Name: enrichment of detergent-insoluble protein aggregates from human postmortem brain
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Os autores agradecer os Drs Jim Lah e Allan Levey, Emory departamento de Neurologia, sugestões e comentários úteis. Este trabalho foi parcialmente financiado pela parceria de medicina acelerando subvenção (U01AG046161-02), o Emory Alzheimer centro de pesquisa da doença (P50AG025688) e um Instituto Nacional sobre envelhecimento concedem (R01AG053960-01) para N.T.S. Esta pesquisa também foi apoiada em parte pelo núcleo neuropatologia da subvenção Emory neurociência NINDS núcleo instalações, P30NS055077.

declaração de ética: todos os tecidos do cérebro foram obtidos a partir da Emory Alzheimer &#39; s doença Research Center (ADRC) banco de cérebro. Tecidos humanos após a morte foram adquiridos ao abrigo de protocolos apropriados institucional Review Board (IRB).
 1. homogeneização e fracionamento <ol><li>seleção de tecido Nota: congelado o tecido do córtex frontal após a morte de controle saudável (Ctl) e patologicamente casos confirmados de AD foram seleccionados a partir do Emory Banco de cérebro ADRC (n = 2). Post-mortem neuropatológica avaliação da distribuição de placa amiloide foi realizada de acordo com o consórcio para estabelecer um registro para Alzheimer &#39; s doença semi-quantitativa marcando critérios 24, embora patologia do tangles emaranhado foi avaliada em conformidade com o sistema de preparo de Braak. 16 <ol><li>obter congelado o tecido cerebral post-mortem de controle saudável (Ctl) e patologicamente confirmado casos de AD. Utilizando recipientes de gelo seco, fórceps e uma lâmina de barbear, impostos especiais de consumo ~ porções de 250 mg de matéria cinzenta de cada amostra de tecido na tarada descartável pesam barcos, tendo o cuidado de evitar que o tecido descongelar. Registrar o peso de cada peça para ser homogeneizado.</li>
		<li>Impostos ~ 250 mg de matéria cinzenta, usando a pinça e navalha, inspecionando visualmente para localizar a interface entre a substância cinzenta externa e interna de substância branca. Evitar a substância branca e, mais importante, qualquer grandes vasos sanguíneos ou regiões sangrentas, meninges ou aracnoide. Mantenha o tecido congelado durante o processo de corte a trabalhar rapidamente e frequentemente colocando o barco pesagem com tecido cerebral em recipientes de isopor com gelo seco</li>
		<li>Gravar o peso exato da matéria cinzenta extirpado da cada pedaço congelado usando uma balança analítica. Calcular o volume de tampão baixo sal (LS) (ver tabela 1) necessário para homogeneização de homogenizacao (5 mL/g ou 20% w/v).</li>
		<li>Preparar 10 mL LS de tampão com 1x do inibidor de protease e fosfatase cocktail e calma na Ice.</li>
		<li>Remover tecido pesado e pesagem do barco de gelo seco e corte em pedaços de 2 x 2 mm aproximadamente, enquanto ele derrete. Transferência para um tubo de homogeneizador de pre-refrigerados homogenizacao 2ml no gelo</li>
		<li>Adicionar 5 mL/g (20% w/v) do buffer de sal baixo gelada (LS) com 1 X coquetel de inibidor de protease e fosfatase.</li>
		<li>Homogenize no tecido cerebral no gelo usando aproximadamente 10 traços de alta apuramento pilão A e 15 golpes de pilão de depuração baixa B.</li>
		<li>Transferir tudo homogeneizado para um tubo de polipropileno 2 mL, usando um pipeta Pasteur de vidro. Rótulo do tubo com &#8220; TH &#8221; (Homogenate Total), o tecido caso número e homogeneizado volume. alíquota 0,8 mL num tubo rotulado 1,5 mL. Qualquer excesso homogenate pode ser similarmente aliquotadas e armazenado a -80 ° C.</li>
		<li>a cada 0,8 mL alíquota, adicionar 100 µ l cada de 5 M NaCl e sarkosyl de 10% (p/v) para as concentrações de 0,5 M e 1% p/v, respectivamente. Misturar os tubos bem pela inversão e incubar no gelo por 15 min. de tubos de rótulo com &#34; TH-S &#34; (Total homogeneizado-Sarkosyl) para indicar que o homogenates estão no buffer de sarkosyl (tabela 1).</li>
		<li>Proceda à sonicação cada tubo para pulsos de três 5 s em 30% amplitude no gelo (intensidade máxima = 40%) usando uma sonda de microtip.</li> <li>determinar as concentrações de proteína do homogenates usando o ácido bicinchoninic (BCA) ensaio 25 método. 
		Nota: A concentração de proteína média de TH-S homogenates preparado em 5 mL LS por grama de tecido é de 15-20 mg/mL. O homogenates pode ser fracionada imediatamente ou armazenado a-80 ° C até o uso.</li>
		<li>Homogenates TH-S diluir a 10 mg/mL, usando gelada sark buffer (tabela 1) com 1 x inibidores de protease e fosfatase.</li>
		<li>Transferir 5 mg proteína (0,5 mL) de cada homogenate TH-S em 500 tubos de se µ l em policarbonato e par-equilíbrio com buffer de sark. Carregar os tubos em um rotor pre-refrigerado e se a 180.000 x g durante 30 min a 4 ° C. transferência do sarkosyl-solúvel fracções sobrenadantes (S1) para tubos de 1,5 mL e armazene a -80 ° C. 
		Nota: (Lavagem opcional) adicionar 200 µ l de sark-buffer para o se tubos contendo as frações insolúveis em detergente (P1) e desalojar as pelotas (P1) da parte inferior dos tubos se utilize uma ponta de pipeta de 200 µ l.</li>
		Tubos <li>brevemente de pulso-gire o invertido para 2-3 s (&#8804; 2.500 x g) em um microcentrifuge para transferir o buffer e pelotas (P1) para os tubos de 1,5 mL abaixo. Resuspenda as pelotas insolúveis (P1) no buffer de sark pipetando para cima e para baixo para garantir a pelota é interrompida.</li>
		<li>Par-equilíbrio e centrifugar 180.000 x g por um adicional 30 min a 4 ° C.</li>
		<li>Desprezar o sobrenadante de lavagem opcional (S2) e incube as pelotas de sarkosyl insolúveis (P2) em 50-75 µ l de tampão de ureia (tabela 1) com 1 x PIC (protease e fosfatase inibidor cocktail) por 30 min à temperatura ambiente para solubilizar o pelota. 
		Nota: Buffer de ureia quente a temperatura ambiente antes do uso para evitar a precipitação de SDS. Para aplicações sensíveis ao detergente, omitir SDS de buffer de ureia e considerar lavar o sedimento insolúvel (P2) no baixo sal buffer antes de resuspending no buffer de ureia.</li>
		<li>Transferir as ressuspensa Pelotas (P2) para tubos de 0,5 mL e usar breve (1 s) microtip sonication em amplitude de 20% (intensidade máxima = 40%) para solubilizar totalmente as pelotas.</li>
		<li>Determinar as concentrações de proteína de sarkosyl-solúvel (S1) e insolúveis (P2) frações usando o BCA método de ensaio. Use estas frações imediatamente ou armazenar a-80 ° C até o uso.</li>
	</ol></li></ol> 2. Immunoblotting <ol><li>mancha ocidental Nota: mancha ocidental foi realizada de acordo com os procedimentos anteriormente relatados com ligeiras alterações. 5 , 10 <ol><li>preparar 40 µ g amostras de homogenates total (TH-S), sark-solúvel (S1) e frações de sark insolúveis (P2) em 1 amortecedor da amostra X Laemmli SDS-page com pH TCEP 5 mM 7. Incubar as amostras a 95 ° C por 5 min.</li>
		Amostras de <li>carga em SDS-PAGE um pré-moldado bem 10 4-12% Bis-Tris gel. Electrophorese em 80 V para o primeiro centímetro (10 min) e 120 V para o restante (60 min), ou até que a tintura de seguimento atinge a parte inferior do gel.</li>
		<li>Use uma lâmina de barbear fresca e gel de faca para dividir-abrir a gaveta do gel de pre-cast. Delicadamente cortar fora os pentes e inferior do gel com uma lâmina de barbear fresco.</li>
		<li>Resuspenda as pelotas insolúveis (P1) em 200 µ l sark-buffer com 1x PIC pipetando cima e para baixo.</li>
		<li>Transferência para uma membrana de nitrocelulose de 0,2 µm usando um método de transferência de seco em uma máquina de mancha (consulte a tabela de materiais).</li>
		<li>Bloquear a membrana em tampão de bloqueio (BB) sem Tween 20 por 30 min, seguida com BB com 0,05% Tween 20 (BB/T) por 30 min. Após o bloqueio, lavar a membrana em TBS/T (0.1% Tween 20) por 5 min remover o tampão de bloqueio excesso.</li>
		<li>Preparar diluições de 1:1,000 de pT231 os anticorpos primários, Tau-2 (pan tau), AT8 e EEA1 no BB/T (0,05% Tween 20).</li>
		<li>Incubar as membranas em solução de anticorpo primário durante a noite a 4 ° C, com agitação circular. Lavar a membrana em TBS/T 3 x 15 min.</li>
		<li>Sonda a membrana com uma diluição de 1: 20.000 de fluorescente-etiquetadas infravermelho próximo anticorpo secundário no BB/T para 60 min à temperatura ambiente na escuridão agitador. Lavar a membrana para 3 x 10 min na TBS/T e 2 x 5 min em TBS</li>
		<li>Scan a membrana usando um sistema no comprimento de onda de excitação apropriada (por exemplo, 700 nm (canal vermelho) para tintura infravermelho 680 cabra anti-rato IgG (H + L) secundário e 800 nm (canal verde) para tintura infravermelho 790 burro anti-rabbit IgG (H + L de imagens de infravermelho secundária)).</li>
		<li>Usar o software de imagem para quantificar a intensidade do sinal e executar medidas de densitometria conforme o fabricante &#39; instruções de s, garantir autoconfiguração de fundo é definido como média o fundo todo.</li>
	</ol>
	</li>
</ol>

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Neste documento vamos apresentar e discutir um protocolo de detergente-fracionamento de etapa única Abreviado que é aplicável a uma ampla variedade de aplicações experimentais que variam de análise proteômica de espectrometria de massa-baseado a proteína funcional ensaios de enrolamento e mancha ocidental. 5 , 6 , 7 , 10 esta metodologia é talvez mais eficaz quando usado para estudar p...

Doenças neurodegenerativas, como a doença de Alzheimer (AD), a doença de Parkinson (PD), a doença de Huntington (HD), esclerose lateral amiotrófica (ela) e as doenças de príon estreitamente relacionadas são proteinopathies caracterizadas pelo acúmulo gradual de agregados de proteína insolúvel em detergente no cérebro com acompanhamento cognitivo declinam. 1 , 2 este recurso compartilhado patológico é pensado para jogar um papel central na etiologia e fisiopatologia dessas doenças neurodegenerativas. 2 estes agregados, normalmente, compostos de fibras de amiloides poliméricas, que são compostas de repetir unidades de misfolded proteínas exibindo Cruz β-estrutura. 1 , 2 , 3 , 4 bioquimicamente, agregados amiloides são altamente resistentes à desnaturação química ou térmica e solubilização,3 que apresenta desafios únicos para a sua purificação, análise e estudo através de técnicas bioquímicas tradicionais. 2 , 5 , 6 , 7 , 8 , 9 , 10 , 11 não é novidade, a fração de proteína insolúvel em detergente tem sido o foco de muita pesquisa em fisiopatologia das doenças neurodegenerativas que envolvem o acúmulo de misfolded proteínas. 6 , 12 , 13 , 14
Técnicas de fracionamento bioquímico frequentemente têm sido utilizadas para enriquecer a fração insolúvel em detergente de homogenates cerebral post-mortem. 6 , 12 , 13 , 14 um dos métodos mais comuns envolve a extração sequencial de tecido homogenates com buffers e detergentes de aumentar o rigor, seguido por ultracentrifugação para particionar as frações solúveis e insolúveis. Um protocolo comumente usado fracionamento sequencial6,14 envolve a homogeneização de amostras de tecido congelado em um buffer de detergente livre baixo sal (LS) e as pelotas insolúveis resultantes então são extraídas sequencialmente com amortecedores que contêm sal elevado, detergentes não-iônicos, alta sacarose, detergentes iônicos e finalmente chaotropes como ureia. 6 , 14 uma desvantagem óbvia de tais protocolos um fracionamento sequencial é o tempo substancial e compromisso de trabalho necessário para concluí-las. Incluindo a homogeneização e ultracentrifugação, um protocolo típico de cinco etapas de fracionamento pode levar várias horas ou mesmo dias para completar. 4 , 6 , 7 , 10 , 15 , 16 , 17 , 18 além disso, como muitos agregados de proteína patológica permanecem insolúveis em todos, mas as mais adversas condições19,20 a maioria das frações geradas são de valor limitado. Assim, as etapas de fracionamento menos rígidas utilizando altas concentrações de sal e detergentes não-iônicos são em grande parte redundantes.
Estudos anteriores mostraram que o detergente iônico N-Lauril-sarcosina (sarkosyl) é um excelente candidato para um protocolo simplificado fracionamento de detergente de etapa única. 5 , 6 , 12 , 13 , 14 , 21 , 22 , 23 como um detergente de desnaturação, sarkosyl é suficientemente rigorosa para solubilizar a maioria vasta das proteínas nativamente dobradas no cérebro sem solubilizing agregados de misfolded proteínas compostos por beta-amiloide (Aβ),6,11 tau fosforilada (pTau),6 TAR ADN-proteína obrigatória 43 (TDP-43),14 alfa-synuclein,12,13 do tremor epizoótico,23 ou U1 pequenas ribonucleoproteínas nucleares (snRNPs U1) como U1 - 70 K. 5 , 21 , 22 Como sarkosyl é menos rigoroso do sulfato dodecyl de sódio detergente aniônico onipresente (SDS), preserva menos robustas oligoméricas formas de agregados de misfolded proteínas que não podem suportar o tratamento de SDS. 9
Anteriormente, nós descrevemos um protocolo de detergente-fracionamento Abreviado que obtiveram resultados comparáveis aos métodos de fracionamento sequencial mais trabalhosos. 5 , omitindo as menos rigorosas etapas de fracionamento, fomos capazes de desenvolver um protocolo facile fracionamento de etapa única para o enriquecimento dos agregados de proteína insolúvel em detergente do cérebro humano após a morte. 5 este protocolo detalhado aqui descrito é bem adequado para uma ampla gama de aplicações experimentais que variam de mancha ocidental e ensaios de espectrometria de massa-baseado proteomics para enrolamento de proteína funcional e propagação de agregação. 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

Neste estudo, descrevemos um protocolo abreviado fracionamento de etapa única para o enriquecimento dos agregados de proteína insolúvel em detergente do cérebro humano após a morte. O detergente iônico N-Lauril-sarcosina (sarkosyl) efetivamente solubiliza proteínas dobradas nativamente no tecido cerebral, permitindo o enriquecimento de agregados da proteína insolúvel em detergente de uma ampla gama de proteinopathies neurodegenerativas, tais como A doença de Alzheimer (AD), doença de Parkinson e esclerose lateral amiotrófica e doenças de príon. Controle humano e tecidos do cérebro após a morte de AD foram homogeneizadas e sedimentada por ultracentrifugação na presença de sarkosyl para enriquecer agregados de proteína insolúvel em detergente incluindo tau fosforilada patológica, o componente do núcleo do tangles emaranhados em AD. Mancha ocidental demonstrou a solubilidade diferencial de agregados-tau fosforilada e a proteína solúvel em detergente, Early Endosome antígeno 1 (EEA1) no controle e no cérebro do AD. Análise proteômica também revelou o enriquecimento de β-amiloide (Aβ), tau, snRNP70 (U1 - 70 K) e apolipoproteína E (APOE) nas fracções sarkosyl insolúveis do cérebro AD comparados de controle, consistente com estratégias de fracionamento de tecido anterior . Assim, o presente protocolo simples enriquecimento é ideal para uma ampla gama de aplicações experimentais variando de mancha ocidental e proteína funcional ensaios de agregação co a espectrometria de massa-baseado proteomics.

O protocolo de sarkosyl de etapa única abreviado-fracionamento foi usado para enriquecer agregados de proteína insolúvel em detergente de controle e o cérebro após a morte de AD (Figura 1). Proteínas de frações TH-S, S1, S2 e P2 foram resolvidas por SDS-PAGE, fixado por 15 min em Coomassie azul reserva fixador seguida de coloração suave com buffer de coloração azul brilhante de Coomassie G-250. A etapa de ressuspensão é opcional, desde que não ...

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

Um protocolo de fracionamento abreviado para o enriquecimento dos agregados de proteína insolúvel em detergente do cérebro humano após a morte é descrito.

Enriquecimento de agregados da proteína insolúvel em detergente do cérebro humano após a morte

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 visualizações.  Emory School of Medicine. Um protocolo de fracionamento abreviado para o enriquecimento dos agregados de proteína insolúvel em detergente do cérebro humano após a morte é descrito.

Vídeo: 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.

SDS-PAGE/Immunoblot Detecção de multímeros Aâ no tecido cortical Humanos homogeneizados utilizando antígeno Epitope-Retrieval

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

Neurociência

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.

Isolamento de Oligómeros PrP solúveis e insolúveis no cérebro 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.

Isolamento Núcleos neuronais de Tecidos Humanos Postmortem Cérebro

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

Biologia

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.

Proteína Consenso Brain-derivada, protocolo de extração para o Estudo do cérebro humano e murino Proteome Usando ambas 2D DIGE e 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.

Extração seqüencial de solúveis e insolúveis alfa-sinucleína de Brains 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.

Um ensaio baseado em espectroscopia de fluorescência com eficiência temporal para avaliar o status de polimerização de actina em roedores e tecidos cerebrais 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.

Purificação Bioquímica e Caracterização Proteômica de Núcleos de Fibril Amiloides do Cérebro

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.

Isolamento de fibrilas amilóides do extrato de tecido cerebral

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

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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.

Isolamento de agregados de proteínas insolúveis em detergente do tecido 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 ...