RESUMO
BAX is a Bcl-2 family protein acting on apoptosis. It also promotes mitochondrial fusion by interacting with the mitochondrial fusion protein Mitofusin (Mfn1 and Mfn2). Neuronal mitochondria are important for the development and modification of dendritic spines, which are subcellular compartments accommodating excitatory synapses in postsynaptic neurons. The abundance of dendritic mitochondria influences dendritic spine development. Mitochondrial fusion is essential for mitochondrial homeostasis. Here, we show that in the hippocampal neuron of BAX knockout mice, mitochondrial fusion is impaired, leading to decreases in mitochondrial length and total mitochondrial mass in dendrites. Notably, BAX knockout mice also have fewer dendritic spines and less cellular Adenosine 5'triphosphate (ATP) in dendrites. The spine and ATP changes are abolished by restoring mitochondria fusion via overexpressing Mfn1 and Mfn2. These findings indicate that BAX-mediated mitochondrial fusion in neurons is crucial for the development of dendritic spines and the maintenance of cellular ATP levels.
Assuntos
Espinhas Dendríticas , Dinâmica Mitocondrial , Trifosfato de Adenosina , Animais , Espinhas Dendríticas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Camundongos , Proteínas Mitocondriais/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Cisplatin (DDP), a widely used chemotherapeutic drug in cancer treatment, causes oxidative stress, resulting in cancer cachexia and skeletal muscle atrophy. This study investigated the effects and activity of silibinin (SLI) in reducing DDP-induced oxidative stress and skeletal muscle atrophy in vivo and in vitro. SLI alleviated weight loss, food intake, muscle wasting, adipose tissue depletion, and organ weight reduction induced by DDP and improved the reduction of grip force caused by DDP. SLI can attenuated the increase in reactive oxygen species (ROS) levels, the decrease in Nrf2 expression, the decrease in the fiber cross-sectional area, and changes in fiber type induced by DDP. SLI regulated the ERK/FoxO and JNK/FoxO pathways by downregulating the abnormal increase in ROS and Nrf2 expression in DDP-treated skeletal muscle and C2C12 myotube cells. Further, SLI inhibited the upregulation of MAFbx and Mstn, the downregulation of MyHC and MyoG, the increase in protein degradation, and the decrease of protein synthesis. The protective effects of SLI were reversed by cotreatment with JNK agonists and ERK inhibitors. These results suggest that SLI can reduce DDP-induced skeletal muscle atrophy by reducing oxidative stress and regulating ERK/FoxO and JNK/FoxO pathways.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Sistema de Sinalização das MAP Quinases/imunologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/tratamento farmacológico , Estresse Oxidativo/fisiologia , Silibina/uso terapêutico , Animais , Antineoplásicos Fitogênicos/farmacologia , Humanos , Masculino , Camundongos , Silibina/farmacologiaRESUMO
Myosin 18Aα is a myosin 2-like protein containing unique N- and C-terminal protein interaction domains that co-assembles with myosin 2. One protein known to bind to myosin 18Aα is ß-Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42 that has been shown to promote dendritic spine maturation by activating the assembly of actin and myosin filaments in spines. Here, we show that myosin 18A⺠concentrates in the spines of cerebellar Purkinje neurons via co-assembly with myosin 2 and through an actin binding site in its N-terminal extension. miRNA-mediated knockdown of myosin 18A⺠results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18Aâº. Importantly, ß-Pix co-localizes with myosin 18A⺠in spines, and its spine localization is lost upon myosin 18A⺠knockdown or when its myosin 18A⺠binding site is deleted. Finally, we show that the spines of myosin 18A⺠knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data argue that mixed filaments of myosin 2 and myosin 18A⺠form a complex with ß-Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of ß-Pix's GEF activity.
Assuntos
Espinhas Dendríticas/metabolismo , Miosinas/metabolismo , Células de Purkinje/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Animais , Espinhas Dendríticas/genética , Deleção de Genes , Camundongos , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Miosinas/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genéticaRESUMO
Sideroflexin 1 (Sfxn1) is a mitochondrial serine transporter involved in one-carbon metabolism in blood and cancer cell lines. The expression of other Sfxn homologs varies across tissues implying that each homolog may have tissue-specific functions. RNA databases suggest that among the Sfxns, Sfxn3 may have a specific function in the brain. Here, we systematically analyzed the level, cellular distribution, and subcellular localization of Sfxn3 protein in the developing and adult rodent brain. We found that, in the cortex and hippocampus, Sfxn3 protein level is low at birth but increases during development and remains at a high level in the mature brains. Similarly, in cultured hippocampal neurons, Sfxn3 protein level is low in young neurons but increases as neurons mature. Sfxn3 protein level is much higher in neurons than in astrocytes. Within neurons, Sfxn3 localizes to mitochondria in all major neuronal compartments. Our results establish that Sfxn3 is a mitochondrial protein enriched in neurons wherein it is developmentally expressed. These findings provide a foundation for future research aimed at understanding the functions of Sfxn3 and one-carbon metabolism in neurons.
Assuntos
Química Encefálica , Proteínas de Transporte de Cátions/fisiologia , Proteínas Mitocondriais/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Proteínas de Transporte de Cátions/análise , Células Cultivadas , Córtex Cerebral/química , Córtex Cerebral/crescimento & desenvolvimento , Feminino , Hipocampo/química , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Ratos , Frações Subcelulares/químicaRESUMO
TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.
Assuntos
Audição/fisiologia , Proteínas dos Microfilamentos/fisiologia , Estereocílios/fisiologia , Actinas/fisiologia , Animais , Surdez/etiologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/deficiência , Isoformas de Proteínas/fisiologia , Estereocílios/ultraestruturaRESUMO
Of the seven P2X receptor subtypes, P2X4 receptor (P2X4R) is widely distributed in the central nervous system, including in neurons, astrocytes, and microglia. Accumulating evidence supports roles for P2X4R in the central nervous system, including regulating cell excitability, synaptic transmission, and neuropathic pain. However, little information is available about the distribution and function of P2X4R in the peripheral nervous system. In this study, we find that P2X4R is mainly localized in the lysosomes of Schwann cells in the peripheral nervous system. In cultured Schwann cells, TNF-a not only enhances the synthesis of P2X4R protein but also promotes P2X4R trafficking to the surface of Schwann cells. TNF-a-induced BDNF secretion in Schwann cells is P2X4R dependent. in vivo experiments reveal that expression of P2X4R in Schwann cells of injured nerves is strikingly upregulated following nerve crush injury. Moreover, overexpression of P2X4R in Schwann cells by genetic manipulation promotes motor and sensory functional recovery and accelerates nerve remyelination via BDNF release following nerve injury. Our results suggest that enhancement of P2X4R expression in Schwann cells after nerve injury may be an effective approach to facilitate the regrowth and remyelination of injured nerves.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Receptores Purinérgicos P2X4/biossíntese , Recuperação de Função Fisiológica/fisiologia , Remielinização/fisiologia , Células de Schwann/metabolismo , Animais , Animais Recém-Nascidos , Fator Neurotrófico Derivado do Encéfalo/agonistas , Células Cultivadas , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismos dos Nervos Periféricos/patologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X4/genética , Recuperação de Função Fisiológica/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Células de Schwann/efeitos dos fármacos , Células de Schwann/patologia , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination following peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.
RESUMO
Angiogenesis is a key process in regenerative medicine generally, as well as in the specific field of nerve regeneration. However, no convenient and objective method for evaluating the angiogenesis of tissue-engineered nerves has been reported. In this study, tissue-engineered nerves were constructed in vitro using Schwann cells differentiated from rat skin-derived precursors as supporting cells and chitosan nerve conduits combined with silk fibroin fibers as scaffolds to bridge 10-mm sciatic nerve defects in rats. Four weeks after surgery, three-dimensional blood vessel reconstructions were made through MICROFIL perfusion and micro-CT scanning, and parameter analysis of the tissue-engineered nerves was performed. New blood vessels grew into the tissue-engineered nerves from three main directions: the proximal end, the distal end, and the middle. The parameter analysis of the three-dimensional blood vessel images yielded several parameters, including the number, diameter, connection, and spatial distribution of blood vessels. The new blood vessels were mainly capillaries and microvessels, with diameters ranging from 9 to 301 µm. The blood vessels with diameters from 27 to 155 µm accounted for 82.84% of the new vessels. The microvessels in the tissue-engineered nerves implanted in vivo were relatively well-identified using the MICROFIL perfusion and micro-CT scanning method, which allows the evaluation and comparison of differences and changes of angiogenesis in tissue-engineered nerves implanted in vivo.
RESUMO
The presence of Sonic Hedgehog (Shh) and its signaling components in the neurons of the hippocampus raises a question about what role the Shh signaling pathway may play in these neurons. We show here that activation of the Shh signaling pathway stimulates axon elongation in rat hippocampal neurons. This Shh-induced effect depends on the pathway transducer Smoothened (Smo) and the transcription factor Gli1. The axon itself does not respond directly to Shh; instead, the Shh signal transduction originates from the somatodendritic region of the neurons and occurs in neurons with and without detectable primary cilia. Upon Shh stimulation, Smo localization to dendrites increases significantly. Shh pathway activation results in increased levels of profilin1 (Pfn1), an actin-binding protein. Mutations in Pfn1's actin-binding sites or reduction of Pfn1 eliminate the Shh-induced axon elongation. These findings indicate that Shh can regulate axon growth, which may be critical for development of hippocampal neurons. SIGNIFICANCE STATEMENT: Although numerous signaling mechanisms have been identified that act directly on axons to regulate their outgrowth, it is not known whether signals transduced in dendrites may also affect axon outgrowth. We describe here a transcellular signaling pathway in embryonic hippocampal neurons in which activation of Sonic Hedgehog (Shh) receptors in dendrites stimulates axon growth. The pathway involves the dendritic-membrane-associated Shh signal transducer Smoothened (Smo) and the transcription factor Gli, which induces the expression of the gene encoding the actin-binding protein profilin 1. Our findings suggest scenarios in which stimulation of Shh in dendrites results in accelerated outgrowth of the axon, which therefore reaches its presumptive postsynaptic target cell more quickly. By this mechanism, Shh may play critical roles in the development of hippocampal neuronal circuits.
Assuntos
Axônios/fisiologia , Dendritos/metabolismo , Proteínas Hedgehog/metabolismo , Hipocampo/citologia , Neurônios/citologia , Transdução de Sinais/fisiologia , Animais , Axônios/ultraestrutura , Células Cultivadas , Dendritos/efeitos dos fármacos , Dendritos/ultraestrutura , Embrião de Mamíferos , Regulação da Expressão Gênica/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/farmacologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Neurônios/efeitos dos fármacos , Profilinas/genética , Profilinas/metabolismo , Transporte Proteico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Receptor Smoothened , Fatores de Tempo , Proteína GLI1 em Dedos de ZincoRESUMO
Acetylcholine is a neuromodulatory transmitter that controls synaptic plasticity and sensory processing in many brain regions. The dorsal cochlear nucleus (DCN) is an auditory brainstem nucleus that integrates auditory signals from the cochlea with multisensory inputs from several brainstem nuclei and receives prominent cholinergic projections. In the auditory periphery, cholinergic modulation serves a neuroprotective function, reducing cochlear output under high sound levels. However, the role of cholinergic signaling in the DCN is less understood. Here we examine postsynaptic mechanisms of cholinergic modulation at glutamatergic synapses formed by parallel fiber axons onto cartwheel cells (CWCs) in the apical DCN circuit from mouse brainstem slice using calcium (Ca) imaging combined with two-photon laser glutamate uncaging onto CWC spines. Activation of muscarinic acetylcholine receptors (mAChRs) significantly increased the amplitude of both uncaging-evoked EPSPs (uEPSPs) and spine Ca transients. Our results demonstrate that mAChRs in CWC spines act by suppressing large-conductance calcium-activated potassium (BK) channels, and this effect is mediated through the cAMP/protein kinase A signaling pathway. Blocking BK channels relieves voltage-dependent magnesium block of NMDA receptors, thereby enhancing uEPSPs and spine Ca transients. Finally, we demonstrate that mAChR activation inhibits L-type Ca channels and thus may contribute to the suppression of BK channels by mAChRs. In summary, we demonstrate a novel role for BK channels in regulating glutamatergic transmission and show that this mechanism is under modulatory control of mAChRs.
Assuntos
Cálcio/metabolismo , Neurônios Colinérgicos/metabolismo , Núcleo Coclear/fisiologia , Espinhas Dendríticas/metabolismo , Potenciais Pós-Sinápticos Excitadores , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Neurônios Colinérgicos/fisiologia , Núcleo Coclear/citologia , Núcleo Coclear/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espinhas Dendríticas/fisiologia , Ácido Glutâmico/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos CBA , Bloqueadores dos Canais de Potássio/farmacologia , Receptores Muscarínicos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Sinapses/fisiologiaRESUMO
OBJECTIVE: Serum p53 protein overexpression was detected in population exposed to traffic exhaust gas to study the relation between traffic exhaust gas and the increased risk in p53 gene mutation. METHODS: Serum p53 protein expression was measured by enzyme-linked immunosorbent assay. Relationship between different types of job and serum p53 protein overexpression were studied by pearson Chi-square tests. RESULTS: Results on serum p53 protein overexpression on jobs outside of office (5.74%) were not significantly higher than jobs inside the office. However, it suggested that traffic police men (12.12%) working outside of office, with whose length of service longer than 30 years had a significant overexpression of serum p53 protein than the others (5.36%) whose length of service was less than 30 years (P < 0.05, OR = 2.43, 95% CI: 1.11 - 5.33). Overexpression rate of p53 protein appeared to be 6.89% in the group whose average weekly exposure hours were more than 40 hours, which was significant higher than the group whose exposed hours were less than 40 hours (P < 0.05, OR = 1.71, 95% CI: 1.03 - 2.81). CONCLUSION: The result suggested that traffic exhaust gas was likely to cause mutation of p53 gene and increasing the incidence of lung cancer.
Assuntos
Neoplasias Pulmonares/etiologia , Exposição Ocupacional , Proteína Supressora de Tumor p53/sangue , Emissões de Veículos/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Genes p53 , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
OBJECTIVE: To explore the pathogenic mechanisms of enterotoxigenic Escherichia coli (ETEC) in guinea pigs. METHODS: Pathological examination of the intestines was performed in ETEC-infected guinea pigs, and the intracellular free Ca2+ concentration, cytoplasmic pH, cell membrane potential and mitochondria membrane potential were determined immunohistochemically and by means of flow cytometry. RESULTS: The guinea pigs were sensitive to the human-origin ETEC, which caused pathological changes in the small intestines such as edema, hyperemia and lymphocyte infiltration, but no bacteria invasion into the epithelial cells was identified. Under transmission electron microscope, the ileal epithelial cells were shown with vacuolar degeneration and evidence of mitochondrial proliferation; heat-labile (LT) and heat-stable (ST) toxins pervaded in the ileal tissue, resulting in significant increase of intracellular free Ca2+ concentration, cytoplasmic pH value and cell membrane potential, with concomitance of significant decrease in mitochondrial membrane potential. CONCLUSIONS: ETEC disturbs the absorption and secretion functions of the intestinal epithelial cells, and induces extensive inflammation in the small intestines of guinea pigs. Besides their action on intestinal epithelial cells, LT and ST also affect the myocytes in the muscularis, and their action sites and mechanism might be similar. ETEC-induced diarrhea is correlated with excessive water excretion from the cells.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/patogenicidade , Animais , Toxinas Bacterianas/análise , Cálcio/metabolismo , Enterotoxinas/análise , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Feminino , Cobaias , Imuno-Histoquímica , Mucosa Intestinal/ultraestrutura , Masculino , Microscopia Eletrônica , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
OBJECTIVE: To isolate immunoglobulin (IgY) from egg yolk of p53 protein-immunized hens and to study its reactivity to the antigens. METHOD: Immunization of egg-laying hens was performed for 3 times at the interval of 14 days with purified p53 protein that had been expressed in E.coli. The eggs laid by these immunized hens were then collected during the whole period of the experiment to prepare IgY from egg yolk by ammonium sulfate precipitation. Final purification and identification of the IgY were performed using SDS-PAGE, enzyme-linked immunosorbent assay and Western blotting. RESULT: All immunized hens developed specific antibodies to p53 protein in contrast to the control ones. The highest titers of the IgY occurred 4 weeks after the first immunization and reached 1:10(6), which remained stable for up to 3 months. CONCLUSION: IgY technology is less costly, non-invasive, fast, simple and highly efficient to generate polyclonal antibodies.