Os autores gostariam de agradecer Damien Gaillard para inoculações e acompanhamento de experiências com animais. Este trabalho foi apoiado por ANSES (Agência Francesa de Alimentos, Saúde Ambiental e Ocupacional e Segurança) e por uma bolsa da Fundação France Parkinson.

Todos os procedimentos e protocolos que envolvam animais estavam em conformidade com a Directiva CE 86/609 / CEE do Conselho e ratificado pelo vem, o comitê nacional francês para a consideração de Ética em Experimentação Animal (protocolo 11-0043). Os animais foram alojados e tratados em ANSES de aprovado instalações experimentais em Lyon (aprovação B 69387 0801). 1. Preparação de Ratos <ol><li> Eutanásia ratinhos por injecção intraperitoneal de uma dose letal de pentobarbital de sódio. </li><li> Recuperar todo o cérebro do crânio do rato e colocá-lo em um 35 milímetros de plástico placa de Petri sobre gelo até a extração. </li><li> Extrai-se a medula espinal cervical. NOTA: aS Extrair quer a partir de uma das metades do cérebro após a secção sagital ou a partir de cérebros de rato dissecadas, disponível após as experiências enumeradas na Tabela 1. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Experiment </td><td> Ratos Inóculo (equivalente cérebro) </td><td> Período de sobrevivência (Dpi) </td><td> Median / sobrevivência máxima (dias de idade) </td><td> aS detecção d por ELISA / WB / IHC </td></tr><tr><td> 1 </td><td> Ratos não inoculadas </td><td> 441 ± 166 </td><td> 419/736 </td><td> 8/8 </td></tr><tr><td> 2 </td><td> Camundongos inoculados (0,2 mg) </td><td> 150 ± 52 </td><td> 140/241 </td><td> 9/9 </td></tr></tbody></table> Tabela 1. Lista de experiências realizadas em ratos M83. As inoculações foram realizadas com 6 semanas de experiência 2 na área do estriato-cortical com 20 ul de um homogenato de cérebro de um rato doente (1% peso / vol em glucose a 5%), depois anestesia de seis semanas de idade ratos M83 homozigotos 3% inalação de isoflurano. dpi: dias após inoculção. 2. Extracção de aS Cérebro Metades <ol><li> Sagital cortar o cérebro para obter duas metades. Pesar cada metade em um tubo ribolysis contendo bolas de moagem. </li><li> Prepare alta Sal (SH) de tampão contendo 50 mM de Tris-HCl, pH 7,5, NaCl 750 mM, EDTA 5 mM, DTT 1 mM, 1% de fosfatase e inibidores da protease cocktails. Adicionar tampão alto de sal para as metades do cérebro para se obter 20% (peso / volume) homogeneizados. </li><li> Preparar as amostras de as metades cerebrais usando um homogeneizador mecânico em 6,0 m / s durante 23 seg duas vezes. Após os primeiros 23 segundos de homogeneização, colocar os tubos que contêm os homogeneizados em gelo durante 2 min antes do segundo ciclo de 23 seg. </li><li> Centrifugar as amostras a 1000 xg durante 5 minutos para eliminar fragmentos de cérebro não moídos. Recuperar os sobrenadantes, dividem-se em 200 mL alíquotas e mantê-los à temperatura de -80 ° C para análise posterior ELISA. </li></ol> 3. aS Extração de Dissecadas regiões do cérebro <ol><li> Dissecar umcérebro inteiro em um 35 milímetros de plástico placa de Petri no gelo com um ampliador de baixa potência (ampliação 8X), utilizando dois forcipes cujas extremidades são mantidos juntos, exceto quando dissecando o hipocampo. Não exceda 10 min para preservar a integridade do cérebro. Coloque o cérebro lado direito para cima e recuperar os seguintes regiões do cérebro, nesta ordem: <ol><li> Separe um dos dois bulbos olfatórios usando uma pinça colocados logo atrás do bulbo. Deslocá-lo do cérebro por um movimento para baixo. Repita esta operação para a segunda lâmpada. </li><li> Suavemente cunha a pinça entre as duas córtices e movê-lo para a frente para facilitar a dissociação das duas córtex. Mantendo o cérebro no lugar com um fórceps, utilizar outro para separar o córtex do hipocampo. </li><li> Posicionar a pinça 2 mm abaixo do córtex. Manter uma pressão suave sobre a pinça até que o topo do hipocampo é visível. Retire a primeira parte do córtex, e repita com a segunda parte. Utilizar as pinças para separar os dois córticescomeçando no hipocampo e que se deslocam para a frente do cérebro. </li><li> Posicione a pinça abertos ao redor um dos hipocampos. Fechar a pinça, na parte inferior do hipocampo, em seguida, removê-lo suavemente, recuperando-se, tanto quanto possível. Repetir o procedimento para a segunda hipocampo. </li><li> Posicionar as pinças abertas abaixo de um dos corpos estriados e separá-lo suavemente a partir do cérebro. Utilizar as pinças para remover qualquer remanescente córtex do striatum. Repita este procedimento para o segundo striatum. </li><li> Utilizar as pinças para deprimir suavemente por 2 mm, o contorno do cerebelo para facilitar a separação do cerebelo a partir do cérebro. Coloque a pinça logo atrás do cerebelo e removê-lo, movendo a pinça para a frente. </li><li> Utilizar a parte larga da pinça para elevar o mesencéfalo, a fim de ver claramente onde se junta o tronco cerebral. Adicione uma incisão vertical na junção, em seguida, remover o tronco cerebral. </li><li> Posicione a pinça atrás do mesencéfalo, que is composta por quatro estruturas arredondadas. Inciso verticalmente até o mesencéfalo foi completamente separado do restante do cérebro. </li></ol></li><li> Prepare homogenatos de variável% (peso / volume) em tampão SH, dependendo da quantidade de tecidos disponíveis, isto é, 5% homogeneizado para um peso entre 10 e 30 mg, 10% para um peso entre 30 e 80 mg, e 20% para um peso acima de 80 mg. <ol><li> Adicionar um volume adequado de tampão de HS para as regiões cerebrais dissecados para se obter o esperado% de homogenato. </li><li> Vortex e verificar que os tecidos são totalmente imersos no buffer HS. </li><li> Homogeneizar amostras preparadas a partir das regiões cerebrais dissecados ou a medula espinhal cervical com um moinho de tecido composto por um tubo de vidro de borosilicato e dois pilões, A e B. </li><li> Pour cada região do cérebro para ser esmagado directamente para dentro do tubo. Inserir um pilão para dentro do tubo e retirá-la. Repetir este movimento cerca de dez vezes para dissociar o tecido. Em seguida, use um pilão B para continue moer o tecido com mais 20 movimentos. Transferir os homogeneizados num tubo de 1,5 ml com uma pipeta de transferência de 1 ml. </li></ol></li><li> Centrifugar as amostras a 1000 xg durante 5 min a 4 ° C para eliminar quaisquer fragmentos cerebrais não moídos. Recuperar os sobrenadantes, dividi-los em 200 mL alíquotas e mantê-los à temperatura de -80 ° C para análise posterior ELISA. </li></ol> 4. Detecção de aS por ELISA <ol><li> Diluir os anticorpos de revestimento para 0,01 ng / ml. Use um anti-soro policlonal de coelho ou aS monoclonal clone de anticorpo em 42 mM de Na 2 CO / 3 tampão NaHCO 3 50 (pH 9,6). </li><li> Revestir a microplacas de 96 poços com 100 ul por poço de solução de revestimento, esta e deixar a 4 ° CO / N. Utilizar o anticorpo policlonal de coelho anti-aS na solução de revestimento para ELISA, utilizando anticorpos de detecção syn514, clone 42, LB509, AS11, 4d6 ou 8A5. Use o anti-aS anticorpo monoclonal clone 42 como uma solução de revestimentoem combinação com o anti-pSer129 aS anticorpo de detecção. NOTA: Se necessário, as placas podem ser mantidas a 4 ° C durante uma semana antes do ELISA é realizado. </li><li> Usar um lavador de placas para lavar as placas cinco vezes com 300 ul de solução salina tamponada com fosfato com 0,05% de Tween 20 (PBST) por poço. A partir deste passo em diante, a incubação é a TA. </li><li> Adicionar 200 ul de tampão PBS T20 por cavidade de bloqueio. Agita-se durante 1 hora a 150 rpm. Lavam-se as placas cinco vezes com PBST. </li><li> Diluir os homogenatos cerebrais (diluição 1: 100 dos homogenatos de 20%, 01:50 10% dos homogenatos e 01:25 dos homogenatos de 5% em PBST BSA a 1%), e adicionar 100 ul a cada poço. Em seguida incubar durante 2 horas enquanto se agitava a 150 rpm. Lavam-se as placas cinco vezes com PBST. </li><li> Adicione os diferentes anticorpos de detecção aS em PBST com BSA 1% nas diluições mencionados na Lista de Materiais. Incubar durante 1 hora a 150 rpm. Lavam-se as placas cinco vezes com PBST. </li><li> Adicionar tanto anti-Mouse ou anti-coelho conjugados HRP de IgG diluído 1: 8000 em PBST suplementado com BSA a 1% durante 1 hora, agitando a 150 rpm. Lavam-se as placas cinco vezes com PBST. </li><li> Adicionar 100 ul de 3,3 &#39;, 5,5&#39; solução-tetrametilbenzidina (TMB) a cada poço e incubar durante 15 min no escuro, com agitação a 150 rpm. </li><li> Parar a reacção por adição de 100 ul de HCl 1 N por poço, em seguida, medir a absorvência a 450 nm com um leitor de microplacas. </li><li> Para análise de dados, subtrair o valor de DO obtidos com um bem com todos os reagentes, excepto as amostras do cérebro do rato (bem em branco) a partir dos valores de DO medido para cada uma das amostras analisadas. </li></ol> 5. Mapeamento de Epitopos <ol><li> Realizar mapeamento de epítopos de acordo com o método descrito por Osman 16. Resumidamente, os péptidos da sequência no local de α-sinucleína humano contendo 12 aminoácidos de nitrocelulose com 10 aminoácidos que se sobrepõem. </li><li> Bloco com 50 mM de Tris / NaCI 150 mM, pH 10 contendo 0,05% de Tween 20 e 5% de leite em pó. Incubar o anticorpo em solução a uma concentração de 2 ug de anticorpo por ml de bloqueio a 2-10 ° CO / N. </li><li> Lava-se a membrana três vezes, utilizando Tris a 50 mM / NaCl 150 mM, pH 10, que contém 0,05% de Tween 20. Incubar, com o anticorpo de cabra anti-IgG de ratinho conjugado com HRP. Lava-se a membrana mais cinco vezes, utilizando o mesmo tampão, em seguida, a mancha usando um kit de coloração de Western blot de TMB. </li></ol> 6. Análise Estatística <ol><li> Use o pacote de software e R nlme para executar as regressões de efeitos mistos para modelar OD. Para cada comparação, realizar um modelo de regressão de efeitos mistos. Use um efeito fixo para distinguir sintomático de grupos assintomáticos. </li><li> Usar um efeito aleatório para reflectir a variabilidade de repetições para um dado rato. Verifique homoscedasticidade examinando os resíduos e, se necessário, use as funções de variância para modelar a estrutura de variância de erros dentro do grupo de acordo com Pinheiro e Bates 17. Defina 0,05 como limite de significância de P. </li></ol>

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The authors have no competing interests to disclose.

Foi demonstrada a utilização de um ELISA para detectar especificamente aS D directamente a partir de homogenatos do cérebro do rato no decorrer da doença no modelo de ratinho transgénico para M83. Na verdade, este ELISA poderia facilmente distinguir os ratos doentes M83 M83 de camundongos saudáveis ​​utilizando homogeneizados única inteiras cerebrais em tampão de alta sal. Os passos mais críticos para os resultados bem sucedidos utilizando este ELISA são: dissecting ...

Most current methods for detecting disease-associated &#945;-synuclein (&#945;SD) in experimental models of Parkinson's disease (PD), such as immunohistochemistry or Western blot, are time-consuming and not quantitative. This neurodegenerative disease is characterized by alpha-synuclein aggregation mainly in the form of inclusions containing an aggregated form of the normally soluble presynaptic protein &#945;S1,2 (Lewy bodies and Lewy neurites). Normally only marginally phosphorylated, &#945;S is hyperphosphorylated at its serine 129 residue in these inclusions3 and can be monitored by antibodies specifically directed against Ser129 phosphorylated &#945;S, thus providing a reliable marker of the pathology. 
Recent research suggests that a &#8220;prion-like&#8221; mechanism could be involved in the propagation of &#945;S aggregation within the nervous system of an affected patient4,5. These studies reported the acceleration of a synucleinopathy by inoculating brain extracts containing &#945;SD into a transgenic mouse model (M83) expressing an A53T mutated human &#945;S protein associated with a severe motor impairment occurring as the mice age6. In the same manner, intra-cerebral inoculation of aggregated recombinant &#945;S in the same M83 mouse model confirmed the acceleration of aggregation5. The induction of deposits of phosphorylated &#945;S has also been reported after inoculation of C57Bl/6 wild-type mice with either fibrillar recombinant &#945;S or brain extracts from human DLB patients7,8. Sacino et al.9 recently pointed out that after injection of fibrillar human &#945;S, a widespread and progressive cerebral &#945;S inclusion formation could be induced in M83 mice, but not in E46K transgenic mice or non-transgenic mice in which induced &#945;S inclusions were transient, and mainly restricted to the site of injection. Recent studies on monkeys confirmed propagation of &#945;S aggregates after inoculation of PD-derived extracts in species closer to humans10.
The link between &#945;S alterations and Parkinson&#8217;s disease suggest that &#945;SD is a potential biomarker for Parkinson&#8217;s disease11. A recent study showed the detection of oligomeric soluble aggregates of &#945;-synuclein in human cerebro-spinal fluid (CSF) and plasma as a potential biomarker for Parkinson&#8217;s disease based on a conventional sandwich system ELISA using the same antibody to capture and detect &#945;S12. Based on the same method, multimeric proteins were recognized in biological samples, including the brain, because there are multiple copies of epitopes present in the assembled forms13. Very recently, pathological &#945;S in the CSF of patients with a proven Lewy body pathology was detected using both an ELISA kit with a highly specific antibody against &#945;SD (5G4) and an immunoprecipitation assay14. These methods could differentiate patients with PD/DLB from other types of dementia.
The &#8220;prion-like&#8221; propagation of &#945;S aggregation was further studied in transgenic mouse model M83 using an ELISA approach that was designed to specifically identify &#945;SD15. In this study, we report the detailed ELISA protocol used to quantitatively detect &#945;SD in sick mice (whether or not inoculated with &#945;SD from sick M83 mice) and more especially in the brain regions specifically targeted by the pathological process in this M83 transgenic mouse model4.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="2" />
	</colgroup>
	<tbody>
		<tr>
			<td>LB509&#160;</td>
			<td></td>
			<td>Abcam</td>
			<td>&#160;ab27766</td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>AS11</td>
			<td></td>
			<td>Produced at Anses</td>
			<td></td>
			<td>Detection antibody 1/1000</td>
		</tr>
		<tr>
			<td>4D6&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>ab1903</td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>PSer129&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>&#160;ab59264</td>
			<td>Detection antibody 1/3000</td>
		</tr>
		<tr>
			<td>PSer129&#160;EP1536Y</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>ab51253</td>
			<td>Detection antibody 1/1000</td>
		</tr>
		<tr>
			<td>syn514&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>&#160;ab24717</td>
			<td>Detection antibody 1/500</td>
		</tr>
		<tr>
			<td>clone 42</td>
			<td></td>
			<td>BD Biosciences&#160;</td>
			<td>610787</td>
			<td>Coating and detection antibody (1/2000)</td>
		</tr>
		<tr>
			<td>8A5&#160;</td>
			<td></td>
			<td>Provided by Dr. Anderson</td>
			<td></td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>polyclonal anti-&#945;syn antibody</td>
			<td></td>
			<td>Millipore</td>
			<td>&#160;AB5038P</td>
			<td>Coating&#160; antibody</td>
		</tr>
		<tr>
			<td>Anti-mouse&#160; IgG HRP conjugate</td>
			<td></td>
			<td>Southern Biotech</td>
			<td>1010-05</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Anti-rabbit IgG HRP conjugate</td>
			<td></td>
			<td>Southern Biotech</td>
			<td>4010-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse&#160; IgG HRP conjugate</td>
			<td></td>
			<td>Dianova</td>
			<td>115-035-164</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="4">HS buffer</td>
			<td>Tris-HCl 50 mM&#160;</td>
			<td>Euromedex</td>
			<td>26-128-3094-B</td>
			<td rowspan="4">Adjust at pH 7.5 and keep at 4&#176;C</td>
		</tr>
		<tr>
			<td>NaCl 750 mM&#160;</td>
			<td>Euromedex</td>
			<td>1112-A</td>
		</tr>
		<tr>
			<td>EDTA 5 mM&#160;</td>
			<td>Euromedex</td>
			<td>EU0007-B</td>
		</tr>
		<tr>
			<td>DTT 1 mM&#160;</td>
			<td>Sigma</td>
			<td>43815</td>
		</tr>
		<tr>
			<td rowspan="4">PBS</td>
			<td>Na2HPO4 1 mM</td>
			<td>Euromedex</td>
			<td>1309</td>
			<td rowspan="4">Adjust at pH 7.5</td>
		</tr>
		<tr>
			<td>KH2PO4 1,5 mM</td>
			<td>Euromedex</td>
			<td>2018</td>
		</tr>
		<tr>
			<td>NaCl&#160; 137 mM</td>
			<td>Euromedex</td>
			<td>1112-A</td>
		</tr>
		<tr>
			<td>KCl 2,7 mM</td>
			<td>Euromedex</td>
			<td>P017</td>
		</tr>
		<tr>
			<td>Tween 20&#160;</td>
			<td></td>
			<td>Euromedex</td>
			<td>2001-C</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT 1 mM</td>
			<td></td>
			<td>Sigma</td>
			<td>43815</td>
			<td>stock solution 100 mM, toxic</td>
		</tr>
		<tr>
			<td>1% phosphatase cocktail</td>
			<td></td>
			<td>Pierce</td>
			<td>78428</td>
			<td></td>
		</tr>
		<tr>
			<td>1% protease inhibitor cocktail</td>
			<td></td>
			<td>Roche</td>
			<td>04&#160;693&#160;132&#160;001</td>
			<td>50 X concentrated</td>
		</tr>
		<tr>
			<td>Microplate MaxiSorpTM</td>
			<td></td>
			<td>Thermo Scientific&#160;</td>
			<td>442404</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Tampon carbonate 50 mM pH 9.6&#160;</td>
			<td>&#160;Na2CO3, 10H2O&#160;</td>
			<td>Sigma</td>
			<td>71360</td>
			<td>2.86 g/L</td>
		</tr>
		<tr>
			<td>&#160;NaHCO3&#160;</td>
			<td>Merk</td>
			<td>6329</td>
			<td>3.36 g/L, pH9.6</td>
		</tr>
		<tr>
			<td>Superblock T20 PBS blocking buffer&#160;</td>
			<td></td>
			<td>Pierce</td>
			<td>E6423H</td>
			<td>10 X concentrated</td>
		</tr>
		<tr>
			<td>TMB&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>T0440</td>
			<td>Used for ELISA</td>
		</tr>
		<tr>
			<td>TMB&#160;</td>
			<td></td>
			<td>Analytik Jena AG</td>
			<td>847-0104200302</td>
			<td>Used for epitope mapping</td>
		</tr>
		<tr>
			<td>HCl 1N&#160;</td>
			<td></td>
			<td>Chimie plus</td>
			<td>40030</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribolyser</td>
			<td></td>
			<td>Thermo</td>
			<td>Fast prep FP120</td>
			<td>keep on ice at this step</td>
		</tr>
		<tr>
			<td>Grinding tubes</td>
			<td></td>
			<td>Biorad</td>
			<td>355-1197</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate washer</td>
			<td></td>
			<td>Tecan</td>
			<td>Columbus Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td></td>
			<td>Biorad</td>
			<td>Model 680</td>
			<td></td>
		</tr>
		<tr>
			<td>Low power magnifier&#160;</td>
			<td></td>
			<td>VWR</td>
			<td>630-1062</td>
			<td>X8 magnification</td>
		</tr>
		<tr>
			<td>Forceps Dumont#7</td>
			<td></td>
			<td>WPI</td>
			<td>14097</td>
			<td>For dissection steps</td>
		</tr>
		<tr>
			<td>Transfer pipette 1ml Samso</td>
			<td></td>
			<td>Samso</td>
			<td>043231</td>
			<td></td>
		</tr>
		<tr>
			<td>1,5 ml tubes</td>
			<td></td>
			<td>Dutscher</td>
			<td>033290</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of disease-associated &#945;-synuclein by enhanced elisa in the brain of transgenic mice overexpressing human a53t mutated &#945;-synuclein

Além dos métodos estabelecidos como Western blot, são necessários novos métodos para quantificar rapidamente e facilmente associada a doença α-sinucleína (D aS) em modelos experimentais de synucleopathies. Uma linha de ratinhos transgénicos (M83) que sobre-expressam os aS A53T humanos e o desenvolvimento de um fenótipo clínico espontaneamente dramática entre oito e 22 meses de idade, caracterizada por sintomas que incluem a perda de peso, prostração, e deficiência motora grave, foi utilizada neste estudo. Para análises moleculares de aS D (aS associada a doença) nestes ratos, um ensaio ELISA foi desenvolvido para quantificar especificamente aS D em ratinhos doentes. Análise do sistema nervoso central neste modelo de ratinho revelou a presença de D aS principalmente nas regiões do cérebro caudal e da medula espinhal. Não houve diferenças na distribuição aS D entre diferentes condições experimentais que conduzem à doença clínica, isto é, em uninoculATED e normalmente envelhecimento ratinhos transgénicos e em ratinhos inoculados com extractos de cérebro de ratinhos doentes. A detecção específica de aS D imunorreactividade usando um anticorpo contra a Ser129 fosforilada aS por ELISA essencialmente correlacionados com os valores obtidos por transferência de Western e imuno-histoquímica. Inesperadamente, resultados semelhantes foram observados com vários outros anticorpos contra a parte C-terminal de aS. A propagação de aS D, sugerindo o envolvimento de um mecanismo de &quot;prião-like&quot;, pode, assim, ser facilmente controlado e quantificado neste modelo de ratinho utilizando uma abordagem de ELISA.

Neste estudo, as ELISAs utilizados especificamente identificados aS associados à doença (D aS) em homogenatos de cérebro preparados num tampão de alto de sal a partir de ratinhos doentes M83. Usando um anticorpo que reconhece especificamente pSer129 aS (p = 0,0074), o ELISA facilmente distingue antigos, camundongos doentes (&gt; 8 meses de idade) de jovens (2-5 meses de idade), os camundongos saudáveis ​​M83 (Figura 1). Vários outros anticorpos mostraram sinais igualmente elevados (...

Watch this Scientific Journal Video about Detection of Disease-associated Î±-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated Î±-synuclein at JoVE.com

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

Detecção de doença associada α-sinucleína por ELISA reforçada no cérebro de ratinhos transgénicos que sobre-expressam A53T Humano Mutante α-sinucleína

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

detection-disease-associated-synuclein-enhanced-elisa-brain

Research

JoVE Journal

Medicine

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

10.6K visualizações.  ANSES - French Agency for Food, Environmental and Occupational Health & Safety. An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated α-synuclein (αSD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated αS form or the C-terminal part of the protein.

Vídeo: Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

A utilização de recursos primários fibroblastos humanos de Monitoramento Fenótipos mitocondriais no Campo da Doença de Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Medicina

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

Indução de carcinoma da bexiga invasivo de células transicionais em imunológico intacto Humanos MUC1 Ratos transgênicos: um modelo de desenvolvimento Imunoterapia

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

Imagem-guiado Entrega reforçada-Convecção em Modelos gel de agarose do Cérebro

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Aguda Cérebro Trauma em camundongos seguiu por Longitudinal dois fótons de imagem

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Minimal procedimento cirúrgico invasivo de induzir Infarto do Miocárdio em Ratos

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Indução de Aterosclerose Acelerada em Camundongos: O Modelo de "Lesão por Fio"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo de deteção de Mitophagy em células humanas, C. Eleganse os ratos

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Estudo do metabolismo in vivo da glicose em camundongos alimentados com dieta de alto teor em gordura usando o teste oral de tolerância à glicose (OGTT) e o teste de tolerância à insulina (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.