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1.
Int J Mol Sci ; 23(4)2022 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-35216491

RESUMO

Systemic inflammation has been implicated in the progression of Alzheimer's disease (AD); however, less is understood about how existing AD pathology contributes to adverse outcomes following acute inflammatory insults. In the present study, our goal was to determine how AD-associated amyloid beta (Aß) pathology influences the acute neuroinflammatory and behavioral responses to a moderate systemic inflammatory insult. We treated 16-18-month-old female Tg2576 (Tg) mice, which overproduce human Aß and develop plaques, and age-matched wild-type (WT) littermate mice with an intraperitoneal injection of 0.33 mg/kg lipopolysaccharide (LPS) or saline. Mice were then evaluated over the next 28 h for sickness/depressive-like behaviors (food intake, weight loss, locomotion, and sucrose preference), systemic inflammation (serum amyloid A, SAA), blood-brain barrier (BBB) disruption, astrogliosis (glial fibrillary acidic protein/GFAP), Aß, and cytokine levels in the brain. We found that LPS caused a larger reduction in body weight in Tg vs. WT mice, but that other behavioral responses to LPS did not differ by genotype. BBB disruption was not apparent in either genotype following LPS. Concentrations of the systemic inflammatory marker, SAA, in the blood and brain were significantly increased with LPS but did not significantly differ by genotype. GFAP was increased in Tg mice vs. WT but was not significantly affected by LPS in either genotype. Finally, LPS-induced increases of eight cytokines (IL-1ß, IL-6, IL-12 (p40), IL-10, IL-17A, MIP-1α/CCL3, MIP-1ß/CCL4, and RANTES/CCL5) were found to be significantly higher in Tg mice vs. WT. In summary, our data show that Aß pathology exacerbates the neuroinflammatory response to LPS and identifies cytokines that are selectively regulated by Aß. The association of worse neuroinflammation with greater weight loss in Tg mice suggests that Aß pathology could contribute to poor outcomes following a systemic inflammatory insult.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Citocinas/metabolismo , Hipocampo/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos Transgênicos/metabolismo , Redução de Peso/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Gliose/metabolismo , Gliose/patologia , Hipocampo/patologia , Inflamação/metabolismo , Camundongos , Microglia/metabolismo , Microglia/patologia , Placa Amiloide/metabolismo , Placa Amiloide/patologia
2.
Sci Rep ; 12(1): 496, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017633

RESUMO

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.


Assuntos
Vesículas Extracelulares/metabolismo , Proteínas de Fluorescência Verde/genética , Camundongos Transgênicos/genética , Animais , Células Epiteliais/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/genética , Genes Reporter , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Túbulos Renais Distais/citologia , Túbulos Renais Distais/metabolismo , Camundongos , Camundongos Transgênicos/metabolismo , Miócitos Cardíacos/metabolismo , Especificidade de Órgãos , Transgenes
3.
Int J Mol Sci ; 22(24)2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34948212

RESUMO

CCN5/WISP2 is a matricellular protein, the expression of which is under the regulation of Wnt signaling and IGF-1. Our initial characterization supports the notion that CCN5 might promote the proliferation and survival of pancreatic ß-cells and thus improve the metabolic profile of the animals. More recently, the roles of endogenous expression of CCN5 and its ectopic, transgenic overexpression on metabolic regulation have been revealed through two reports. Here, we attempt to compare the experimental findings from those studies, side-by-side, in order to further establish its roles in metabolic regulation. Prominent among the discoveries was that a systemic deficiency of CCN5 gene expression caused adipocyte hypertrophy, increased adipogenesis, and lipid accumulation, resulting in insulin resistance and glucose intolerance, which were further exacerbated upon high-fat diet feeding. On the other hand, the adipocyte-specific and systemic overexpression of CCN5 caused an increase in lean body mass, improved insulin sensitivity, hyperplasia of cardiomyocytes, and increased heart mass, but decreased fasting glucose levels. CCN5 is clearly a regulator of adipocyte proliferation and maturation, affecting lean/fat mass ratio and insulin sensitivity. Not all results from these models are consistent; moreover, several important aspects of CCN5 physiology are yet to be explored.


Assuntos
Proteínas de Sinalização Intercelular CCN/genética , Proteínas de Sinalização Intercelular CCN/metabolismo , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Animais , Expressão Gênica/genética , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Miócitos Cardíacos/metabolismo
4.
Int J Mol Sci ; 22(21)2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34769048

RESUMO

(1) Background: Amyotrophic lateral sclerosis (ALS) is an incurable, neurodegenerative disease. In some cases, ALS causes behavioral disturbances and cognitive dysfunction. Swimming has revealed a neuroprotective influence on the motor neurons in ALS. (2) Methods: In the present study, a SOD1-G93A mice model of ALS were used, with wild-type B6SJL mice as controls. ALS mice were analyzed before ALS onset (10th week of life), at ALS 1 onset (first symptoms of the disease, ALS 1 onset, and ALS 1 onset SWIM), and at terminal ALS (last stage of the disease, ALS TER, and ALS TER SWIM), and compared with wild-type mice. Swim training was applied 5 times per week for 30 min. All mice underwent behavioral tests. The spinal cord was analyzed for the enzyme activities and oxidative stress markers. (3) Results: Pre-symptomatic ALS mice showed increased locomotor activity versus control mice; the swim training reduced these symptoms. The metabolic changes in the spinal cord were present at the pre-symptomatic stage of the disease with a shift towards glycolytic processes at the terminal stage of ALS. Swim training caused an adaptation, resulting in higher glutathione peroxidase (GPx) and protection against oxidative stress. (4) Conclusion: Therapeutic aquatic activity might slow down the progression of ALS.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Glutationa Peroxidase/metabolismo , Locomoção/fisiologia , Neurônios Motores/fisiologia , Medula Espinal/metabolismo , Natação/fisiologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Masculino , Camundongos , Camundongos Transgênicos/metabolismo , Camundongos Transgênicos/fisiologia , Microglia/metabolismo , Microglia/fisiologia , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Neurônios Motores/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Estresse Oxidativo/fisiologia , Medula Espinal/fisiopatologia , Superóxido Dismutase/metabolismo
5.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638584

RESUMO

Skeletal muscle is a vital organ for a healthy life, but its mass and function decline with aging, resulting in a condition termed sarcopenia. The etiology of sarcopenia remains unclear. We recently demonstrated that interstitial mesenchymal progenitors are essential for homeostatic muscle maintenance, and a diminished expression of the mesenchymal-specific gene Bmp3b is associated with sarcopenia. Here, we assessed the protective function of Bmp3b against sarcopenia by generating conditional transgenic (Tg) mice that enable a forced expression of Bmp3b specifically in mesenchymal progenitors. The mice were grown until they reached the geriatric stage, and the age-related muscle phenotypes were examined. The Tg mice had significantly heavier muscles compared to control mice, and the type IIB myofiber cross-sectional areas were preserved in Tg mice. The composition of the myofiber types did not differ between the genotypes. The Tg mice showed a decreasing trend of fibrosis, but the degree of fat infiltration was as low as that in the control mice. Finally, we observed the preservation of innervated neuromuscular junctions (NMJs) in the Tg muscle in contrast to the control muscle, where the NMJ degeneration was conspicuous. Thus, our results indicate that the transgenic expression of Bmp3b in mesenchymal progenitors alleviates age-related muscle deterioration. Collectively, this study strengthens the beneficial role of mesenchymal Bmp3b against sarcopenia and suggests that preserving the youthfulness of mesenchymal progenitors may be an effective means of combating sarcopenia.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Junção Neuromuscular/metabolismo , Envelhecimento/metabolismo , Animais , Fator 10 de Diferenciação de Crescimento/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/metabolismo , Sarcopenia/metabolismo
6.
Biochim Biophys Acta Biomembr ; 1863(8): 183616, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33872576

RESUMO

Aquaporin-4 (AQP4) water channels and gap junction proteins (connexins) are two classes of astrocytic membrane proteins critically involved in brain water and ion homeostasis. AQP4 channels are anchored by α1-syntrophin to the perivascular astrocytic endfoot membrane domains where they control water flux at the blood-brain interface while connexins cluster at the lateral aspects of the astrocytic endfeet forming gap junctions that allow water and ions to dissipate through the astrocyte syncytium. Recent studies have pointed to an interdependence between astrocytic AQP4 and astrocytic gap junctions but the underlying mechanism remains to be explored. Here we use a novel transgenic mouse line to unravel whether ß1-syntrophin (coexpressed with α1-syntrophin in astrocytic plasma membranes) is implicated in the expression of AQP4 isoforms and formation of gap junctions in brain. Our results show that while the effect of ß1-syntrophin deletion is rather limited, double knockout of α1- and ß1-syntrophin causes a downregulation of the novel AQP4 isoform AQP4ex and an increase in the number of astrocytic gap junctions. The present study highlight the importance of syntrophins in orchestrating specialized functional domains of brain astrocytes.


Assuntos
Aquaporina 4/genética , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/genética , Conexina 43/genética , Proteínas Associadas à Distrofina/genética , Proteínas de Membrana/genética , Proteínas Musculares/genética , Animais , Membrana Celular/genética , Junções Comunicantes/genética , Regulação da Expressão Gênica/genética , Homeostase/genética , Íons/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Água/metabolismo
7.
Int J Mol Sci ; 22(5)2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33802349

RESUMO

Since NLRP3 inflammasome plays a pivotal role in several neurodegenerative disorders, we hypothesized that levels of inflammasome components could help in diagnosis or prognosis of amyotrophic lateral sclerosis (ALS). Gene and protein expression was assayed by RT-PCR and Western blot. Spearman's correlation coefficient was used to determine the linear correlation of transcriptional expression levels with longevity throughout disease progression in mice models. Kaplan-Meier analysis was performed to evaluate MCC950 effects (NLRP3 inhibitor) on lifespan of SOD1G93A mice. The results showed significant alterations in NLRP3 inflammasome gene and protein levels in the skeletal muscle of SOD1G93A mice. Spearman's correlation coefficient revealed a positive association between Nlrp3 transcriptional levels in skeletal muscle and longevity of SOD1G93A mice (r = 0.506; p = 0.027). Accordingly, NLRP3 inactivation with MCC950 decreased the lifespan of mice. Furthermore, NLRP3 mRNA levels were significantly elevated in the blood of ALS patients compared to healthy controls (p = 0.03). In conclusion, NLRP3 could be involved in skeletal muscle pathogenesis of ALS, either through inflammasome or independently, and may play a dual role during disease progression. NLRP3 gene expression levels could be used as a biomarker to improve diagnosis and prognosis in skeletal muscle from animal models and also to support diagnosis in clinical practice with the blood of ALS patients.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Biomarcadores/metabolismo , Inflamassomos/metabolismo , Músculo Esquelético/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Idoso , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Progressão da Doença , Feminino , Furanos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Indenos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/metabolismo , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , Prognóstico , Sulfonamidas , Sulfonas/farmacologia , Superóxido Dismutase-1/metabolismo
9.
Int J Mol Sci ; 22(1)2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33466445

RESUMO

The lack of effective disease-modifying therapeutics to tackle Alzheimer's disease (AD) is unsettling considering the actual prevalence of this devastating neurodegenerative disorder worldwide. Intermittent hypoxic conditioning (IHC) is a powerful non-pharmacological procedure known to enhance brain resilience. In this context, the aim of the present study was to investigate the potential long-term protective impact of IHC against AD-related phenotype, putting a special focus on cognition and mitochondrial bioenergetics and dynamics. For this purpose, six-month-old male triple transgenic AD mice (3×Tg-AD) were submitted to an IHC protocol for two weeks and the behavioral assessment was performed at 8.5 months of age, while the sacrifice of mice occurred at nine months of age and their brains were removed for the remaining analyses. Interestingly, IHC was able to prevent anxiety-like behavior and memory and learning deficits and significantly reduced brain cortical levels of amyloid-ß (Aß) in 3×Tg-AD mice. Concerning brain energy metabolism, IHC caused a significant increase in brain cortical levels of glucose and a robust improvement of the mitochondrial bioenergetic profile in 3×Tg-AD mice, as mirrored by the significant increase in mitochondrial membrane potential (ΔΨm) and respiratory control ratio (RCR). Notably, the improvement of mitochondrial bioenergetics seems to result from an adaptative coordination of the distinct but intertwined aspects of the mitochondrial quality control axis. Particularly, our results indicate that IHC favors mitochondrial fusion and promotes mitochondrial biogenesis and transport and mitophagy in the brain cortex of 3×Tg-AD mice. Lastly, IHC also induced a marked reduction in synaptosomal-associated protein 25 kDa (SNAP-25) levels and a significant increase in both glutamate and GABA levels in the brain cortex of 3×Tg-AD mice, suggesting a remodeling of the synaptic microenvironment. Overall, these results demonstrate the effectiveness of the IHC paradigm in forestalling the AD-related phenotype in the 3×Tg-AD mouse model, offering new insights to AD therapy and forcing a rethink concerning the potential value of non-pharmacological interventions in clinical practice.


Assuntos
Doença de Alzheimer/fisiopatologia , Transtornos Cognitivos/fisiopatologia , Cognição/fisiologia , Metabolismo Energético/fisiologia , Hipóxia/fisiopatologia , Camundongos Transgênicos/fisiologia , Mitocôndrias/fisiologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Transtornos Cognitivos/metabolismo , Modelos Animais de Doenças , Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Transgênicos/metabolismo , Mitocôndrias/metabolismo
10.
Int J Mol Sci ; 22(2)2021 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-33477525

RESUMO

Adult humans and mice possess significant classical brown adipose tissues (BAT) and, upon cold-induction, acquire brown-like adipocytes in certain depots of white adipose tissues (WAT), known as beige adipose tissues or WAT browning/beiging. Activating thermogenic classical BAT or WAT beiging to generate heat limits diet-induced obesity or type-2 diabetes in mice. Adiponectin is a beneficial adipokine resisting diabetes, and causing "healthy obese" by increasing WAT expansion to limit lipotoxicity in other metabolic tissues during high-fat feeding. However, the role of its receptors, especially adiponectin receptor 1 (AdipoR1), on cold-induced thermogenesis in vivo in BAT and in WAT beiging is still elusive. Here, we established a cold-induction procedure in transgenic mice over-expressing AdipoR1 and applied a live 3-D [18F] fluorodeoxyglucose-PET/CT (18F-FDG PET/CT) scanning to measure BAT activity by determining glucose uptake in cold-acclimated transgenic mice. Results showed that cold-acclimated mice over-expressing AdipoR1 had diminished cold-induced glucose uptake, enlarged adipocyte size in BAT and in browned WAT, and reduced surface BAT/body temperature in vivo. Furthermore, decreased gene expression, related to thermogenic Ucp1, BAT-specific markers, BAT-enriched mitochondrial markers, lipolysis and fatty acid oxidation, and increased expression of whitening genes in BAT or in browned subcutaneous inguinal WAT of AdipoR1 mice are congruent with results of PET/CT scanning and surface body temperature in vivo. Moreover, differentiated brown-like beige adipocytes isolated from pre-adipocytes in subcutaneous WAT of transgenic AdipoR1 mice also had similar effects of lowered expression of thermogenic Ucp1, BAT selective markers, and BAT mitochondrial markers. Therefore, this study combines in vitro and in vivo results with live 3-D scanning and reveals one of the many facets of the adiponectin receptors in regulating energy homeostasis, especially in the involvement of cold-induced thermogenesis.


Assuntos
Tecido Adiposo Bege/metabolismo , Tecido Adiposo Marrom/metabolismo , Receptores de Adiponectina/genética , Termogênese/genética , Proteína Desacopladora 1/genética , Adipócitos Bege/metabolismo , Tecido Adiposo Bege/diagnóstico por imagem , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Branco/diagnóstico por imagem , Tecido Adiposo Branco/metabolismo , Animais , Metabolismo Energético/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Mitocôndrias/genética , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Tomografia por Emissão de Pósitrons
11.
Sci Rep ; 10(1): 16424, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009465

RESUMO

Trigonelline (TGN; 1-methylpyridin-1-ium-3-carboxylate) is a widely distributed alkaloid derived from plants. Since we previously found a neurite outgrowth effect of TGN, we hypothesised that TGN might help to improve memory deficits. Here, the efficacy of TGN in restoring amyloid ß (Aß)-induced axonal degeneration and in improving memory function was investigated in Alzheimer's disease 5XFAD model mice that overexpress mutated APP and PS1 genes. Exposure of Aß25-35 for 3 days induced atrophy of axons and dendrites. Post treatment of TGN recovered the lengths of axons and dendrites. Following oral administration of TGN in mice, TGN itself was detected in the plasma and cerebral cortex. Oral administration of TGN to 5XFAD mice for 14 days showed significant improvement in object recognition memory (P < 0.001) and object location memory (P < 0.01). TGN administration also normalised neurofilament light levels in the cerebral cortex (P < 0.05), which is an axonal damage-associated biomarker. Analysis of target proteins of TGN in neurons by a drug affinity responsive target stability (DARTS) method identified that creatine kinase B-type (CKB) is a direct binding protein of TGN. Treatment with a CKB inhibitor cancelled the TGN-induced axonal and dendritic growth. In conclusion, we found for the first time that TGN penetrates the brain and may activate CKB, leading to axonal formation. This study shows the potential of TGN as a new drug candidate, and a new target molecule, CKB, in memory recovery signalling.


Assuntos
Alcaloides/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Córtex Cerebral/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Memória/efeitos dos fármacos , Alcaloides/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Atrofia/tratamento farmacológico , Atrofia/metabolismo , Axônios/efeitos dos fármacos , Axônios/metabolismo , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Feminino , Transtornos da Memória/metabolismo , Camundongos , Camundongos Transgênicos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo
12.
Int J Mol Sci ; 21(15)2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32756522

RESUMO

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role.


Assuntos
Esclerose Lateral Amiotrófica/genética , Axônios/metabolismo , Neurônios Motores/metabolismo , Superóxido Dismutase/genética , Vesículas Sinápticas/genética , Esclerose Lateral Amiotrófica/sangue , Esclerose Lateral Amiotrófica/patologia , Animais , Axônios/patologia , Cálcio/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Neurônios Motores/patologia , Mutação/genética , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Vesículas Sinápticas/patologia
13.
Theranostics ; 9(26): 8155-8170, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31754387

RESUMO

Lung epithelial sodium channel (ENaC) encoded by Scnn1 genes is essential for maintaining transepithelial salt and fluid homeostasis in the airway and the lung. Compared to α, ß, and γ subunits, the role of respiratory δ-ENaC has not been studied in vivo due to the lack of animal models. Methods: We characterized full-length human δ802-ENaC expressed in both Xenopus oocytes and humanized transgenic mice. AT2 proliferation and differentiation in 3D organoids were analysed with FACS and a confocal microscope. Both two-electrode voltage clamp and Ussing chamber systems were applied to digitize δ802-ENaC channel activity. Immunoblotting was utilized to analyse δ802-ENaC protein. Transcripts of individual ENaC subunits in human lung tissues were quantitated with qPCR. Results: The results indicate that δ802-ENaC functions as an amiloride-inhibitable Na+ channel. Inhibitory peptide α-13 distinguishes δ802- from α-type ENaC channels. Modified proteolysis of γ-ENaC by plasmin and aprotinin did not alter the inhibition of amiloride and α-13 peptide. Expression of δ802-ENaC at the apical membrane of respiratory epithelium was detected with biophysical features similar to those of heterologously expressed channels in oocytes. δ802-ENaC regulated alveologenesis through facilitating the proliferation of alveolar type 2 epithelial cells. Conclusion: The humanized mouse line conditionally expressing human δ802-ENaC is a novel model for studying the expression and function of this protein in vivo .


Assuntos
Canais Epiteliais de Sódio/genética , Modelos Animais , Células Epiteliais Alveolares/metabolismo , Animais , Canais Epiteliais de Sódio/metabolismo , Expressão Gênica , Humanos , Transporte de Íons/genética , Transporte de Íons/fisiologia , Camundongos , Camundongos Transgênicos/metabolismo , Oócitos , Células-Tronco/metabolismo , Xenopus
14.
PLoS One ; 14(10): e0223052, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31613887

RESUMO

To further investigate the role of the phosphate (Pi) transporter PIT1 in Pi homeostasis and tissue mineralization, we developed a transgenic mouse expressing the C-terminal influenza hemagglutinin (HA) epitope-tagged human PIT1 transporter under control of the cytomegalovirus/chicken beta actin/rabbit beta-globin gene (CAG) promotor and a loxP-stop-loxP (LSL) cassette permitting conditional activation of transgene expression (LSL-HA-hPITtg/+). For an initial characterization of this conditional mouse model, germline excision of the LSL cassette was performed to induce expression of the transgene in all mouse tissues (HA-hPIT1tg/+). Recombination was confirmed using genomic DNA obtained from blood samples of these mice. Furthermore, expression of HA-hPIT1 was found to be at least 10-fold above endogenous mouse Pit1 in total RNA isolated from multiple tissues and from cultured primary calvaria osteoblasts (PCOB) estimated by semi-quantitative RT-PCR. Robust expression of the HA-hPIT1 protein was also observed upon immunoblot analysis in most tissues and permits HA-mediated immunoprecipitation of the transporter. Characterization of the phenotype of HA-hPIT1tg/+ mice at 80 days of age when fed a standard chow (0.7% Pi and 1% calcium) showed elevated plasma Pi, but normal plasma iPTH, iFGF23, serum calcium, BUN, 1,25-dihydroxy vitamin D levels and urine Pi, calcium and protein excretion when compared to WT littermates. Likewise, no change in bone mineral density was observed upon uCT analysis of the distal femur obtained from these mice. In conclusion, heterozygous overexpression of HA-hPIT1 is compatible with life and causes hyperphosphatemia while bone and mineral metabolism of these mice are otherwise normal.


Assuntos
Efeito Fundador , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Camundongos Transgênicos/genética , Fosfatos/metabolismo , Fator de Transcrição Pit-1/genética , Transgenes , Actinas/genética , Actinas/metabolismo , Animais , Transporte Biológico , Densidade Óssea , Calcitriol/sangue , Galinhas , Citomegalovirus/genética , Citomegalovirus/metabolismo , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Crânio/citologia , Crânio/metabolismo , Fator de Transcrição Pit-1/metabolismo , Globinas beta/genética , Globinas beta/metabolismo
15.
PLoS One ; 14(8): e0220818, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31393940

RESUMO

Cytokeratin 19 (KRT19) protein is highly expressed in the epithelium of the gastrointestinal (GI) tract, hepatobiliary tissues, and pancreas of humans and mice. In the present study, we used an improved Cre (iCre) gene to enhance the efficiency of Cre expression in mammalian cells. We established a new transgenic Krt19-iCre bacterial artificial chromosome (BAC) mouse model using the BAC recombineering strategy. Site-specific iCre expression pattern was examined in embryos, adults, and elderly Krt19-iCre mice crossed with Tomato or LacZ reporter mice. Both iCre and reporter protein expressions in adult Krt19-iCre;Tomatoflox/+ (Krt19-iCre Tomato reporter) mice were observed mainly in the epithelial cells of the GI tract, hepatobiliary tissues, and pancreas. However, the expression in the intrahepatic and small pancreatic duct were lower than those in the common bile and large pancreatic duct. In the Krt19-iCre; LacZ reporter embryos, ß-galactosidase for the LacZ reporter was expressed in the glandular epithelial cells of the GI tract in 9.5-day embryos, 12-day embryos, and newborn mice. The reporter protein expression in Krt19-iCre-Tomato reporter mice was consistent with the KRT19 expression in human GI tissues. In conclusion, Krt19-iCre BAC transgenic mice can be used to investigate developmental and pathological conditions using the iCre-loxP system.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Sistema Digestório/metabolismo , Integrases/metabolismo , Queratina-19/metabolismo , Camundongos Transgênicos/metabolismo , Animais , Genes Reporter , Óperon Lac , Camundongos , Modelos Animais
16.
FASEB J ; 33(3): 3549-3561, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30423260

RESUMO

Excessive iron increases the incidence of diabetes and worsens diabetic complications. Reciprocally, diabetes induces iron loading, partially attributable to elevated intestinal iron export according to a recent report. Herein, we show that iron uptake and the mRNA expression of iron importer divalent metal transporter 1 (DMT1) were significantly increased in the duodenum of streptozotocin-induced diabetic mice. Immunofluorescence staining of human intestinal biopsies revealed increased brush border membrane (BBM) and decreased cytoplasmic DMT1 expression in patients with diabetes, suggesting translocation of DMT1. This pattern of DMT1 regulation was corroborated by immunoblotting results in diabetic mice showing that BBM DMT1 expression was increased by 210%, in contrast to a 60% increase in total DMT1. PKC mediates many diabetic complications, and PKCα activity was increased in diabetic mouse intestine. Intriguingly, diabetic mice with PKCα deficiency did not show increases in iron uptake and BBM DMT1 expression. High-glucose treatment increased plasma membrane DMT1 expression via the activation of PKCα in cultured IECs. Inhibition of PKCα potentiated the ubiquitination and degradation of DMT1 protein. We further showed that high glucose suppressed membrane DMT1 internalization. These findings demonstrate that PKCα promotes microvillus membrane DMT1 expression and intestinal iron uptake, contributing to diabetic iron loading.-Zhao, L., Bartnikas, T., Chu, X., Klein, J., Yun, C., Srinivasan, S., He, P. Hyperglycemia promotes microvillus membrane expression of DMT1 in intestinal epithelial cells in a PKCα-dependent manner.


Assuntos
Duodeno/metabolismo , Células Epiteliais/metabolismo , Hiperglicemia/metabolismo , Microvilosidades/metabolismo , Proteína Quinase C-alfa/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Idoso , Animais , Transporte Biológico/fisiologia , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Ferro/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos/metabolismo , Pessoa de Meia-Idade , Ubiquitinação/fisiologia
17.
Heart Fail Rev ; 24(2): 269-277, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30284070

RESUMO

Iron deficiency (ID) is a common and ominous comorbidity in heart failure (HF) and predicts worse outcomes, independently of the presence of anaemia. Accumulated data from animal models of systemic ID suggest that ID is associated with several functional and structural abnormalities of the heart. However, the exact role of myocardial iron deficiency irrespective of systemic ID and/or anaemia has been elusive. Recently, several transgenic models of cardiac-specific ID have been developed to investigate the influence of ID on cardiac tissue. In this review, we discuss structural and functional cardiac consequences of ID in these models and summarize data from clinical studies. Moreover, the beneficial effects of intravenous iron supplementation are specified.


Assuntos
Anemia Ferropriva/complicações , Insuficiência Cardíaca/fisiopatologia , Coração/fisiopatologia , Deficiências de Ferro , Ferro/sangue , Administração Intravenosa , Animais , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Comorbidade , Feminino , Coração/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/mortalidade , Hepcidinas/metabolismo , Homeostase/fisiologia , Humanos , Ferro/administração & dosagem , Ferro/uso terapêutico , Distúrbios do Metabolismo do Ferro/complicações , Masculino , Camundongos , Camundongos Transgênicos/metabolismo , Modelos Animais , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Receptores da Transferrina/metabolismo
18.
Stem Cells Transl Med ; 8(3): 236-246, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30474937

RESUMO

Circulating angiogenic cells (CACs) have been implicated in the repair of ischemic tissues, and their mobilization from bone marrow is known to be regulated by the activations of chemokine receptors, including CXCR2 and CXCR4. This study was conducted to investigate the role of N-acetylated proline-glycine-proline (Ac-PGP; a collagen-derived chemotactic tripeptide) on CAC mobilization and its therapeutic potential for the treatment of peripheral artery diseases. Ac-PGP was administered daily to a murine hind limb ischemia model, and the effects of Ac-PGP on blood perfusion and CAC mobilization (Sca1+ Flk1+ cells) into peripheral blood were assessed. Intramuscular administration of Ac-PGP significantly improved ischemic limb perfusion and increased limb salvage rate by increasing blood vessel formation, whereas Ac-PGP-induced blood perfusion and angiogenesis in ischemic limbs were not observed in CXCR2-knockout mice. In addition, Ac-PGP-induced CAC mobilization was found to occur in wild-type mice but not in CXCR2-knockout mice. Transplantation of bone marrow from green fluorescent protein (GFP) transgenic mice to wild-type mice showed bone marrow-derived cells homed to ischemic limbs after Ac-PGP administration and that GFP-positive cells contributed to the formation of ILB4-positive capillaries and α smooth muscle actin (α-SMA)-positive arteries. These results suggest CXCR2 activation in bone marrow after Ac-PGP administration improves blood perfusion and reduces tissue necrosis by inducing CAC mobilization. These findings suggest a new pharmaceutical basis for the treatment of critical limb ischemia. Stem Cells Translational Medicine 2019;8:236&246.


Assuntos
Neovascularização Fisiológica/fisiologia , Peptídeos/metabolismo , Receptores de Interleucina-8B/metabolismo , Animais , Capilares/metabolismo , Membro Posterior/metabolismo , Isquemia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos/metabolismo
19.
FASEB J ; 33(3): 3575-3589, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30452882

RESUMO

Soluble receptor for advanced glycation end products (sRAGE), shed from cell surfaces, is found in human circulation and has been implicated in cardiovascular disease. Its pathophysiological regulation and underlying mechanisms are scarcely understood. In endothelium-specific human RAGE transgenic mice, human sRAGE was detected in circulation, whereas its level was markedly increased after LPS treatment. That increase was preceded by a rapid rise in TNF-α level. Treatment with TNF-α also significantly increased serum sRAGE. In human microvascular endothelial cells or human umbilical vein endothelial cells with RAGE overexpression, TNF-α markedly induced RAGE shedding, which was dependent on MMP9 and ADAM10. TNF-α-stimulated MMP9 expression was completely dependent on JNK activation, with its inhibition partially effective in suppressing TNF-α-induced RAGE shedding. In contrast, TNF-α transiently induced activation transcription factor (ATF)4, a major component in unfolded protein response (UPR), whereas knockdown of ATF4 abrogated TNF-α-stimulated RAGE shedding. Protein levels of the pro and activated forms of ADAM10 were also decreased by ATF4 knockdown, whereas inhibition of other components of UPR, including XBP1 and ATF6, failed to block TNF-α-stimulated RAGE shedding. Although the endoplasmic reticulum stressors thapsigargin and tunicamycin induced markedly and sustained expression of ATF4 and XBP-1, they did not induce RAGE shedding to the same level as TNF-α, suggesting that ATF4 is necessary but not sufficient alone for TNF-α-mediated RAGE shedding. ATF4 inhibition did not affect TNF-α-stimulated MMP9 expression, whereas inhibition of JNK activity did not influence ADAM10 activation. Thus, inflammatory cascades including TNF-α induced RAGE shedding in endothelial cells in vivo and in vitro. JNK and ATF4 may be 2 platforms for regulation of TNF-α-stimulated RAGE shedding.-Miyoshi, A., Koyama, S., Sasagawa-Monden, M., Kadoya, M., Konishi, K., Shoji, T., Inaba, M., Yamamoto, Y., Koyama, H. JNK and ATF4 as two important platforms for tumor necrosis factor-α-stimulated shedding of receptor for advanced glycation end products.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos/metabolismo
20.
JCI Insight ; 3(21)2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385721

RESUMO

TGF-ß signals through a receptor complex composed of 2 type I and 2 type II (TGF-ßRII) subunits. We investigated the role of macrophage TGF-ß signaling in fibrosis after AKI in mice with selective monocyte/macrophage TGF-ßRII deletion (macrophage TGF-ßRII-/- mice). Four weeks after injury, renal TGF-ß1 expression and fibrosis were higher in WT mice than macrophage TGF-ßRII-/- mice, which had decreased renal macrophages. The in vitro chemotactic response to f-Met-Leu-Phe was comparable between bone marrow-derived monocytes (BMMs) from WT and macrophage TGF-ßRII-/- mice, but TGF-ßRII-/- BMMs did not respond to TGF-ß. We then implanted Matrigel plugs suffused with either f-Met-Leu-Phe or TGF-ß1 into WT or macrophage TGF-ßRII-/- mice. After 6 days, f-Met-Leu-Phe induced similar macrophage infiltration into the Matrigel plugs of WT and macrophage TGF-ßRII-/- mice, but TGF-ß induced infiltration only in WT mice. We further determined the number of labeled WT or TGF-ßRII-/- BMMs infiltrating into WT kidneys 20 days after ischemic injury. There were more labeled WT BMMs than TGF-ßRII-/- BMMs. Therefore, macrophage TGF-ßRII deletion protects against the development of tubulointerstitial fibrosis following severe ischemic renal injury. Chemoattraction of macrophages to the injured kidney through a TGF-ß/TGF-ßRII axis is a heretofore undescribed mechanism by which TGF-ß can mediate renal fibrosis during progressive renal injury.


Assuntos
Injúria Renal Aguda/patologia , Fibrose/metabolismo , Rim/metabolismo , Macrófagos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Injúria Renal Aguda/complicações , Animais , Células da Medula Óssea/citologia , Fatores Quimiotáticos/metabolismo , Fatores Quimiotáticos/fisiologia , Fibrose/etiologia , Rim/patologia , Masculino , Camundongos , Camundongos Transgênicos/metabolismo , Monócitos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
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