RESUMO
BACKGROUND: PALMD (palmdelphin) belongs to the family of paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis and predict severity of the disease. METHODS: Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, coimmunoprecipitation, and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in small interferring RNA-treated EC cultures, knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the single nucleotide polymorphism rs7543130 informed about gene regulatory changes. RESULTS: ECs express the cytosolic PALMD-KKVI splice variant, which associated with RANGAP1 (RAN GTP hydrolyase activating protein 1). RANGAP1 regulates the activity of the GTPase RAN and thereby nucleocytoplasmic shuttling via XPO1 (Exportin1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently in gene regulation because of the effect on localization of transcriptional regulators. Changes in EC responsiveness on loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd-/- mouse aorta, and human aortic valve samples derived from patients with calcific aortic valve stenosis. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs. CONCLUSIONS: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD's contribution to calcific aortic valve stenosis pathology.
Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Proteínas de Membrana/genética , Estresse Mecânico , Idoso , Animais , Comunicação Celular/genética , Linhagem Celular , Movimento Celular/genética , Células Cultivadas , Biologia Computacional/métodos , Bases de Dados Genéticas , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Ontologia Genética , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Transporte ProteicoRESUMO
Lipopolysaccharide-responsive beige-like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain-containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein-truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Células Ciliadas Auditivas/metabolismo , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana/genética , Fosfoproteínas/genética , Trocadores de Sódio-Hidrogênio/genética , Estereocílios/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Adulto , Animais , Proteínas do Citoesqueleto/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/patologia , Audição/fisiologia , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fosfoproteínas/metabolismo , Domínios Proteicos , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/metabolismo , Gânglio Espiral da Cóclea/metabolismo , Gânglio Espiral da Cóclea/patologia , Estereocílios/patologiaRESUMO
Loss of Ostm1 leads to the most severe form of osteopetrosis in mice and humans. Because functional rescue of the osteopetrotic defect in these mice extended their lifespan from â¼3 weeks to 6 weeks, this unraveled a second essential role of Ostm1. We discovered that Ostm1 is highly expressed in the mouse brain in neurons, microglia, and astrocytes. At 3-4 weeks of age, mice with Ostm1 loss showed 3-10-fold stimulation of reactive gliosis, with an increased astrocyte cell population and microglia activation. This inflammatory response was associated with marked retinal photoreceptor degeneration and massive neuronal loss in the brain. Intracellular characterization of neurons revealed abnormal storage of carbohydrates, lipids, and ubiquitinated proteins, combined with marked accumulation of autophagosomes that causes frequent axonal swelling. Stimulation of autophagy was provided by specific markers and by significant down-regulation of the mammalian target of rapamycin signaling, identifying a cellular pathologic mechanism. A series of transgenic mouse lines specifically targeted to distinct central nervous system cell subpopulations determined that Ostm1 has a primary and autonomous role in neuronal homeostasis. Complete functional complementation demonstrated that the development of severe and rapid neurodegeneration in these mice is independent of the hematopoietic lineage and has clinical implications for treatment of osteopetrosis. Importantly, this study establishes a novel neurodegenerative mouse model critical for understanding the multistep pathogenic cascade of cellular autophagy disorders toward therapeutic strategy design.
Assuntos
Autofagia , Proteínas de Membrana/deficiência , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Ubiquitina-Proteína Ligases/deficiência , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Hematopoese , Homeostase , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Doenças Neurodegenerativas/genética , Neurônios/metabolismo , Neurônios/patologia , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Glycogen is the storage form of glucose in animal cells. Its degradation can rapidly provide fuel for energy production (particularly important in muscle), or replenish blood glucose during fasting by the liver. Genetic defects of glycogen metabolism give rise to glycogen storage diseases (GSDs), manifesting histologically in abnormal quantity or quality of glycogen in the cells. GSDs can be caused by defects of proteins participating in the synthesis or degradation of glycogen itself, in the glycolytic degradation of glucose phosphates in muscle and erythrocytes, in the release of glucose from liver and kidney into the bloodstream, in the clearance of glycogen from lysosomes (all, "primary GSDs"), or in the control of these pathways ("secondary GSDs"). Most genes responsible for classical, primary GSDs have probably been identified, and future progress in understanding the biochemical and genetic defects underlying unsolved disorders presenting with glycogen storage abnormalities will perhaps be predominantly in the field of secondary GSDs.
Assuntos
Doença de Depósito de Glicogênio/genética , Glicogênio/genética , Glicólise/genética , Músculos/patologia , Animais , Genética Médica , HumanosRESUMO
Neurobeachin (Nbea) regulates neuronal membrane protein trafficking and is required for the development and functioning of central and neuromuscular synapses. In homozygous knockout (KO) mice, Nbea deficiency causes perinatal death. Here, we report that heterozygous KO mice haploinsufficient for Nbea have higher body weight due to increased adipose tissue mass. In several feeding paradigms, heterozygous KO mice consumed more food than wild-type (WT) controls, and this consumption was primarily driven by calories rather than palatability. Expression analysis of feeding-related genes in the hypothalamus and brainstem with real-time PCR showed differential expression of a subset of neuropeptide or neuropeptide receptor mRNAs between WT and Nbea+/- mice in the sated state and in response to food deprivation, but not to feeding reward. In humans, we identified two intronic NBEA single-nucleotide polymorphisms (SNPs) that are significantly associated with body-mass index (BMI) in adult and juvenile cohorts. Overall, data obtained in mice and humans suggest that variation of Nbea abundance or activity critically affects body weight, presumably by influencing the activity of feeding-related neural circuits. Our study emphasizes the importance of neural mechanisms in body weight control and points out NBEA as a potential risk gene in human obesity.
Assuntos
Índice de Massa Corporal , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Comportamento Alimentar , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Obesidade/genética , Tecido Adiposo/metabolismo , Adolescente , Animais , Tronco Encefálico/metabolismo , Criança , Privação de Alimentos , Regulação da Expressão Gênica/genética , Estudos de Associação Genética , Humanos , Hipotálamo/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The protein machinery of neurotransmitter exocytosis requires efficient orchestration in space and time, for speed and precision of neurotransmission and also for synaptic ontogeny and plasticity. However, its spatial organization in situ is virtually unknown. Aczonin/Piccolo is a putative organizer protein of mammalian active zones. We determined by immunogold electron microscopy (EM) (i) the spatial arrangement (i.e., topology) of 11 segments of the Aczonin polypeptide in situ, and correlated it to (ii) the positioning of Aczonin-interacting domains of Bassoon, CAST/ELKS, Munc13, and RIM and (iii) the ultrastructurally defined presynaptic macromolecular aggregates known as dense projections and synaptic ribbons. At conventional synapses, Aczonin assumes a compact molecular topology within a layer 35 to 80 nm parallel to the plasma membrane (PM), with a "trunk" sitting on the dense projection top and a C-terminal "arm" extending down toward the PM and sideward to the dense projection periphery. At ribbon synapses, Aczonin occupies the whole ribbon area. Bassoon colocalizes with Aczonin at conventional synapses but not at ribbon synapses. At both conventional and ribbon synapses, CAST, Munc13, and RIM are segregated from Aczonin, closer to the PM, and Aczonin is positioned such that it may control the access of neurotransmitter vesicles to the fusion site.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas de Ligação ao GTP/metabolismo , Immunoblotting , Microscopia Imunoeletrônica , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Sinapses/ultraestruturaRESUMO
ABSTRACT: Hematopoietic stem cells (HSCs) can generate all blood cells. This ability is exploited in HSC transplantation (HSCT) to treat hematologic disease. A clear understanding of the molecular mechanisms that regulate HSCT is necessary to continue improving transplant protocols. We identified the Beige and Chediak-Higashi domain-containing protein (BDCP), Neurobeachin (NBEA), as a putative regulator of HSCT. Here, we demonstrated that NBEA and related BDCPs, including LPS Responsive Beige-Like Anchor Protein (LRBA), Neurobeachin Like 1 (NBEAL1) and Lysosomal Trafficking Regulator (LYST), are required during HSCT to efficiently reconstitute the hematopoietic system of lethally irradiated mice. Nbea knockdown in mouse HSCs induced apoptosis and a differentiation block after transplantation. Nbea deficiency in hematopoietic progenitor cells perturbed the expression of genes implicated in vesicle trafficking and led to changes in NOTCH receptor localization. This resulted in perturbation of the NOTCH transcriptional program, which is required for efficient HSC engraftment. In summary, our findings reveal a novel role for NBEA in the control of NOTCH receptor turnover in hematopoietic cells and supports a model in which BDCP-regulated vesicle trafficking is required for efficient HSCT.
Assuntos
Diferenciação Celular , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Receptores Notch , Animais , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Receptores Notch/metabolismo , Sobrevivência Celular , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , ApoptoseRESUMO
Mutations in LRBA, a BEACH domain protein, cause severe immune deficiency in humans. LRBA is expressed in many tissues and organs according to biochemical analysis, but little is known about its cellular and subcellular localization, and its deficiency phenotype outside the immune system. By LacZ histochemistry of Lrba gene-trap mice, we performed a comprehensive survey of LRBA expression in numerous tissues, detecting it in many if not all epithelia, in exocrine and endocrine cells, and in subpopulations of neurons. Immunofluorescence microscopy of the exocrine and endocrine pancreas, salivary glands, and intestinal segments, confirmed these patterns of cellular expression and provided information on the subcellular localizations of the LRBA protein. Immuno-electron microscopy demonstrated that in neurons and endocrine cells, which co-express LRBA and its closest relative, neurobeachin, both proteins display partial association with endomembranes in complementary, rather than overlapping, subcellular distributions. Prominent manifestations of human LRBA deficiency, such as inflammatory bowel disease or endocrinopathies, are believed to be primarily due to immune dysregulation. However, as essentially all affected tissues also express LRBA, it is possible that LRBA deficiency enhances their vulnerability and contributes to the pathogenesis.
Assuntos
Glândulas Endócrinas , Epitélio , Glândulas Exócrinas , Síndromes de Imunodeficiência , Neurônios , Animais , Humanos , Camundongos , Glândulas Endócrinas/metabolismo , Epitélio/metabolismo , Glândulas Exócrinas/metabolismo , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Síndromes de Imunodeficiência/patologia , Mutação , Neurônios/metabolismoRESUMO
Postsynaptic proteins play crucial roles in synaptic function and plasticity. During brain development, alterations in synaptic number, shape, and stability occur, known as synapse maturation. However, the postsynaptic protein composition changes during development are not fully understood. Here, we show the trajectory of the postsynaptic proteome in developing male mice and common marmosets. Proteomic analysis of mice at 2, 3, 6, and 12 weeks of age shows that proteins involved in synaptogenesis are differentially expressed during this period. Analysis of published transcriptome datasets shows that the changes in postsynaptic protein composition in the mouse brain after 2 weeks of age correlate with gene expression changes. Proteomic analysis of marmosets at 0, 2, 3, 6, and 24 months of age show that the changes in the marmoset brain can be categorized into two parts: the first 2 months and after that. The changes observed in the first 2 months are similar to those in the mouse brain between 2 and 12 weeks of age. The changes observed in marmoset after 2 months old include differential expression of synaptogenesis-related molecules, which hardly overlap with that in mice. Our results provide a comprehensive proteomic resource that underlies developmental synapse maturation in rodents and primates.
Assuntos
Fenômenos Biológicos , Callithrix , Animais , Camundongos , Masculino , Proteoma/metabolismo , Proteômica , Sinapses/metabolismoRESUMO
Introduction: Lrba is a cytoplasmic protein involved in vesicular trafficking. Lrba-deficient (Lrba-/-) mice exhibit substantially higher levels of IgA in both serum and feces than wild-type (WT) mice. Transforming growth factor ß1 (TGFß1) and its receptors (TGFßR I and II) is essential for differentiating IgA+ B cells. Furthermore, increased IgA production suggests a potential connection between Lrba and the TGFßR signaling pathway in IgA production. However, the specific function of Lrba in B cell biology remains unknown. Aim: Given the increased IgA levels in Lrba-/- mice, the goal in this work was to explore the lymph organs where the switch to IgA occurs, and if TGFßR function is affected. Methods: Non-immunized Lrba-/- mice were compared with Lrba+/+ mice. IgA levels in the serum and feces, as well as during peripheral B cell development, were determined. IgA+ B cells and plasma cells were assessed in the small intestine and secondary lymphoid organs, such as the spleen, mesenteric lymph nodes, and Peyer's patches. The TGFßR signaling pathway was evaluated by determining the expression of TGFßR on B cells. Additionally, SMAD2 phosphorylation was measured under basal conditions and in response to recombinant TGFß. Finally, confocal microscopy was performed to investigate a possible interaction between Lrba and TGFßR in B cells. Results: Lrba-/- mice exhibited significantly higher levels of circulating IgA, IgA+ B, and plasma cells than in peripheral lymphoid organs those in WT mice. TGFßR expression on the membrane of B cells was similar in both Lrba-/- and Lrba+/+ mice. However, intracellular TGFßR expression was reduced in Lrba-/- mice. SMAD2 phosphorylation showed increased levels under basal conditions; stimulation with recombinant TGFß elicited a poorer response than in that in Lrba+/+ B cells. Finally, we found that Lrba colocalizes with TGFßR in B cells. Conclusion: Lrba is essential in controlling TGFßR signaling, subsequently regulating SMAD2 phosphorylation on B cells. This mechanism may explain the increased differentiation of IgA+ B cells and production of IgA-producing plasma cells.
Assuntos
Linfócitos B , Diferenciação Celular , Imunoglobulina A , Transdução de Sinais , Animais , Camundongos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular/imunologia , Imunoglobulina A/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteína Smad2/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Introduction: Lipopolysaccharide-responsive and beige-like anchor (LRBA) is a scaffolding protein that interacts with proteins such as CTLA-4 and PKA, the importance of which has been determined in various cell types, including T regulatory cells, B cells, and renal cells. LRBA deficiency is associated with an inborn error in immunity characterized by immunodeficiency and autoimmunity. In addition to defects in T regulatory cells, patients with LRBA deficiency also exhibit B cell defects, such as reduced cell number, low memory B cells, hypogammaglobulinemia, impaired B cell proliferation, and increased autophagy. Although Lrba-/- mice do not exhibit the immunodeficiency observed in humans, responses to B cell receptors (BCR) in B cells have not been explored. Therefore, a murine model is for elucidating the mechanism of Lrba mechanism in B cells. Aim: To compare and evaluate spleen-derived B cell responses to BCR crosslinking in C57BL6 Lrba-/- and Lrba+/+ mice. Materials and methods: Spleen-derived B cells were obtained from 8 to 12-week-old mice. Subpopulations were determined by immunostaining and flow cytometry. BCR crosslinking was assessed by the F(ab')2 anti-µ chain. Activation, proliferation and viability assays were performed using flow cytometry and protein phosphorylation was evaluated by immunoblotting. The nuclear localization of p65 was determined using confocal microscopy. Nur77 expression was evaluated by Western blot. Results: Lrba-/- B cells showed an activated phenotype and a decreased proportion of transitional 1 B cells, and both proliferation and survival were affected after BCR crosslinking in the Lrba-/- mice. The NF-κB pathway exhibited a basal activation status of several components, resulting in increased activation of p50, p65, and IκBα, basal p50 activation was reduced by the Plcγ2 inhibitor U73122. BCR crosslinking in Lrba-/ - B cells resulted in poor p50 phosphorylation and p65 nuclear localization. Increased levels of Nur77 were detected. Discussion: These results indicate the importance of Lrba in controlling NF-κB activation driven by BCR. Basal activation of NF-κB could impact cellular processes, such as, activation, differentiation, proliferation, and maintenance of B cells after antigen encounter.
Assuntos
Linfócitos B , NF-kappa B , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Lipopolissacarídeos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
The lymphatic system, the network of lymphatic vessels and lymphoid organs, maintains the body fluid balance and ensures the immunological surveillance of the body. In the adult organism, the de novo formation of lymphatic vessels is mainly observed in pathological conditions. In contrast to the molecular mechanisms governing the generation of the lymphatic vasculature during embryogenesis, the processes underlying pathological lymphangiogenesis are less well understood. A genome-wide screen comparing the transcriptome of tumor-derived lymphatic endothelial cells with that of blood vessel endothelial cells identified paralemmin-1 as a protein prominently expressed in lymphatic endothelial cells. Paralemmin-1 is a lipid-anchored membrane protein that in fibroblasts and neurons plays a role in the regulation of cell shape, plasma membrane dynamics and cell motility. Here, we show that paralemmin-1 is expressed in tumor-derived lymphatic endothelial cells as well as in lymphatic endothelial cells of normal, non-tumorigenic tissue. Paralemmin-1 represses cell migration and delays the formation of tube-like structures of lymphatic endothelial cells in vitro by modulating cell-substrate adhesion, filopodia formation and plasma membrane blebbing. While constitutive genetic ablation of paralemmin-1 expression in mice has no effect on the development and physiological function of the lymphatic system, the loss of paralemmin-1 impaired tumor-associated lymphangiogenesis. Together, these results newly identify paralemmin-1 as a protein highly expressed in lymphatic endothelial cells. Similar to its function in neurons, it may link the cytoskeleton to the plasma membrane and thereby modulate lymphatic endothelial cell adhesion, migration and lymphangiogenesis.
Assuntos
Células Endoteliais/metabolismo , Insulinoma/patologia , Linfangiogênese/fisiologia , Vasos Linfáticos/citologia , Proteínas de Membrana/fisiologia , Neoplasias Pancreáticas/patologia , Fosfoproteínas/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Adesão Celular , Movimento Celular , Extensões da Superfície Celular/ultraestrutura , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Insulinoma/metabolismo , Insulinoma/secundário , Ilhotas Pancreáticas/metabolismo , Metástase Linfática , Vasos Linfáticos/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/biossíntese , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Paralemmin-1 is a phosphoprotein lipid-anchored to the cytoplasmic face of membranes where it functions in membrane dynamics, maintenance of cell shape, and process formation. Expression of paralemmin-1 and its major splice variant (Δ exon 8) as well as the extent of posttranslational modifications are tissue- and development-specific. Paralemmin-1 expression in normal breast and breast cancer tissue has not been described previously. RESULTS: Paralemmin-1 mRNA and protein expression was evaluated in ten breast cell lines, 26 primary tumors, and 10 reduction mammoplasty (RM) tissues using real time RT-PCR. Paralemmin-1 splice variants were assessed in tumor and RM tissues using a series of primers and RT-PCR. Paralemmin-1 protein expression was examined in cell lines using Western Blots and in 31 ductal carcinomas in situ, 65 infiltrating ductal carcinomas, and 40 RM tissues using immunohistochemistry. Paralemmin-1 mRNA levels were higher in breast cancers than in RM tissue and estrogen receptor (ER)-positive tumors had higher transcript levels than ER-negative tumors. The Δ exon 8 splice variant was detected more frequently in tumor than in RM tissues. Protein expression was consistent with mRNA results showing higher paralemmin-1 expression in ER-positive tumors. CONCLUSIONS: The differential expression of paralemmin-1 in a subset of breast cancers suggests the existence of variation in membrane dynamics that may be exploited to improve diagnosis or provide a therapeutic target.
RESUMO
Inhibitory glycine receptors (GlyRs) are densely packed in the postsynaptic membrane due to a high-affinity interaction of their ß-subunits with the scaffolding protein gephyrin. Here, we used an affinity-based proteomic approach to identify the trafficking proteins Vacuolar Protein Sorting 35 (Vps35) and Neurobeachin (Nbea) as novel GlyR ß-subunit (GlyRß) interacting proteins in rat brain. Recombinant Vps35 and a central fragment of Nbea bound to the large intracellular loop of GlyRß in glutathione-S-transferase pull-downs; in addition, Vps35 displayed binding to gephyrin. Immunocytochemical staining of spinal cord sections revealed Nbea immunoreactivity apposed to and colocalizing with marker proteins of inhibitory synapses. Our data are consistent with roles of Vps35 and Nbea in the retrieval and post-Golgi trafficking of synaptic GlyRs and possibly other neurotransmitter receptors.
Assuntos
Encéfalo/metabolismo , Receptores de Glicina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Transporte Proteico , Proteômica/métodos , Ratos , Receptores de Glicina/genética , Medula Espinal , Sinapses/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
Multidomain scaffolding proteins organize the molecular machinery of neurotransmitter vesicle dynamics during synaptogenesis and synaptic activity. We find that domains of five active zone proteins converge on an interaction node that centers on the N-terminal region of Munc13-1 and includes the zinc-finger domain of Rim1, the C-terminal region of Bassoon, a segment of CAST1/ELKS2, and the third coiled-coil domain (CC3) of either Aczonin/Piccolo or Bassoon. This multidomain complex may constitute a center for the physical and functional integration of the protein machinery at the active zone. An additional connection between Aczonin and Bassoon is mediated by the second coiled-coil domain of Aczonin. Recombinant Aczonin-CC3, expressed in cultured neurons as a green fluorescent protein fusion protein, is targeted to synapses and suppresses vesicle turnover, suggesting involvements in synaptic assembly as well as activity. Our findings show that Aczonin, Bassoon, CAST1, Munc13, and Rim are closely and multiply interconnected, they indicate that Aczonin-CC3 can actively participate in neurotransmitter vesicle dynamics, and they highlight the N-terminal region of Munc13-1 as a hub of protein interactions by adding three new binding partners to its mechanistic potential in the control of synaptic vesicle priming.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Animais , Sequência de Bases , Camundongos , Dados de Sequência Molecular , Neurotransmissores/metabolismo , Sinapses/metabolismoRESUMO
During embryonic development of the peripheral nervous system (PNS), the adhesion molecule neuronal cadherin (N-cadherin) is expressed by Schwann cell precursors and associated with axonal growth cones. N-cadherin expression levels decrease as precursors differentiate into Schwann cells. In this study, we investigated the distribution of N-cadherin in the developing postnatal and adult rat peripheral nervous system. N-cadherin was found primarily in ensheathing glia throughout development, concentrated at neuron-glial or glial-glial contacts of the sciatic nerve, dorsal root ganglia (DRG), and myenteric plexi. In the sciatic nerve, N-cadherin decreases with age and progress of myelination. In adult animals, N-cadherin was found exclusively in nonmyelinating Schwann cells. The distribution of N-cadherin in developing E17 DRG primary cultures is similar to what was observed in vivo. Functional studies of N-cadherin in these cultures, using the antagonist peptide INPISGQ, show a disruption of the attachment between Schwann cells, but no interference in the initial or long-term contact between Schwann cells and axons. We suggest that N-cadherin acts primarily in the adhesion between glial cells during postnatal development. It may form adherents/junctions between nonmyelinating glia, which contribute to the stable tubular structure encapsulating thin caliber axons and thus stabilize the nerve structure as a whole.
Assuntos
Caderinas/metabolismo , Caderinas/fisiologia , Células de Schwann/metabolismo , Células de Schwann/fisiologia , Envelhecimento/fisiologia , Animais , Western Blotting , Caderinas/antagonistas & inibidores , Adesão Celular/fisiologia , Células Cultivadas , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Microscopia Imunoeletrônica , Plexo Mientérico/citologia , Plexo Mientérico/metabolismo , Neuroglia/fisiologia , Sistema Nervoso Periférico/crescimento & desenvolvimento , Sistema Nervoso Periférico/fisiologia , Gravidez , Ratos , Gânglio Estrelado/citologia , Gânglio Estrelado/fisiologiaRESUMO
The development of neuronal networks in the brain requires the differentiation of functional synapses. Neurobeachin (Nbea) was identified as a putative regulator of membrane protein trafficking associated with tubulovesicular endomembranes and postsynaptic plasma membranes. Nbea is essential for evoked transmission at neuromuscular junctions, but its role in the central nervous system has not been characterized. Here, we have studied central synapses of a newly generated gene-trap knockout (KO) mouse line at embryonic day 18, because null-mutant mice are paralysed and die perinatally. Although the overall brain architecture was normal, we identified major abnormalities of synaptic function in mutant animals. In acute slices from the brainstem, both spontaneous excitatory and inhibitory postsynaptic currents were clearly reduced and failure rates of evoked inhibitory responses were markedly increased. In addition, the frequency of miniature excitatory and both the frequency and amplitudes of miniature inhibitory postsynaptic currents were severely diminished in KO mice, indicating a perturbation of both action potential-dependent and -independent transmitter release. Moreover, Nbea appears to be important for the formation and composition of central synapses because the area density of mature asymmetric contacts in the fetal brainstem was reduced to 30% of wild-type levels, and the expression levels of a subset of synaptic marker proteins were smaller than in littermate controls. Our data demonstrate for the first time a function of Nbea at central synapses that may be based on its presumed role in targeting membrane proteins to synaptic contacts, and are consistent with the 'excitatory-inhibitory imbalance' model of autism where Nbea gene rearrangements have been detected in some patients.
Assuntos
Transtorno Autístico/fisiopatologia , Tronco Encefálico/embriologia , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Transporte Proteico/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Tronco Encefálico/fisiologia , Células Cultivadas , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The evolutionary expansion of the mammalian neocortex (Ncx) is thought to be linked to increased proliferative capacity of basal progenitors (BPs) and their neurogenic capacity. Here, by quantifying BP morphology in the developing Ncx of mouse, ferret, and human, we show that increased BP proliferative capacity is linked to an increase in BP process number. We identify human membrane-bound PALMDELPHIN (PALMD-Caax) as an underlying factor, and we show that it drives BP process growth and proliferation when expressed in developing mouse and ferret Ncx. Conversely, CRISPR/Cas9-mediated disruption of PALMD or its binding partner ADDUCIN-γ in fetal human Ncx reduces BP process numbers and proliferation. We further show that PALMD-induced processes enable BPs to receive pro-proliferative integrin-dependent signals. These findings provide a link between BP morphology and proliferation, suggesting that changes in BP morphology may have contributed to the evolutionary expansion of the Ncx.
Assuntos
Neocórtex/anatomia & histologia , Neocórtex/citologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Animais , Proliferação de Células , Células Cultivadas , Furões , Humanos , Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Transdução de SinaisRESUMO
We have generated mice that carry a neuron-specific leptin receptor (LEPR) transgene whose expression is driven by the rat synapsin I promoter synapsin-LEPR B (SYN-LEPR-B). We have also generated mice that are compound hemizygotes for the transgenes SYN-LEPR-B and neuron-specific enolase-LEPR B (NSE-LEPR-B). We observed a degree of correction in db/db mice that are hemizygous (Syn db/db) and homozygous (Syn/Syn db/db) for the SYN-LEPR-B transgene similar to that previously reported for the NSE-LEPR-B transgene. We also show complete correction of the obesity and related phenotypes of db/db mice that are hemizygous for both NSE-LEPR-B and SYN-LEPR-B transgenes (Nse+Syn db/db). Body composition, insulin sensitivity, and cold tolerance were completely normalized in Nse+Syn db/db mice at 12 weeks of age compared with lean controls. In situ hybridization for LEPR B isoform expression in Nse+Syn db/db mice showed robust expression in the energy homeostasis-relevant regions of the hypothalamus. Expression of 3 neuropeptide genes, agouti-related peptide (Agrp), neuropeptide Y (Npy), and proopiomelanocortin (Pomc), was fully normalized in dual transgenic db/db mice. The 2 transgenes in concert conferred normal fertility to male and female db/db mice. Male mice with partial peripheral deletion of Lepr, induced in the periweaning phase, did not show alterations in body composition or mass. In summary, we show that brain-specific leptin signaling is sufficient to reverse the obesity, diabetes, and infertility of db/db mice.
Assuntos
Diabetes Mellitus/terapia , Terapia Genética/métodos , Infertilidade/terapia , Neurônios/metabolismo , Obesidade/terapia , Receptores de Superfície Celular/genética , Proteína Relacionada com Agouti , Alelos , Animais , Glicemia/metabolismo , Composição Corporal , Peso Corporal , Temperatura Baixa , DNA Complementar/metabolismo , Diabetes Mellitus/genética , Feminino , Fertilidade , Regulação da Expressão Gênica , Genótipo , Glucose/metabolismo , Homeostase , Homozigoto , Hipotálamo/patologia , Hibridização In Situ , Infertilidade/genética , Infertilidade Feminina/terapia , Infertilidade Masculina/terapia , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Transgênicos , Neuropeptídeo Y/genética , Obesidade/genética , Peptídeos/química , Fenótipo , Fosfopiruvato Hidratase/genética , Reação em Cadeia da Polimerase , Pró-Opiomelanocortina/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas , Proteínas/genética , Ratos , Receptores para Leptina , Transdução de Sinais , Sinapsinas/genética , Fatores de Tempo , Distribuição Tecidual , TransgenesRESUMO
Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology.