RESUMO
PURPOSE OF REVIEW: The aim of this study was to present a new regulation system in the hippocampus constituted by the neuronal surface P antigen (NSPA) and the tyrosine phosphatase PTPMEG/PTPN4, which provides mechanistic and therapeutic possibilities for cognitive dysfunction driven by antiribosomal P protein autoantibodies in patients with systemic lupus erythematosus (SLE). RECENT FINDINGS: Mice models lacking the function of NSPA as an E3 ubiquitin ligase show impaired glutamatergic synaptic plasticity, decreased levels of NMDAR at the postsynaptic density in hippocampus and memory deficits. The levels of PTPMEG/PTPN4 are increased due to lower ubiquitination and proteasomal degradation, resulting in dephosphorylation of tyrosines that control endocytosis in GluN2 NMDAR subunits. Adult hippocampal neurogenesis (AHN) that normally contributes to memory processes is also defective in the absence of NSPA. SUMMARY: NSPA function is crucial in memory processes controlling the stability of NMDAR at PSD through the ubiquitination of PTPMEG/PTPN4 and also through AHN. As anti-P autoantibodies reproduce the impairments of glutamatergic transmission, plasticity and memory performance seen in the absence of NSPA, it might be expected to perturb the NSPA/PTPMEG/PTPN4 pathway leading to hypofunction of NMDAR. This neuropathogenic mechanism contrasts with that of anti-NMDAR antibodies also involved in lupus cognitive dysfunction. Testing this hypothesis might open new therapeutic possibilities for cognitive dysfunction in SLE patients bearing anti-P autoantibodies.
Assuntos
Encefalopatias , Lúpus Eritematoso Sistêmico , Animais , Anticorpos Antinucleares , Autoanticorpos , Encéfalo , Encefalopatias/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Plasticidade Neuronal , Proteína Tirosina Fosfatase não Receptora Tipo 4/metabolismo , Receptores de N-Metil-D-AspartatoRESUMO
The insulin-IGF-1/DAF-2 pathway has a central role in the determination of aging and longevity in Caenorhabditis elegans and other organisms. In this paper, we measured neuronal insulin secretion (using INS-22::Venus) during C. elegans lifespan and monitored how this secretion is modified by redox homeostasis. We showed that INS-22::Venus secretion fluctuates during the organism lifetime reaching maximum levels in the active reproductive stage. We also demonstrate that long-lived daf-2 insulin receptor mutants show remarkable low levels of INS-22::Venus secretion. In contrast, we found that short-lived mutant worms that lack the oxidation repair enzyme MSRA-1 show increased levels of INS-22::Venus secretion, specifically during the reproductive stage. MSRA-1 is a target of the insulin-IGF-1/DAF-2 pathway, and the expression of this antioxidant enzyme exclusively in the nervous system rescues the mutant insulin release phenotype and longevity. The msra-1 mutant phenotype can also be reverted by antioxidant treatment during the active reproductive stage. We showed for the first time that there is a pattern of neuronal insulin release with a noticeable increment during the peak of reproduction. Our results suggest that redox homeostasis can modulate longevity through the regulation of insulin secretion, and that the insulin-IGF-1/DAF-2 pathway could be regulated, at least in part, by a feedback loop. These findings highlight the importance of timing for therapeutic interventions aimed at improving health span.
Assuntos
Envelhecimento/fisiologia , Caenorhabditis elegans/fisiologia , Homeostase , Insulina/metabolismo , Neurônios/metabolismo , Acetilcisteína/farmacologia , Envelhecimento/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/metabolismo , Regulação para Baixo/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Modelos Biológicos , Atividade Motora/efeitos dos fármacos , Mutação/genética , Neurônios/efeitos dos fármacos , Oxirredução , Reprodução/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
OBJECTIVE: Central nervous system disease occurs in over 20% of patients with systemic lupus erythematosus (SLE) resulting in major morbidity and damage. Cognitive dysfunction is common in SLE, but the cause remains uncertain and treatment options are limited. This study explores the influence of clinical, neuropsychological factors and anti-neuronal antibodies on lupus damage accrual. METHOD: A prospective cohort with 99 SLE patients recruited between 2008 and 2013 and followed up in 2016 was established. Baseline evaluations were depression (MINI-Plus), cognitive function evaluating attention, visuospatial memory and executive functions, and anti-neuronal antibodies. Activity index (SLEDAI-2K) and SLICC/ACR Damage Index (SDI) were assessed at baseline and last follow-up. RESULTS: At baseline, median (interquartile range) age was 36.0 years (27.0-45.0), disease duration 3.7 years (0.4-12.4), SLEDAI-2K 6.0 (3.0-12.0), and SDI score 1.0 (0-1.0). Major depression was present in 23%, cognitive deficit in 18%, and received immunomodulators in 36%. Anti-dsDNA/N-methyl-D-aspartate receptor antibodies were present in 19%, anti-ribosomal P in 12%, and anti-neuronal surface P antigen (NSPA) in 5%. After a median follow-up of 55 months (interquartile range 39-78), 11% had damage accrual. In a multivariate analysis, baseline SDI, SLEDAI-2K, and immunomodulators use were associated with final damage, whereas SLEDAI-2K and immunomodulator use were also associated with accrual damage. Models including anti-NSPA showed impact on final and accrual damage. Cognitive deficit, depression, and other autoantibodies were not predictors. CONCLUSIONS: Disease activity and immunomodulator use associate with lupus damage. Of the anti-neuronal antibodies examined, anti-NSPA emerged as a potential poor prognostic factor, probably related to severe SLE onset requiring elevated corticosteroid doses. Key Points ⢠Anti-NSPA may be a worse prognostic factor in SLE. ⢠Other neuropsychological factors do not influence damage.
Assuntos
Lúpus Eritematoso Sistêmico/psicologia , Neurônios/imunologia , Adulto , Disfunção Cognitiva/etiologia , Depressão/etiologia , Feminino , Humanos , Fatores Imunológicos/efeitos adversos , Estudos Longitudinais , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Pessoa de Meia-IdadeRESUMO
Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial-mesenchymal-transition (EMT). The glycan-binding protein galectin-8 (Gal-8) activates selective ß1-integrins involved in EMT and is overexpressed by certain carcinomas. Here we show that Gal-8 overexpression or exogenous addition promotes proliferation, migration, and invasion in nontumoral Madin-Darby canine kidney (MDCK) cells, involving focal-adhesion kinase (FAK)-mediated transactivation of the epidermal growth factor receptor (EGFR), likely triggered by α5ß1integrin binding. Under subconfluent conditions, Gal-8-overexpressing MDCK cells (MDCK-Gal-8H) display hallmarks of EMT, including decreased E-cadherin and up-regulated expression of vimentin, fibronectin, and Snail, as well as increased ß-catenin activity. Changes related to migration/invasion included higher expression of α5ß1 integrin, extracellular matrix-degrading MMP13 and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) protease systems. Gal-8-stimulated FAK/EGFR pathway leads to proteasome overactivity characteristic of cancer cells. Yet MDCK-Gal-8H cells still develop apical/basolateral polarity reverting EMT markers and proteasome activity under confluence. This is due to the opposite segregation of Gal-8 secretion (apical) and ß1-integrins distribution (basolateral). Strikingly, MDCK-Gal-8H cells acquired tumorigenic potential, as reflected in anchorage-independent growth in soft agar and tumor generation in immunodeficient NSG mice. Therefore, Gal-8 can promote oncogenic-like transformation of epithelial cells through partial and reversible EMT, accompanied by higher proliferation, migration/invasion, and tumorigenic properties.
Assuntos
Transição Epitelial-Mesenquimal , Receptores ErbB/metabolismo , Galectinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Animais , Caderinas/metabolismo , Carcinogênese , Cães , Quinase 1 de Adesão Focal/metabolismo , Humanos , Integrina beta1/metabolismo , Células Madin Darby de Rim Canino , Masculino , Camundongos , Neoplasias Experimentais , Proteínas Recombinantes/metabolismo , Transfecção , Regulação para Cima , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
AIMS: To examine the role of the enzyme methionine sulfoxide reductase A-1 (MSRA-1) in amyloid-ß peptide (Aß)-peptide aggregation and toxicity in vivo, using a Caenorhabditis elegans model of the human amyloidogenic disease inclusion body myositis. RESULTS: MSRA-1 specifically reduces oxidized methionines in proteins. Therefore, a deletion of the msra-1 gene was introduced into transgenic C. elegans worms that express the Aß-peptide in muscle cells to prevent the reduction of oxidized methionines in proteins. In a constitutive transgenic Aß strain that lacks MSRA-1, the number of amyloid aggregates decreases while the number of oligomeric Aß species increases. These results correlate with enhanced synaptic dysfunction and mislocalization of the nicotinic acetylcholine receptor ACR-16 at the neuromuscular junction (NMJ). INNOVATION: This approach aims at modulating the oxidation of Aß in vivo indirectly by dismantling the methionine sulfoxide repair system. The evidence presented here shows that the absence of MSRA-1 influences Aß aggregation and aggravates locomotor behavior and NMJ dysfunction. The results suggest that therapies which boost the activity of the Msr system could have a beneficial effect in managing amyloidogenic pathologies. CONCLUSION: The absence of MSRA-1 modulates Aß-peptide aggregation and increments its deleterious effects in vivo.
Assuntos
Metionina Sulfóxido Redutases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Imunoprecipitação , Locomoção/fisiologia , Metionina , Oxirredução , Receptores Nicotínicos/metabolismoRESUMO
OBJECTIVE: To assess whether autoantibodies against ribosomal P (anti-P), which are possibly pathogenic in neuropsychiatric systemic lupus erythematosus (NPSLE), alter glutamatergic synaptic transmission and to what extent the cross-reacting neuronal surface P antigen (NSPA) is involved. METHODS: We analyzed glutamatergic transmission and long-term potentiation (LTP) mediated by AMPA receptor (AMPAR) and N-methyl-d-aspartate receptor (NMDAR) by field excitatory postsynaptic potential (EPSP) at the CA3-CA1 synapse. AMPAR activation by patch-clamp recordings in primary ventral spinal cord neurons was analyzed. In primary hippocampal neurons, NSPA distribution was assessed by double immunofluorescence, and intracellular calcium changes were evaluated using Fura-2 AM. NSPA-LacZ reporter-knockin mice expressing a truncated NSPA were used to assess NSPA expression pattern and function in the brain using ß-galactosidase staining and comparative electrophysiology, calcium responses, and water maze memory tests. RESULTS: NSPA was expressed in the brain in hippocampal CA1, dentate gyrus and ventral, but not dorsal, CA3 regions, encompassing postsynaptic regions and partial colocalization with NMDAR. Notably, NSPA-LacZ reporter-knockin mice showed impaired memory, and decreased NMDAR activity and LTP, with neurons insensitive to anti-P autoantibodies. Anti-P autoantibodies enhanced CA1 postsynaptic transmission, increasing AMPAR and NMDAR activity and leading to LTP abrogation after prolonged (20-minute) incubation. CONCLUSION: Our findings indicate that the neuronal cell surface target of anti-P, NSPA, is involved in glutamatergic synaptic transmission and plasticity related to memory in the hippocampus, and mediates the deleterious effects of anti-P on these processes. Cognitive impairment, as well as other diffuse NPSLE manifestations, may develop when anti-P autoantibodies have access to brain regions coexpressing NSPA, AMPAR, and NMDAR.
Assuntos
Autoanticorpos/imunologia , Hipocampo/metabolismo , Potenciação de Longa Duração , Lúpus Eritematoso Sistêmico/imunologia , Neurônios/metabolismo , Proteínas Ribossômicas/imunologia , Transmissão Sináptica , Adulto , Animais , Antígenos de Superfície , Região CA1 Hipocampal/metabolismo , Região CA3 Hipocampal/metabolismo , Giro Denteado/metabolismo , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores , Feminino , Técnicas de Introdução de Genes , Ácido Glutâmico/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Memória , Camundongos , Plasticidade Neuronal , Neurônios/imunologia , Técnicas de Patch-Clamp , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Ribossômicas/metabolismo , Medula Espinal/citologia , Adulto JovemRESUMO
OBJECTIVE: To define whether anti-ribosomal P (anti-P) autoantibodies from patients with neuropsychiatric systemic lupus erythematosus (NPSLE) impair the function of hippocampal neurons that express the neuronal surface P antigen (NSPA) when accessing the brain via circulating blood. METHODS: We used anti-P antibodies from patients with NPSLE and rabbit-generated anti-P and anti-NSPA antibodies. Primary hippocampal neurons from mice were analyzed to determine antibody cell surface binding (double immunofluorescence), intracellular calcium variations (Fura 2 AM), and apoptosis (caspase 3 activation). Hippocampal-dependent spatial flexible memory was assessed in mice subjected to a water maze test 24 hours after an intravenous injection of anti-P or anti-NSPA, using lipopolysaccharide (LPS) to permeate the blood-brain barrier. Presence of antibodies and apoptosis in the hippocampus was studied using immunohistochemistry and TUNEL assays. RESULTS: Hippocampal neurons expressed NSPA on the cell surface, as revealed by anti-P and anti-NSPA staining colocalization, and responded to both anti-P and anti-NSPA by exhibiting increased intracellular calcium levels. Neuronal apoptosis was induced when anti-P was directly injected by stereotaxis into the hippocampus or added to primary cultures. Upon LPS treatment, intravenously injected anti-P impaired memory but did not elicit neuronal apoptosis in the hippocampus, where it was detectable in low amounts. Anti-NSPA antibodies also impaired memory. CONCLUSION: Anti-P antibodies interact with NSPA on the surface of hippocampal neurons leading to apoptotic death or to functional perturbations, results that are likely dependent on the concentration of these antibodies. Circulating anti-P can access the hippocampus and impair memory without requiring neuronal death when the blood-brain barrier is disrupted. NSPA can mediate antibody-driven diffuse brain dysfunction, and anti-P might contribute to the cognitive impairment that is frequently observed in SLE.
Assuntos
Autoanticorpos/efeitos adversos , Vasculite Associada ao Lúpus do Sistema Nervoso Central/imunologia , Transtornos da Memória/etiologia , Transtornos da Memória/imunologia , Proteínas Ribossômicas/imunologia , Adolescente , Animais , Antígenos de Superfície/metabolismo , Apoptose/efeitos dos fármacos , Autoanticorpos/sangue , Autoanticorpos/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Vasculite Associada ao Lúpus do Sistema Nervoso Central/metabolismo , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Proteínas Ribossômicas/metabolismo , Adulto JovemRESUMO
Epidermal growth factor receptor (EGFR) exaggerated (oncogenic) function is currently targeted in cancer treatment with drugs that block receptor ligand binding or tyrosine kinase activity. Because endocytic trafficking is a crucial regulator of EGFR function, its pharmacological perturbation might provide a new anti-tumoral strategy. Inhibition of phosphatidic acid (PA) phosphohydrolase (PAP) activity has been shown to trigger PA signaling towards type 4 phosphodiesterase (PDE4) activation and protein kinase A inhibition, leading to internalization of empty/inactive EGFR. Here, we used propranolol, its l- and d- isomers and desipramine as PAP inhibitors to further explore the effects of PAP inhibition on EGFR endocytic trafficking and its consequences on EGFR-dependent cancer cell line models. PAP inhibition not only made EGFR inaccessible to stimuli but also prolonged the signaling lifetime of ligand-activated EGFR in recycling endosomes. Strikingly, such endocytic perturbations applied in acute/intermittent PAP inhibitor treatments selectively impaired cell proliferation/viability sustained by an exaggerated EGFR function. Phospholipase D inhibition with FIPI (5-fluoro-2-indolyl des-chlorohalopemide) and PDE4 inhibition with rolipram abrogated both the anti-tumoral and endocytic effects of PAP inhibition. Prolonged treatments with a low concentration of PAP inhibitors, although without detectable endocytic effects, still counteracted cell proliferation, induced apoptosis and decreased anchorage-independent growth of cells bearing EGFR oncogenic influences. Overall, our results show that PAP inhibitors can counteract EGFR oncogenic traits, including receptor overexpression or activating mutations resistant to current tyrosine kinase inhibitors, perturbing EGFR endocytic trafficking and perhaps other as yet unknown processes, depending on treatment conditions. This puts PAP activity forward as a new suitable target against EGFR-driven malignancy.
Assuntos
Endocitose , Inibidores Enzimáticos/uso terapêutico , Receptores ErbB/metabolismo , Neoplasias/tratamento farmacológico , Fosfatidato Fosfatase/antagonistas & inibidores , Desipramina/farmacologia , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Ligantes , Fosforilação , Propranolol/farmacologiaRESUMO
Epidermal growth factor receptor (EGFR) function is transregulated by a variety of stimuli, including agonists of certain G-protein-coupled receptors (GPCRs). One of the most ubiquitous GPCRs is the P2Y(1) receptor (P2RY1, hereafter referred to as P2Y(1)R) for extracellular nucleotides, mainly ADP. Here, we show in tumoral HeLa cells and normal FRT epithelial cells that P2Y(1)R broadcasts mitogenic signals by transactivating the EGFR. The pathway involves PKC, Src and cell surface metalloproteases. Stimulation of P2Y(1)R for as little as 15-60 minutes triggers mitogenesis, mirroring the half-life of extracellular ADP. Apyrase degradation of extracellular nucleotides and drug inhibition of P2Y(1)R, both reduced basal cell proliferation of HeLa and FRT cells, but not MDCK cells, which do not express P2Y(1)R. Thus, cell-released nucleotides constitute strong mitogenic stimuli, which act via P2Y(1)R. Strikingly, MDCK cells ectopically expressing P2Y(1)R display a highly proliferative phenotype that depends on EGFR activity associated with an increased level of EGFR, thus disclosing a novel aspect of GPCR-mediated regulation of EGFR function. These results highlight a role of P2Y(1)R in EGFR-dependent epithelial cell proliferation. P2Y(1)R could potentially mediate both trophic stimuli of basally released nucleotides and first-line mitogenic stimulation upon tissue damage. It could also contribute to carcinogenesis and serve as target for antitumor therapies.
Assuntos
Proliferação de Células , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Receptores Purinérgicos P2/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Cães , Células Epiteliais/citologia , Receptores ErbB/genética , Células HeLa , Humanos , Agonistas do Receptor Purinérgico P2 , Ratos , Ratos Endogâmicos F344 , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Transdução de Sinais , Ativação Transcricional , TransfecçãoRESUMO
The interesting observation was made 20 years ago that psychotic manifestations in patients with systemic lupus erythematosus are associated with the production of antiribosomal-P protein (anti-P) autoantibodies. Since then, the pathogenic role of anti-P antibodies has attracted considerable attention, giving rise to long-term controversies as evidence has either contradicted or confirmed their clinical association with lupus psychosis. Furthermore, a plausible mechanism supporting an anti-P-mediated neuronal dysfunction is still lacking. We show that anti-P antibodies recognize a new integral membrane protein of the neuronal cell surface. In the brain, this neuronal surface P antigen (NSPA) is preferentially distributed in areas involved in memory, cognition, and emotion. When added to brain cellular cultures, anti-P antibodies caused a rapid and sustained increase in calcium influx in neurons, resulting in apoptotic cell death. In contrast, astrocytes, which do not express NSPA, were not affected. Injection of anti-P antibodies into the brain of living rats also triggered neuronal death by apoptosis. These results demonstrate a neuropathogenic potential of anti-P antibodies and contribute a mechanistic basis for psychiatric lupus. They also provide a molecular target for future exploration of this and other psychiatric diseases.
Assuntos
Apoptose , Autoanticorpos/química , Cálcio/química , Membrana Celular/metabolismo , Vasculite Associada ao Lúpus do Sistema Nervoso Central/imunologia , Neurônios/metabolismo , Proteínas/química , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Sistema Nervoso Central/metabolismo , Epitopos/química , Humanos , Vasculite Associada ao Lúpus do Sistema Nervoso Central/metabolismo , Modelos Biológicos , Peptídeos/química , Ratos , Ribossomos/metabolismo , Sinaptossomos/metabolismoRESUMO
Integrin-mediated encounters of T cells with extracellular cues lead these cells to adhere to a variety of substrates and acquire a spread phenotype needed for their tissue incursions. We studied the effects of galectin-8 (Gal-8), a beta-galactoside binding lectin, on Jurkat T cells. Immobilized Gal-8 bound alpha1beta1, alpha3beta1 and alpha5beta1 but not alpha2beta1 and alpha4beta1 and adhered these cells with similar kinetics to immobilized fibronectin (FN). Function-blocking experiments with monoclonal anti-integrin antibodies suggested that alpha5beta1 is the main mediator of cell adhesion to this lectin. Gal-8, but not FN, induced extensive cell spreading frequently leading to a polarized phenotype characterized by an asymmetric lamellipodial protrusion. These morphological changes involved actin cytoskeletal rearrangements controlled by PI3K, Rac-1 and ERK1/2 activity. Gal-8-induced Rac-1 activation and binding to alpha1 and alpha5 integrins have not been described in any other cellular system. Strikingly, Gal-8 was also a strong stimulus on Jurkat cells in suspension, triggering ERK1/2 activation that in most adherent cells is instead dependent on cell attachment. In addition, we found that patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disorder, produce Gal-8 autoantibodies that impede both its binding to integrins and cell adhesion. These are the first function-blocking autoantibodies reported for a member of the galectin family. These results indicate that Gal-8 constitutes a novel extracellular stimulus for T cells, able to bind specific beta1 integrins and to trigger signaling pathways conducive to cell spreading. Gal-8 could modulate a wide range of T cell-driven immune processes that eventually become altered in autoimmune disorders.