RESUMEN
In contrast to stage-specific transcription factors, the role of ubiquitous transcription factors in neuronal development remains a matter of scrutiny. Here, we demonstrated that a ubiquitous factor NF-Y is essential for neural progenitor maintenance during brain morphogenesis. Deletion of the NF-YA subunit in neural progenitors by using nestin-cre transgene in mice resulted in significant abnormalities in brain morphology, including a thinner cerebral cortex and loss of striatum during embryogenesis. Detailed analyses revealed a progressive decline in multiple neural progenitors in the cerebral cortex and ganglionic eminences, accompanied by induced apoptotic cell death and reduced cell proliferation. In neural progenitors, the NF-YA short isoform lacking exon 3 is dominant and co-expressed with cell cycle genes. ChIP-seq analysis from the cortex during early corticogenesis revealed preferential binding of NF-Y to the cell cycle genes, some of which were confirmed to be downregulated following NF-YA deletion. Notably, the NF-YA short isoform disappears and is replaced by its long isoform during neuronal differentiation. Forced expression of the NF-YA long isoform in neural progenitors resulted in a significant decline in neuronal count, possibly due to the suppression of cell proliferation. Collectively, we elucidated a critical role of the NF-YA short isoform in maintaining neural progenitors, possibly by regulating cell proliferation and apoptosis. Moreover, we identified an isoform switch in NF-YA within the neuronal lineage in vivo, which may explain the stage-specific role of NF-Y during neuronal development.
Asunto(s)
Factor de Unión a CCAAT , Corteza Cerebral , Animales , Ratones , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Regulación de la Expresión Génica , Neurogénesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
We reviewed 20 randomized controlled trials concerning the intervention methods and effects of lavender essential oil on adults' sleep quality. Fourteen showed positive intervention effects. A mixture of subjective and objective indicators was used. Lavender essential oil was associated with improved sleep quality before insomnia or other sleep disorders occurred.
Asunto(s)
Lavandula , Aceites Volátiles , Trastornos del Inicio y del Mantenimiento del Sueño , Trastornos del Sueño-Vigilia , Adulto , Humanos , Aceites Volátiles/farmacología , Aceites Volátiles/uso terapéutico , Sueño , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Calidad del SueñoRESUMEN
Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.
Asunto(s)
Endopeptidasa K/metabolismo , Agregación Patológica de Proteínas/metabolismo , Sinucleinopatías/metabolismo , alfa-Sinucleína/metabolismo , Anciano , Animales , Encéfalo/metabolismo , Encéfalo/patología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Agregado de Proteínas , Agregación Patológica de Proteínas/patología , Sinucleinopatías/patologíaRESUMEN
The bone is the main storage site for Ca2+ and Mg2+ ions in the mammalian body. Although investigations into Ca2+ signaling have progressed rapidly and led to better understanding of bone biology, the Mg2+ signaling pathway and associated molecules remain to be elucidated. Here, we investigated the role of a potential Mg2+ signaling-related lysosomal molecule, two-pore channel subtype 2 (TPC2), in osteoclast differentiation and bone remodeling. Previously, we found that under normal Mg2+ conditions, TPC2 promotes osteoclastogenesis. We observed that under low-Mg2+ conditions, TPC2 inhibited, rather than promoted, the osteoclast differentiation and that the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) signaling pathway played a role in the TPC2 activation under low-Mg2+ conditions. Furthermore, PI(3,5)P2 depolarized the membrane potential by increasing the intracellular Na+ levels. To investigate how membrane depolarization affects osteoclast differentiation, we generated a light-sensitive cell line and developed a system for the light-stimulated depolarization of the membrane potential. The light-induced depolarization inhibited the osteoclast differentiation. We then tested the effect of myo-inositol supplementation, which increased the PI(3,5)P2 levels in mice fed a low-Mg2+ diet. The myo-inositol supplementation rescued the low-Mg2+ diet-induced trabecular bone loss, which was accompanied by the inhibition of osteoclastogenesis. These results indicate that low-Mg2+-induced osteoclastogenesis involves changes in the role of TPC2, which are mediated through the PI(3,5)P2 pathway. Our findings also suggest that myo-inositol consumption might provide beneficial effects in Mg2+ deficiency-induced skeletal diseases.
Asunto(s)
Canales de Calcio/metabolismo , Magnesio/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Animales , Remodelación Ósea/efectos de los fármacos , Remodelación Ósea/fisiología , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Resorción Ósea/patología , Señalización del Calcio , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Inositol/farmacología , Lisosomas/metabolismo , Deficiencia de Magnesio/tratamiento farmacológico , Deficiencia de Magnesio/metabolismo , Deficiencia de Magnesio/patología , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Fosfatos de Fosfatidilinositol/metabolismo , Células RAW 264.7 , Sodio/metabolismoRESUMEN
Background and Purpose: Visualizing the thought processes of nurses is useful in forming evidence to prevent falls. This study aimed to quantify nursing judgment by comparing the choices made by nurses with different experiences regarding fall prevention. Methods: Questionnaires were administered to participants with <9 and ≥10 years of nursing experience to examine their importance ratings regarding fall prevention using an analytic hierarchy process (AHP). Results: Compared with the group with <9 years of experience, the group with ≥10 years of experience viewed habitual behavior in unstable activity as the most important fall risk. They also viewed early detection and alleviation of symptoms that lead to fall risk due to side effects of drugs and diseases as an important nursing practice. Conclusion: Since differences in nursing judgment between experienced and inexperienced nurses were revealed, it is possible that nursing judgment can be measured using AHP.
RESUMEN
Amyloid fibril deposits are a main source of pathology in neurodegenerative diseases. Normal proteins such as tau, alpha-synuclein, TDP-43 and others could form specific conformational fibrils called amyloid, which deposited in the brains of neurodegenerative diseases. Although the pathological roles of amyloids in cell death have been discussed a lot, their other functions have not been investigated well. Here, we studied the effect of amyloids on DNA transfection in vivo. We injected quantum dot labeled or non-labeled amyloid-preformed fibrils (PFFs) and a green fluorescent protein (EGFP) expression vector into organs including brain, testis, liver and calf muscle. GFP expression patterns were examined by immunohistochemistry and western blotting. At 24 h after injection, EGFP was predominantly expressed in the neurons in the cortex and the striatum, Leydig cells in testis, hepatocytes in the liver and muscle cells. EGFP expression was inhibited by an endocytosis inhibitor, sertraline in the brain and testis. The amyloid-PFFs potentiated Ca2+ transients shown by calcium imaging and EGFP expression in the brain was blocked by Ca blocker, cilnidipine. Our results show that amyloid-PFFs facilitate DNA transfection and can be used for a new gene delivery system in vivo.
Asunto(s)
Amiloide , alfa-Sinucleína , ADN/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Neuronas/metabolismo , Transfección , alfa-Sinucleína/metabolismoRESUMEN
Synucleinopathies are neurodegenerative disorders including Parkinson disease (PD), dementia with Lewy body (DLB), and multiple system atrophy (MSA) that involve deposits of the protein alpha-synuclein (α-syn) in the brain. The inoculation of α-syn aggregates derived from synucleinopathy or preformed fibrils (PFF) formed in vitro induces misfolding and deposition of endogenous α-syn. This is referred to as prion-like transmission, and the mechanism is still unknown. In this study, we label α-syn PFF with quantum dots and visualize their movement directly in acute slices of brain tissue inoculated with α-syn PFF seeds. Using this system, we find that the trafficking of α-syn seeds is dependent on fast axonal transport and the seed spreading is dependent on endocytosis and neuronal activity. We also observe pharmacological effects on α-syn seed spreading; clinically available drugs including riluzole are effective in reducing the spread of α-syn seeds and this effect is also observed in vivo. Our quantum-dot-labeled α-syn seed assay system combined with in vivo transmission experiment reveals an early phase of transmission, in which uptake and spreading of seeds occur depending on neuronal activity, and a later phase, in which seeds induce the propagation of endogenous misfolded α-syn.
Asunto(s)
Enfermedad de Parkinson , Priones , Puntos Cuánticos , Sinucleinopatías , Encéfalo/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Priones/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
The pathological form of a-synuclein (a-syn) is transmitted through neural circuits in the brains of Parkinson disease (PD) patients and amplifies misfolded a-syn, further forming intracellular deposits. However, the details of a-syn pre-formed fibrils (PFFs) transmission in vivo have not been fully elucidated. By inoculating Quantum dots (QD)-labeled a-syn PFFs (QD-a-syn PFFs) into the unilateral striatum, we detected QD-a-syn PFFs in brain homogenates obtained from the ipsilateral and contralateral sides of the inoculated site and further obtained QD-a-syn PFFs enriched-particles with fluorescence-activated organelle sorting. Proteomic analysis suggested that QD-a-syn PFFs-enriched particles in the contralateral side were associated with component proteins of synapse. In contrast, QD-a-syn PFFs-enriched particles in the ipsilateral side were associated with proteins belonging to ER components. Immunostaining of brain sections confirmed that QD-a-syn PFFs in the contralateral side were co-localized with synaptic vesicle marker proteins in the cortex and striatum. Additionally, QD-a-syn PFFs in the ipsilateral side were more co-localized with ER marker proteins compared to the contralateral side. These results correspond to proteomic analysis. This study provides potential candidates for the subcellular localization of a-syn PFFs in vivo during the dissemination phase of seeds. These subcellular compartments could be involved in the transmission of seeds.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Encéfalo/metabolismo , Humanos , Proteómica , Vesículas Sinápticas/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
Zinc is a trace element in the mammalian body, and increasing evidence shows its critical role in bone development and osteoclastogenesis. The relationships between zinc and voltage-gated ion channels have been reported; however, the effects of zinc on membrane potential and the related ion channels remain unknown. In this study, we found that zinc-induced hyperpolarization in RAW264.7 cells (RAW) was promoted by inhibition of hyperpolarization-activated cyclic nucleotide modulated channels (HCNs). In electrophysiological experiments with RAW-derived osteoclasts, HCNs were functional and generated hyperpolarization-activated inward currents (Ih) with properties similar to the Ih recorded in excitable cells such as neurons and cardiomyocytes. Quantitative PCR of HCN subunits HCN1 and HCN4 in RAW cells showed detectable levels of HCN1 mRNA and HCN4 expression was the highest of all four subunits. HCN4 knockdown decreased osteoclastic Ih and promoted osteoclastogenesis in the presence of zinc, but not in the absence of zinc. To determine the effect of membrane hyperpolarization on osteoclastogenesis, we developed a light-controllable membrane potential system in RAW cells by stably expressing the light-driven outward proton pump, Archaerhodopsin3 (Arch). Arch activation by yellow-green light hyperpolarizes the cell membrane. Light-induced hyperpolarization accelerated osteoclast differentiation in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL). Thus, HCN activation reduced the hyperpolarization-related promotion of osteoclast differentiation in the presence of zinc. This study revealed the novel role of HCN and membrane potential in non-excitable osteoclasts.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Potenciales de la Membrana , Osteoclastos/citología , Zinc/química , Animales , Diferenciación Celular , Membrana Celular , Electrofisiología , Proteínas Fluorescentes Verdes/metabolismo , Luz , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Neuronas/metabolismo , Ligando RANK/metabolismo , Oligoelementos/químicaRESUMEN
Parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (VD3) are important factors in Ca(2+) homeostasis, and promote osteoclastogenesis by modulating receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression. However, their contribution to RANKL intracellular transport (RANKLiT), including the trigger for RANKL lysosomal vesicle (RANKL-lv) fusion to the cell membrane, is unclear. In neurons, depolarization of membrane potential increases the intracellular Ca(2+) level ([Ca(2+)]i) and promotes neurotransmitter release via fusion of the synaptic vesicles to the cell membrane. To determine whether membrane depolarization also regulates cellular processes such as RANKLiT in MC3T3-E1 osteoblasts (OBs), we generated a light-sensitive OB cell line and developed a system for altering their membrane potential via delivery of a blue light stimulus. In the membrane fraction of RANKL-overexpressing OBs, PTH and VD3 increased the membrane-bound RANKL (mbRANKL) level at 10 min after application without affecting the mRNA expression level, and depolarized the cell membrane while transiently increasing [Ca(2+)]i. In our novel OB line stably expressing the channelrhodopsin-wide receiver, blue light-induced depolarization increased the mbRANKL level, which was reversed by treatment of blockers for L-type voltage-gated Ca(2+) channels and Ca(2+) release from the endoplasmic reticulum. In co-cultures of osteoclast precursor-like RAW264.7 cells and light-sensitive OBs overexpressing RANKL, light stimulation induced an increase in tartrate-resistant acid phosphatase activity and promoted osteoclast differentiation. These results indicate that depolarization of the cell membrane is a trigger for RANKL-lv fusion to the membrane and that membrane potential contributes to the function of OBs. In addition, the non-genomic action of VD3-induced RANKL-lv fusion included the membrane-bound VD3 receptor (1,25D3-MARRS receptor). Elucidating the mechanism of RANKLiT regulation by PTH and VD3 will be useful for the development of drugs to prevent bone loss in osteoporosis and other bone diseases.