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Por que usar neurônios embrionários para estudar nocautes / mutantes de proteínas na potenciação de longo prazo?

Por que usar neurônios embrionários para estudar nocautes / mutantes de proteínas na potenciação de longo prazo?


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Estou me perguntando se alguém tem alguma ideia sobre a pergunta do título. Estou apenas me perguntando por que alguns artigos usam culturas embrionárias de regiões cerebrais específicas para neurônios para testar os efeitos de nocautes / mutações na plasticidade sináptica (por exemplo, potenciação de longo prazo, LTP) em vez de culturas adultas?

Muito Obrigado


Algumas sugestões, pode haver mais:

1) O nocaute pode não ser viável na idade adulta (os animais morrem). Talvez os heterozigotos sejam viáveis, mas para testar o nocaute completo você precisa de um homozigoto.

2) Mesmo que o nocaute seja viável na idade adulta, o cérebro pode desenvolver adaptações ao nocaute que não são específicas do próprio nocaute: regulação para cima ou para baixo de certos canais, por exemplo.

3) A plasticidade é regulada positivamente em cérebros em desenvolvimento em comparação com cérebros adultos, portanto, os efeitos podem ser mais pronunciados no tecido jovem.

4) Culturas de tecidos mais jovens melhor - você pode manter as células vivas por mais tempo, com mais facilidade.

Também é bom notar que as células-tronco indiferenciadas tendem a ser "padrão de neurônios" - isto é, se você apenas cultivar células-tronco embrionárias e não fizer nada de especial com elas, elas tenderão a gerar células semelhantes a neurônios. No adulto, os neurônios sempre estarão ao lado de outros tipos de células: glia, tecido vascular, etc.


Disrupção genômica direcionada de H-ras e n-ras, Individualmente ou em combinação, revela a dispensabilidade de ambos os locais para crescimento e desenvolvimento de camundongos

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular da Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Carlos Vicario-Abej & # x000f3n

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular, Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Pedro Fern & # x000e1ndez-Salguero

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular da Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Alberto Fern & # x000e1ndez-Medarde

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular da Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Nalini Swaminathan

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular da Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Kate Yienger

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular da Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Eva Lopez

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular da Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Marcos Malumbres

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular da Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Ron McKay

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular da Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Jerrold M. Ward

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular da Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Angel Pellicer

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular da Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5

Eugenio Santos

Centro de Investigaci & # x000f3n del C & # x000e1ncer, IBMCC, CSIC-USAL, Universidade de Salamanca, Salamanca, 1 e Departamento Bioqu & # x000edmica y Biolog & # x000eda Molecular, Universidad de Extremadura, Badajoz, 4 Laboratório Nacional de Biologia Celular e Molecular, Espanha Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland e Departamento de Patologia e Kaplan Cancer Center, New York University Medical Center , Nova York, Nova York 5


1. Introdução

L1cam é um gene ligado ao X que codifica a molécula de adesão de células neurais L1. L1cam é um membro da superfamília de genes de imunoglobulina transmembrana e um dos quatro genes no L1cam subfamília (Chl1, Nrcam, Nfasc, e L1cam em si). L1cam e seus outros membros da subfamília são amplamente expressos no sistema nervoso em desenvolvimento e adulto (Allen Brain Atlas). L1, em particular, é conhecido por desempenhar papéis essenciais durante o neurodesenvolvimento (Kenwrick et al., 2000 Hortsch et al., 2014 Sytnyk et al., 2017).

O domínio extracelular de L1 pode interagir com muitos outros ligantes e receptores da superfície celular e seu domínio intracelular pode ativar cascatas de sinalização. Em humanos, a perda projetada de L1CAM a expressão em neurônios cultivados derivados de ES leva a déficits na arborização axonal e dendrítica (Patzke et al., 2016). Mais de 350 mutações hereditárias e espontâneas em L1CAM, que se prevê levar à perda da função L1, foram identificados na população humana (NIH, Biblioteca Nacional de Medicina dos EUA, Referência de Genética). Essas mutações costumam levar à síndrome & # x0201cL1 & # x0201d, quase exclusivamente em homens devido à natureza ligada ao X de L1CAM. Os indivíduos afetados exibem uma variedade de defeitos do sistema nervoso com as manifestações mais comuns, incluindo hidrocefalia devido a estenose, paraplegia espástica tipo 1, marcha anormal, agenesia do corpo caloso, afasia e deficiência intelectual (incluindo autismo e esquizofrenia) (Wong et al., 1995 Christaller et al., 2017).

Este espectro de defeitos do sistema nervoso humano se sobrepõe ao espectro observado em L1cam camundongos e ratos knockout. L1cam roedores nocauteados exibem hidrocefalia e ventrículos aumentados, controle motor dos membros posteriores prejudicado, corpo caloso e tratos corticospinais reduzidos, aumento da letalidade perinatal, tamanho corporal menor, defeitos cerebelares e outros déficits neurológicos (Dahme et al., 1997 Cohen et al., 1997 Fransen et al., 1998, Emmert et al., 2019). A importância de L1 para o desenvolvimento do sistema nervoso é conservada além dos vertebrados. No verme C. elegans, por exemplo, o L1 homólogo SAX-7 é necessário para o posicionamento do neurônio e ramificação da neurite (Dong et al., 2013 Salzberg et al., 2013 Diaz-Balzac et al., 2015 Zou et al., 2016 Yang et al., 2019).

Em vermes e moscas-das-frutas, o crescimento e a ramificação dos axônios e dendritos são, em alguns casos, mediados por uma interação entre L1 e os homólogos dos receptores do fator de crescimento de fibroblastos (FGFRs) (Diaz-Balzac et al., 2015 Forni et al., 2004 ) Uma interação L1-FGFR para promover a maturação de neurônios se estende às células de mamíferos, como mostrado em vários ensaios de cultura definidos (Doherty e Walsh, 1996 Saffell et al., 1997) e na proliferação e mobilização de células de glioblastoma (Anderson e Galileo, 2016) . L1 pode atuar como um ligante que ativa não apenas FGFRs, mas também receptores de neurotrofinas (Colombo et al., 2014). FGFRs e receptores de neurotrofina desempenham vários papéis em todo o neurodesenvolvimento (Guillemot e Zimmer, 2011 H & # x000e9bert, 2011 Park e Poo, 2013). Além disso, os receptores de FGF e de neurotrofina são importantes na promoção de várias etapas da neurogênese hipocampal no giro dentado adulto (DG), onde a atividade de FGFR é necessária e suficiente para promover a neurogênese e a dendritogênese e a atividade do receptor de tirosina quinase B (TRKB) promove a maturação dendrítica ( Kang e H & # x000e9bert, 2015 de Vincenti et al., 2019).

No entanto, a extensão em que L1 desempenha um papel coincidente com a dos FGFRs e TRKs no GD adulto permanece desconhecida. Com base nos papéis essenciais de L1 & # x02032s durante o neurodesenvolvimento entre as espécies e, em alguns casos, sua ação direta através dos receptores de tirosina quinase, formulamos a hipótese de que L1 é necessária para uma ou mais etapas na linhagem de diferenciação de neurônios recém-nascidos na DG do hipocampo adulto. Usando várias linhas do mouse do driver Cre para excluir condicionalmente L1cam em diferentes tipos de células da linhagem neurogênica e neurônios circundantes, encontramos defeitos na produção de neurônios, dendritogênese e comportamento, sugerindo que L1 é crítica não apenas durante a neurogênese do desenvolvimento, mas também durante a neurogênese hipocampal adulta.


Capítulo 184 - Efeitos de nocautes de proteína quinase dependentes de cGMP

Proteína quinases cíclicas dependentes de GMP (cGKs) são serina / treonina quinases que são ativadas pelo segundo mensageiro cGMP. O terminal amino de cGKI é codificado por dois exões usados ​​alternativamente, resultando na produção de duas isoformas de cGKI, cGKIα e cGKIβ. O óxido nítrico (NO) e o peptídeo natriurético atrial (ANP) estimulam a síntese de cGMP e relaxam pequenas artérias e arteríolas, resultando em uma diminuição da pressão arterial. A inativação direcionada do gene do receptor de NO-sintase III (NOS III), ANP ou ANP causou hipertensão. O NO é de grande importância para a homeostase do endotélio plaquetário e das interações plaquetas-plaquetas por meio da inibição da adesão das plaquetas ao endotélio lesado e da ativação e agregação plaquetária. A cGKI plaquetária é essencial para prevenir a adesão intravascular e agregação de plaquetas após isquemia, provavelmente por inibir a ativação do receptor de fibrinogênio plaquetário, a glicoproteína IIb-IIIa. Esses efeitos de cGKIβ são provavelmente mediados por IRAG e uma liberação reduzida de Ca 2+ dos depósitos intracelulares. cGKI desempenha um papel importante para a homeostase cardiovascular e gastrointestinal e tem funções discretas no sistema nervoso central e periférico. A deleção do gene cGKI prejudica o relaxamento induzido por NO / cGMP do músculo liso peniano, levando à disfunção erétil, mas não afeta a motilidade e a fertilidade dos espermatozoides. Além disso, o relaxamento dependente de NO / cGMP do músculo liso do trato urinário é abolido em mutantes nulos de cGKI. A cGKII regula o crescimento ósseo longitudinal, o transporte intestinal de íons e a liberação de renina no rim e pode estar envolvida na geração de comportamentos complexos como ansiedade e dependência.


Resultados

Expressão e caracterização de isoformas mutantes de efrinaB2

Em uma comunicação anterior (Makinen et al., 2005), mostramos que as isoformas mutantes efrinB2 5Y e efrinB2 & # x00394V foram expressas em níveis iguais em lisados ​​de cérebro adulto, foram tirosina fosforilada em graus semelhantes em lisados ​​de proteína de embrião e foram expressos na superfície celular de células transfectadas em níveis semelhantes em comparação com efrinaB2 de tipo selvagem. Embora esta fosse uma forte evidência para o direcionamento subcelular normal da proteína efrinaB2 mutante, agora investigamos a expressão de superfície e o comportamento de agrupamento da efrinaB2 endógena em neurônios em cultura. Culturas de neurônios do hipocampo foram estabelecidas a partir de embriões homozigotos knock-in (em E16.5) que expressam ephrinB2 de tipo selvagem (ephrinB2 WT / WT ), ou isoformas mutantes de efrinaB2 (efrinaB2 5Y / 5Y e ephrinB2 & # x00394V /& # x00394V ) Após 2 DIV, usamos a proteína de fusão EphB4-Fc solúvel para induzir especificamente os agrupamentos de proteína ephrinB2. Na presença da proteína Fc de controle, nenhum agrupamento de ephrinB2 foi induzido (dados não mostrados). Aglomerados de proteína ephrinB2 endógena foram visualizados por coloração contra a porção Fc de EphB4-Fc e quantificados usando software MetaMorph. Não detectamos quaisquer diferenças marcadas nas densidades e tamanhos dos agrupamentos de efrinaB2 em neurônios que expressam as isoformas mutantes de efrinaB2 em comparação com efrinaB2 de tipo selvagem knock-in (Fig. 1 A & # x02013E) Coloração de ephrinB2 nocautes condicionais [ephrinB2-Nestincre (Grunwald et al., 2004)] com EphB4-Fc não revelou aglomerados fluorescentes, indicando ligação específica a ephrinB2 (Fig. S1 suplementar, disponível em www.jneurosci.org como material suplementar). Esses achados sugerem que a maioria das proteínas mutantes ephrinB2 foram transportadas para os locais subcelulares apropriados em neuritos do hipocampo. Para visualizar diretamente o ephrinB2 endógeno na sinapse CA3 & # x02013CA1, aplicamos a análise microscópica imunoeletrônica usando um anticorpo específico ephrinB2 (Fig. 1 F & # x02013H) Usando esta técnica, tínhamos mostrado anteriormente que a rotulagem ephrinB2 no lado pós-sináptico caiu quase quatro vezes em ephrinB2 nocautes condicionais (ephrinB2-CamKIIcre) (Grunwald et al., 2004). Aqui, descobrimos que o número de partículas de ouro nas sinapses positivas para ephrinB2 e o número de sinapses marcadas foram menores nos mutantes do que nos controles (Fig. 1 eu) Juntamente com os resultados de trabalhos anteriores (Makinen et al., 2005), esses dados sugerem que o direcionamento de efrinaB2 para sítios subcelulares não foi afetado principalmente no efrinaB2 5Y / 5Y e ephrinB2 & # x00394V /& # x00394V ratos (ver discussão).

Expressão e caracterização bioquímica de proteínas mutantes ephrinB2. A & # x02013C, Imagens representativas de dendritos de neurônios do hipocampo cultivados derivados do indicado ephrinB2 ratos knock-in. As células foram incubadas com EphB4-Fc e os agrupamentos positivos para ephrinB2 foram visualizados por imunocoloração anti-Fc. D, Quantificação da densidade de aglomerados anti-Fc-positivos por micrômetro de dendrito. E, Quantificação dos tamanhos dos clusters anti-Fc-positivos. F & # x02013H, Sinapses da região CA1 imunomarcadas com anticorpo ephrinB2 e analisadas por microscopia eletrônica. Os asteriscos indicam o lado pré-sináptico de cada sinapse mostrada. As pontas de seta apontam para as densidades pós-sinápticas. As partículas de ouro imune aparecem como pequenos pontos pretos. eu, Quantificação de microscopia imunoeletrônica ephrinB2. Os números de grãos de ouro por sinapse são mostrados em comparação com o número de sinapses marcadas em um pool de 300 sinapses. Uma proporção maior de sinapses não é marcada nos dois camundongos mutantes. O número de partículas de ouro nas sinapses positivas para efrinaB2 são semelhantes entre mutantes e controles (n = 2 ratos por grupo *p & # x0003c 0,05, t teste). J, Coimunoprecipitação (IP) de ephrinB2 com GRIP1. Células HeLa transitoriamente transfectadas com construtos que codificam ephrinB2 marcado com GFP de tipo selvagem (wt), deficiente no local PDZ (& # x00394V) ou mutante de tirosina (5Y) foram lisadas e imunoprecipitadas usando anticorpo anti-GFP. As proteínas foram imunotransferidas com anticorpos anti-GFP (meio) ou anti-GRIP (topo). Uma pequena quantidade do lisado total foi testada com anticorpos anti-GRIP para visualizar os níveis de GRIP nas células (parte inferior). Observe que GRIP não é coimmunoprecipitado com ephrinB2 & # x00394V. K, Quantificação da coimunoprecipitação de ephrinB2 (eB2) com GRIP1 [razão entre GRIP e eB2 densidade óptica (OD)]. eu, Fosforilação de tirosina de proteínas ephrinB2 mutantes em fatias de hipocampo adulto. Fatias derivadas de ephrinB2 WT / WT ao controle, efrinaB2 5Y / 5Y , e ephrinB2 & # x00394V /& # x00394V os camundongos foram deixados sem estimulação ou foram estimulados com o inibidor geral da proteína tirosina fosfatase ortovanadato. As fatias foram lisadas e a efrinaB2 foi imunoprecipitada com anticorpos anti-efrinaB2. As proteínas foram imunotransferidas com anticorpos anti-efrinaB2 (parte inferior) ou anti-fosfotirosina (parte superior 4G10). A seta aponta para ephrinB2. Barras de erro indicam SEM.

Para avaliar a ligação de PDZ por proteínas mutantes ephrinB2, investigamos a interação com GRIP1, uma proteína contendo sete domínios PDZ enriquecida em sinapses excitatórias (Dong et al., 1997) e com probabilidade de interagir com ephrinB2 (Bruckner et al., 1999 Lin et al., 1999). Expressamos isoformas de ephrinB2 em células HeLa e comparamos os níveis de proteína relativos por análise de Western blot contra GFP, que foi anexado ao seu terminal N extremo, uma modificação que não interfere na expressão de superfície e ligação ao receptor (Zimmer et al., 2003 Makinen et al. ., 2005) (Fig. 1 J) As isoformas EphrinB2 WT e ephrinB2 5Y coimmunoprecipitaram com GRIP1, demonstrando que a ligação dependente do domínio PDZ de GRIP1 não foi afetada pela remoção de resíduos de tirosina em efrinB2. Em contraste, GRIP1 não foi detectado em imunoprecipitados ephrinB2 & # x00394V, indicando que a ligação de PDZ de GRIP1 foi destruída [como mostrado anteriormente para sintenina (Makinen et al., 2005)]. A quantificação de seis experimentos separados revelou que a capacidade de ephrinB2 & # x00394V de coimmunoprecipitar GRIP foi 10 vezes menor em comparação com as isoformas ephrinB2 WT e ephrinB2 5Y (Fig. 1 K) Para avaliar a fosforilação da tirosina das isoformas endógenas de efrinB2 em fatias agudas do hipocampo de camundongos adultos, tratamos as fatias com o inibidor de tirosina fosfatase vanadato para permitir a atividade máxima da tirosina quinase. Na presença de vanadato, a proteína efrinaB2 & # x00394V foi fosforilada por tirosina em uma extensão semelhante à efrinaB2 de tipo selvagem, sugerindo que pode ser um substrato para tirosina quinases no hipocampo adulto (Fig. 1 eu) Como esperado, a isoforma mutante efrinaB2 5Y não conseguiu obter tirosina fosforilada sob essas condições (Fig. 1 eu) Esses resultados também confirmaram que os níveis gerais de proteínas ephrinB2 no hipocampo adulto mutante eram normais nos mutantes knock-in. Juntos, esses resultados indicam que as proteínas efrinaB2 mutantes são direcionadas para corrigir os locais subcelulares e se comportam como esperado em relação à ligação dependente do domínio PDZ e suscetibilidade à fosforilação da tirosina.

Morfologia ultraestrutural normal de efrinaB2 5Y / 5Y camundongos

Relatórios anteriores indicaram que os receptores neuronais para ephrinB2, nomeadamente EphB1-B3 e EphA4, têm funções (parcialmente redundantes) na formação de sinapses e espinhas (Dalva et al., 2000 Henkemeyer et al., 2003 Murai et al., 2003 Kayser et al., 2006). Em análises EM de ephrinB2 nocautes condicionais, não havíamos encontrado anteriormente diferenças nos números de sinapses de CA1 em comparação com controles de companheiros de ninhada (Grunwald et al., 2004). Porque o ephrinB2 Os knock-ins de isoformas são não condicionais e as proteínas ephrinB2 mutantes estão presentes ao longo do desenvolvimento, é possível que ocorram efeitos no número de sinapses que não são vistos nos knock-outs condicionais. Portanto, quantificamos o número de sinapses na região CA1 e avaliamos a morfologia dos PSDs. Encontramos uma redução modesta, mas significativa, nos números de sinapses CA1 em ambos ephrinB2 mutantes knock-in em comparação com ephrinB2 WT / WT controles (20% para ephrinB2 & # x00394V /& # x00394V e 15% para efrinaB2 5Y / 5Y às 6 semanas de idade) (Fig. 2 A & # x02013D) Esta redução no número de sinapses foi mais branda no 15º dia pós-natal (P15 14% para ephrinB2 & # x00394V /& # x00394V e 10% para efrinaB2 5Y / 5Y ) (Fig. S2 suplementar, disponível em www.jneurosci.org como material suplementar). Um relatório anterior havia indicado uma redução modesta do comprimento de PSD nas regiões CA1 e CA3 de triplo EphB ratos knock-out (EphB1 & # x02212 / & # x02212 B2 & # x02212 / & # x02212 B3 & # x02212 / & # x02212) (Henkemeyer et al., 2003). Encontramos uma diminuição modesta (10%) do comprimento do PSD em ephrinB2 & # x00394V /& # x00394V mas não em efrinaB2 5Y / 5Y ratos, em comparação com ephrinB2 WT / WT controles (Fig. 2 E) Além disso, notamos um aumento significativo na largura do PSD em ephrinB2 & # x00394V /& # x00394V mas não em efrinaB2 5Y / 5Y ratos, em comparação com ephrinB2 WT / WT controles (32 e 7%, respectivamente) (Fig. 2 F) O padrão de dendritos do hipocampo no estrato radiatum do CA1 foi semelhante com base na coloração de MAP2 em todas as três linhas (Fig. S3 suplementar, disponível em www.jneurosci.org como material suplementar).

Morfologias de sinapses derivadas de ephrinB2 camundongos mutantes. A & # x02013C, Micrografias eletrônicas mostrando a morfologia das sinapses assimétricas na região CA1 do hipocampo dos camundongos indicados. PSDs são indicados por setas. D, Números de sinapses por 100 & # x003bcm 2 na região CA1 (n = 3 camundongos por genótipo & # x0003e2000 sinapses por animal contado) de camundongos de 2 meses de idade. Observe uma redução pequena, mas significativa em efrinaB2 5Y / 5Y e ephrinB2 & # x00394V /& # x00394V ratos em comparação com ephrinB2 WT / WT controles (*p & # x0003c 0.01). E, Quantificação do comprimento PSD. Observe uma redução pequena, mas significativa em ephrinB2 & # x00394V /& # x00394V ratos em comparação com ephrinB2 WT / WT controles (*p & # x0003c 0.01) e nenhuma mudança em efrinaB2 5Y / 5Y camundongos. F, Quantificação da largura PSD. Observe um pequeno, mas significativo aumento em ephrinB2 & # x00394V /& # x00394V ratos em comparação com ephrinB2 WT / WT controles (*p & # x0003c 0.01) e nenhuma mudança em efrinaB2 5Y / 5Y camundongos. Barras de erro indicam SEM.

Como efrinaB2 está predominantemente localizada pós-sinapticamente nas sinapses CA3 & # x02013CA1, perguntamos a seguir se a modificação do domínio citoplasmático de efrinaB2 alteraria o número de especializações pós-sinápticas em vitro. Cultivamos neurônios do hipocampo de ephrinB2 mutantes (como descrito para a Fig. 1) e pontos imunorreativos PSD-95 pós-sinápticos visualizados por imunofluorescência após 20 DIV. Não encontramos diferenças significativas nos números de pontos positivos para PSD-95 entre ephrinB2 mutantes e ephrinB2 WT / WT controles (Fig. 3). Juntos, esses resultados sugerem que a proteína mutante efrinaB2 sem todos os resíduos de tirosina (efrinaB2 5Y) medeia a morfologia normal da sinapse na Vivo e especializações pós-sinápticas em neurônios cultivados. A expressão da proteína ephrinB2 & # x00394V leva a uma morfologia da sinapse ligeiramente alterada, mas a especializações pós-sinápticas normais em neurônios em cultura.

Análise de fluorescência de pontos PSD-95 em cultura de neurônios do hipocampo. A & # x02013C, Imagens representativas de células foram derivadas de embriões E16.5 das linhas de camundongos indicadas, cultivadas por 20 DIV e imunocoradas usando anticorpos anti-PSD-95. D, Quantificação de pontos PSD-95 por 100 & # x003bcm 2. Não foram observadas diferenças significativas (n = 3 embriões e 90 & # x02013130 ​​neurônios por genótipo p & # x0003e 0,05). Barras de erro indicam SEM.

Os parâmetros sinápticos basais permanecem inalterados em ephrinB2 mutantes citoplasmáticos

Em seguida, examinamos as consequências funcionais das mutações no domínio citoplasmático ephrinB2. Realizamos uma série de testes eletrofisiológicos comparando ephrinB2 & # x00394V /& # x00394V e efrinaB2 5Y / 5Y ratos para ephrinB2 WT / WT controles, que não mostraram quaisquer diferenças significativas para os camundongos do tipo selvagem (dados não mostrados). Primeiro, comparamos a transmissão sináptica basal entre ephrinB2 WT / WT e camundongos mutantes. As fEPSPs foram evocadas com intensidades de estímulo crescentes nas sinapses CA1, estimulando colaterais de Schaffer, uma das três principais vias excitatórias. Os tamanhos dos FVs pré-sinápticos, que são proporcionais ao número de axônios pré-sinápticos recrutados pela estimulação, foram comparados com a inclinação da resposta fEPSP, para estabelecer relações de entrada e saída (Fig. 4 UMA) Não observamos diferenças entre ephrinB2 WT / WT e ephrinB2 & # x00394V /& # x00394V ou efrinaB2 5Y / 5Y ratos, respectivamente (ephrinB2 & # x00394V /& # x00394V camundongos: inclinação e porcentagem do controle, 93 & # x000b1 5,6% efrinaB2 5Y / 5Y camundongos: inclinação e porcentagem do controle, 115 & # x000b1 13,2% p & # x0003e 0.2, t teste).

Os parâmetros sinápticos basais permanecem inalterados em ephrinB2 mutantes citoplasmáticos. UMA, inclinação de fEPSP em várias intensidades de estímulo (FV). Na faixa de 0,1 & # x020130,6 mV, os três grupos de camundongos mostraram valores semelhantes (ephrinB2 WT / WT , n = 17 fatias ephrinB2 & # x00394V /& # x00394V , n = 15 fatias efrinaB2 5Y / 5Y , n = 19 fatias p & # x0003e 0,05, t teste). B, Razão NMDA / AMPA. EPSCs do receptor AMPA foram evocados em um potencial de membrana de & # x0221270 mV, e EPSCs do receptor NMDA foram evocados a +40 mV. Para os neurônios de controle, a razão NMDA / AMPA foi de 1,6 & # x000b1 0,1 (n = 10 fatias). A proporção de NMDA para AMPA não foi significativamente alterada nos camundongos mutantes (ephrinB2 & # x00394V /& # x00394V : 1.7 e # x000b1 0.3, n = 11 fatias efrinaB2 5Y / 5Y : 1.8 e # x000b1 0.2, n = 9 fatias p & # x0003e 0,05, t teste). C, PPF do EPSP em um intervalo interestímulo (ISI) de 40 ms dos três grupos de camundongos não foi significativamente diferente (ephrinB2 WT / WT : 1,5 e # x000b1 0,1, n = 9 fatias ephrinB2 & # x00394V /& # x00394V : 1,6 e # x000b1 0,2, n = 9 fatias efrinaB2 5Y / 5Y : 1.6 e # x000b1 0.1, n = 8 fatias p & # x0003e 0,05, t teste). D, Gráfico de probabilidade cumulativa das amplitudes mEPSC (D1, D2) e frequências (D3, D4) nos ratos controle e mutantes. Nem as amplitudes de mEPSC nem as frequências de mEPSC mudaram significativamente nos camundongos mutantes (AV50 amplitude para mEPSCs: ephrinB2 WT / WT , & # x0221213.3 & # x000b1 0,9 pA, n = 19 fatias efrinaB2 5Y / 5Y , & # x0221214.4 & # x000b1 0,8 pA, n = 19 fatias ephrinB2 & # x00394V /& # x00394V , & # x0221214.2 & # x000b1 4 pA, n = 18 fatias p & # x0003e 0,05, t teste de frequência de eventos: ephrinB2 WT / WT , 0,9 e # x000b1 0,2 Hz, n = 19 fatias efrinaB2 5Y / 5Y , 1,0 e # x000b1 0,2 Hz, n = 19 fatias ephrinB2 & # x00394V /& # x00394V , 1,0 e # x000b1 0,2 Hz, n = 18 fatias p & # x0003e 0,05, t teste). Barras de erro indicam SEM.

Para avaliar se o número de receptores sinápticos AMPA e / ou NMDA eram diferentes em camundongos mutantes, comparamos os tamanhos dos EPSCs do receptor AMPA com os do receptor EPSCs do NMDA por registros de células inteiras de neurônios CA1 em fatias do hipocampo. EPSCs do receptor AMPA foram evocados em um potencial de membrana de & # x0221270 mV, e EPSCs do receptor NMDA foram evocados a +40 mV para aliviar o bloqueio de magnésio. A magnitude do EPSC do receptor NMDA foi determinada medindo a amplitude dos EPSCs 70 ms após o estímulo. Para os neurônios de controle, a razão NMDA / AMPA foi de 1,6 & # x000b1 0,1 (n = 10) (Fig. 4 B) A proporção de NMDA para AMPA não foi alterada significativamente nos camundongos mutantes (ephrinB2 & # x00394V /& # x00394V : 1.7 e # x000b1 0.3, n = 11 efrinaB2 5Y / 5Y : 1.8 e # x000b1 0.2, n = 9 p & # x0003e 0,05, t teste).

Em seguida, medimos o PPF, uma medida sensível de mudanças na probabilidade de liberação do transmissor (Pr). Para avaliar os efeitos das mutações do domínio citoplasmático de efrinaB2 no Pr, medimos o PPF usando as razões do segundo e do primeiro declive EPSP em um intervalo interpulso de 40 ms. Conforme mostrado na Figura 4 C, descobrimos que as sinapses em camundongos do tipo selvagem e mutantes exibiram PPFs muito semelhantes durante a transmissão sináptica basal. Esses resultados indicam que as funções pré-sinápticas nesses camundongos eram normais.

Finalmente, projetamos experimentos para procurar mudanças nos números das sinapses funcionais. A amplitude de mEPSCs é uma medida para o número do receptor AMPA por sinapse, enquanto uma mudança na frequência de mEPSC reflete uma mudança no número de sinapses funcionais. Comparação das amplitudes e frequências EPSC cumulativas (Fig. 4 D) para o tipo selvagem e os camundongos mutantes não revelaram diferenças significativas. As frequências de mEPSC foram medidas em camundongos de 2 a 3 semanas de idade, em um momento em que a microscopia eletrônica revelou reduções leves no número de sinapses (14% para ephrinB2 & # x00394V /& # x00394V e 10% para efrinaB2 5Y / 5Y camundongos). Because various parameters of mEPSC (such as probability of spontaneous vesicle fusion, detection level, attenuation in the dendrite) are heterogeneous from synapse to synapse, it is not surprising that small changes in synapse number are not detected as a change in mEPSC frequency. Together, these findings indicate that the ephrinB2 cytoplasmic domain mutations caused no apparent changes in excitatory circuitry.

EphrinB2–PDZ interaction and tyrosine phosphorylation sites participate in CA3� hippocampal LTP

Using two different protocols to induce LTP (tetanic and theta burst see Materials and Methods), we investigated the consequences of ephrinB2 cytoplasmic domain mutations in CA3� hippocampal LTP. Using acute slices from adult mice, either tetanic stimulation ( Fig. 5 UMA) or TBS ( Fig. 5 B) were applied to fibers of the CA3 presynaptic neurons, and LTP was recorded from CA1 neurons for up to 1 h after stimulation. In both stimulation protocols, the magnitude of the potentiation was different between controls and mutants. After tetanic stimulation ( Fig. 5 UMA), the mean EPSP slope as a percentage of baseline 55� min after stimulation was 156.4 ± 6.9% in the ephrinB2 WT/WT controls (n = 13 slices), whereas ephrinB2 ΔV/ΔV e ephrinB2 5Y/5Y mice showed significantly lower normalized slopes (117.7 ± 3.8 and 120.6 ± 8.9%, respectively p < 0.01, t teste). Similar differences were observed after TBS ( Fig. 5 B), indicating that the impairments in LTP were not specific to a single induction protocol. We next investigated slices taken from P14–P20 animals. As shown in Figure 5 C, both ephrinB2 ΔV/ΔV e ephrinB2 5Y/5Y mice displayed reduced LTP compared with ephrinB2 WT/WT controls, although the difference was less dramatic than in adult slices (ephrinB2 WT/WT , 141.5 ± 5.7% ephrinB2 ΔV/ΔV , 120.8 ± 5.5% ephrinB2 5Y/5Y , 120.9 ± 4.6% p < 0.01, t teste). These data indicate that hippocampal LTP requires both intact PDZ target and tyrosine phosphorylation sites in the ephrinB2 cytoplasmic domain.

EphrinB2 tyrosine phosphorylation sites are not required for LTD and depotentiation

Because LTD at CA3� hippocampal synapses requires the presence of ephrinB2 (Grunwald et al., 2004), we analyzed hippocampal LTD in slices taken from ephrinB2 WT/WT control knock-in mice and ephrinB2 signaling mutants. After recording stable baseline responses (see Materials and Methods), LTD was induced by low-frequency stimulation (LFS) of 900 stimuli at 1 Hz (15 min). As shown in Figure 6 UMA, ephrinB2 WT/WT controls showed a persistent decrease in fEPSPs, which lasted for at least 60 min. EphrinB2 ΔV/ΔV slices were defective in LTD and returned to baseline at 40 min after LTD induction (ephrinB2 ΔV/ΔV slices, 97.9 ± 2.2% vs ephrinB2 WT/WT slices, 84.1 ± 1.7% p < 0.01, t teste). Unexpectedly, ephrinB2 5Y/5Y slices showed LTD nearly indistinguishable from ephrinB2 WT/WT controls (ephrinB2 5Y/5Y , 82.3 ± 3.4% vs ephrinB2 WT/WT slices, 84.1 ± 1.7% p = 0.56, t teste).

EphrinB2 tyrosine phosphorylation sites are not required for LTD and depotentiation. UMA, EphrinB2–PDZ interaction but not tyrosine phosphorylation sites are required for LTD in young hippocampal slices (P14–P20). LFS induced a significant long-lasting decrease in fEPSPs in ephrinB2 WT/WT controle e ephrinB2 ΔV/ΔV slices but not in ephrinB2 5Y/5Y slices (97.9 ± 2.2% in ephrinB2 ΔV/ΔV mutants compared with 82.3 ± 3.4% in ephrinB2 5Y/5Y mutants and 84.1 ± 1.7% in ephrinB2 WT/WT controls at 55� min after LFS p < 0.05). B, Depotentiation is impaired in ephrinB2 ΔV/ΔV mas não ephrinB2 5Y/5Y mutantes. Ten minutes after tetanus (arrow) to produce the initial phase of LTP, slices were subjected to a 15 min LFS train (horizontal line 1 Hz). The fEPSPs from ephrinB2 WT/WT controle e ephrinB2 5Y/5Y slices returned to baseline (ephrinB2 WT/WT slices, 104.0 ± 7.6% ephrinB2 5Y/5Y slices, 98.9 ± 6.2%), whereas ephrinB2 ΔV/ΔV slices remained potentiated (126.9 ± 4.6% p < 0.05, compared with ephrinB2 WT/WT controls).

We additionally analyzed another form of long-term plasticity known as depotentiation, the depression of synaptic efficacy at synapses that have recently undergone LTP (O'Dell and Kandel, 1994). Although the induction protocol for depotentiation is identical to LTD, they may represent distinct processes (Montgomery and Madison, 2002, and references within). After recording a stable baseline, we applied a tetanic stimulation to induce LTP, followed (after 10 min) by a depotentiation stimulus (LFS of 900 stimuli at 1 Hz for 15 min). Slices derived from ephrinB2 WT/WT controls showed a normal initiation phase of LTP (first 10 min after tetanus application) and complete depotentiation after 15 min of LFS ( Fig. 6 B) As expected from the LTP results, both ephrinB2 ΔV/ΔV e ephrinB2 5Y/5Y slices displayed reduced early LTP, but their reaction to LFS varied: ephrinB2 ΔV/ΔV slices were defective in depotentiation and remained potentiated 55� min after LFS application (fEPSP slope size: ephrinB2 ΔV/ΔV slices, 126.9 ± 4.6% vs ephrinB2 WT/WT slices, 104.0% ± 7.7% p < 0.05, t teste). Em contraste, ephrinB2 5Y/5Y slices displayed complete depotentiation 1 h after the application of the LFS protocol (98.9 ± 6.2% fEPSP slope) ( Fig. 6 B), following a time course indistinguishable from controls. These findings suggest that the required function of ephrinB2 in LTD as well as in depotentiation is independent of phosphotyrosine signaling.

EphrinB2 reverse signaling in cultured neurons induces phosphorylation of NMDA receptors

In a number of different cell types, including excitatory neurons, ephrinB reverse signaling involves the rapid recruitment and activation of SFKs to ephrinB clusters (Palmer et al., 2002 Georgakopoulos et al., 2006). SFKs bind to the ephrinB cytoplasmic domain and phosphorylate ephrinB on tyrosine residues. SFKs are also the major protein tyrosine kinases that upregulate the activity of NMDA receptors (reviewed in (Salter and Kalia, 2004 Lee, 2006). SFK-mediated tyrosine phosphorylation of NMDA receptors is critical for induction of NMDA receptor-dependent LTP (Lu et al., 1998). Tyrosine phosphorylation of NMDA receptor subunits regulates their membrane trafficking. To begin addressing the biochemical events mediated by ephrinB2 reverse signaling, we investigated the degree of tyrosine phosphorylation of the NR2A subunit of NMDA receptors in cultured forebrain neurons. Stimulation of neurons derived from ephrinB2 WT/WT mice with EphB4-Fc led to a robust increase in NR2A tyrosine phosphorylation compared with control Fc-treated cultures ( Fig. 7 A & # x02013C) Interestingly, NR2A tyrosine phosphorylation was abnormal in mutant neurons: in the presence of the ephrinB2 ΔV isoform, basal NR2A tyrosine phosphorylation was elevated and was not further enhanced by EphB4-Fc stimulation ( Fig. 7 B) In the presence of the ephrinB2 5Y isoform, basal NR2A tyrosine phosphorylation was comparable to wild-type neurons, but EphB4-Fc stimulation did not lead to an increase in NR2A tyrosine phosphorylation. The changes in NR2A tyrosine phosphorylation correlated with similar changes in Src phosphorylation as revealed by a phospho-Src ELISA ( Fig. 7 D) These results demonstrate NR2A tyrosine phosphorylation as a consequence of ephrinB activation in cultured neurons and that both ephrinB2 ΔV and ephrinB2 5Y isoforms fail to regulate NR2A tyrosine phosphorylation.

EphrinB2 activation causes NR2A and Src tyrosine phosphorylation. Dissociated cultures of embryonic forebrain neurons (8 DIV) derived from wild-type (UMA) or from the indicated mutant mice (B𠄽) were stimulated with control Fc or EphB4-Fc for the indicated times. UMA, Note the EphB4-Fc-induced increase in NR2A tyrosine phosphorylation in wild-type neurons. B, Independent experiment using ephrinB2 WT/WT e ephrinB2 ΔV/ΔV neurons. Note the elevated basal and lack of stimulated NR2A tyrosine phosphorylation in ephrinB2 ΔV/ΔV neurons. C, Independent experiment using ephrinB2 WT/WT e ephrinB2 5Y/5Y neurons. There is no EphB4-Fc-induced increase in NR2A tyrosine phosphorylation in ephrinB2 5Y/5Y neurons. D, Cell lysates were subjected to a phospho-Src ELISA. EphB4-Fc-stimulation of ephrinB2 WT/WT neurons led to a significant induction of Src phosphorylation at position 418, which is required for Src activation (*p < 0.05, t teste). Changes in Src phosphorylation observed in ephrinB2 ΔV/ΔV e ephrinB2 5Y/5Y neurons were not significant. p-NR2A, Phosphorylated NR2A.


Brain Extracellular Matrix in Health and Disease

Oleg Senkov , . Alexander Dityatev , in Progress in Brain Research , 2014

7 Reelin

Reelin is a large secreted ECM glycoprotein (400 kDa, gene RELN) that controls neuronal migration and brain development ( Cooper, 2008 Dɺrcangelo et al., 1995 Rice and Curran, 2001 Soriano and Del Rio, 2005 ). Reelin binds to the lipoprotein family receptors apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR) ( Dɺrcangelo et al., 1999 Hiesberger et al., 1999 ) and induces the phosphorylation of the adaptor protein Dab1 ( Howell et al., 1997 Howell et al., 1999 ). The downstream Reelin cascade includes several signaling pathways, including distinct members of the Src kinase family ( Arnaud et al., 2003 ), Erk1/2 ( Simo et al., 2007 ), AKT/GSK3 ( Beffert et al., 2002 ), and ubiquitination/degradation of phosphorylated mDab1 triggered by Cul5 ( Simo and Cooper, 2013 Simo et al., 2010 ).

In addition to developmental stages, Reelin is expressed in the adult cerebral cortex mainly by γ-amino-butyric acid (GABA)-positive interneurons ( Alcantara et al., 1998 ) where it has been proposed to regulate plasticity processes ( Dityatev et al., 2010a Herz and Chen, 2006 ). For instance, it has been shown that Reelin potentiates glutamatergic neurotransmission, LTP, and synaptic maturation increases the expression of AMPA and NMDA receptor subunits and favors the trafficking and substitution of NR2B subunits by NR2A subunits ( Beffert et al., 2005 Chen et al., 2005 Groc et al., 2007 Qiu and Weeber, 2007 Qiu et al., 2006b ). Moreover, Reelin has been suggested to regulate the density and stabilization of dendritic spines ( Niu et al., 2008 Ventruti et al., 2011 ). Two models have been used to address specifically the role of Reelin in the adult brain. Local na Vivo injections of Reelin increase spine density, modify spine morphology, and enhance LTP ( Rogers et al., 2011 Rogers et al., 2013 ). Similarly, transgenic mice overexpressing Reelin in the adult forebrain (Reelin-OE mice) show hypertrophy of dendritic spines and enhanced glutamatergic neurotransmission and LTP ( Pujadas et al., 2010 Teixeira et al., 2011 ). Moreover, both acute administration of Reelin in wild-type mice and studies in Reelin-OE mice show enhanced associative and spatial learning and memory ( Pujadas et al., 2010 Pujadas et al., 2014 Rogers et al., 2011 Rogers et al., 2013 ). Conversely, administration of recombinant receptor-associated protein (as a Reelin signaling blocking tool) is associated with impaired performance in a hippocampus-dependent MWM test ( Stranahan et al., 2011 ).

Reelin is also highly expressed in neurogenic niches, the subventricular zone (SVZ) and the dentate gyrus, and in rostral migratory stream and olfactory bulb ( Courtes et al., 2011 Dityatev et al., 2010b ). In the SVZ, Reelin was shown to control the behavior of SVZ-derived migrating neurons, triggering them to leave prematurely the rostral migratory stream leading to ectopic neurons both along the rostral migratory pathway and in the olfactory bulb ( Courtes et al., 2011 Pujadas et al., 2010 ). In the dentate gyrus, Reelin overexpression results in increased neurogenesis and accelerated dendritic maturation, likely reflecting increased functional integration into adult circuits. Conversely, inactivation of the Reelin signaling pathway specifically in adult neuroprogenitor cells resulted in aberrant migration, decreased dendrite development, and formation of ectopic dendrites in the hilus and in the establishment of aberrant circuits ( Teixeira et al., 2012 ). These studies support a critical role for the Reelin pathway in regulating adult neurogenesis and dendritic development of adult-generated neurons. Taken together with the above data, Reelin emerges as a key regulator of adult plasticity processes important for learning and memory including synaptic plasticity and remodeling and adult neurogenesis.

Reelin is also known to be involved in a spectrum of cognitive pathological conditions, for example, its expression is compromised in the brain of schizophrenic patients and in autism, bipolar disorder, major depression, AD, and several polymorphisms, and rare variants in the RELN gene have been associated with these disorders ( Botella-Lopez et al., 2006 Chin et al., 2007 Fatemi et al., 2000 Kramer et al., 2011 Liu et al., 2010 ). However, experimental evidence in heterozygous reeler mice, expressing a half of normal Reelin protein levels, has been contradictory. While some studies show that heterozygous reeler mice have cognitive ( Brigman et al., 2006 Qiu et al., 2006a ) and sensorimotor deficits as measured in the prepulse inhibition test (PPI) ( Barr et al., 2008 ), other studies have failed to find differences ( Krueger et al., 2006 Podhorna and Didriksen, 2004 Teixeira et al., 2011 ). Moreover, additional tests associated to psychiatric-related behavioral dysfunctions (e.g., OF, BW, and FST tests) also failed to demonstrate clear differences between heterozygous reeler e wt mice ( Podhorna and Didriksen, 2004 Teixeira et al., 2011 ). Finally, relatively mild behavioral deficits have been found in VLDLR or ApoER2 mutant mice ( Barr et al., 2007 ). Taking advantage of an experimentally different approach, Teixeira et al. (2011) investigated whether Reelin overexpression may modify psychiatric-related phenotypes. While increased Reelin expression does not alter mood-related behaviors under basal conditions, Reelin overexpression was found to be protective against PPI deficits induced by NMDA antagonists, cocaine sensitization (as a model of maniac disorder), and chronic stress-induced depression phenotypes.

In relation to AD pathology, it is noteworthy that Reelin is present in amyloid plaques ( Doehner et al., 2010 ), controls APP processing ( Hoe et al., 2006 ), and reduces tau phosphorylation by inhibiting glycogen synthase kinase 3 (GSK3 Ohkubo et al., 2003 ). Importantly, Reelin has been consistently found to counteract Aβ42-induced synaptic dysfunction including LTP ( Durakoglugil et al., 2009 ). In addition, AD brain samples show altered levels of Reelin and RELN polymorphisms have been associated with this disease ( Botella-Lopez et al., 2006 Botella-Lopez et al., 2010 Chin et al., 2007 Kramer et al., 2011 ). These studies suggest that dysfunction of the Reelin pathway may be at the root of the neuropathologic mechanisms leading to sporadic, late-onset AD ( Krstic and Knuesel, 2013 Krstic et al., 2012 ). This hypothesis has been experimentally addressed. Indeed, the reduction of Reelin in an AD mouse model crossed with heterozygous reeler accelerates the onset of plaque formation and tau pathology ( Kocherhans et al., 2010 ). Conversely, Reelin overexpression in hAPPSwe/ind (J20) mice reduces amyloid plaque load, rescues dendritic spine loss in J20 mice, and enhances cognitive performance in both aged wild-type and J20 mice. At the molecular level, Reelin delays Aβ42 fibril formation—by interacting with Aβ42 soluble species including oligomers—until it is sequestered into amyloid fibrils. Importantly, Reelin overcomes the toxicity of Aβ42 oligomers in neuronal cultures ( Pujadas et al., 2014 ). Taken together with Reelin's role in plasticity, these data support a model in which the Reelin pathway may exert beneficial effects on both AD pathology and cognition by at least two complementary mechanisms: in addition to extracellular Reelin delaying amyloid fibril formation and reducing neurotoxicity by interacting with Aβ42, the activation of the Reelin cascade itself would potentiate adult plasticity events, including synaptic plasticity and adult neurogenesis, and lead to decreased GSK3 activity and tau phosphorylation. It is thus likely that activation of the Reelin pathway might represent a therapeutic strategy for ameliorating the cognitive decline and neuropathologic hallmarks associated with AD.


Comentários

This is a very interesting study that provides several important advances and insights for the field. It is particularly intriguing to see that BACE1 inhibition not only reduces plaque growth, but may even shrink existing plaques. If it also happens in patients this would be fantastic news for the clinical trials with BACE inhibitors. Because AD mouse models, including the 5xFAD model used in this study, typically mimic presymptomatic AD, it would appear possible that secondary prevention trials with BACE inhibitors may not only yield reduced growth or number of plaques, but even a shrinking of pre-existing plaques and a reduction of neuroinflammation—provided that the drugs are given early enough before the symptoms start. This should and will be tested in the trials’ participants using PET imaging. These new findings contradict a recent study that demonstrated that plaques remain stable in size when BACE1 is inhibited pharmacologically. I am sure that this discrepancy will be resolved with future experiments and may depend on the mouse line used, or the level or duration of BACE1 inhibition.

We also need to consider that the amazing effects of the mouse study were accompanied by an intriguingly gradual reduction of BACE1 protein levels. It is difficult to predict exactly which inhibition level of BACE1 would be needed in humans to achieve the same results. Potentially, the levels currently used in trials are already sufficient.

Another take-home message of the study is that memory and LTP deficits were improved in the AD mice as BACE1 was suppressed. While this is good news, the study also demonstrated that LTP did not fully recover. This indicates that the therapeutically desired BACE inhibition in adult mice may interfere with physiological BACE1 functions. Besides LTP, this includes muscle spindle formation/maintenance and dendritic spine densities in adult mice. Translated to humans, this could indicate a larger number of falls or psychiatric symptoms in individuals treated with BACE inhibitors. Whether this is a realistic concern will be seen from the results of the currently ongoing and recently terminated BACE inhibitor trials.

Constitutive BACE1-deficient mice show a number of different phenotypes. Another major insight of the new study is that adult deletion of BACE1 overcomes at least one of the symptoms—the hypomyelination. The new conditional BACE1-deficient mice are an excellent tool to analyze whether the other BACE1-deficient mouse phenotypes are also of developmental origin and which functions of BACE1 are relevant in adult mice. Taken together, the new study is an excellent basis both for basic and translational BACE1 research.

I am interested to know why, under BACE1 cKO, there is, in addition to an expected decrease in C99, a decrease in C83 as well. The authors suggest that a cKO of BACE1 improves autophagy, increasing turnover of C-terminal fragments. However, there is no change in C83 in the original KO (Cai, 2001). Have the authors looked at APP trafficking in their model? I would be interested to know if the amount of sAPPα is the same, i.e. is APP still being trafficked to the cell surface?


Jin-Yu Lee and Li-Jen Lee: These authors contributed equally to this work.

Afiliações

Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan

Jin-Yu Lee, Hsin-An Shih & Mau-Sun Chang

Graduate Institute of Anatomy and Cell Biology, National Taiwan University, Taipei, Taiwan

Institute of Brain and Mind Sciences National Taiwan University, Taipei, Taiwan

Neurobiology and Cognitive Science Center, National Taiwan University, Taipei, Taiwan

Department of Superintendent Office, Mackay Memorial Hospital, Taipei, Taiwan

Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu, Taiwan

Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan

Department of Life Science, College of Life Science, National Taiwan University, Taipei, Taiwan


Resultados

SynGAP mutant mice die shortly after birth

To explore the possible role of SynGAP na Vivo, the SynGAP gene was disrupted in mice. The genomic DNA containing the 5′ end of the mouse SynGAP gene was isolated and analyzed for targeting vector construction. SynGAP is extensively spliced at the 5′ end, leading to splice variants: SynGAP-a, -b, -c, and -d (Chen et al., 1998 Kim et al., 1998 Li et al., 2001) (Fig.1UMA) Therefore, the exon cassette containing the first common methionine present in the shortest splice variant, SynGAP-c, was chosen for deletion, along with an adjacent exon encoding a portion of the C2 domain by replacing it with a neo R gene cassette in the reverse orientation (Fig. 1B) The normal splicing events in the targeted SynGAP gene are predicted to yield transcripts with premature stop codons. The targeted and the wild-type alleles can be differentiated by performing Southern blotting after theKpnI digestion using the inner and outer probes (Fig.1B), and the genotyping result can also be confirmed by PCR using the primers shown (Fig. 1C,direito).

SynGAP splice variants and domain structure and gene targeting strategy. UMA, N-terminal splicing leads to different start sites and sequences in SynGAP-a, -b, -c, and -d. SynGAP protein contains a pleckstrin homology (PH) domain, a phospholipid-dependent Ca 2+ binding motif (C2) domain, a Ras GTPase-activation protein (RasGAP) domain, and a C-terminal sequence PSD-95/discs large/zona occludens-1 domain binding motif (QTRV) Alternative splicing occurs also at the C terminal with C-terminal sequences other than −QTRV.B, The SynGAP gene structure is shown (not drawn to scale) of the region analyzed for gene targeting. Targeting of the SynGAP gene was performed by replacing the SacoII andEcoRI fragments of the SynGAP gene containing two exons with the neo R cassette. The targeting construct spanning the Xhoeu e HindIII fragment of the SynGAP gene is 11.5 kb long. Outer and inner probes were used for Southern blotting. The PCR primers used for genotyping and the predicted amplified sizes are shown. TK, Thymidine kinase. C, Genotype analyses of tail DNA of second filial generation (F2) mice by PCR and Southern blotting. The wild-type allele is detected by Southern blotting after digestion with the KpnI restriction enzyme (deixou) The size of the detected wild-type band is 1.8 kb longer than that of the targeted allele. Two alleles can be distinguished using PCR primer sets (direito).

The chimeric male mice from two independent ES clones (17.28 and 18.8) were mated with C57BL/6 female mice to generate heterozygotes, and sibling mating of heterozygotes produced homozygotes. O F2 mice genotypes exhibited a Mendelian ratio of 1:2:1, indicating that there is no embryonic lethality caused by the null mutation of the SynGAP protein or abnormal segregation of the gene. A database search revealed that the SynGAP gene is on mouse chromosome 17. A sample Southern blotting analysis of the F2 mice is shown in Figure 1C,deixou, where the mobility of the targeted allele differs from that of the wild type after the KpnI digestion because the neo R gene does not contain the restriction site. The heterozygotes are indistinguishable from the wild types in their size and activity, and they breed normally. The homozygotes are indistinguishable from the wild types and the heterozygotes for the first 2 d after birth. By the third day the mutant mice begin to show less movement and do not feed from the mother mice. Between P4 and P6, the pups stay small in size, and they die between P5 and P7. These observations were confirmed in the two independent mutant mouse lines.

SynGAP gene targeting abolishes the expression of the wild-type SynGAP protein

Immunoblot analysis of mouse brain homogenates at P5 with the anti-GAP SynGAP antibody showed that expression of the 130 kDa SynGAP protein is abolished in the mutant mice (Fig.2UMA) However, overexposure of the immunoblot showed that a very low level of a smear of smaller proteins (∼120 kDa) could be detectable using the anti-GAP antibody. These are most likely protein products of the SynGAP gene from cryptic start sites downstream of the deleted exons in the targeted gene. These protein products are present at <2% the level of the wild-type SynGAP protein. To analyze whether deletion of SynGAP affected the expression of other neuronal proteins, various synaptic proteins were surveyed in the SynGAP mice at P5 (Fig.2B) NMDA receptor subunits, NR1 and NR2B, and associated proteins, PSD-95 and SAP102, were similar in expression level in all genotypes. Also, the level of the AMPA receptor subunit GluR1 and the AMPA receptor-associated proteins GRIP1 and GRASP1 were indistinguishable in the wild types, heterozygotes, and homozygotes.

A proper expression of SynGAP protein is abolished in the mutant mice, whereas other synaptic proteins are not affected at P5. UMA, Mouse brain homogenates were prepared and immunoblotted with the anti-GAP SynGAP antibodies at P5 and compared with that of rat brain homogenate at P4. With an equal amount of protein loaded in each faixa, SynGAP protein expression is absent in the sample from a homozygous (Homoz) mouse.Heteroz, Heterozygous. B, The expression of proteins at synapses was examined in the SynGAP mice using the antibodies to the proteins indicated, and no detectable change in the expression was seen. C, Tissue distribution of SynGAP protein in the mutant and the wild-type mice at P5 was examined using the α-GAP domain antibody. In wild-type mice, a prominent band of ∼130 kDa was detected in the cortex and the cerebellum. In contrast, the ∼130 kDa protein was not detected in the homozygotes or in non-neuronal tissues.

Brain development in the SynGAP mutant mice

Examination of the SynGAP mutant mice at a gross anatomical level reveals typical development of tissues and organs, similar to the wild-type mice. Because SynGAP protein expression is selectively expressed in the brain (Fig. 2C), it seems likely that the prenatal development of non-neuronal tissues is not affected by the absence of SynGAP protein. Brain development at the gross anatomical level also seems normal in the mutant mice. The formation and organization of the forebrain appears to be similar in mice of all three genotypes at P5, as revealed by Nissl staining (data not shown). However, the size of the mutant mouse brain is significantly smaller, indicating that SynGAP may be crucial to the proliferation and development of neuronal tissues after birth, especially around P3. It is not clear why the SynGAP mutant mice die (see Discussion).

Decreased number of silent synapses in neurons cultured from homozygous mice

To investigate the role of SynGAP during synaptogenesis, cortical neuronal cultures were prepared from SynGAP mice and analyzed after 18–20 d em vitro (DIV). The neurons were fixed, and immunocytochemistry was performed using an anti-synaptophysin antibody to identify synapses, anti-GluR1 and anti-GluR2/3 antibodies to identify AMPA receptors, and anti-NR1 antibody for NMDA receptors. In heterozygote and homozygous neurons, synapses, identified by the anti-synaptophysin antibody, were present in similar numbers [heterozygote mice, 94.1 ± 4.7 (SD) of wild-type mice,p < 0.68 homozygote mice 109.4 ± 4.9%,p < 0.49 of wild-type mice] and pattern to those in wild-type neurons. Interestingly, AMPA receptor clusters, identified by the anti-GluR1 antibody, were present in a greater number in the homozygotes than in the heterozygotes and the wild-types (Fig.3UMA) A similar result was obtained using the anti-GluR2/3 antibody. Quantitation of the number of AMPA receptor puncta is shown in Figure 3B. The number of GluR1-positive clusters was increased in the homozygotes by 32.1 ± 9.0% (p < 0.05 ANOVA) compared with the wild types (Fig. 3) and was also higher in the heterozygotes (21.4 ± 8.6%). There was a slight increase in the number of NMDA receptor puncta, although this was not statistically significant (p > 0.05 ANOVA data not shown). Because the number of AMPA receptor clusters increased more than the number of NMDA receptor clusters, we determined whether the number of morphological silent synapses (synapses that contain NMDA receptors but not AMPA receptors) (Liao et al., 1999, 2001) was increased in the mutant mouse. The cultures from the SynGAP mutant mice had significantly fewer morphological silent synapses than their wild-type littermates (Fig.3C).

The number of AMPA receptor clusters in the SynGAP mutant mice is increased. UMA, Primary cortical cultures from the SynGAP mutant mice and their wild-type and heterozygous littermates were immunostained with anti-GluR1 antibodies after 18–20 DIV. There was an increase in the number of GluR1-positive clusters in the cultures prepared from the homozygous pups. B, Quantitation of GluR1-positive puncta in SynGAP mouse neuronal cultures at 18–20 DIV (n = 14, n = 19, and n = 13, respectively p < 0.05 ANOVA F = 3.52). C, The number of morphological silent synapses in the cultures was quantitated by comparing the number of AMPA receptor cluster/NMDA receptor cluster puncta (n = 9, n = 13, andn = 7, respectively) at 18–20 DIV.

Synaptic plasticity in SynGAP knock-out mice

We tested the role of SynGAP in hippocampal synaptic plasticity by comparing the magnitude of LTP and LTD in the CA1 region of adult wild-type and heterozygous mice. LTP induced by TBS was significantly decreased in slices from heterozygous mice (140 ± 6% of baseline at 1 hr after TBS n = 20 slices from five animals) compared with their wild-type littermates (174 ± 9% of baseline n = 16 slices from five animalsp < 0.01 Student's t test) (Fig.4UMA) Next we tested whether LTD is affected in the SynGAP heterozygotes. To examine LTD, we used paired pulses at an interstimulus interval of 50 msec repeated at 1 Hz for 15 min (PP-1 Hz), which has been used previously to induce LTD in hippocampal slices from adult rats (Kemp et al., 2000). As shown in Figure 4B, there was no difference in the magnitude of LTD in the heterozygous animals (80 ± 3% of baseline measured 1 hr after the start of PP-1 Hz n = 21 slices from four animals) compared with wild-type littermates (82 ± 4% of baseline n = 18 slices from four animalsp > 0.4 Student's t teste). In mice, LTD induced by the PP-1 Hz protocol is completely blocked by bath application of the NMDA receptor antagonist APV (data not shown).

UMA, Schaffer collateral to CA1 LTP in adult SynGAP heterozygotes (● n = 20 slices from 5 animals) are significantly reduced compared with wild-type littermates (○ n = 16 slices from 5 animals). FP traces taken just before and 1 hr after TBS for wild types and heterozygotes are shown to the direito. B, No significant difference in PP-LTD (PP-1Hz) in SynGAP heterozygotes (● n = 21 slices from 4 animals) and wild types (○ n = 18 slices from 4 animals). FP traces taken just before and 1 hr after the initiation of PP-1 Hz are shown to the direito. C, AMPA receptor-mediated synaptic transmission measured as the initial FP slope plotted against fiber volley amplitude. Plots of both wild types (○ n = 32 slices from 8 animals) and heterozygotes (● n = 33 slices from 8 animals) essentially overlap, suggesting that synaptic transmission is normal in heterozygotes. D, No difference was observed in presynaptic function as monitored by paired-pulse facilitation between wild types (○ n = 14 slices from 5 animals) and heterozygotes (● n = 14 slices from 5 animals). Paired pulses were given at interstimulus intervals of 25, 50 100, 200, 400, 800, and 1600 msec at baseline stimulus intensity.E, Pharmacologically isolated NMDA receptor-mediated synaptic transmission does not differ much between SynGAP heterozygous and wild-type littermates. NMDA receptor-mediated synaptic responses were pharmacologically isolated by bath application of ACSF with 0 m m Mg 2+ and 10 μ m NBQX. An input–output curve was generated by plotting the amplitude of NMDA receptor (NR)-mediated FP against fiber volley amplitude. At the end of each experiment, 100 μ m d,l -APV was added to the bath, completely abolishing the responses (data not shown). Dashed lines indicate normalized FP and paired-pulse facilitation ratio.

The phenotype seen in heterozygotes was not attributable to changes in AMPA receptor-mediated synaptic transmission, because there were no detectable differences in the input–output curve (Fig. 4C) Presynaptic function measured by the paired-pulse facilitation ratio at interstimulus intervals ranging from 25 to 1600 msec were also normal in the heterozygotes (Fig. 4D) To rule out the possibility that the reduced LTP in the SynGAP heterozygotes is attributable to alterations in NMDA receptor-mediated synaptic responses, we pharmacologically isolated NMDA receptor-mediated components of synaptic transmission by recording in ACSF with 0 m m Mg 2+ and 10 μ m NBQX. The magnitude of the NMDA receptor-mediated response was measured by generating an input–output curve. We plotted NMDA receptor-mediated FP amplitude against the fiber volley amplitude to correct for variability in recruiting presynaptic fibers. As shown in Figure 4E, there is no significant effect on NMDA receptor-mediated responses in the heterozygotes.


Conclusões

The functioning of the CPEB family proteins is essential at all stages of ontogeny. CPEBs play an important role in the formation and maintenance of cell polarity, participating in mRNA transport and localization, translational repression or activation of target mRNAs [30, 81,82,83]. In the nervous system, this function is manifested in the participation of the CPEB proteins in neurogenesis and the functioning of neurons. Much attention is devoted now to the role that the prion-like conformation of these proteins plays in the formation of long-term memory.

The CPEB proteins participate in the translational control of a wide range of mRNAs and, therefore, are involved in pathologies of the nervous system. Moreover, disturbances in the functioning of the CPEB proteins cause other pathological processes, including carcinogenesis, tumor invasion, and angiogenesis. In the case of rectal cancer, breast cancer, and gliomas, the expression levels of several CPEB proteins change simultaneously, which is indicative of interactions between them in the oncological process [67, 84,85,86]. The role of the CPEB proteins in certain liver diseases and metabolic disorders (e.g., hepatosteatosis) was also revealed [87].

Thus, investigation of the role of the CPEB proteins is an extremely important fundamental task that opens up prospects for understanding the molecular mechanisms of the formation and functioning of the nervous system and other body systems, as well as for finding ways to treat a wide range of diseases.


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