Los autores desean agradecer a Damien Gaillard para inoculaciones y el seguimiento de los experimentos con animales. Este trabajo fue apoyado por la ANSES (Agencia Francesa para la Alimentación, Medio Ambiente y Salud Ocupacional y Seguridad) y por una beca de la Fundación France Parkinson.

Todos los procedimientos y protocolos con animales estaban en conformidad con la Directiva 86/609 / CEE del Consejo y ratificado por venir, el Comité Nacional Francés para la consideración de la ética en la experimentación animal (protocolo de 11 a 0043) de la CE. Los animales fueron alojados y atendidos en ANSES aprobada de instalaciones experimentales en Lyon (aprobación B 69387 0801). 1. Preparación de ratones <ol><li> La eutanasia a los ratones mediante una inyección intraperitoneal de dosis letal de pentobarbital de sodio. </li><li> Recuperar todo el cerebro del cráneo del ratón y colocarlo en un 35 mm placa Petri de plástico en el hielo hasta la extracción. </li><li> Se extrae la médula espinal cervical. NOTA: aS Extraer ya sea desde una de las mitades del cerebro después de la sección sagital o de cerebros de ratón disecados, disponible después de los experimentos enumerados en la Tabla 1. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Experiment </td><td> Ratones Inóculo (cerebro equivalente) </td><td> Período de supervivencia (Dpi) </td><td> La mediana / supervivencia máxima (días de edad) </td><td> aS d detección por ELISA / BM / IHC </td></tr><tr><td> 1 </td><td> Los ratones inoculados </td><td> 441 ± 166 </td><td> 419/736 </td><td> 8/8 </td></tr><tr><td> 2 </td><td> Ratones inoculados (0,2 mg) </td><td> 150 ± 52 </td><td> 140/241 </td><td> 9/9 </td></tr></tbody></table> Tabla 1. Lista de experimentos realizados en ratones M83. Las inoculaciones se realizaron a las 6 semanas para el experimento 2 en la zona cortical-estriado con 20 l de un homogeneizado de cerebro de un ratón enfermo (1% peso / vol en glucosa al 5%), después de anestesia de 6 semanas de edad ratones homocigotos M83 en un 3% la inhalación de isoflurano. dpi: días después inoculación. 2. aS Extracción de Brain Mitades <ol><li> Sagitalmente cortó el cerebro para obtener dos mitades. Pesar cada mitad en un tubo que contiene ribolysis bolas de molienda. </li><li> Preparar elevado de sal (HS) tampón que contiene 50 mM Tris-HCl, pH 7,5, NaCl 750 mM, EDTA 5 mM, DTT 1 mM, 1% de fosfatasa inhibidores de la proteasa y cócteles. Añadir tampón elevado de sal a las mitades del cerebro para obtener 20% (peso / volumen) homogeneizados. </li><li> Preparar las muestras de las mitades del cerebro utilizando un homogeneizador mecánico a 6,0 m / s para 23 seg dos veces. Después de la primera homogeneización 23 seg, colocar los tubos que contienen los homogeneizados en hielo durante 2 minutos antes de que el segundo ciclo de 23 seg. </li><li> Centrifugar las muestras a 1000 xg durante 5 min para eliminar fragmentos cerebrales no molidos. Recuperar los sobrenadantes, se dividen en 200 ml de alícuotas y mantenerlos a -80 ° C para el análisis ELISA posterior. </li></ol> 3. aS Extracción de diseccionado cerebrales Regiones <ol><li> Diseccionar untodo el cerebro en un 35 mm placa Petri de plástico en el hielo con una lupa de baja potencia (ampliación 8X) utilizando dos forcipes cuyos extremos se mantienen juntos, excepto cuando la disección del hipocampo. No exceda de 10 minutos para preservar la integridad del cerebro. Coloque el cerebro boca arriba y recuperar las regiones cerebrales siguientes en este orden: <ol><li> Separar una de las dos bulbos olfatorios utilizando fórceps colocados justo detrás de la bombilla. Separe desde el cerebro por un movimiento hacia abajo. Repita esta operación para el segundo foco. </li><li> Cuña suavemente las pinzas entre las dos cortezas y moverlo hacia delante para facilitar la disociación de las dos cortezas. Mantener el cerebro en su lugar con uno fórceps, utilizar otra para separar la corteza del hipocampo. </li><li> Coloque la pinza 2 mm por debajo de la corteza. Mantener una presión suave sobre las pinzas hasta la parte superior del hipocampo es visible. Despegar la primera parte de la corteza, y repetir con la segunda parte. Utilice las pinzas para separar las dos cortezascomenzando en el hipocampo y moviéndose hacia la parte delantera del cerebro. </li><li> Coloque las pinzas abiertas alrededor de uno de los hipocampos. Cierre la pinza en la parte inferior del hipocampo luego retire suavemente, recuperando tanto como sea posible. Repita el procedimiento para el segundo hipocampo. </li><li> Coloque las pinzas abiertas debajo de uno de los cuerpos estriados y suavemente separarlo del cerebro. Utilice las pinzas para quitar cualquier corteza restante del cuerpo estriado. Repita este procedimiento para el segundo cuerpo estriado. </li><li> Usa los fórceps para deprimir suavemente por 2 mm el contorno del cerebelo para facilitar la separación del cerebelo del cerebro. Coloque la pinza justo detrás del cerebelo y quitarlo moviendo la pinza hacia adelante. </li><li> Utilice la parte ancha de las pinzas para levantar el mesencéfalo con el fin de ver claramente donde se une con el tallo cerebral. Hacer una incisión vertical en la unión luego retire el tallo cerebral. </li><li> Coloque la pinza detrás del mesencéfalo, que is compuesta de cuatro estructuras redondeadas. Una incisión vertical hasta el mesencéfalo ha sido completamente separado del cerebro restante. </li></ol></li><li> Preparar homogeneizados de% variable (peso / volumen) en tampón HS, dependiendo de la cantidad de tejidos disponibles, es decir, 5% homogeneizado para un peso entre 10 y 30 mg, 10% para un peso entre 30 y 80 mg, y 20% para un peso por encima de 80 mg. <ol><li> Añadir un volumen adecuado de tampón HS a las regiones cerebrales disecadas para obtener el% esperado de homogeneizado. </li><li> Vortex y comprobar que los tejidos están completamente inmersos en el búfer HS. </li><li> Homogeneizar las muestras preparadas a partir de las regiones cerebrales disecadas o la médula espinal cervical con un triturador de tejidos compuesta de un tubo de vidrio de borosilicato y dos manos de mortero, A y B. </li><li> Verter cada región del cerebro de ser aplastado directamente en el tubo. Inserte mano de mortero A en el tubo y retraerlo. Repita este movimiento alrededor de diez veces para disociar el tejido. A continuación, utilice la maja B para continuac moliendo el tejido con 20 movimientos. La transferencia de los homogeneizados en un tubo de 1,5 ml con una pipeta de transferencia 1 ml. </li></ol></li><li> Centrifugar las muestras a 1000 xg durante 5 min a 4 ° C para eliminar cualquier fragmento cerebrales no molidos. Recuperar los sobrenadantes, dividirlos en 200 ml de alícuotas y mantenerlos a -80 ° C para el análisis ELISA posterior. </li></ol> 4. Detección de aS por ELISA <ol><li> Diluir los anticuerpos de recubrimiento de 0,01 ng / ml. Utilice-aS policlonal anti conejo o monoclonal clon 42 de anticuerpos en 50 mM Na 2 CO 3 / NaHCO3 (pH 9,6). </li><li> Escudo del microplacas de 96 pocillos con 100 l por pocillo de esta solución de revestimiento, y dejar a 4 ° CO / N. Utilice el anticuerpo policlonal de conejo anti-aS en la solución de recubrimiento para ELISA utilizando anticuerpos de detección syn514, clonar 42, LB509, AS11, 4D6 o 8A5. Utilice el clon de anticuerpo monoclonal anti-aS 42 como una solución de revestimientoen combinación con el anticuerpo de detección anti-pSer129 aS. NOTA: Si es necesario, las placas pueden mantenerse a 4 ° C durante una semana antes de la prueba ELISA se lleva a cabo. </li><li> Utilice un lavador de placas para lavar las placas cinco veces con 300 l de solución salina tamponada con fosfato con 0,05% de Tween 20 (PBST) por pocillo. A partir de este paso hacia adelante, la incubación es a TA. </li><li> Añadir 200 l de PBS T20 tampón de bloqueo por pocillo. Agitar durante 1 hora a 150 rpm. Lavar las placas cinco veces con PBST. </li><li> Diluir los homogeneizados de cerebro (dilución 1: 100 de los homogeneizados de 20%, 01:50 de los homogeneizados de 10% y 1:25 de los homogeneizados de 5% en PBST BSA 1%), y añadir 100 l a cada pocillo. A continuación se incuba durante 2 horas con agitación a 150 rpm. Lavar las placas cinco veces con PBST. </li><li> Añadir los diferentes anticuerpos de detección aS en PBST con BSA 1% en las diluciones mencionadas en la Lista de materiales. Incubar durante 1 hora a 150 rpm. Lavar las placas cinco veces con PBST. </li><li> Añadir o bien contra-Ratón o anti-conejo conjugados HRP IgG diluyeron 1: 8000 en PBST suplementado con BSA 1% durante 1 h con agitación a 150 rpm. Lavar las placas cinco veces con PBST. </li><li> Añadir 100 l de 3,3 &#39;, solución de 5,5&#39;-tetrametilbencidina (TMB) a cada pocillo e incubar durante 15 min en la oscuridad con agitación a 150 rpm. </li><li> Detener la reacción mediante la adición de 100 l de HCl 1 N por pocillo luego medir la absorbancia a 450 nm con el lector de microplacas. </li><li> Para el análisis de datos, restar el valor de DO obtenido en un pozo con todos los reactivos excepto las muestras de cerebro de ratón (así en blanco) de los valores de la DO medidos para cada una de las muestras analizadas. </li></ol> 5. Epitope Mapping <ol><li> Realizar el mapeo de epítopos de acuerdo con el método descrito por Osman 16. En pocas palabras, los péptidos del punto de la secuencia de α-sinucleína humana contiene 12 aminoácidos de nitrocelulosa con 10 aminoácidos que se solapan. </li><li> Bloque con 50 mM Tris / tampón NaCl 150 mM pH 10 que contiene 0,05% de Tween 20 y 5% de leche en polvo. Incubar el anticuerpo en solución de bloqueo a una concentración de 2 mg de anticuerpo por ml a 2-10 ° CO / N. </li><li> Se lava la membrana tres veces con 50 mM Tris / NaCl 150 mM pH del tampón 10 que contiene 0,05% de Tween 20. Incubar con el anticuerpo de cabra anti-IgG de ratón conjugado HRP. Lavar la membrana otros cinco veces con el mismo tampón a continuación mancha usando un kit de tinción blot TMB occidental. </li></ol> 6. Análisis estadístico <ol><li> Utilice el paquete de software de R y nlme para realizar regresiones de efectos mixtos para modelar OD. Para cada comparación, lleve a cabo un modelo de regresión efecto mixto. Use un efecto fijo para distinguir los síntomas de los grupos asintomáticos. </li><li> Utilice un efecto aleatorio para reflejar la variabilidad de repeticiones para un ratón dado. Compruebe homocedasticidad mediante el examen de los residuos y, si es necesario, utilice las funciones de varianza para modelar la estructura de la varianza de los errores dentro de los grupos de acuerdo con Pinheiro y Bates 17. Establecer 0,05 como el umbral de significación de P. </li></ol>

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The authors have no competing interests to disclose.

El uso de un ELISA se demostró para detectar específicamente aS D directamente a partir de homogeneizados de cerebro de ratón durante la enfermedad en el modelo de ratón transgénico M83. De hecho, este ELISA podría distinguir fácilmente los ratones M83 enfermos de ratones sanos M83 utilizando homogeneizados de cerebro solamente enteros en tampón elevado de sal. Los pasos más importantes para los resultados exitosos que utilizan este ELISA son: disección correctamente las...

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

Además de los métodos establecidos como Western blot, se necesitan nuevos métodos para cuantificar rápida y fácilmente asociado a la enfermedad α-sinucleína (aS D) en modelos experimentales de synucleopathies. Una línea de ratón transgénico (M83) sobre-expresión de los aS A53T humanos y desarrollar espontáneamente un fenotipo clínico dramática entre ocho y 22 meses de edad, que se caracterizan por síntomas que incluyen pérdida de peso, postración, y deterioro motor severa, se utilizó en este estudio. Para los análisis moleculares de aS D (aS asociado a la enfermedad) en estos ratones, un ELISA fue diseñado para cuantificar específicamente aS D en ratones enfermos. Análisis del sistema nervioso central en este modelo de ratón mostró la presencia de aS D principalmente en las regiones cerebrales caudal y la médula espinal. No hubo diferencias en la distribución aS D entre diferentes condiciones experimentales que conducen a la enfermedad clínica, es decir, en uninoculado y normalmente envejecimiento ratones transgénicos y en ratones inoculados con extractos de cerebro de ratones enfermos. La detección específica de aS D inmunorreactividad utilizando un anticuerpo contra la Ser129 fosforilada aS por ELISA esencialmente correlacionados con la obtenida por Western blot e inmunohistoquímica. Inesperadamente, se observaron resultados similares con varios otros anticuerpos contra la parte C-terminal de aS. La propagación de aS D, lo que sugiere la participación de un mecanismo de &quot;-prión como&quot;, de este modo se puede monitorizar y cuantificar en este modelo de ratón utilizando un enfoque ELISA fácilmente.

En este estudio, las pruebas ELISA utilizadas específicamente identificados aS enfermedades asociadas (aS D) en homogeneizados de cerebro preparados en un tampón de alta sal de ratones enfermos M83. El uso de un anticuerpo que reconoce específicamente pSer129 aS (p = 0,0074), el ELISA distingue fácilmente de edad, ratones enfermos (&gt; 8 meses de edad) de los jóvenes (2-5 meses de edad), los ratones sanos M83 (Figura 1). Varios otros anticuerpos mostraron igualmente altos señales (&gt;...

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.

La detección de la enfermedad asociada-α-sinucleína por ELISA mejorada en el cerebro de ratones transgénicos que sobreexpresan A53T humano mutado 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 vistas.  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.

Video: 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.

El uso de fibroblastos humanos primarios para la Supervisión de fenotipos mitocondriales en el campo de la enfermedad 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 ...

Investigación

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.

La inducción de la transición de vejiga invasivo Carcinoma en inmune intacto humanos MUC1 Ratones Transgénicos: Un Modelo para el Desarrollo Inmunoterapia

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.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

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.

Guiada por imagen potenciada por convección Entrega en Modelos en gel de agarosa del Cerebro

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.

Trauma cerebral aguda en ratones seguido de Longitudinal dos fotones de imágenes

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).

Mínimo procedimiento quirúrgico invasivo de inducir infarto de miocardio en ratones

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.

Inducción de la Aterosclerosis Acelerada en Ratones: El Modelo de "Lesiones por Alambre"

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 la detección de la mitofagia en células humanas, C. Elegansy los ratones

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.

Estudio In Vivo del metabolismo de glucosa en ratones alimentados con dieta alta en grasas mediante prueba de tolerancia a la glucosa Oral (OGTT) y prueba de tolerancia a 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.

Inducción de las placas ateroscleróticas a través de la activación de los receptores de mineralocorticoides en ratones deficientes en apolipoproteína E