Questo lavoro è stato sostenuto da Inserm, dell&#39;Università di Lille 2, MEDIALZ, LABEX (laboratorio di eccellenza, programma di investire per il futuro) e DISTALZ (sviluppo di strategie innovative per un approccio transdisciplinare alla malattia di Alzheimer). FJ.FG è attualmente una borsa destinatario di ANR (francese Agenzia Nazionale di Ricerca / NeuroSplice de Tau Projet ANR-2010-BLAN-1114 01), ma questo lavoro è stato anche sotto il supporto di una sovvenzione da parte del JCCM (Spagna).

1. Omogeneizzazione e Total estrazione di proteine ​​da umano e mouse tessuto cerebrale <ol><li> Cervello omogeneizzazione del tessuto. Omogeneizzare il tessuto cerebrale 10% (w / v) in 8 M urea, 2 M tiourea e 1% w / v tampone SDS (UTS) utilizzando una lente potter (per campioni umani) o Teflon omogeneizzatore (per topi campioni). Ultrasuoni a 60 Hz 30 impulsi con un generatore di ultrasuoni applicazione 0.5 sec per la disintegrazione del tessuto totale 13, 14. <ol><li> Determinare la concentrazione di proteine ​​con saggio Bradford, utilizzando BSA come standard. Questo UTS estrazione buffer è completamente compatibile con una dimensione SDS-PAGE purché non lisati sono sovra-riscaldato per evitare carbamilazione 15. </li><li> Aggiungere 1% (w / v) di Amberlite e incubare con miscelazione delicata per 10 min a temperatura ambiente. Successivamente filtrare su 0.22 micron filtri per siringa e infine aggiungere la SDS per la soluzione di urea / tiourea. Questo passaggio riduce la quantità di acido urico e acido isocianico, che sono in equilibrIUM con urea in soluzione e facilitare la sua degradazione. </li></ol></li><li> Cloroformio / metanolo precipitazione delle proteine. Precipitare tra 100-150 mg di proteine ​​per 11 centimetri strisce IPG e 1-1,5 mg per 18 centimetri quelli (indipendentemente sulla gamma pH selezionato per eseguire l&#39;IEF). <ol><li> Eseguire tutti i passaggi su ghiaccio oppure a 4 ° C. Fresco cloroformio e metanolo a 4 ° C per 30 min prima di iniziare la procedura di precipitazione. </li><li> Aggiungere tre volumi di metanolo e un volume di cloroformio per un volume di UTS lisato. Agitare manualmente la miscela e aggiungere tre volumi di acqua fredda. Vortex per 1 min e agglomerare le proteine ​​mediante centrifugazione a 12.000 xg per 30 min a 4 ° C. </li><li> Eliminare il surnatante con una pipetta Pasteur e aggiungere tre volumi di freddo MeOH. Nuovamente Vortex e centrifugare a 12.000 xg per 30 min a 4 ° C. Infine, scartare il surnatante e asciugare il pellet sotto N 2. </li></ol></li><li> Preparazione di proteine ​​per analisi 2D.Risospendere il pellet di proteine-essiccati in 2D-buffer (8 M urea, 2 M tiourea integrato con 4% CHAPS). Lavare la soluzione con Amberlite come indicato sopra e cercare di mantenere la proporzione di 500 mcg di proteine ​​per 200 ml di tampone per 2D 2D-DIGE. Nota: tampone 2D può essere conservato a -80 ° C o mantenuta a 4 ° C per una settimana (per evitare degradazione dell&#39;urea). <ol><li> Sonicare e conservare i campioni a -80 ° C fino effettuare il passaggio seguente. Degno di nota, questo protocollo può essere usato dopo solubilizzazione di acido formico di proteine ​​aggregati 10. In breve, far evaporare l&#39;acido formico sotto N 2 e risospendere il pellet come descritto. </li></ol></li></ol> 2. 2D-DIGE <ol><li> Campioni di prova di qualità. Campioni di dose ancora utilizzando il metodo di Bradford. Diluire 15 pg di proteine ​​in tampone campione LDS, riscaldare a 37 ° C e 15 caricare sul 4-12% gel di poliacrilammide. </li><li> Colorazione con Coomassie. Macchiare gel di poliacrilammide con 0.1% (w / v) CoomassieBlu G-250, 50% EtOH e 10% (v / v) di acido acetico per almeno 1 ora. Sia sfondo Destain notte in 7% di acido acetico e 10% (v / v) di etanolo. </li><li> Etichettatura pre-elettroforetica dei campioni. Prima di eseguire l&#39;etichettatura colorante cianina, verificare il pH del campione, che dovrebbe essere di base o anche neutro poiché la sostituzione N-idrossisuccinimmide è più efficiente a pH basico e somministrato essere ottimale a pH 8,6. <ol><li> Etichettare minimamente i due campioni di studio con coloranti fluorescenti Cy3 o Cy5 con un rapporto di 50 ug protein/400 pmol colorante e mantenere per 1 ora a 4 ° C al buio per facilitare il gruppo estere attivo NHS delle cianine con l&#39;ammina senza protoni gruppi di lisine. </li><li> Ridurre le variazioni di esempio facendo un cross-etichettatura con Cy3 e Cy5 coloranti, evitando un accoppiamento preferenziale di un campione a un particolare cianina. Si noti che il cianina-colorante marcatura minima può essere adattato per piccole quantità di proteine ​​con mantenere la proporzione di 1 ug protein per 8 pmol Cyanine-dye. Se è così, il sistema mini-2DE può essere usata al posto della grande sistema gel 2D. Ciò consentirà l&#39;analisi 2D-DIGE per una piccola quantità di tessuto cerebrale. </li><li> Etichettare un pool di entrambi i campioni con la stessa quantità di proteina (50 mg in totale) con colorante fluorescente Cy2 e usato come standard interno. Utilizzare le stesse condizioni come precedentemente indicato. </li><li> Completa in un volume totale di 350 microlitri di tampone 2D contenente 1,1% di reagente Destreak, 1,2% di tampone IPG e tracce di blu di bromofenolo totale 150 mcg di proteine ​​marcate (50 mg di Cy2, 50 mg di Cy3 e 50 ug di Cy2). Eseguire questo passaggio in quadruplice copia utilizzando quattro strisce indipendenti. Inoltre, preparare due ulteriori strisce non etichettati (uno per ogni campione) con 350-450 mg di proteine ​​per i gel preparative. </li><li> Lasciare le sei strisce cariche ricoperte di pernottamento oli minerali per la reidratazione passiva. </li></ol></li><li> Isoelettrofocalizzazione. Effettuare la IEF a 206; C. Per un pH 3-11NL, 18 centimetri striscia il protocollo è: l&#39;impostazione corrente massima è di 50 ìA / strip durante le 8 tappe: 1) 150 V step per 1 ora, 2) 200 V fase per 5 ore, 3) 500 V con pendenza 2 ore, 4) gradiente di 1,000 V per 2 ore, 5) 2,000 V pendenza per 2 ore, 6) 4,000 V pendenza per 2 ore, 7) 8,000 V pendenza per 2 ore e 8) per un totale di 24.000 V · h passo . Dopo IEF, inviare l&#39;eccesso di olio minerale con carta cromatografica e memorizzare le strisce a -20 ° C fino alla seconda dimensione. </li><li> IPG Strips equilibratura. Equilibrare strisce in 6 M urea, 2% SDS, 30% glicerolo, 50 mM Tris-HCl pH 8,6 tampone con 1% di DTT durante e dopo 15 min con 4,7% di iodoacetamide nello stesso tampone ma senza DTT. </li><li> Seconda dimensione o SDS-PAGE elettroforesi. Effettuare le sei 12% gel SDS-PAGE (1,5 M Tris-HCl pH 8,6, 12% di acrilammide / bisacrylamide, 0.1% (w / v) di SDS, 0,072% (w / v) APS e 0,1% (v / v) TEMED ) Allo stesso tempo utilizzando un grande sistema 2D, con quattro lastre di vetro a bassa fluorescenza per la strips con campioni di fluorescenza e altri due lastre di vetro con 1,5 mm di spessore per i gel preparative, al fine di asportare le macchie proteiche. <ol><li> Caricare i sei IPG strisce sulla cima dei gel e più strati con 0,5% (w / v) di agarosio. Tampone di corsa è composta da 25 mM Tris, glicina 192 mM e 0,1% (w / v) di SDS. Eseguire la migrazione a 2,5 W / gel durante la notte con un sistema di refrigerazione impostato a 4 ° C 16,17. </li></ol></li><li> Immagini di fluorescenza acquisizione e l&#39;analisi. Utilizzare uno scanner Typhoon 9400 per ottenere le immagini gel di fluorescenza (12 immagini per un esperimento). Scan Cy2, Cy3 e Cy5 immagini per ogni gel a 488/520 nm, 532/580 nm e 630/670 nm di eccitazione / lunghezze d&#39;onda, rispettivamente. Utilizzare il software informatico per analizzare le immagini. I dati sono rappresentativi delle variazioni di volume posto associati alla distribuzione del segnale di tutti i quattro gel tra i due campioni studiati. </li><li> Coomassie colorazione per i grandi gel. Fissare il gel di preparazione in 50% (v / v) EtOH e 3% (v / v) di acido ortofosforico per almeno 24 ore. Poi, dopo, lavare due volte con acqua ultra pura per 20 min. Eseguire colorazione in 34% (v / v) metanolo, 17% (w / v) di solfato di ammonio e acido ortofosforico 2% per 1 ora prima di aggiungere 0,1% (w / v) Coomassie Blu G-250. Tenere i gel nella colorazione per almeno 48 ore e decolorati in acqua ultra pura. <ol><li> Spot per l&#39;identificazione MS. Una volta che le immagini di fluorescenza vengono analizzate, il software fornisce un elenco di punti la cui espressione è statisticamente diverso. Asportare manualmente queste macchie dai gel di preparazione. Questa procedura richiede pratica e una certa destrezza manuale per evitare contaminazione cheratina. Poi i campioni possono essere mantenuti in acqua a -20 ° C fino invio di MS. Identificazione Spot è perfettamente compatibile con spettrometria di massa tandem (MS / MS) e la rilevanza delle identità di proteine ​​è giudicata in base al punteggio Mowse basato calcolato con un p-value di 0,05 (p &lt;0,05) 18 probabilità. </li> &lt;/ Ol&gt; </li></ol> 3. Qualitativa Mini-2DE Assay <ol><li> Risospendere massimo 100 pg di proteina in tampone campione 2D fino a un volume finale di 200 microlitri di una striscia IPG 11 cm. Nel caso di sistema sovraespressione o proteina purificata, l&#39;importo di partenza può essere abbassata a 20 mg. </li><li> Aggiungere 1,1% (v / v) Destreak reagente, 0,55% (v / v) di tampone di IPG e tracce di blu di bromofenolo in un volume finale di 200 microlitri. Poi, vortice, fare un giro veloce e caricare in una striscia IPG per la reidratazione passiva (per portare il nastro al suo spessore originale) durante la notte coperto con olio minerale. Nota: la percentuale di buffer di IPG è la stessa per tutti l&#39;intervallo di pH desiderato (pH 3-11, 4-7, 7-11, ecc), ma la sua composizione varia in funzione del campo di pH selezionato. </li><li> Isoelectrofocalisation. Eseguire IEF a 20 ° C. La durata del programma dipende dall&#39;intervallo di pH selezionato. Degno di nota, parametri come elettroendosmosi possono disturbare il profilo IEF e in questi casi,ottimizzazione in tempo electrofocalisation è necessaria 19. Nota: non è necessariamente un IEF più che fornisce risultati migliori ma accorciare il tempo di IEF può anche migliorare la risoluzione. Dopo IEF, strisce IPG possono essere conservati a -20 ° C per mesi. </li><li> IPG strisce di equilibrio. Equilibrare le strisce in un tampone contenente 25 mM Tris-HCl pH 6,8, 20 mM DTT, 10% glicerolo, 5% SDS, 0,05% blu di bromofenolo. Fai tre equilibratura bagna per 15 minuti con il cambiamento la soluzione tampone tra ogni bagno. </li><li> SDS-PAGE. Strato strip IPG su gel prefabbricato (IPG +1 ben 11 centimetri striscia IPG) con una percentuale di poliacrilammide scelto a seconda del MW della proteina di interesse. Poi, asciugare il gel su una membrana di nitrocellulosa / PVDF a 100 mV durante 33 min. </li><li> Incubare per una notte con l&#39;anticorpo richiesta e visualizzare per attività perossidasi. Eseguire gli allineamenti di immunoblots usando polipeptidi indipendenti rivelate dal anticorpo secondario. Raggiungere un semi-quantitative analisi densitometria del volume e l&#39;intensità delle tracce / spot con il software ImageJ. </li></ol>

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D., Marcus, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+advancement+of+neuroproteomics.">Multidimensional advancement of neuroproteomics.</a> Expert Rev Proteomics. 5, 149-151 (2008).</li><li>Hernandez-Hernandez, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myotonic+dystrophy+CTG+expansion+affects+synaptic+vesicle+proteins,+neurotransmission+and+mouse.">Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse.</a> 136, 957-970 (2013).</li><li>Deramecourt, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+staging+of+synucleinopathy+and+amyloid+deposition+in+dementia+with+Lewy+bodies.">Biochemical staging of synucleinopathy and amyloid deposition in dementia with Lewy bodies.</a> J Neuropathol Exp Neurol. 65, 278-288 (2006).</li><li>Tofaris, G. K., Razzaq, A., Ghetti, B., Lilley, K. S., Spillantini, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+of+alpha-synuclein+in+Lewy+bodies+is+a+pathological+event+not+associated+with+impairment+of+proteasome+function.">Ubiquitination of alpha-synuclein in Lewy bodies is a pathological event not associated with impairment of proteasome function.</a> J Biol Chem. 278, 44405-44411 (2003).</li><li>Sergeant, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Truncated+beta-amyloid+peptide+species+in+pre-clinical+Alzheimer's+disease+as+new+targets+for+the+vaccination+approach.">Truncated beta-amyloid peptide species in pre-clinical Alzheimer's disease as new targets for the vaccination approach.</a> J Neurochem. 85, 1581-1591 (2003).</li><li>Le Freche, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+phosphorylation+and+sevoflurane+anesthesia:+an+association+to+postoperative+cognitive+impairment.">Tau phosphorylation and sevoflurane anesthesia: an association to postoperative cognitive impairment.</a> Anesthesiology. 116, 779-787 (2012).</li><li>Leboucher, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detrimental+effects+of+diet-induced+obesity+on+tau+pathology+are+independent+of+insulin+resistance+in+tau+transgenic+mice.">Detrimental effects of diet-induced obesity on tau pathology are independent of insulin resistance in tau transgenic mice.</a> Diabetes. 62, 1681-1688 (2013).</li><li>Deramecourt, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical,+neuropathological,+and+biochemical+characterization+of+the+novel+tau+mutation+P332S.">Clinical, neuropathological, and biochemical characterization of the novel tau mutation P332S.</a> J Alzheimers Dis. 31, 741-749 (2012).</li><li>Birdsill, A. C., Walker, D. G., Lue, L., Sue, L. I., Beach, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmortem+interval+effect+on+RNA+and+gene+expression+in+human+brain+tissue.">Postmortem interval effect on RNA and gene expression in human brain tissue.</a> Cell Tissue Bank. 12, 311-318 (2011).</li><li>Bell, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+a+twenty-first+century+brain+bank:+experience+in+the+BrainNet+Europe+consortium.">Management of a twenty-first century brain bank: experience in the BrainNet Europe consortium.</a> Acta Neuropathol. 115, 497-507 (2008).</li><li>Crecelius, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+quantitative+post-mortem+changes+in+the+gray+matter+of+the+human+frontal+cortex+proteome+by+2-D+DIGE.">Assessing quantitative post-mortem changes in the gray matter of the human frontal cortex proteome by 2-D DIGE.</a> Proteomics. 8, 1276-1291 (2008).</li><li>Swatton, J. E., Prabakaran, S., Karp, N. A., Lilley, K. S., Bahn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+profiling+of+human+postmortem+brain+using+2-dimensional+fluorescence+difference+gel+electrophoresis+(2-D+DIGE).">Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE).</a> Mol Psychiatry. 9, 128-143 (2004).</li><li>Viswanathan, S., Unlu, M., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+difference+gel+electrophoresis.">Two-dimensional difference gel electrophoresis.</a> Nat Protoc. 1, 1351-1358 (2006).</li><li>Tannu, N. S., Hemby, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+fluorescence+difference+gel+electrophoresis+for+comparative+proteomics+profiling.">Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling.</a> Nat Protoc. 1, 1732-1742 (2006).</li></ol>

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Scoprire cambiamenti di espressione di proteine ​​patologiche, la ricerca dei biomarcatori e la modulazione dei potenziali percorsi di bersagli farmacologici sono tra gli obiettivi delle neuroproteomics avvicina 30. Tra gli strumenti emergenti, il campo 2DE aggiunge un expectative promettente. Tuttavia, un consenso dovrebbe essere raggiunto per minimizzare la variabilità e aumentare la riproducibilità negli esperimenti. In linea di questa idea un protocollo standardizzato effettuare 2D-DIGE (Fig...

Disturbi mentali e neurologici rappresentano il 13% del carico globale di malattia, nuove sfide come meccanismi fisiopatologici, fattori di rischio e biomarcatori prodromici devono essere esplorate 1. In linea con questo obiettivo, proteomica studi del cervello umano diventano indispensabili per scoprire i meccanismi molecolari coinvolti nei processi come la memoria, il comportamento, le emozioni e la plasticità neuronale per esempio, non solo per motivi fisiologici, ma anche per le condizioni patologiche. Pertanto, l&#39;uso di modelli animali e più specificamente i topi transgenici, porta una vasta gamma di possibilità di imitare l&#39;eziologia delle malattie neurodegenerative umane 2. Proteomica approcci sono oggi disponibili per realizzare queste nuove prospettive nel campo delle neuroscienze. Gel elettroforesi bidimensionale (2DE) è un metodo semplice e potente probabile che permette di confrontare il proteoma di una vasta gamma di campioni. Inoltre, è anche unpotente metodo per isolare una proteina da una miscela complessa al fine di identificare e analizzare ulteriormente mediante spettrometria di massa. Questa tecnica consiste essenzialmente in due fasi successive: 1) la separazione delle proteine ​​in base al loro punto isoelettrico (pI) da isoelettrofocalizzazione (IEF). Più precisamente, un potenziale elettrico viene applicato attraverso le strisce di acrilammide Immobiline tra un gradiente di pH e quindi proteine ​​migreranno e concentrarsi su un pI determinata in funzione della loro carica netta globale. 2) proteine ​​Isoelectrically focalizzati sono denaturati e caricati negativamente con l&#39;aggiunta di sodio dodecil solfato (SDS), in tal modo le proteine ​​nella loro prima struttura sono separati in funzione del loro peso molecolare apparente (MW) mediante SDS-PAGE 3. Queste due proprietà distinte ci permettono di affrontare un valore doppio di andare oltre nello studio del proteoma. Da un lato questo approccio offre la possibilità di effettuare una analisi quantitativa utilizzando coloranti minime metodo 2D-DIGE, e dall&#39;altro un qualitative analisi mini-2DE accoppiato al western blotting. Analisi quantitativa da 2D-DIGE conferisce espressione della proteina cambia tutto il proteoma del campione. In breve, i campioni vengono etichettati con tre cianine (CY2, Cy3 e Cy5) che emettono a tre lunghezze d&#39;onda distinte (blu, verde e rosso). Questi coloranti minime fluor contenenti N-idrossi gruppo estere succinimidyl reagiscono con il gruppo ε-amminico lysines residui di proteine ​​conseguente covalenti legami ammidici 4. Residui di lisina sono etichettati solo tra 1-3% e impedendo così più etichette, oltre a proteine ​​e importanti modifiche di carica al netto 5, 6. Cy3 e Cy5 sono spesso utilizzati per etichettare due campioni indipendenti mentre Cy2 tag un mix di uguale percentuale dei campioni da confrontare. I due vantaggi principali sono che tutti i campioni etichettati sono miscelati e IEF e SDS-PAGE sono svolte in un gel in una sola volta per ogni passo, evitando l&#39;inter variabilità tra esperimenti a causa di gel confronto. Inoltre, presenta una soglia alta rilevamento di circa 1 femtomole di proteine ​​7. Gel vengono analizzati e software 2D a confronto le immagini 2D fluorescenza gel, dove Cy2 serve come standard interno consentendo l&#39;identificazione delle differenze statistiche tra i punti per la loro identificazione posteriore mediante spettrometria di massa. Il software di analisi 2D utilizzando lo standard interno raggiunge un rilevamento rapido di meno del 10% delle differenze tra i campioni con più del 95% di confidenza statistica 8. Analisi qualitativa da mini-2DE è un passo cruciale per la caratterizzazione delle proteine. Il principio è lo stesso come precedentemente descritto per pI e separazione MW, ma in questo caso le proteine ​​vengono trasferite da una piccola gel di poliacrilammide ad una membrana e immunoblotting viene eseguita dopo. Mentre una elettroforesi su gel di dimensione fornisce cambiamenti nell&#39;espressione di proteine ​​per uno o più epitopi proteici in funzione della anticorpo, il information di mini-2DE dota di due parametri aggiuntivi. In primo luogo, le isovariants proteine ​​cambiano in funzione del pI, indicando che modificazioni post-traduzionali potrebbero aver luogo. In secondo luogo, l&#39;identificazione spettrometria di massa può essere indicativo di zimogeni plausibili e prodotti catabolici di proteine. Pertanto, le modifiche osservate da 2D-DIGE sono probabilmente indicativi dei meccanismi alla base cambiamenti nel profilo proteoma globale. Allineamenti di immunoblots tra i diversi campioni per la stessa proteina epitopo / s in mini-2DE possono riflettere i cambiamenti di acidità che faccia luce sulle variazioni post-traslazionali leggermente osservate o addirittura non da immunoblotting monodimensionale 9, 10. Inoltre, questa analisi informa circa i potenziali siti di taglio dovuti alla conoscenza del pI e MW dei residui metabolici 11,12. La combinazione di queste due tecniche fornisce un complementare analisi proteomica. Da un lato 2D-DIGE offre un tipicoe di differenze che permettono un isolamento precisa di polipeptidi la cui espressione è diversa. Queste differenze consistono essenzialmente nella comparsa o scomparsa di un punto o di aumento / diminuzione dell&#39;intensità di un dato da una analisi software dei gel fluorescenti. Tuttavia, queste osservazioni da soli sono difficilmente spiegare la natura della modifica osservato. Per queste ragioni, una volta che il polipeptide è isolato e identificato mediante spettrometria di massa, l&#39;uso di mini-2DE permette di confermare precisamente 1) l&#39;identità della proteina isolata e 2) la natura della differenza: variazione del livello isovariant / isoforma espressione, modificazioni post-traslazionali e processi di scissione per esempio. Tuttavia, è necessario sviluppare un tampone di lisi a partire sia compatibile con 2D-DIGE e mini-2DE per limitare i potenziali dispersioni derivanti dall&#39;uso di protocolli di estrazione che sono molto dissimili. Nel presente articolo, DEdescritto un protocollo adattato per la preparazione, estrazione e prestazioni delle tecniche mini-2DE per proteine ​​cerebrali provenienti da tessuti umani e mouse 2D-DIGE e.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">CyDye DIGE FLUOR MIN KIT 5nmol 1 * 5 Nmo</td>
			<td style="width:113px;">GE Healtcare</td>
			<td style="width:119px;">25-8010-65</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Immobiline DryStrip</td>
			<td style="width:113px;">GE Healtcare</td>
			<td style="width:119px;"></td>
			<td>Reference in function of the pH interval/size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">IPG Buffer, pH X-X</td>
			<td style="width:113px;">GE Healtcare</td>
			<td style="width:119px;"></td>
			<td>Reference in function of the pH interval desired</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">AMBERLITE IRN-150L MIXED BED RESIN 1&#160;</td>
			<td style="width:113px;">GE Healtcare</td>
			<td style="width:119px;">551797N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DRYSTRIP COVER FLUID IMMOBILINE 1 * 1 l</td>
			<td style="width:113px;">GE Healtcare</td>
			<td>17-1335-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KIT BOX IPG 1 * 1 KIT</td>
			<td style="width:113px;">GE Healtcare</td>
			<td style="width:119px;">28-9334-92</td>
			<td>Plastic box to make the passive rehydration&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">MILLEX GS Filter</td>
			<td style="width:113px;">Millipore</td>
			<td style="width:119px;">SLGS033SB</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Criterion XT Precast Gels 13.3 x 8.7 cm (W x L)</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td>Reference in function of the MW to separate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">IPGphor III Isoelectric Focusing Unit</td>
			<td style="width:113px;">GE Healtcare</td>
			<td style="width:119px;">11-0033-64</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Ettan DALTsix Large Electrophoresis System</td>
			<td style="width:113px;">GE Healtcare</td>
			<td style="width:119px;">80-6485-08&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OneTouch 2D Gel SpotPicker 1.5 mm&#160;</td>
			<td style="width:113px;">Gel Company</td>
			<td style="width:119px;">P2D1.5</td>
			<td></td>
		</tr>
	</tbody>
</table>

consensus brain-derived protein, extraction protocol for the study of human and murine brain proteome using both 2d-dige and mini 2de immunoblotting

Gel elettroforesi bidimensionale (2DE) è un potente strumento per scoprire le modifiche del proteoma potenzialmente correlati alle diverse condizioni fisiologiche o patologiche. Fondamentalmente, questa tecnica si basa sulla separazione delle proteine ​​in base al loro punto isoelettrico in una prima fase, e in secondo luogo in base al loro peso molecolare mediante SDS elettroforesi su gel di poliacrilammide (SDS-PAGE). In questo rapporto viene descritto un protocollo di preparazione del campione ottimizzato per la piccola quantità di tessuto post mortem e di cervello di topo umano. Questo metodo consente di eseguire sia l&#39;elettroforesi bidimensionale differenza di fluorescenza gel (2D-DIGE) e mini 2DE immunoblotting. La combinazione di questi approcci permette non solo di trovare nuove proteine ​​e / o modifiche proteine ​​nella loro espressione grazie alla compatibilità con rilevazione spettrometria di massa, ma anche una nuova comprensione convalida marcatori. Quindi, mini-2DE accoppiato ai permessi di Western blotting per identificare e convalidare postale-Traduzionali modifiche, proteine ​​catabolismo e fornisce un confronto qualitativo tra condizioni e / o trattamenti diversi. Qui, forniamo un metodo per studiare i componenti di aggregati proteici presenti in AD e demenza Lewy, come il peptide beta-amiloide e l&#39;alfa-sinucleina. Il nostro metodo può quindi essere adattato per l&#39;analisi del proteoma e proteine ​​insolubili estrarre dai modelli di tessuto cerebrale e topi umani. Parallelamente, essa può fornire informazioni utili per lo studio dei meccanismi molecolari e cellulari coinvolti nelle malattie neurodegenerative, nonché i potenziali nuovi biomarcatori e bersagli terapeutici.

Proteomica sul tessuto cerebrale rimane difficile poiché nessun tampone ideale esiste per recuperare il 100% di proteine, proteine ​​del citoscheletro soprattutto associata alla membrana o. La prima serie di esperimenti si sono concentrati sulla ricerca di un tampone di lisi adatto compatibile con i due approcci e che consente di recuperare un grande pannello di proteine. Così, tre buffer di lisi sono stati analizzati per determinare la più appropriata. In primo luogo, è stato utilizzato il buffer comune biologi...

Watch this Scientific Journal Video about Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting at JoVE.c

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

Un protocollo di estrazione proteina comune con tampone di urea / tiourea / SDS per il tessuto cerebrale umano e topi permette indentification delle proteine ​​da 2D-DIGE e la loro successiva caratterizzazione mediante mini 2DE immunoblotting. Questo metodo permette di ottenere risultati più riproducibili e affidabili da biopsie umane e modelli sperimentali.

Protein Consenso Brain-derived, estrazione protocollo per lo studio dei diritti dell&#39;uomo e murini Cervello Proteome Uso Sia 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 ...

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Neuroscience

15.9K Views.  Inserm UMR 837.  Un protocollo di estrazione proteina comune con tampone di urea / tiourea / SDS per il tessuto cerebrale umano e topi permette indentification delle proteine ​​da 2D-DIGE e la loro successiva caratterizzazione mediante mini 2DE immunoblotting. Questo metodo permette di ottenere risultati più riproducibili e affidabili da biopsie umane e modelli sperimentali. 

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

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: Utilizzo del Theta Burst protocollo Esplora Mechasnism di plasticità in individui con Sindrome X Fragile e autismo

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

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Neuroscienze

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

Metodi per lo studio della morfogenesi neuronale:<em&gt; Ex vivo</em&gt; Elettroporazione RNAi in Embryonic Cerebral Cortex Murine

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential Photo-sbiancante per Delineare cellule di Schwann singoli sulla giunzione neuromuscolare

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipolazione genetica delle cerebellare granuli neuroni<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Per studiare neuronale Morfologia e migrazione

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

Nuovi metodi per studiare la codifica gustativa

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

High-throughput analisi del comportamento motorio nell'analisi di Isola della drosofila

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

Isolamento e l'estrazione di RNA dei neuroni, macrofagi e Microglia da cervelli di Zebrafish larvale

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Extraction and Dissection of the Domesticated Pig Brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

Estrazione e dissezione del cervello di maiale addomesticato

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. Besides neuromelanin, NMGs contain a variety of proteins, lipids, and metals. Although NMGs-containing dopaminergic neurons are preferentially lost in neurodegenerative diseases like Parkinson&#39;s disease and dementia with Lewy bodies, only little is known about the mechanism of NMG formation and the role of NMGs in health and disease. Thus, further research on the molecular characterization of NMGs is essential. Unfortunately, standard protocols for the isolation of proteins are based on density gradient ultracentrifugation and therefore require high amounts of human tissue. Thus, an automated laser microdissection (LMD)-based protocol is established here which allows the collection of NMGs and surrounding substantia nigra (SN) tissue using minimal amounts of tissue in an unbiased, automatized way. Excised samples are subsequently analyzed by mass spectrometry to decipher their proteomic composition. With this workflow, 2,079 proteins were identified of which 514 proteins were exclusively identified in NMGs and 181 in SN. The present results have been compared with a previous study using a similar LMD-based approach reaching an overlap of 87.6% for both proteomes, verifying the applicability of the revised and optimized protocol presented here. To validate current findings, proteins of interest were analyzed by targeted mass spectrometry, e.g., parallel reaction monitoring (PRM)-experiments.

A robust protocol is presented here for isolating neuromelanin granules from human post-mortem substantia nigra pars compacta tissue via laser microdissection. This revised and optimized protocol massively minimizes the required time for sample collection, reduces the required sample amount, and enhances the identification and quantification of proteins by LC-MS/MS analysis.

Protocollo basato sulla microdissezione laser per l'analisi LC-MS/MS del profilo proteomico dei granuli di neuromelanina

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. ...

Rivoluzionare gli studi fNIRS: un nuovo approccio all'accoppiamento neurale

New Framework for Understanding Cross-Brain Coherence in Functional Near-Infrared Spectroscopy (fNIRS) Hyperscanning Studies

Despite the growing body of functional near-infrared spectroscopy (fNIRS) hyperscanning studies, the assessment of coupling between two neural signals using wavelet transform coherence (WTC) seems to ignore the directionality of the interaction. The field is currently lacking a framework that allows researchers to determine whether a high coherence value obtained using a WTC function reflects in-phase synchronization (i.e., neural activation is seen in both members of the dyad at the same time), lagged synchronization (i.e., neural activation is seen in one member of the dyad prior to the other member), or anti-phase synchronization (i.e., neural activation is increased in one member of the dyad and decreased in the other). To address this need, a complementary and more sensitive approach for analyzing the phase coherence of two neural signals is proposed in this work. The toolbox allows investigators to estimate the coupling directionality by classifying the phase angle values obtained using traditional WTC into in-phase synchronization, lagged synchronization, and anti-phase synchronization. The toolbox also allows researchers to assess how the dynamics of interactions develop and change throughout the task. Using this novel WTC approach and the toolbox will advance our understanding of complex social interactions through their uses in fNIRS hyperscanning studies.

Autore in primo piano: Sbloccare nuove intuizioni negli studi fNIRS - Un nuovo quadro per l'analisi della sincronia intercerebrale 

Wavelet transform coherence (WTC) is a common methodology for assessing the coupling between signals that is used in functional near-infrared spectroscopy (fNIRS) hyperscanning studies. A toolbox for assessing the directionality of the signal interaction is presented in this work.

Nuovo quadro per la comprensione della coerenza cross-brain negli studi di iperscansione della spettroscopia funzionale nel vicino infrarosso (fNIRS)

Despite the growing body of functional near-infrared spectroscopy (fNIRS) hyperscanning studies, the assessment of coupling between two neural signals ...