Name: enrichment of detergent-insoluble protein aggregates from human postmortem brain
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Los autores agradecen a los Drs. Jim Lah y Allan Levey, Departamento de Neurología de Emory, para sugerencias y comentarios. Este trabajo fue financiado en parte por la Asociación de medicina acelerar concesión (U01AG046161-02), el Emory Alzheimer centro de investigación de la enfermedad (P50AG025688) y un Instituto Nacional del envejecimiento conceden (R01AG053960-01) a N.T.S. Esta investigación también fue apoyada en parte por el núcleo de la neuropatología de la donacion de Emory Neurociencia NINDS base instalaciones, P30NS055077.

declaración de ética: todos los tejidos del cerebro fueron obtenidos de la Emory de Alzheimer &#39; s enfermedad investigación centro (ADRC) Banco de cerebros. Tejidos humanos post mortem fueron adquiridos bajo adecuados protocolos de la Junta de revisión institucional (IRB).
 1. homogeneización y fraccionamiento <ol><li>selección de tejido Nota: congelados de tejido post mortem corteza frontal de control sano (Ctl) y patológico casos confirmados de AD fueron seleccionados de la Emory Banco de cerebros ADRC (n = 2). Evaluación neuropathological post mortem de la distribución de la placa amiloide fue realizada según el consorcio establecer un registro de Alzheimer &#39; s enfermedad semicuantitativo de puntuación criterios 24, aunque patología neurofibrilares fue evaluado de acuerdo con el sistema de estadiaje de Braak. 16 <ol><li>obtener congelados de tejido cerebral post mortem de control sano (Ctl) y patológico confirmó casos de AD. Utilizando contenedores de hielo seco, pinzas y una navaja, suprimir ~ porciones de 250 mg de materia gris de cada muestra de tejido en desechables previamente tarado pesan barcos, teniendo cuidado de evitar que el tejido descongelado. Registrar el peso de cada pieza a ser homogeneizado.</li>
		<li>Impuesto especial ~ 250 mg de materia gris usando pinzas y navajas por inspeccionar visualmente para localizar la interfaz entre el exterior materia gris y materia blanca interior. Evitar que la materia blanca y lo más importante, cualquier vasos sanguíneos o regiones sangrientas, meninges o aracnoides. Mantener el tejido congelado durante el proceso de corte trabajando rápidamente y con frecuencia colocando peso barco con tejido cerebral en contenedores de poliestireno con hielo seco</li>
		<li>Registrar el peso exacto de la materia gris de cada pieza congelada utilizando una balanza analítica. Calcular el volumen de tampón bajo en sal (LS) (ver tabla 1) necesarios para la homogeneización de dounce (5 mL/g o 20% w/v).</li>
		<li>Preparar 10 mL LS de tampón 1 x inhibidor de la proteasa y fosfatasa cóctel y frialdad de hielo.</li>
		<li>Eliminar el tejido pesado y pesaje del barco de hielo seco y dados en trozos de aproximadamente 2 x 2 mm como deshiela. Transfiéralo a un tubo de homogeneizador de 2 mL dounce previamente enfriado en hielo.</li>
		<li>Añadir 5 mL/g (20% w/v) de tampón de helado bajo en sal (LS) con 1 X inhibidor de la proteasa y fosfatasa cóctel.</li>
		<li>Homogeneizar el tejido de cerebro en hielo, mediante aproximadamente 10 movimientos de separación alta Maja A y 15 golpes de mortero de baja holgura B.</li>
		<li>Transferencia de homogeneizado de todos a un tubo de polipropileno de 2 mL con un pipeta Pasteur de vidrio. Etiquetar el tubo con &#8220; TH &#8221; (homogenado Total), el tejido del caso número y volumen de homogenado. alícuota 0,8 mL en un tubo de 1,5 mL etiquetado. Cualquier exceso homogeneizado puede ser semejantemente alícuotas y almacenados a -80 ° C.</li>
		<li>a cada 0,8 mL de alícuota, añada 100 μl de 5 M NaCl y sarkosyl 10% (p/v) a concentraciones de 0,5 y 1% w/v, respectivamente. Tubos de mezclar bien por inversión e incubar en hielo por 15 minutos los tubos etiqueta con &#34; TH-S &#34; (homogeneizado Total-Sarkosyl) para indicar que los homogenados en sarkosyl-buffer (tabla 1).</li>
		<li>Someter a ultrasonidos cada tubo de impulsos tres 5 s en amplitud de 30% en el hielo (intensidad máxima = 40%) utilizando una sonda de microtip.</li> <li>determinar las concentraciones de proteína de los homogenados del ácido bicinchoninic (BCA) ensayo 25 método. 
		Nota: La concentración promedio de proteína de TH S homogenados preparados en LS de 5 mL por gramo de tejido es 15-20 mg/mL. Los homogenados pueden ser fraccionada inmediatamente o almacenadas a-80 ° C hasta su uso.</li>
		<li>TH-S diluir homogenados a 10 mg/mL con buffer de sark helada (tabla 1) 1 x inhibidores de la proteasa y fosfatasa.</li>
		Proteína de <li>transferencia 5 mg (0,5 mL) de cada homogenado TH S en 500 μl policarbonato ultracentrífuga tubos y par-equilibrio con tampón de sark. Cargar tubos en un rotor previamente enfriada y ultracentrífuga a 180.000 x g durante 30 min en sobrenadantes de transferencia sarkosyl-soluble 4 ° C. (S1) para tubos de 1,5 mL y guardar a -80 ° C. 
		Nota: (Lavado opcional) Añadir 200 μL de tampón de sark en la ultracentrífuga tubos que contienen las fracciones insolubles en detergente (P1) y desalojar los pellets (P1) de la parte inferior de los tubos de la ultracentrífuga utilizando una pipeta de 200 μl.</li>
		Tubos de <li>brevemente pulso spin la invertida para 2-3 s (&#8804; 2.500 x g) en una microcentrífuga para transferir el pellets (P1) y buffer a los tubos de 1,5 mL a continuación. Resuspender los gránulos insolubles (P1) en el búfer de sark transfiriendo hacia arriba y hacia abajo para asegurarse de la pelotilla se interrumpe.</li>
		<li>El equilibrio de par y centrifugar a 180.000 x g durante un adicional 30 min a 4 ° C.</li>
		<li>Descarte el sobrenadante del lavado opcional (S2) e incubar los pellets sarkosyl insoluble (P2) en 50-75 μl de tampón de urea (cuadro 1) con 1 x PIC (inhibidor de proteasa y fosfatasa cóctel) durante 30 min a temperatura ambiente para solubilizar el pellets de. 
		Nota: Urea caliente la memoria intermedia a la temperatura ambiente antes de su uso para evitar la precipitación de la SDS. Para aplicaciones sensibles al detergente, omitir SDS desde el búfer de urea y considerar lavado el precipitado insoluble (P2) en tampón salino bajo antes de resuspender en tampón urea.</li>
		<li>Transferir las pelotillas resuspendidas (P2) a tubos de 0.5 mL y uso breve (1 s) microtip sonicación en 20% de amplitud (intensidad máxima = 40%) para solubilizar completamente las pelotillas.</li>
		<li>Determinar las concentraciones de proteína del sarkosyl-soluble (S1) y - insolubles fracciones (P2) con el BCA método de ensayo. Utilice estas fracciones inmediatamente o almacenar a-80 ° C hasta su uso.</li>
	</ol></li></ol> 2. Immunoblotting <ol><li>Western blotting Nota: borrar occidental fue realizado según los procedimientos previamente divulgados con leves modificaciones. 5 , 10 <ol><li>preparar 40 μg muestras de homogenados total (TH-S), soluble en sark (S1) y fracciones de sark-insoluble (P2) en el tampón de muestra Laemmli SDS-page de X 1 con pH de 5 mM TCEP 7. Incubar las muestras a 95 ° C por 5 min</li>
		<li>Carga de muestras en un prefabricado 10-pozo 4-12% Bis-Tris SDS-PAGE gel. Electrophorese 80 V para el primer centímetro (10 min) y 120 V para el resto (60 min) o hasta que el tinte de seguimiento alcance la parte inferior del gel.</li>
		<li>Utilizar una nueva hoja de afeitar y un cuchillo de gel para split-abrir el cartucho de gel prefabricado. Con cuidado corte el peines y parte inferior del gel con una cuchilla de afeitar nueva.</li>
		<li>Volver a suspender los pellets insolubles (P1) en 200 μL de tampón de sark con 1 x PIC mediante pipeteo arriba y abajo.</li>
		<li>Transferencia a una membrana de nitrocelulosa de 0.2 μm utilizando un método de transferencia en seco en una máquina de Blot (ver la tabla de materiales).</li>
		<li>Bloquear la membrana en solución amortiguadora de bloqueo (BB) sin Tween 20 durante 30 min, seguida con BB con 0,05% Tween 20 (BB/T) para 30 minutos. Después de bloquear, enjuagar la membrana en TBS/T (0,1% Tween 20) por 5 min eliminar el exceso de tampón bloqueo.</li>
		1:1,000 de <li>preparar diluciones de los anticuerpos primarios pT231, Tau-2 (pan tau), AT8 y EEA1 en BB/T (0,05% Tween 20).</li>
		<li>Incubar las membranas en solución de anticuerpo primario durante la noche a 4 ° C con agitación circular. Enjuagar la membrana en TBS/T 3 x 15 min</li>
		Anticuerpo secundario cercano-IR de <li>punta de prueba de la membrana con una dilución de 1: 20.000 de la marcada con fluorescencia, en BB/T durante 60 min a temperatura ambiente en oscuridad en coctelera. Enjuagar la membrana para 3 x 10 min en TBS/T y 2 x 5 minutos en TBS.</li>
		<li>Analizar la membrana usando un infrarrojo sistema de la longitud de onda de excitación apropiada (por ejemplo 700 nm (canal rojo) para tinte infrarrojo 680 cabra anti-mouse IgG (H+L) secundaria y 800 nm (canal verde) para tinte infrarrojo 790 burro anti-rabbit IgG (H+L de imagen secundaria)).</li>
		<li>Uso del software de proyección de imagen para cuantificar la intensidades de la señal y realizar mediciones de densitometría según el fabricante &#39; instrucciones, asegurando el ajuste de auto-fondo se establece en promedio el fondo entero.</li>
	</ol>
	</li>
</ol>

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En este documento presentamos y discutir un protocolo abreviado de detergente-fraccionamiento de solo paso que es aplicable a una amplia variedad de aplicaciones experimentales que van desde análisis de proteómica basada en espectrometría de masa al mal plegamiento de análisis funcional de la proteína y western blot. 5 , 6 , 7 , 10 esta metodología es más eficaz cuando se utiliza para el...

Enfermedades neurodegenerativas como la enfermedad de Alzheimer (EA), enfermedad de Parkinson (EP), enfermedad de Huntington (EH), esclerosis lateral amiotrófica (ELA) y las enfermedades de prión estrechamente relacionados son proteinopatías caracterizada por la acumulación gradual de agregados de proteína insoluble en detergente en el cerebro con el acompañamiento cognitivo disminución. 1 , 2 esta característica patológica común se cree que desempeñan un papel central en la etiología y la patofisiología de estas enfermedades neurodegenerativas. 2 estos agregados consisten en típicamente poliméricas fibras amiloides, que se componen de unidades de proteínas mal plegadas que Cruz β-estructura. 1 , 2 , 3 , 4 bioquímico, agregados amiloides son altamente resistentes a la desnaturalización térmica o química y solubilización,3 que presenta retos únicos para su purificación, análisis y estudian por medio de técnicas bioquímicas tradicionales. 2 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , la fracción de proteína insoluble en detergente ha sido el foco de mucha investigación en la fisiopatología de las enfermedades neurodegenerativas que implica la acumulación de proteínas mal plegadas. 6 , 12 , 13 , 14
Técnicas de fraccionamiento bioquímico a menudo han sido utilizadas para enriquecer la fracción insoluble en detergente de homogenados de cerebro post mortem. 6 , 12 , 13 , 14 uno de los métodos más comunes consiste en la extracción secuencial de homogenados de tejido con tampones y detergentes de aumentar rigor, seguido por ultracentrifugación para dividir las fracciones solubles e insolubles. Un protocolo comúnmente usado fraccionamiento secuencial6,14 implica la homogeneización de las muestras de tejido congelado en un tampón libre de detergente bajo en sal (LS) y los pellets insolubles resultantes entonces son extraídos secuencialmente con buffers que contengan alta sal, detergentes no iónicos, alta sacarosa, detergentes iónicos y finalmente chaotropes como urea. 6 , 14 un inconveniente obvio de tales protocolos de fraccionamiento secuencial es el substancial del tiempo y el compromiso de la mano de obra necesaria para completarlos. Homogeneización y ultracentrifugación, un protocolo típico fraccionamiento de cinco pasos puede tomar varias horas o incluso días. 4 , 6 , 7 , 10 , 15 , 16 , 17 , 18 además, tantos agregados de proteína patológica permanecen insolubles en casi todos las más duras condiciones19,20 la mayoría de las fracciones generadas son de valor limitado. Así, los pasos de fraccionamiento menos-rigurosos utilizando altas concentraciones de sal y detergentes no iónicos son en gran medida redundantes.
Estudios previos han demostrado que el detergente iónico N-lauril-sarcosina (sarkosyl) es un excelente candidato para un protocolo simplificado solo paso detergente fraccionamiento. 5 , 6 , 12 , 13 , 14 , 21 , 22 , 23 como un desnaturalización detergente sarkosyl es lo suficientemente estricta como para solubilizar la gran mayoría de proteínas de forma nativa plegadas en cerebro sin solubilización agregados de proteínas mal plegadas compuestos de beta-amiloide (Aβ),6,11 tau fosforilada (pTau),6 proteína de unión a ADN TAR 43 (TDP-43),14 alfa-sinucleína,12,13 tembladera,23 o U1 ribonucleoproteínas nucleares pequeñas (snRNPs U1) como U1 - 70 K. 5 , 21 , 22 Como sarkosyl es menos estricto que el dodecil sulfato de sodio detergente aniónico ubicua (SDS), conserva formas oligoméricas menos robustas de agregados de proteínas mal plegadas que no pueden soportar el tratamiento de la SDS. 9
Anteriormente, hemos descrito un protocolo abreviado de detergente-fraccionamiento que logra resultados comparables a la metodología de fraccionamiento secuencial más laboriosa. 5 por omitir las medidas menos estrictas de fraccionamiento, hemos sido capaces de desarrollar un protocolo de fácil fraccionamiento solo paso para el enriquecimiento de los agregados de proteína insoluble en detergente de cerebro humano postmortem. 5 este protocolo detallado descrito en este documento es adecuado para una amplia gama de aplicaciones experimentales que van desde el Western Blot y análisis de proteómica basada en espectrometría de masa mal plegamiento de proteínas funcionales y siembra de agregación. 5 , 6 , 21

<table><tbody><tr><td>Protease and phosphatase inhibitor cocktail, EDTA-free (100X)</td><td>Thermo Fisher</td><td>78441</td><td>protease &amp;amp; phosphatase inhibitor cocktail</td></tr><tr><td>Sonic Dismembrator System (ultrasonicator)</td><td>Fisher Scientific</td><td>FB505110</td><td>microtip ultrasonicator</td></tr><tr><td>Optimax TLX Ultracentrifuge</td><td>Beckman Coulter</td><td>361545</td><td>refrigerated ultracentrifuge</td></tr><tr><td>TLA120.1 rotor</td><td>Beckman Coulter</td><td>362224</td><td>ultracentrifuge rotor</td></tr><tr><td>500 ul (8 x 34 mm) polycarbonate tubes, thickwall</td><td>Beckman Coulter</td><td>343776</td><td>ultracentrifuge tubes for TLA120.1 rotor</td></tr><tr><td>4X SDS sample buffer</td><td>Home-made</td><td>N/A</td><td>SDS-PAGE</td></tr><tr><td>TCEP solution, neutral pH</td><td>Thermo Fisher</td><td>77720</td><td>reducing agent</td></tr><tr><td>(TBS) blocking buffer</td><td>Thermo Fisher</td><td>37542</td><td>blocking buffer</td></tr><tr><td>(TBS) blocking buffer + 0.05% Tween 20</td><td>Thermo Fisher</td><td>37543</td><td>blocking buffer and antibody diluent</td></tr><tr><td>4-12% Bolt Bis-Tris Plus gels, 10-well</td><td>Thermo Fisher</td><td>NW04120BOX</td><td>precast SDS-PAGE gels</td></tr><tr><td>MES SDS Running Buffer (20X)</td><td>Thermo Fisher</td><td>B0002</td><td>SDS-PAGE running buffer</td></tr><tr><td>N-Lauroylsarcosine sodium salt (sarkosyl)</td><td>Sigma Aldrich</td><td>L5777-50G</td><td>detergent</td></tr><tr><td>Anti-Tau-2 (pan tau) antibody</td><td>Chemicon</td><td>MAB375</td><td>antibodies</td></tr><tr><td>Anti-phospho-threonine 231 Tau antibody</td><td>Millipore</td><td>MAB5450</td><td>antibodies</td></tr><tr><td>Anti-phospho-seroine 202 and threonine 205 Tau antibody (AT8)</td><td>Thermo Fisher</td><td>MN1020</td><td>antibodies</td></tr><tr><td>Anti-early endosome antigen 1 (EEA1) antibody</td><td>abcam</td><td>ab2900</td><td>antibodies</td></tr><tr><td>Alexa Fluor 680 goat anti-mouse IgG (H+L) secondary antibody</td><td>Thermo Fisher</td><td>A21058</td><td>antibodies</td></tr><tr><td>Alexa Fluor 790 donkey anti-rabbit IgG (H+L) secondary antibody</td><td>Thermo Fisher</td><td>A11374</td><td>antibodies</td></tr><tr><td>iBlot2 Dry Blotting System</td><td>Thermo Fisher</td><td>IB21001</td><td>Gel transfer</td></tr><tr><td>iBlot2 Transfer Stacks, Nitrocellulose, mini</td><td>Thermo Fisher</td><td>IB23002</td><td>Gel transfer</td></tr></tbody></table>

enrichment of detergent-insoluble protein aggregates from human postmortem brain

En este estudio, se describe un protocolo abreviado fraccionamiento solo paso para el enriquecimiento de los agregados de proteína insoluble en detergente de cerebro humano postmortem. El detergente iónico N-lauril-sarcosina (sarkosyl) efectivamente solubiliza proteínas forma nativa plegadas en el tejido cerebral que permite el enriquecimiento de los agregados de proteína insoluble en detergente de una amplia gama de proteinopatías neurodegenerativas, tales como Enfermedad de Alzheimer (EA), enfermedad de Parkinson y esclerosis lateral amiotrófica y enfermedades priónicas. Control humano y los tejidos del cerebro post mortem AD fueron homogeneizadas y sedimentados por ultracentrifugación en presencia sarkosyl enriquecer agregados de proteína insoluble en detergente incluyendo patológica tau fosforilada, el componente principal de ovillos enredos en AD. Western Blot demostró la solubilidad diferencial de agregados tau fosforilado y la proteína soluble en detergente, temprano Endosome antígeno 1 (EEA1) en control y el cerebro de AD. Análisis proteómico también revelaron enriquecimiento de β-amiloide (Aβ), tau, snRNP70 (U1 - 70 K) y apolipoproteína E (APOE) en las fracciones insolubles en sarkosyl de AD cerebral comparados con los de control, consistente con anteriores estrategias de fraccionamiento de tejido . Así, este Protocolo simple enriquecimiento es ideal para una amplia gama de aplicaciones experimentales que van desde borrar occidental y funcional de la proteína ensayos de agregación Co proteómica basada en espectrometría de masa.

El protocolo abreviado solo paso sarkosyl-fraccionamiento fue utilizado para enriquecer a agregados de proteína insoluble en detergente de control y cerebro post mortem AD (figura 1). Proteínas de fracciones TH S, S1, S2 y P2 fueron resueltas por SDS-PAGE, de 15 min en Coomassie azul fijador tampón seguido de tinción suave con tampón de tinción G-250 azul brillante de Coomassie. El paso de resuspensión es opcional puesto que había niveles indetectable...

Watch this Scientific Journal Video about Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain at JoVE.com

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

Se describe un protocolo abreviado de fraccionamiento para el enriquecimiento de los agregados de proteína insoluble en detergente de cerebro humano postmortem.

Enriquecimiento de los agregados de proteína insoluble en detergente de cerebro humano Postmortem

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

enrichment-detergent-insoluble-protein-aggregates-from-human

Research

JoVE Journal

Biochemistry

12.0K vistas.  Emory School of Medicine. Se describe un protocolo abreviado de fraccionamiento para el enriquecimiento de los agregados de proteína insoluble en detergente de cerebro humano postmortem.

Video: Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

Detección SDS-PAGE/Immunoblot de multímeros Aß en el tejido cortical humano homogeneizados utilizando antígeno de recuperación del epítopo

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

Investigación

Neurociencias

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

Aislamiento de oligómeros de PrP soluble e insoluble en el cerebro humano normal

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Medicina

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Aislamiento de los núcleos neuronales a partir de tejido cerebral humano post-mortem

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Biología

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

La proteína obtenida de cerebro de Consenso, protocolo de extracción para el Estudio de los Derechos Humanos y murino cerebro proteoma Uso de ambas 2D-DIGE y Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Extracción secuencial de soluble e insoluble alfa-sinucleína de cerebros parkinsonianos

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

Un Ensayo Basado En Espectroscopia De Fluorescencia Eficiente En El Tiempo Para Evaluar El Estado De Polimerización De Actina En Roedores Y Tejidos Cerebrales Humanos

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). Collectively, these Nups function to regulate genome organization, gene expression, and nucleocytoplasmic transport (NCT). Recently, defects in NCT and alterations to specific Nups have been identified as early and prominent pathologies in multiple neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer&#39;s Disease (AD)/Frontotemporal Dementia (FTD), and Huntington&#39;s Disease (HD). Advances in both light and electron microscopy allow for a thorough examination of sub-cellular structures, including the NPC and its Nup constituents, with increased precision and resolution. Of the commonly used techniques, super-resolution structured illumination microscopy (SIM) affords the unparalleled opportunity to study the localization and expression of individual Nups using conventional antibody-based labeling strategies. Isolation of nuclei prior to SIM enables the visualization of individual Nup proteins within the NPC and nucleoplasm in fully and accurately reconstructed 3D space. This protocol describes a procedure for nuclei isolation and SIM to evaluate Nup expression and distribution in human iPSC-derived CNS cells and postmortem tissues.

This protocol describes an optimized workflow for nuclei isolation and super-resolution structured illumination microscopy to evaluate individual nucleoporins within the nucleoplasm and NPCs in induced pluripotent stem cell derived neurons and postmortem human tissues.

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Purificación bioquímica y caracterización proteómica de núcleos de fibrillas amiloides del cerebro

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Isolation of Amyloid Fibrils from Brain Tissue Extract

Source: Upadhyay, A. et al., <a href="https://app.jove.com/v/63816">Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.</a> J. Vis. Exp. (2022)
This video demonstrates the protocol for isolating&#160; amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

Aislamiento de fibrillas de amiloide a partir de extracto de tejido cerebral

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Tissue harvesting and ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

Isolation of Detergent-Insoluble Protein Aggregates From Human Postmortem Brain Tissue

Source: Diner, I., et al. <a href="https://app.jove.com/v/55835">Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain.</a> J. Vis. Exp. (2017).
This video explains the process of isolating and enriching detergent-insoluble protein aggregates from frozen postmortem human brain tissue. It demonstrates key steps, including homogenization, detergent treatment, sonication, and ultracentrifugation to separate and purify the insoluble aggregates. The final step involves solubilizing the aggregates in urea buffer for further analysis.

Aislamiento de agregados de proteínas insolubles en detergente a partir de tejido cerebral humano post mortem

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.&#160;&#160;
1. Homogenization and ...