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Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (Aβ, tau), Parkinson's Disease (α-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggr...

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Autores principales: Bader, Verian, Ottis, Philipp, Pum, Martin, Huston, Joseph P., Korth, Carsten
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486765/
https://www.ncbi.nlm.nih.gov/pubmed/22952026
http://dx.doi.org/10.3791/4132
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author Bader, Verian
Ottis, Philipp
Pum, Martin
Huston, Joseph P.
Korth, Carsten
author_facet Bader, Verian
Ottis, Philipp
Pum, Martin
Huston, Joseph P.
Korth, Carsten
author_sort Bader, Verian
collection PubMed
description Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (Aβ, tau), Parkinson's Disease (α-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms. Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease(1, 2), could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia(3). Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C(4), and that DISC1 aggresomes generated in vitro were cell-invasive(5), similar to what had been shown for Aβ(6), tau(7-9), α-synuclein(10), polyglutamine(11), or SOD1 aggregates(12). These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders. Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic α-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.
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spelling pubmed-34867652012-11-06 Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species Bader, Verian Ottis, Philipp Pum, Martin Huston, Joseph P. Korth, Carsten J Vis Exp Molecular Biology Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (Aβ, tau), Parkinson's Disease (α-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms. Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease(1, 2), could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia(3). Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C(4), and that DISC1 aggresomes generated in vitro were cell-invasive(5), similar to what had been shown for Aβ(6), tau(7-9), α-synuclein(10), polyglutamine(11), or SOD1 aggregates(12). These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders. Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic α-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals. MyJove Corporation 2012-08-30 /pmc/articles/PMC3486765/ /pubmed/22952026 http://dx.doi.org/10.3791/4132 Text en Copyright © 2012, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Molecular Biology
Bader, Verian
Ottis, Philipp
Pum, Martin
Huston, Joseph P.
Korth, Carsten
Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
title Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
title_full Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
title_fullStr Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
title_full_unstemmed Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
title_short Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
title_sort generation, purification, and characterization of cell-invasive disc1 protein species
topic Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486765/
https://www.ncbi.nlm.nih.gov/pubmed/22952026
http://dx.doi.org/10.3791/4132
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