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1.
J Biol Chem ; 290(8): 4908-4927, 2015 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-25533468

RESUMO

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Quinase 5 Dependente de Ciclina , Proteínas Ativadoras de GTPase , Proteínas Oncogênicas , Proteínas Proto-Oncogênicas p21(ras) , Sinapses/enzimologia , Proteínas rap de Ligação ao GTP , Proteínas rap1 de Ligação ao GTP , Proteínas Ativadoras de ras GTPase , Proteínas ras , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Quinase 5 Dependente de Ciclina/química , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Neurônios/citologia , Neurônios/enzimologia , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas rap1 de Ligação ao GTP/química , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismo
2.
J Immunol ; 184(2): 816-23, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20018625

RESUMO

Studies of mice lacking MHC class I (MHC I)-associated proteins have demonstrated a role for MHC I in neurodevelopment. A central question arising from these observations is whether neuronal recognition of MHC I has specificity for the MHC I allele product and the peptide presented. Using a well-established embryonic retina explant system, we observed that picomolar levels of a recombinant self-MHC I molecule inhibited neurite outgrowth. We then assessed the neurobiological activity of a panel of recombinant soluble MHC Is, consisting of different MHC I heavy chains with a defined self- or nonself-peptide presented, on cultured embryonic retinas from mice with different MHC I haplotypes. We observed that self-MHC I allele products had greater inhibitory neuroactivity than nonself-MHC I molecules, regardless of the nature of the peptide presented, a pattern akin to MHC I recognition by some innate immune system receptors. However, self-MHC I molecules had no effect on retinas from MHC I-deficient mice. These observations suggest that neuronal recognition of MHC I may be coordinated with the inherited MHC I alleles, as occurs in the innate immune system. Consistent with this notion, we show that MHC I and MHC I receptors are coexpressed by precursor cells at the earliest stages of retina development, which could enable such coordination.


Assuntos
Alelos , Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/genética , Neurônios/imunologia , Peptídeos/imunologia , Animais , Autoantígenos/imunologia , Células-Tronco Embrionárias , Imunidade Inata , Camundongos , Neuritos/imunologia , Neurônios/citologia , Retina/embriologia
3.
J Neurosci Res ; 89(3): 365-72, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21259323

RESUMO

Mice that are deficient in classical major histocompatibility complex class I (MHCI) have abnormalities in synaptic plasticity and neurodevelopment and have more extensive loss of synapses and reduced axon regeneration after sciatic nerve transection, suggesting that MHCI participates in maintaining synapses and axon regeneration. Little is known about the biological consequences of up-regulating MHCI's expression on neurons. To understand MHCI's neurobiological activity better, and in particular its role in neurorepair after injury, we have studied neurorepair in a transgenic mouse model in which classical MHCI expression is up-regulated only on neurons. Using a well-established spinal cord injury (SCI) model, we observed that transgenic mice with elevated neuronal MHCI expression had significantly better recovery of locomotor abilities after SCI than wild-type mice. Although previous studies have implicated inflammation as both deleterious and beneficial for recovery after SCI, our results point directly to enhanced neuronal MHCI expression as a beneficial factor for promoting recovery of locomotor function after SCI.


Assuntos
Regulação da Expressão Gênica/genética , Antígenos de Histocompatibilidade Classe I/genética , Locomoção/fisiologia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Análise de Variância , Animais , Modelos Animais de Doenças , Teste de Esforço/métodos , Lateralidade Funcional , Locomoção/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Fosfopiruvato Hidratase/genética , Recuperação de Função Fisiológica/genética , Traumatismos da Medula Espinal/patologia
4.
PLoS One ; 6(3): e18439, 2011 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-21483793

RESUMO

The neurobiological activities of classical major histocompatibility class I (MHCI) molecules are just beginning to be explored. To further examine MHCI's actions during the formation of neuronal connections, we cultured embryonic mouse retina explants a short distance from wildtype thalamic explants, or thalami from transgenic mice (termed "NSE-Db") whose neurons express higher levels of MHCI. While retina neurites extended to form connections with wildtype thalami, we were surprised to find that retina neurite outgrowth was very stunted in regions proximal to NSE-Db thalamic explants, suggesting that a diffusible factor from these thalami inhibited retina neurite outgrowth. It has been long known that MHCI-expressing cells release soluble forms of MHCI (sMHCI) due to the shedding of intact MHCI molecules, as well as the alternative exon splicing of its heavy chain or the action proteases which cleave off it's transmembrane anchor. We show that the diffusible inhibitory factor from the NSE-Db thalami is sMHCI. We also show that COS cells programmed to express murine MHCI release sMHCI that inhibits neurite outgrowth from nearby neurons in vitro. The neuroinhibitory effect of sMHCI could be blocked by lowering cAMP levels, suggesting that the neuronal MHCI receptor's signaling mechanism involves a cyclic nucleotide-dependent pathway. Our results suggest that MHCI may not only have neurobiological activity in its membrane-bound form, it may also influence local neurons as a soluble molecule. We discuss the involvement of complement proteins in generating sMHCI and new theoretical models of MHCI's biological activities in the nervous system.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Neuritos/metabolismo , Neuritos/fisiologia , Animais , Células COS , Chlorocebus aethiops , Técnicas de Cocultura , Meios de Cultivo Condicionados , Camundongos , Camundongos Endogâmicos C57BL , Retina/citologia , Retina/embriologia , Retina/metabolismo , Tálamo/citologia , Tálamo/embriologia , Tálamo/metabolismo
5.
J Neuroimmunol ; 232(1-2): 8-16, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20950866

RESUMO

Mice deficient in classical major histocompatibility complex class I (MHCI) have aberrations in neurodevelopment. The consequences of upregulated neuronal MHCI expression have not been examined. We found that transgenic C57Bl/6 mice that are engineered to express higher levels of self-D(b) on their CNS neurons have alterations in their hippocampal morphology and retinogeniculate projections, as well as impaired neurorepair responses. Thus, enhanced neuronal classical MHCI expression can lead to aberrations in neural circuitry and neurorepair. These findings complement a growing body of knowledge concerning the neurobiological activities of MHCI and may have potential clinical relevance.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Regeneração Nervosa/fisiologia , Neurogênese/fisiologia , Neurônios/metabolismo , Acetilcolinesterase/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/imunologia , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/imunologia , Neurônios/patologia , Técnicas de Cultura de Órgãos , Transmissão Sináptica/fisiologia
6.
Proc Natl Acad Sci U S A ; 103(30): 11294-9, 2006 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-16868090

RESUMO

Islet transplantation offers a potential therapy to restore glucose homeostasis in type 1 diabetes patients. However, islet transplantation is not routinely successful because most islet recipients gradually lose graft function. Furthermore, serological markers of islet function are insensitive to islet loss until the latter stages of islet graft rejection. A noninvasive method of monitoring islet grafts would aid in the assessment of islet graft survival and the evaluation of interventions designed to prolong graft survival. Here, we show that recombinant adenovirus can engineer isolated islets to express a positron-emission tomography (PET) reporter gene and that these islets can be repeatedly imaged by using microPET after transplantation into mice. The magnitude of signal from engineered islets implanted into the axillary cavity was directly related to the implanted islet mass. PET signals attenuated over the following weeks because of the transient nature of adenovirus-mediated gene expression. Because the liver is the preferred site for islet implantation in humans, we also tested whether islets could be imaged after transfusion into the mouse liver. Control studies revealed that both intrahepatic islet transplantation and hyperglycemia altered the biodistribution kinetics of the PET probe systemically. Although transplanted islets were dispersed throughout the liver, clear signals from the liver region of mice receiving PET reporter-expressing islets were detectable for several weeks. Viral transduction, PET reporter expression, and repeated microPET imaging had no apparent deleterious effects on islet function after implantation. These studies lay a foundation for noninvasive quantitative assessments of islet graft survival using PET.


Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Tomografia por Emissão de Pósitrons/métodos , Animais , Sobrevivência Celular , Genes Reporter , Sobrevivência de Enxerto , Humanos , Fígado/patologia , Camundongos , Ratos , Transdução de Sinais
7.
Nat Rev Neurosci ; 6(6): 423-34, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15928715

RESUMO

Short-term and long-term changes in the strength of synapses in neural networks underlie working memory and long-term memory storage in the brain. These changes are regulated by many biochemical signalling pathways in the postsynaptic spines of excitatory synapses. Recent findings about the roles and regulation of the small GTPases Ras, Rap and Rac in spines provide new insights into the coordination and cooperation of different pathways to effect synaptic plasticity. Here, we present an initial working representation of the interactions of five signalling cascades that are usually studied individually. We discuss their integrated function in the regulation of postsynaptic plasticity.


Assuntos
Espinhas Dendríticas/fisiologia , Plasticidade Neuronal/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Espinhas Dendríticas/química , Espinhas Dendríticas/metabolismo , Humanos , Sinapses/química , Sinapses/metabolismo
8.
J Immunol ; 175(3): 1991-9, 2005 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16034144

RESUMO

As organ-specific autoimmune diseases do not become manifest until well-advanced, interventive therapies must inhibit late-stage disease processes. Using a panel of immunogenic peptides from various beta cell Ags, we evaluated the factors influencing the efficacy of Ag-based therapies in diabetes-prone NOD mice with advanced disease. The ability of the major beta cell autoantigen target determinants (TDs) to prime Th2 responses declined sharply between 6 and 12 wk of age, whereas the ability of immunogenic ignored determinants (IDs) of beta cell Ags to prime Th2 responses was unaffected by the disease process. The different patterns of TD and ID immunogenicity (even from the same beta cell Ag) may be due to the exhaustion of uncommitted TD-reactive, but not ID-reactive, T cell pools by recruitment into the autoimmune cascade. Therapeutic efficacy was associated with a peptide's immunogenicity and ability to promote Th2 spreading late in the disease process but not its affinity for I-Ag7 or its expression pattern (beta cell specific/nonspecific or rare/abundant). Characterization of some IDs revealed them to be "absolute" cryptic determinants. Such determinants have little impact on T cell selection, leaving large precursor T cell pools available for priming by synthetic peptides. Traditional Ag-based therapeutics using whole autoantigens or their TDs cannot prime responses to such determinants. These findings suggest a new strategy for designing more efficacious Ag-based therapeutics for late-stage autoimmune diseases.


Assuntos
Autoantígenos/uso terapêutico , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Epitopos/uso terapêutico , Predisposição Genética para Doença , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Sequência de Aminoácidos , Animais , Autoantígenos/administração & dosagem , Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/genética , Progressão da Doença , Esquema de Medicação , Epitopos/administração & dosagem , Epitopos/imunologia , Feminino , Glutamato Descarboxilase/administração & dosagem , Glutamato Descarboxilase/imunologia , Glutamato Descarboxilase/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Isoenzimas/administração & dosagem , Isoenzimas/imunologia , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Peptídeos/administração & dosagem , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo
9.
Mol Ther ; 9(3): 428-35, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15006610

RESUMO

Islet transplantation offers a potential therapy to restore glucose homeostasis in type 1 diabetes patients. A method to image transplanted islets noninvasively and repeatedly would greatly assist studies of islet transplantation. Using recombinant adenovirus, we show that isolated rodent and human islets can be genetically engineered to express luciferase and then imaged after implantation into NOD-scid mice using a cooled charge-coupled device. The magnitude of the signal was dependent on the islet dose. Adenovirus-directed luciferase expression, however, rapidly attenuated. We next tested lentivirus vectors that should direct the long-term expression of reporter genes in transduced islets. Transplanted lentivirus-transduced islets restored euglycemia long term in streptozotocin-treated NOD-scid mice. The signal from implanted lentivirus-transduced islets was related directly to the implanted islet mass, and the signal did not attenuate over the observation period. Viral transduction, luciferase expression, and repeated imaging had no apparent long-term deleterious effects on islet function after implantation. These data demonstrate that the introduction of reporter genes into an isolated tissue allows the long-term monitoring of its survival following implantation. Such imaging technologies may allow earlier detection of graft rejection and the adjustment of therapies to prolong graft survival posttransplantation.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Sobrevivência de Enxerto , Transplante das Ilhotas Pancreáticas/métodos , Adenoviridae/genética , Animais , Diabetes Mellitus Tipo 1/terapia , Genes Reporter , Vetores Genéticos , Humanos , Imuno-Histoquímica , Ilhotas Pancreáticas/metabolismo , Lentivirus/genética , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos , Estreptozocina/farmacologia , Fatores de Tempo
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