate the aging process itself. The most robust intervention extending lifespan, plausibly impinging on the aging process, involves different modalities of dietary restriction (DR). Lifespan extension by DR is associated with broad protection against diseases (natural and engineered). Here we review potential epigenetic processes that may link lifespan to age-related diseases, particularly in the context of DR and (other) ketogenic diets, focusing on brain and hypothalamic mechanisms.
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Restricción Calórica , Dieta Cetogénica , Epigénesis Genética , Inhibidores de Histona Desacetilasas/farmacología , Hidroxibenzoatos/farmacología , Longevidad/efectos de los fármacos , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Metilación de ADN , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Hipotálamo/citología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Longevidad/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Transducción de Señal , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
We have reported a correlation between hypothalamic expression of Creb-binding protein (Cbp) and lifespan, and that inhibition of Cbp prevents protective effects of dietary restriction during aging, suggesting that hypothalamic Cbp plays a role in responses to nutritional status and energy balance. Recent GWAS and network analyses have also implicated Cbp as the most connected gene in protein-protein interactions in human Type 2 diabetes. The present studies address mechanisms mediating the role of Cbp in diabetes by inhibiting hypothalamic Cbp using a Cre-lox strategy. Inhibition of hypothalamic Cbp results in profound obesity and impaired glucose homeostasis, increased food intake, and decreased body temperature. In addition, these changes are accompanied by molecular evidence in the hypothalamus for impaired leptin and insulin signaling, a shift from glucose to lipid metabolism, and decreased Pomc mRNA, with no effect on locomotion. Further assessment of the significance of the metabolic switch demonstrated that enhanced expression of hypothalamic Cpt1a, which promotes lipid metabolism, similarly resulted in increased body weight and reduced Pomc mRNA.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Hipotálamo/metabolismo , Obesidad/metabolismo , Animales , Temperatura Corporal , Factor Neurotrófico Derivado del Encéfalo/genética , Proteína de Unión a CREB/genética , Carnitina O-Palmitoiltransferasa/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Regulación hacia Abajo , Ingestión de Alimentos , Metabolismo Energético , Femenino , Regulación de la Expresión Génica , Glucosa/metabolismo , Humanos , Hipotálamo/patología , Insulina/metabolismo , Leptina/metabolismo , Metabolismo de los Lípidos , Masculino , Ratones , Obesidad/genética , Obesidad/patología , Proopiomelanocortina/genética , Transducción de Señal , Aumento de PesoRESUMEN
Huntington's disease (HD) is a fatal neurodegenerative disease characterized by metabolic, cognitive, and motor deficits. HD is caused by an expanded CAG repeat in the first exon of the HTT gene, resulting in an expanded polyglutamine section. Dietary restriction (DR) increases lifespan and ameliorates age-related pathologies, including in a model of HD, but the mechanisms mediating these protective effects are unknown. We report metabolic and behavioral effects of DR in the full-length YAC128 HD mouse model, and associated transcriptional changes in hypothalamus and striatum. DR corrected many effects of the transgene including increased body weight, decreased blood glucose, and impaired motor function. These changes were associated with reduced striatal human (but not mouse) HTT expression, as well as alteration in gene expression regulating histone acetylation modifications, particularly Hdac2. Other mRNAs related to Huntington's pathology in striatal tissue showed significant modulation by the transgene, dietary restriction or both. These results establish a protective role of DR in a transgenic model that contains the complete human HTT gene and for the first time suggest a role for DR in lowering HTT level, which correlates with severity of symptoms.
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Ayuno/metabolismo , Histonas/metabolismo , Enfermedad de Huntington/dietoterapia , Enfermedad de Huntington/metabolismo , Acetilación , Animales , Glucemia/fisiología , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Histona Desacetilasa 2/metabolismo , Proteína Huntingtina , Enfermedad de Huntington/genética , Hipotálamo/metabolismo , Ratones Transgénicos , Actividad Motora/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Transcripción Genética/fisiologíaRESUMEN
The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer's disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1(∆exon8)). We previously reported that PS1 L271V increased amyloid beta (Aß) 42/40 ratios, while PS1(∆exon8) reduced Aß42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1(∆exon8) did not rescue Aß generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1(∆exon8) is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1(∆exon8) in vivo by crossing PS1(∆exon8) transgenics with either PS1-null or Dutch APP(E693Q) mice. As a control, we crossed APP(E693Q) with mice expressing a deletion in an adjacent exon (PS1(∆exon9)). PS1(∆exon8) did not rescue embryonic lethality or Notch-deficient phenotypes of PS1-null mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1(∆exon8) interacts with nicastrin, participating in the γ-secretase complex formation. These data support that catalytically inactive PS1(∆exon8) is generated physiologically and participates in protein-protein interactions.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Exones/genética , Glicoproteínas de Membrana/metabolismo , Presenilina-1/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/genética , Animales , Encéfalo/metabolismo , Embrión de Mamíferos/metabolismo , Retículo Endoplásmico/metabolismo , Fluorescencia , Células HEK293 , Humanos , Inmunoprecipitación , Ratones Noqueados , Actividad Motora , Fenotipo , Placa Amiloide/metabolismo , Placa Amiloide/patología , Presenilina-1/deficiencia , Presenilina-1/metabolismo , Unión Proteica , Eliminación de Secuencia/genética , TransgenesRESUMEN
We propose that energy balance, glucose homeostasis, and aging are all regulated largely by the same nutrient-sensing neurons in the ventromedial hypothalamus (VMH). Although the central role of these neurons in regulating energy balance is clear, their role in regulating glucose homeostasis has only recently become more clear. This latter function may be most relevant to aging and lifespan by controlling the rate of glucose metabolism. Specifically, glucose-sensing neurons in VMH promote peripheral glucose metabolism, and dietary restriction, by reducing glucose metabolism in these neurons, reduces glucose metabolism of the rest of the body, thereby increasing lifespan. Here we discuss recent studies demonstrating the key role of hypothalamic neurons in driving aging and age-related diseases.
Asunto(s)
Envejecimiento/metabolismo , Diabetes Mellitus/metabolismo , Hipotálamo/metabolismo , Obesidad/metabolismo , Animales , HumanosRESUMEN
Dietary restriction (DR) can extend lifespan and reduce disease burden across a wide range of animals and yeast but the mechanisms mediating these remarkably protective effects remain to be elucidated despite extensive efforts. Although it has generally been assumed that protective effects of DR are cell-autonomous, there is considerable evidence that many whole-body responses to nutritional state, including DR, are regulated by nutrient-sensing neurons. In this review, we explore the hypothesis that nutrient sensing neurons in the ventromedial hypothalamus hierarchically regulate the protective responses of dietary restriction. We describe multiple peripheral responses that are hierarchically regulated by the hypothalamus and we present evidence for non-cell autonomous signaling of dietary restriction gathered from a diverse range of models including invertebrates, mammalian cell culture, and rodent studies.