RESUMO
Liver mitochondria play a central role in metabolic adaptations to changing nutritional states, yet their dynamic regulation upon anticipated changes in nutrient availability has remained unaddressed. Here, we found that sensory food perception rapidly induced mitochondrial fragmentation in the liver through protein kinase B/AKT (AKT)-dependent phosphorylation of serine 131 of the mitochondrial fission factor (MFFS131). This response was mediated by activation of hypothalamic pro-opiomelanocortin (POMC)-expressing neurons. A nonphosphorylatable MFFS131G knock-in mutation abrogated AKT-induced mitochondrial fragmentation in vitro. In vivo, MFFS131G knock-in mice displayed altered liver mitochondrial dynamics and impaired insulin-stimulated suppression of hepatic glucose production. Thus, rapid activation of a hypothalamus-liver axis can adapt mitochondrial function to anticipated changes of nutritional state in control of hepatic glucose metabolism.
Assuntos
Alimentos , Gluconeogênese , Glucose , Fígado , Proteínas de Membrana , Mitocôndrias Hepáticas , Dinâmica Mitocondrial , Proteínas Mitocondriais , Percepção , Animais , Masculino , Camundongos , Técnicas de Introdução de Genes , Glucose/metabolismo , Hipotálamo/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Neurônios/metabolismo , Fosforilação , Pró-Opiomelanocortina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos TransgênicosRESUMO
BACKGROUND: An active lifestyle is associated with improved cognitive functions in aged people and may prevent or slow down the progression of various neurodegenerative diseases including Alzheimer's disease (AD). To investigate these protective effects, male APPNL-G-F mice were exposed to long-term voluntary exercise. METHODS: Three-month-old AD mice were housed in a cage supplemented with a running wheel for 9 months for long-term exercise. At the age of 12 months, behavioral tests were completed for all groups. After completing behavioral testing, their brains were assessed for amyloid pathology, microgliosis, and cholinergic cells. RESULTS: The results showed that APPNL-G-F mice allowed to voluntarily exercise showed an improvement in cognitive functions. Furthermore, long-term exercise also improved anxiety in APPNL-G-F mice as assessed by measuring thigmotaxis in the Morris water task. We also found reductions in amyloid load and microgliosis, and a preservation of cholinergic cells in the brain of APPNL-G-F mice allowed to exercise in their home cages. These profound reductions in brain pathology associated with AD are likely responsible for the observed improvement of learning and memory functions following extensive and regular exercise. CONCLUSION: These findings suggest the potential of physical exercise to mitigate the cognitive deficits in AD.
Assuntos
Doença de Alzheimer , Amiloidose , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ansiedade/etiologia , Encéfalo/metabolismo , Colinérgicos , Cognição , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Transgênicos , ÁguaRESUMO
OBJECTIVE: The paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ connectivity and its role in other physiological regulations are incompletely understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and test their physiological functions beyond feeding. METHODS: Using a combination of viral tools, we mapped PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermoregulation, cardiovascular control, and other behavioral responses beyond feeding. RESULTS: We found that PVNMC4R+ neurons innervate many different brain regions that are known to be important not only for feeding but also for neuroendocrine and autonomic control of thermoregulation and cardiovascular function, including but not limited to the preoptic area, median eminence, parabrachial nucleus, pre-locus coeruleus, nucleus of solitary tract, ventrolateral medulla, and thoracic spinal cord. Contrary to these broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs mainly from other hypothalamic nuclei (preoptic area, arcuate and dorsomedial hypothalamic nuclei, supraoptic nucleus, and premammillary nucleus), the circumventricular organs (subfornical organ and vascular organ of lamina terminalis), the bed nucleus of stria terminalis, and the parabrachial nucleus. Consistent with their broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure, and brown adipose tissue temperature. These physiological changes accompanied acute transient hyperactivity followed by hypoactivity and resting-like behavior. CONCLUSIONS: Our results elucidate the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermoregulation, cardiovascular function, and biphasic behavioral activation.
Assuntos
Núcleo Hipotalâmico Paraventricular/metabolismo , Receptor Tipo 4 de Melanocortina/metabolismo , Animais , Regulação da Temperatura Corporal/fisiologia , Encéfalo/metabolismo , Núcleo Hipotalâmico Dorsomedial/metabolismo , Técnicas de Introdução de Genes/métodos , Hipotálamo/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Receptor Tipo 4 de Melanocortina/fisiologia , Medula Espinal/metabolismoRESUMO
Huntington's disease (HD) is a progressive, fatal neurodegenerative disorder characterized by motor, cognitive, and psychiatric disturbances. There is no known cure for HD, but its progressive nature allows for early therapeutic intervention. Currently, much of the research has focused on the striatum, however, there is evidence suggesting that disruption of thalamocortical circuits could underlie some of the early symptoms of HD. Loss of both cortical pyramidal neurons (CPNs) and thalamic neurons occurs in HD patients, and cognitive, somatosensory, and attention deficits precede motor abnormalities. However, the role of thalamocortical pathways in HD progression has been understudied. Here, we measured single unit activity and local field potentials (LFPs) from electrode arrays implanted in the thalamus and primary motor cortex of 4-5 month-old male and female Q175 mice. We assessed neuronal activity under baseline conditions as well as during presentation of rewards delivered via actuation of an audible solenoid valve. HD mice showed a significantly delayed licking response to the reward stimulus. At the same time, neuronal activation to the reward was delayed in thalamic neurons, CPNs and fast-spiking cortical interneurons (FSIs) of HD mice. In addition, thalamocortical coherence increased at lower frequencies in HD relative to wildtype mice. Together, these data provide evidence that impaired cortical and thalamic responses to reward stimuli, and impaired thalamocortical coherence, may play an important early role in motor, cognitive, and learning deficits in HD patients.
Assuntos
Doença de Huntington/fisiopatologia , Córtex Motor/fisiopatologia , Tálamo/fisiopatologia , Animais , Córtex Cerebral/fisiopatologia , Cognição , Modelos Animais de Doenças , Progressão da Doença , Técnicas de Introdução de Genes , Interneurônios/fisiologia , Camundongos , Atividade Motora , Vias Neurais/fisiopatologia , Técnicas de Patch-Clamp , Células Piramidais/fisiologiaRESUMO
Astrocytes exist throughout the CNS and affect neural circuits and behavior through intracellular Ca2+ signaling. Studying the function(s) of astrocyte Ca2+ signaling has proven difficult because of the paucity of tools to achieve selective attenuation. Based on recent studies, we generated and used male and female knock-in mice for Cre-dependent expression of mCherry-tagged hPMCA2w/b to attenuate astrocyte Ca2+ signaling in genetically defined cells in vivo (CalExflox mice for Calcium Extrusion). We characterized CalExflox mice following local AAV-Cre microinjections into the striatum and found reduced astrocyte Ca2+ signaling (â¼90%) accompanied with repetitive self-grooming behavior. We also crossed CalExflox mice to astrocyte-specific Aldh1l1-Cre/ERT2 mice to achieve inducible global CNS-wide Ca2+ signaling attenuation. Within 6 d of induction in the bigenic mice, we observed significantly altered ambulation in the open field, disrupted motor coordination and gait, and premature lethality. Furthermore, with histologic, imaging, and transcriptomic analyses, we identified cellular and molecular alterations in the cerebellum following mCherry-tagged hPMCA2w/b expression. Our data show that expression of mCherry-tagged hPMCA2w/b with CalExflox mice throughout the CNS resulted in substantial attenuation of astrocyte Ca2+ signaling and significant behavioral alterations in adult mice. We interpreted these findings candidly in relation to the ability of CalEx to attenuate astrocyte Ca2+ signaling, with regards to additional mechanistic interpretations of the data, and their relation to past studies that reduced astrocyte Ca2+ signaling throughout the CNS. The data and resources provide complementary ways to interrogate the function(s) of astrocytes in multiple experimental scenarios.SIGNIFICANCE STATEMENT Astrocytes represent a significant fraction of all brain cells and tile the entire central nervous system. Unlike neurons, astrocytes lack propagated electrical signals. Instead, astrocytes are proposed to use diverse and dynamic intracellular Ca2+ signals to communicate with other cells. An open question concerns if and how astrocyte Ca2+ signaling regulates behavior in adult mice. We approached this problem by generating a new transgenic mouse line to achieve inducible astrocyte Ca2+ signaling attenuation in vivo We report our data with this mouse line and we interpret the findings candidly in relation to past studies and within the framework of different mechanistic interpretations.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Sinalização do Cálcio/fisiologia , Animais , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson's Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.
Assuntos
Endossomos/efeitos dos fármacos , Indazóis/farmacologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Lisossomos/efeitos dos fármacos , Doença de Parkinson/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Avaliação Pré-Clínica de Medicamentos , Endossomos/fisiologia , Mutação com Ganho de Função , Técnicas de Introdução de Genes , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Lisossomos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Especificidade de Órgãos , Fosforilação/efeitos dos fármacos , Mutação Puntual , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Distribuição Aleatória , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues owing to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2 (also known as ACVR1). From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2, compared with other receptors of the BMP/TGF-ß signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D-transgenic mouse line, which provides a model of heterotopic ossification (HO), as well as an inducible ACVR1R206H-knockin mouse, which serves as a genetically and physiologically faithful FOP model. In both models, saracatinib was well tolerated and potently inhibited the development of HO, even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in FOP treatment, offering an accelerated path to clinical proof-of-efficacy studies and potentially significant benefits to individuals with this devastating condition.
Assuntos
Receptores de Ativinas Tipo I/genética , Benzodioxóis/farmacologia , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Músculos/efeitos dos fármacos , Miosite Ossificante/genética , Quinazolinas/farmacologia , Receptores de Ativinas Tipo I/antagonistas & inibidores , Animais , Benzodioxóis/uso terapêutico , Proteínas Morfogenéticas Ósseas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Técnicas de Introdução de Genes , Camundongos , Camundongos Transgênicos , Músculos/metabolismo , Miosite Ossificante/metabolismo , Miosite Ossificante/patologia , Ossificação Heterotópica/genética , Ossificação Heterotópica/metabolismo , Ossificação Heterotópica/patologia , Quinazolinas/uso terapêutico , Peixe-ZebraRESUMO
RPE65 isomerase, expressed in the retinal pigmented epithelium (RPE), is an enzymatic component of the retinoid cycle, converting all-trans retinyl ester into 11-cis retinol, and it is essential for vision, because it replenishes the photon capturing 11-cis retinal. To date, almost 200 loss-of-function mutations have been identified within the RPE65 gene causing inherited retinal dystrophies, most notably Leber congenital amaurosis (LCA) and autosomal recessive retinitis pigmentosa (arRP), which are both severe and early onset disease entities. We previously reported a mutation, D477G, co-segregating with the disease in a late-onset form of autosomal dominant RP (adRP) with choroidal involvement; uniquely, it is the only RPE65 variant to be described with a dominant component. Families or individuals with this variant have been encountered in five countries, and a number of subsequent studies have been reported in which the molecular biological and physiological properties of the variant have been studied in further detail, including observations of possible novel functions in addition to reduced RPE65 enzymatic activity. With regard to the latter, a human phase 1b proof-of-concept study has recently been reported in which aspects of remaining vision were improved for up to one year in four of five patients with advanced disease receiving a single one-week oral dose of 9-cis retinaldehyde, which is the first report showing efficacy and safety of an oral therapy for a dominant form of RP. Here, we review data accrued from published studies investigating molecular mechanisms of this unique variant and include hitherto unpublished material on the clinical spectrum of disease encountered in patients with the D477G variant, which, in many cases bears striking similarities to choroideremia.
Assuntos
Substituição de Aminoácidos , Genes Dominantes , Mutação de Sentido Incorreto , Mutação Puntual , Retinose Pigmentar/genética , cis-trans-Isomerases/genética , Idade de Início , Animais , Coroideremia , Ensaios Clínicos Fase I como Assunto , DNA Complementar/administração & dosagem , DNA Complementar/genética , Terapia de Reposição de Enzimas , Feminino , Técnicas de Introdução de Genes , Terapia Genética , Vetores Genéticos/uso terapêutico , Humanos , Amaurose Congênita de Leber/enzimologia , Amaurose Congênita de Leber/genética , Masculino , Camundongos , Linhagem , Estudo de Prova de Conceito , Isoformas de Proteínas/genética , Retinaldeído/uso terapêutico , Retinose Pigmentar/diagnóstico por imagem , Retinose Pigmentar/enzimologia , Retinose Pigmentar/terapia , cis-trans-Isomerases/deficiência , cis-trans-Isomerases/fisiologia , cis-trans-Isomerases/uso terapêuticoRESUMO
Proximity-based in situ labeling techniques offer a unique way to capture both stable and transient protein-protein and protein-organelle interactions. Combining this technology with mass spectrometry (MS)-based proteomics allows us to obtain snapshots of molecular microenvironments with nanometer resolution, facilitating the discovery of complex and dynamic protein networks. However, a number of technical challenges still exist, such as interferences from endogenously biotinylated proteins and other highly abundant bystanders, how to select the proper controls to minimize false discoveries, and experimental variations among biological/technical replicates. Here, we developed a new method to capture the proteomic microenvironment of the neuronal endolysosomal network by knocking in (KI) an engineered ascorbate peroxidase (APEX) gene to the endogenous locus of lysosome-associated membrane protein 1 (LAMP1). We found that normalizing proximity labeling proteomics data to the endogenously biotinylated protein (PCCA) can greatly reduce variations and enable fair comparisons among different batches of APEX labeling and different APEX probes. We conducted a comparative evaluation between this KI-LAMP1-APEX method and our two overexpression LAMP1-APEX probes, achieving complementary coverage of both known and new lysosomal membrane and lysosomal-interacting proteins in human iPSC-derived neurons. To summarize, this study demonstrated new analytical tools to characterize lysosomal functions and microenvironment in human neurons and filled critical gaps in the field for designing and optimizing proximity labeling proteomic experiments.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Fagossomos/metabolismo , Proteômica/métodos , Ascorbato Peroxidases/genética , Técnicas de Introdução de Genes , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Neurônios/metabolismo , Coloração e RotulagemRESUMO
BACKGROUND: Elevated blood homocysteine levels, termed hyperhomocysteinemia (HHcy), is a prevalent risk factor for Alzheimer's disease (AD) in elderly populations. While dietary supplementation of B-vitamins is a generally effective method to lower homocysteine levels, there is little if any benefit to cognition. In the context of amyloid pathology, dietary-induced HHcy is known to enhance amyloid deposition and certain inflammatory responses. Little is known, however, about whether there is a more specific effect on microglia resulting from combined amyloid and HHcy pathologies. METHODS: The present study used a knock-in mouse model of amyloidosis, aged to 12 months, given 8 weeks of B-vitamin deficiency-induced HHcy to better understand how microglia are affected in this comorbidity context. RESULTS: We found that HHcy-inducing diet increased amyloid plaque burden, altered the neuroinflammatory milieu, and upregulated the expression of multiple damage-associated and "homeostatic" microglial genes. CONCLUSIONS: Taken together, these data indicate complex effects of comorbid pathologies on microglial function that are not driven solely by increased amyloid burden. Given the highly dynamic nature of microglia, their central role in AD pathology, and the frequent occurrence of various comorbidities in AD patients, it is increasingly important to understand how microglia respond to mixed pathological processes.
Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Técnicas de Introdução de Genes/métodos , Hiper-Homocisteinemia/metabolismo , Microglia/metabolismo , Placa Amiloide/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Hiper-Homocisteinemia/genética , Hiper-Homocisteinemia/patologia , Camundongos , Camundongos Transgênicos , Microglia/patologia , Placa Amiloide/genética , Placa Amiloide/patologiaRESUMO
BACKGROUND: Lipid dysregulation is associated with several key characteristics of Alzheimer's disease (AD), including amyloid-ß and tau neuropathology, neurodegeneration, glucose hypometabolism, as well as synaptic and mitochondrial dysfunction. The ß-site amyloid precursor protein cleavage enzyme 1 (BACE1) is associated with increased amyloidogenesis, and has been affiliated with diabetes via its role in metabolic regulation. METHODS: The research presented herein investigates the role of hBACE1 in lipid metabolism and whether specific brain regions show increased vulnerability to lipid dysregulation. By utilising advanced mass spectrometry techniques, a comprehensive, quantitative lipidomics analysis was performed to investigate the phospholipid, sterol, and fatty acid profiles of the brain from the well-known PLB4 hBACE1 knock-in mouse model of AD, which also shows a diabetic phenotype, to provide insight into regional alterations in lipid metabolism. RESULTS: Results show extensive region - specific lipid alterations in the PLB4 brain compared to the wild-type, with decreases in the phosphatidylethanolamine content of the cortex and triacylglycerol content of the hippocampus and hypothalamus, but increases in the phosphatidylcholine, phosphatidylinositol, and diacylglycerol content of the hippocampus. Several sterol and fatty acids were also specifically decreased in the PLB4 hippocampus. CONCLUSION: Collectively, the lipid alterations observed in the PLB4 hBACE1 knock-in AD mouse model highlights the regional vulnerability of the brain, in particular the hippocampus and hypothalamus, to lipid dysregulation, hence supports the premise that metabolic abnormalities have a central role in both AD and diabetes.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Diabetes Mellitus Experimental/metabolismo , Hipocampo/metabolismo , Hipotálamo/metabolismo , Metabolismo dos Lipídeos/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diglicerídeos/metabolismo , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Feminino , Expressão Gênica , Técnicas de Introdução de Genes , Hipocampo/patologia , Humanos , Hipotálamo/patologia , Lipidômica/métodos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos , Fosfatidilcolinas/metabolismo , Fosfatidilinositóis/metabolismo , Esteróis/metabolismo , TransgenesRESUMO
Na+/K+-ATPase is a transmembrane ion pump that is essential for the maintenance of ion gradients and regulation of multiple cellular functions. Na+/K+-ATPase has been associated with nuclear factor kappa B (NFκB) signalling, a signal associated with lipopolysaccharides (LPSs)-induced immune response in connection with activated Toll-like receptor 4 (TLR4) signalling. However, the contribution of Na+/K+-ATPase to regulating inflammatory responses remains elusive. We report that mice haploinsufficient for the astrocyte-enriched α2Na+/K+-ATPase isoform (α2+/G301R mice) have a reduced proinflammatory response to LPS, accompanied by a reduced hypothermic reaction compared to wild type litter mates. Following intraperitoneal injection of LPS, gene expressions of Tnf-α, Il-1ß, and Il-6 was reduced in the hypothalamus and hippocampus from α2+/G301R mice compared to α2+/+ littermates. The α2+/G301R mice experienced increased expression of the gene encoding an antioxidant enzyme, NRF2, in hippocampal astrocytes. Our findings indicate that α2Na+/K+-ATPase haploinsufficiency negatively modulates LPS-induced immune responses, highlighting a rational pharmacological target for reducing LPS-induced inflammation.
Assuntos
Hipocampo/patologia , Hipotálamo/patologia , Lipopolissacarídeos/toxicidade , Enxaqueca com Aura/enzimologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Animais , Astrócitos/metabolismo , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Introdução de Genes , Heterozigoto , Hipocampo/metabolismo , Hipotálamo/metabolismo , Hipotermia/induzido quimicamente , Hipotermia/enzimologia , Hipotermia/genética , Interleucina-1beta/biossíntese , Interleucina-1beta/sangue , Interleucina-1beta/genética , Interleucina-6/biossíntese , Interleucina-6/sangue , Interleucina-6/genética , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Enxaqueca com Aura/genética , Mutação de Sentido Incorreto , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/genética , ATPase Trocadora de Sódio-Potássio/deficiência , ATPase Trocadora de Sódio-Potássio/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genéticaRESUMO
Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease) is a lysosomal storage disease characterized by progressive blindness, seizures, cognitive and motor failures, and premature death. JNCL is caused by mutations in the Ceroid Lipofuscinosis, Neuronal 3 (CLN3) gene, whose function is unclear. Although traditionally considered a neurodegenerative disease, CLN3 disease displays eye-specific effects: Vision loss not only is often one of the earliest symptoms of JNCL, but also has been reported in non-syndromic CLN3 disease. Here we described the roles of CLN3 protein in maintaining healthy retinal pigment epithelium (RPE) and normal vision. Using electroretinogram, fundoscopy and microscopy, we showed impaired visual function, retinal autofluorescent lesions, and RPE disintegration and metaplasia/hyperplasia in a Cln3 ~ 1 kb-deletion mouse model [1] on C57BL/6J background. Utilizing a combination of biochemical analyses, RNA-Seq, Seahorse XF bioenergetic analysis, and Stable Isotope Resolved Metabolomics (SIRM), we further demonstrated that loss of CLN3 increased autophagic flux, suppressed mTORC1 and Akt activities, enhanced AMPK activity, and up-regulated gene expression of the autophagy-lysosomal system in RPE-1 cells, suggesting autophagy induction. This CLN3 deficiency induced autophagy induction coincided with decreased mitochondrial oxygen consumption, glycolysis, the tricarboxylic acid (TCA) cycle, and ATP production. We also reported for the first time that loss of CLN3 led to glycogen accumulation despite of impaired glycogen synthesis. Our comprehensive analyses shed light on how loss of CLN3 affect autophagy and metabolism. This work suggests possible links among metabolic impairment, autophagy induction and lysosomal storage, as well as between RPE atrophy/degeneration and vision loss in JNCL.
Assuntos
Cegueira/genética , Glicoproteínas de Membrana/deficiência , Lipofuscinoses Ceroides Neuronais/genética , Epitélio Pigmentado da Retina/patologia , Animais , Atrofia/genética , Atrofia/patologia , Autofagia , Cegueira/patologia , Linhagem Celular , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Glicogênio/metabolismo , Humanos , Lisossomos/patologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Chaperonas Moleculares/genética , Mutação , Lipofuscinoses Ceroides Neuronais/complicações , Lipofuscinoses Ceroides Neuronais/patologia , RNA Interferente Pequeno/metabolismo , Epitélio Pigmentado da Retina/ultraestruturaRESUMO
The effective material basis of traditional Chinese medicine(TCM) is an indispensable part of studies on TCM, and each technology has its advantages and disadvantages. The target constituent knock-out/knock-in technology has attracted much attention since it was proposed because of its unique advantages of regarding the extract of the formula as a whole, which can better reflect the characteristics of multi-component and multi-target integration and regulation of TCM. This method investigated the contribution of target constituent to the overall efficacy of a TCM by analyzing the changes in efficacy of the remaining formula before and after knock-out/knock-in of the target constitution. The application of this model not only facilitates studies of the effective constituents of TCM, but also help to develop the quality control standard of TCM. However, the application of this model is restricted due to the limitation of target constituent separation technology. By reviewing the literatures in recent years, this study summarized the research process and application of this method for a reference.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Medicina Tradicional Chinesa , Animais , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Controle de QualidadeRESUMO
Leptin stimulates the sympathetic nervous system (SNS), energy expenditure, and weight loss; however, the underlying molecular mechanism remains elusive. Here, we uncover Sh2b1 in leptin receptor (LepR) neurons as a critical component of a SNS/brown adipose tissue (BAT)/thermogenesis axis. LepR neuron-specific deletion of Sh2b1 abrogates leptin-stimulated sympathetic nerve activation and impairs BAT thermogenic programs, leading to reduced core body temperature and cold intolerance. The adipose SNS degenerates progressively in mutant mice after 8 weeks of age. Adult-onset ablation of Sh2b1 in the mediobasal hypothalamus also impairs the SNS/BAT/thermogenesis axis; conversely, hypothalamic overexpression of human SH2B1 has the opposite effects. Mice with either LepR neuron-specific or adult-onset, hypothalamus-specific ablation of Sh2b1 develop obesity, insulin resistance, and liver steatosis. In contrast, hypothalamic overexpression of SH2B1 protects against high fat diet-induced obesity and metabolic syndromes. Our results unravel an unrecognized LepR neuron Sh2b1/SNS/BAT/thermogenesis axis that combats obesity and metabolic disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fígado Gorduroso/patologia , Resistência à Insulina , Neurônios/metabolismo , Obesidade/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Tecido Adiposo Marrom/inervação , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/patologia , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Feminino , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Humanos , Hipotálamo/patologia , Leptina/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Transgênicos , Obesidade/etiologia , Receptores para Leptina/metabolismo , Sistema Nervoso Simpático/fisiologia , Termogênese/fisiologiaRESUMO
BACKGROUND: ß-Glucans (BGs), a group of complex dietary polysaccharides (CDPs), are available as dietary supplements. However, the effects of orally administered highly purified BGs on gut inflammation are largely unknown. OBJECTIVES: The aim of this study was to investigate the impact of orally administering highly purified, yeast-derived BG (YBG; ß-1,3/1,6-d-glucan) on susceptibility to colitis. METHODS: Eight-week-old C57BL/6 (B6) mice were used in a series of experiments. Experiment (Expt) 1: male and female mice were treated every day, for 40 d, with saline (control) or 250 µg YBG, followed by 2.5% (wt:vol) dextran sulfate sodium (DSS) in drinking water during days 30-35; and colitis severity and intestinal immune phenotype were determined. Expt 2: female B6 mice were treated with saline or YBG for 30 d and intestinal immune phenotype, gut microbiota composition, and fecal SCFA concentrations were determined. Expt 3: female B6 mice were treated as in Expt 2, given drinking water with or without antibiotics [Abx; ampicillin (1 g/L), vancomycin (0.5 g/L), neomycin (1 g/L), and metronidazole (1 g/L)] during days 16-30, and gut immune phenotype and fecal SCFA concentrations were determined. Expt 4: female B6 Foxp3-green fluorescent protein (-GFP) reporter mice were treated as in Expt 3, and intestinal T-regulatory cell (Treg) frequencies and immune phenotypes were determined. Expt 5: female mice were treated as in Expt 1, given drinking water with or without antibiotics during days 16-40, and colitis severity and intestinal cytokine production were determined. RESULTS: Compared with controls, the YBG group in Expt 1 exhibited suppressive effects on features of colitis, such as loss of body weight (by 47%; P < 0.001), shortening of colon (by 24%; P = 0.016), and histopathology severity score (by 45%; P = 0.01). The YBG group of Expt 2 showed a shift in the abundance of gut microbiota towards Bacteroides (by 16%; P = 0.049) and Verrucomicrobia (mean ± SD: control = 7.8 ± 0.44 vs. YBG = 21.0 ± 9.6%) and a reduction in Firmicutes (by 66%; P < 0.001). The YBG group also showed significantly higher concentrations of fecal SCFAs such as acetic (by 37%; P = 0.016), propionic (by 47%; P = 0.026), and butyric (by 57%; P = 0.013) acids. Compared with controls, the YBG group of Expt 2 showed higher frequencies of Tregs (by 32%; P = 0.043) in the gut mucosa. Depletion of gut microbiota in the YBG group of mice caused diminished fecal SCFA concentrations (Expt 3) and intestinal Treg frequencies (Expt 4). Compared with the YBG group, the YBG-(Abx) group of Expt 5 showed aggravated colitis features including loss of body weight (by >100%; P < 0.01) and colonic inflammation score (by 42%; P = 0.04). CONCLUSIONS: Studies using B6 mice show that dietary BGs are beneficial for promoting intestinal health when the gut microbiota is intact. However, these CDPs may produce adverse effects if gut microbiota is compromised.
Assuntos
Colite/prevenção & controle , Microbioma Gastrointestinal/efeitos dos fármacos , Polissacarídeos/administração & dosagem , Saccharomyces cerevisiae/química , beta-Glucanas/administração & dosagem , Animais , Colite/induzido quimicamente , Colite/imunologia , Sulfato de Dextrana/farmacologia , Ácidos Graxos Voláteis/análise , Fezes/química , Fezes/microbiologia , Feminino , Fatores de Transcrição Forkhead/genética , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/genética , Imunidade/efeitos dos fármacos , Intestinos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Sorafenib is the standard treatment for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common. By using genome-wide CRISPR/Cas9 library screening, we identify phosphoglycerate dehydrogenase (PHGDH), the first committed enzyme in the serine synthesis pathway (SSP), as a critical driver for Sorafenib resistance. Sorafenib treatment activates SSP by inducing PHGDH expression. With RNAi knockdown and CRISPR/Cas9 knockout models, we show that inactivation of PHGDH paralyzes the SSP and reduce the production of αKG, serine, and NADPH. Concomitantly, inactivation of PHGDH elevates ROS level and induces HCC apoptosis upon Sorafenib treatment. More strikingly, treatment of PHGDH inhibitor NCT-503 works synergistically with Sorafenib to abolish HCC growth in vivo. Similar findings are also obtained in other FDA-approved tyrosine kinase inhibitors (TKIs), including Regorafenib or Lenvatinib. In summary, our results demonstrate that targeting PHGDH is an effective approach to overcome TKI drug resistance in HCC.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hepáticas/tratamento farmacológico , Fosfoglicerato Desidrogenase/genética , Sorafenibe/uso terapêutico , Apoptose , Sistemas CRISPR-Cas , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Humanos , Neoplasias Hepáticas/genética , Compostos de Fenilureia/uso terapêutico , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Piridinas/uso terapêutico , Quinolinas/uso terapêutico , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ambiguity regarding the role of glucose-dependent insulinotropic polypeptide (GIP) in obesity arises from conflicting reports asserting that both GIP receptor (GIPR) agonism and antagonism are effective strategies for inhibiting weight gain. To enable identification and manipulation of Gipr-expressing (Gipr) cells, we created Gipr-Cre knockin mice. As GIPR-agonists have recently been reported to suppress food intake, we aimed to identify central mediators of this effect. Gipr cells were identified in the arcuate, dorsomedial, and paraventricular nuclei of the hypothalamus, as confirmed by RNAscope in mouse and human. Single-cell RNA-seq identified clusters of hypothalamic Gipr cells exhibiting transcriptomic signatures for vascular, glial, and neuronal cells, the latter expressing somatostatin but little pro-opiomelanocortin or agouti-related peptide. Activation of Gq-DREADDs in hypothalamic Gipr cells suppressed food intake in vivo, which was not obviously additive with concomitant GLP1R activation. These data identify hypothalamic GIPR as a target for the regulation of energy balance.
Assuntos
Ingestão de Alimentos/fisiologia , Hipotálamo/citologia , Neurônios/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Idoso de 80 Anos ou mais , Animais , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Polipeptídeo Inibidor Gástrico/metabolismo , Técnicas de Introdução de Genes , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/tratamento farmacológico , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/genéticaRESUMO
Iron deficiency augments hypoxic pulmonary arterial pressure in healthy individuals and exacerbates pulmonary arterial hypertension (PAH) in patients, even without anemia. Conversely, iron supplementation has been shown to be beneficial in both settings. The mechanisms underlying the effects of iron availability are not known, due to lack of understanding of how cells of the pulmonary vasculature respond to changes in iron levels. The iron export protein ferroportin (FPN) and its antagonist peptide hepcidin control systemic iron levels by regulating release from the gut and spleen, the sites of absorption and recycling, respectively. We found FPN to be present in pulmonary arterial smooth muscle cells (PASMCs) and regulated by hepcidin cell autonomously. To interrogate the importance of this regulation, we generated mice with smooth muscle-specific knock in of the hepcidin-resistant isoform fpn C326Y. While retaining normal systemic iron levels, this model developed PAH and right heart failure as a consequence of intracellular iron deficiency and increased expression of the vasoconstrictor endothelin-1 (ET-1) within PASMCs. PAH was prevented and reversed by i.v. iron and by the ET receptor antagonist BQ-123. The regulation of ET-1 by iron was also demonstrated in healthy humans exposed to hypoxia and in PASMCs from PAH patients with mutations in bone morphogenetic protein receptor type II. Such mutations were further associated with dysregulation of the HAMP/FPN axis in PASMCs. This study presents evidence that intracellular iron deficiency specifically within PASMCs alters pulmonary vascular function. It offers a mechanistic underpinning for the known effects of iron availability in humans.
Assuntos
Deficiências de Ferro , Miócitos de Músculo Liso/patologia , Hipertensão Arterial Pulmonar/etiologia , Artéria Pulmonar/patologia , Administração Intravenosa , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Modelos Animais de Doenças , Antagonistas do Receptor de Endotelina A/administração & dosagem , Endotelina-1/metabolismo , Técnicas de Introdução de Genes , Hepcidinas/metabolismo , Humanos , Ferro/administração & dosagem , Masculino , Camundongos , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/patologia , Hipertensão Arterial Pulmonar/prevenção & controle , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , Receptor de Endotelina A/metabolismo , Regulação para CimaRESUMO
Cytochrome P450s CYP1A1 and CYP1A2 can metabolize a broad range of foreign compounds and drugs. However, these enzymes have significantly overlapping substrate specificities. To establish their relative contribution to drug metabolism in vivo, we used a combination of mice humanized for CYP1A1 and CYP1A2 together with mice nulled at the Cyp1a1 and Cyp1a2 gene loci. CYP1A2 was constitutively expressed in the liver, and both proteins were highly inducible by 2,3,7,8-tetrachlorodibenzodioxin (TCDD) in a number of tissues, including the liver, lung, kidney, and small intestine. Using the differential inhibition of the human enzymes by quinidine, we developed a method to distinguish the relative contribution of CYP1A1 or CYP1A2 in the metabolism of drugs and foreign compounds. Both enzymes made a significant contribution to the hepatic metabolism of the probe compounds 7-methoxy and 7-ehthoxyresorufin in microsomal fractions from animals treated with TCDD. This enzyme kinetic approach allows modeling of the CYP1A1, CYP1A2, and non-CYP1A contribution to the metabolism of any substrate at any substrate, inhibitor, or enzyme concentration and, as a consequence, can be integrated into a physiologically based pharmacokinetics model. The validity of the model can then be tested in humanized mice in vivo. SIGNIFICANCE STATEMENT: Human CYP1A1 and CYP1A2 are important in defining the efficacy and toxicity/carcinogenicity of drugs and foreign compounds. In light of differences in substrate specificity and sensitivity to inhibitors, it is of central importance to understand their relative role in foreign compound metabolism. To address this issue, we have generated mice humanized or nulled at the Cyp1a gene locus and, through the use of these mouse lines and selective inhibitors, developed an enzyme kinetic-based model to enable more accurate prediction of the fate of new chemicals in humans and which can be validated in vivo using mice humanized for cytochrome P450-mediated metabolism.