Meso scale discovery-based assays for the detection of aggregated huntingtin
Huntington's disease (HD) is a monogenic neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin (HTT) gene, leading to an expanded poly-glutamine (polyQ) stretch in the HTT protein. This mutant HTT (mHTT) protein is highly prone to intracellul...
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| Veröffentlicht in: | PloS one 2019-03, Vol.14 (3), p.e0213521-e0213521 |
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| creator | Reindl, Wolfgang Baldo, Barbara Schulz, Jana Janack, Isabell Lindner, Ilka Kleinschmidt, Markus Sedaghat, Yalda Thiede, Christina Tillack, Karsten Schmidt, Christina Cardaun, Isabell Schwagarus, Tom Herrmann, Frank Hotze, Madlen Osborne, Georgina F Herrmann, Simone Weiss, Andreas Zerbinatti, Celina Bates, Gillian P Bard, Jonathan Munoz-Sanjuan, Ignacio Macdonald, Douglas |
| description | Huntington's disease (HD) is a monogenic neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin (HTT) gene, leading to an expanded poly-glutamine (polyQ) stretch in the HTT protein. This mutant HTT (mHTT) protein is highly prone to intracellular aggregation, causing significant damage and cellular loss in the striatal, cortical, and other regions of the brain. Therefore, modulation of mHTT levels in these brain regions in order to reduce intracellular mHTT and aggregate levels represents a direct approach in the development of HD therapeutics. To this end, assays that can be used to detect changes in HTT levels in biological samples are invaluable tools to assess target engagement and guide dose selection in clinical trials. The Meso Scale Discovery (MSD) ELISA-based assay platform is a robust and sensitive method previously employed for the quantification of HTT. However, the currently available MSD assays for HTT are primarily detecting the monomeric soluble form of the protein, but not aggregated species. In this study, we describe the development of novel MSD assays preferentially detecting mHTT in an aggregated form. Recombinant monomeric HTT(1-97)-Q46, which forms aggregates in a time-dependent manner, was used to characterize the ability of each established assay to distinguish between HTT monomers and HTT in a higher assembly state. Further validation of these assays was performed using brain lysates from R6/2, zQ175 knock-in, and BACHD mouse models, to replicate a previously well-characterized age-dependent increase in brain aggregate signals, as well as a significant reduction of aggregate levels in the striatum following mHTT knockdown with a CAG-directed allele-specific zinc-finger repressor protein (ZFP). Lastly, size exclusion chromatography was used to separate and characterize HTT species from brain tissue lysates to demonstrate specificity of the assays for the fractions containing aggregated HTT. In summary, we demonstrate that the newly developed assays preferentially detect aggregated HTT with improved performance in comparison to previous assay technologies. These assays complement the existing MSD platform assays specific for soluble HTT monomers, allowing for a more comprehensive analysis of disease-relevant HTT species in preclinical models of HD. |
| doi_str_mv | 10.1371/journal.pone.0213521 |
| format | Article |
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This mutant HTT (mHTT) protein is highly prone to intracellular aggregation, causing significant damage and cellular loss in the striatal, cortical, and other regions of the brain. Therefore, modulation of mHTT levels in these brain regions in order to reduce intracellular mHTT and aggregate levels represents a direct approach in the development of HD therapeutics. To this end, assays that can be used to detect changes in HTT levels in biological samples are invaluable tools to assess target engagement and guide dose selection in clinical trials. The Meso Scale Discovery (MSD) ELISA-based assay platform is a robust and sensitive method previously employed for the quantification of HTT. However, the currently available MSD assays for HTT are primarily detecting the monomeric soluble form of the protein, but not aggregated species. In this study, we describe the development of novel MSD assays preferentially detecting mHTT in an aggregated form. Recombinant monomeric HTT(1-97)-Q46, which forms aggregates in a time-dependent manner, was used to characterize the ability of each established assay to distinguish between HTT monomers and HTT in a higher assembly state. Further validation of these assays was performed using brain lysates from R6/2, zQ175 knock-in, and BACHD mouse models, to replicate a previously well-characterized age-dependent increase in brain aggregate signals, as well as a significant reduction of aggregate levels in the striatum following mHTT knockdown with a CAG-directed allele-specific zinc-finger repressor protein (ZFP). Lastly, size exclusion chromatography was used to separate and characterize HTT species from brain tissue lysates to demonstrate specificity of the assays for the fractions containing aggregated HTT. In summary, we demonstrate that the newly developed assays preferentially detect aggregated HTT with improved performance in comparison to previous assay technologies. These assays complement the existing MSD platform assays specific for soluble HTT monomers, allowing for a more comprehensive analysis of disease-relevant HTT species in preclinical models of HD.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0213521</identifier><identifier>PMID: 30913220</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Age ; Alzheimer's disease ; Alzheimers disease ; Amino acids ; Animal models ; Animals ; Assaying ; Biological Assay ; Biological properties ; Biological samples ; Biology and Life Sciences ; Brain ; Brain - metabolism ; Change detection ; Chromatography ; Clinical trials ; Cortex ; Dementia ; Diagnosis ; Discovery and exploration ; Disease Models, Animal ; Drug development ; Economic models ; Enzyme-linked immunosorbent assay ; Genes ; Glutamine ; Humans ; Huntingtin ; Huntingtin Protein - genetics ; Huntingtin Protein - metabolism ; Huntington Disease - genetics ; Huntington Disease - metabolism ; Huntington Disease - pathology ; Huntington's disease ; Huntingtons disease ; Intracellular ; Lysates ; Medical research ; Medicine and Health Sciences ; Mice ; Mice, Transgenic ; Monomers ; Neostriatum ; Nervous system diseases ; Neurodegenerative diseases ; Neuropathology ; Peptides - genetics ; Peptides - metabolism ; Physical Sciences ; Polyglutamine ; Protein Aggregation, Pathological - genetics ; Protein Aggregation, Pathological - metabolism ; Protein Aggregation, Pathological - pathology ; Proteins ; Research and Analysis Methods ; Size exclusion chromatography ; Species ; Supervision ; Technology ; Therapeutics ; Time dependence ; Trinucleotide repeats ; University colleges ; Zinc ; Zinc finger proteins</subject><ispartof>PloS one, 2019-03, Vol.14 (3), p.e0213521-e0213521</ispartof><rights>COPYRIGHT 2019 Public Library of Science</rights><rights>2019 Reindl et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2019 Reindl et al 2019 Reindl et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-d0a88a44ed1334e6eef34c417ec43ea323804af8a912df9e80a5e7fb303ee1d53</citedby><cites>FETCH-LOGICAL-c692t-d0a88a44ed1334e6eef34c417ec43ea323804af8a912df9e80a5e7fb303ee1d53</cites><orcidid>0000-0001-8308-3179 ; 0000-0001-5592-5594</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435127/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435127/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,724,777,781,861,882,2096,2915,23847,27905,27906,53772,53774,79349,79350</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30913220$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Li, Xiao-Jiang</contributor><creatorcontrib>Reindl, Wolfgang</creatorcontrib><creatorcontrib>Baldo, Barbara</creatorcontrib><creatorcontrib>Schulz, Jana</creatorcontrib><creatorcontrib>Janack, Isabell</creatorcontrib><creatorcontrib>Lindner, Ilka</creatorcontrib><creatorcontrib>Kleinschmidt, Markus</creatorcontrib><creatorcontrib>Sedaghat, Yalda</creatorcontrib><creatorcontrib>Thiede, Christina</creatorcontrib><creatorcontrib>Tillack, Karsten</creatorcontrib><creatorcontrib>Schmidt, Christina</creatorcontrib><creatorcontrib>Cardaun, Isabell</creatorcontrib><creatorcontrib>Schwagarus, Tom</creatorcontrib><creatorcontrib>Herrmann, Frank</creatorcontrib><creatorcontrib>Hotze, Madlen</creatorcontrib><creatorcontrib>Osborne, Georgina F</creatorcontrib><creatorcontrib>Herrmann, Simone</creatorcontrib><creatorcontrib>Weiss, Andreas</creatorcontrib><creatorcontrib>Zerbinatti, Celina</creatorcontrib><creatorcontrib>Bates, Gillian P</creatorcontrib><creatorcontrib>Bard, Jonathan</creatorcontrib><creatorcontrib>Munoz-Sanjuan, Ignacio</creatorcontrib><creatorcontrib>Macdonald, Douglas</creatorcontrib><title>Meso scale discovery-based assays for the detection of aggregated huntingtin</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Huntington's disease (HD) is a monogenic neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin (HTT) gene, leading to an expanded poly-glutamine (polyQ) stretch in the HTT protein. This mutant HTT (mHTT) protein is highly prone to intracellular aggregation, causing significant damage and cellular loss in the striatal, cortical, and other regions of the brain. Therefore, modulation of mHTT levels in these brain regions in order to reduce intracellular mHTT and aggregate levels represents a direct approach in the development of HD therapeutics. To this end, assays that can be used to detect changes in HTT levels in biological samples are invaluable tools to assess target engagement and guide dose selection in clinical trials. The Meso Scale Discovery (MSD) ELISA-based assay platform is a robust and sensitive method previously employed for the quantification of HTT. However, the currently available MSD assays for HTT are primarily detecting the monomeric soluble form of the protein, but not aggregated species. In this study, we describe the development of novel MSD assays preferentially detecting mHTT in an aggregated form. Recombinant monomeric HTT(1-97)-Q46, which forms aggregates in a time-dependent manner, was used to characterize the ability of each established assay to distinguish between HTT monomers and HTT in a higher assembly state. Further validation of these assays was performed using brain lysates from R6/2, zQ175 knock-in, and BACHD mouse models, to replicate a previously well-characterized age-dependent increase in brain aggregate signals, as well as a significant reduction of aggregate levels in the striatum following mHTT knockdown with a CAG-directed allele-specific zinc-finger repressor protein (ZFP). Lastly, size exclusion chromatography was used to separate and characterize HTT species from brain tissue lysates to demonstrate specificity of the assays for the fractions containing aggregated HTT. In summary, we demonstrate that the newly developed assays preferentially detect aggregated HTT with improved performance in comparison to previous assay technologies. These assays complement the existing MSD platform assays specific for soluble HTT monomers, allowing for a more comprehensive analysis of disease-relevant HTT species in preclinical models of HD.</description><subject>Age</subject><subject>Alzheimer's disease</subject><subject>Alzheimers disease</subject><subject>Amino acids</subject><subject>Animal models</subject><subject>Animals</subject><subject>Assaying</subject><subject>Biological Assay</subject><subject>Biological properties</subject><subject>Biological samples</subject><subject>Biology and Life Sciences</subject><subject>Brain</subject><subject>Brain - metabolism</subject><subject>Change detection</subject><subject>Chromatography</subject><subject>Clinical trials</subject><subject>Cortex</subject><subject>Dementia</subject><subject>Diagnosis</subject><subject>Discovery and exploration</subject><subject>Disease Models, Animal</subject><subject>Drug development</subject><subject>Economic models</subject><subject>Enzyme-linked immunosorbent assay</subject><subject>Genes</subject><subject>Glutamine</subject><subject>Humans</subject><subject>Huntingtin</subject><subject>Huntingtin Protein - genetics</subject><subject>Huntingtin Protein - metabolism</subject><subject>Huntington Disease - genetics</subject><subject>Huntington Disease - metabolism</subject><subject>Huntington Disease - pathology</subject><subject>Huntington's disease</subject><subject>Huntingtons disease</subject><subject>Intracellular</subject><subject>Lysates</subject><subject>Medical research</subject><subject>Medicine and Health Sciences</subject><subject>Mice</subject><subject>Mice, Transgenic</subject><subject>Monomers</subject><subject>Neostriatum</subject><subject>Nervous system diseases</subject><subject>Neurodegenerative diseases</subject><subject>Neuropathology</subject><subject>Peptides - genetics</subject><subject>Peptides - metabolism</subject><subject>Physical Sciences</subject><subject>Polyglutamine</subject><subject>Protein Aggregation, Pathological - genetics</subject><subject>Protein Aggregation, Pathological - metabolism</subject><subject>Protein Aggregation, Pathological - pathology</subject><subject>Proteins</subject><subject>Research and Analysis Methods</subject><subject>Size exclusion chromatography</subject><subject>Species</subject><subject>Supervision</subject><subject>Technology</subject><subject>Therapeutics</subject><subject>Time dependence</subject><subject>Trinucleotide repeats</subject><subject>University colleges</subject><subject>Zinc</subject><subject>Zinc finger 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scale discovery-based assays for the detection of aggregated huntingtin</title><author>Reindl, Wolfgang ; Baldo, Barbara ; Schulz, Jana ; Janack, Isabell ; Lindner, Ilka ; Kleinschmidt, Markus ; Sedaghat, Yalda ; Thiede, Christina ; Tillack, Karsten ; Schmidt, Christina ; Cardaun, Isabell ; Schwagarus, Tom ; Herrmann, Frank ; Hotze, Madlen ; Osborne, Georgina F ; Herrmann, Simone ; Weiss, Andreas ; Zerbinatti, Celina ; Bates, Gillian P ; Bard, Jonathan ; Munoz-Sanjuan, Ignacio ; Macdonald, Douglas</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-d0a88a44ed1334e6eef34c417ec43ea323804af8a912df9e80a5e7fb303ee1d53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Age</topic><topic>Alzheimer's disease</topic><topic>Alzheimers disease</topic><topic>Amino acids</topic><topic>Animal models</topic><topic>Animals</topic><topic>Assaying</topic><topic>Biological 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P</creatorcontrib><creatorcontrib>Bard, Jonathan</creatorcontrib><creatorcontrib>Munoz-Sanjuan, Ignacio</creatorcontrib><creatorcontrib>Macdonald, Douglas</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical 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Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - Academic</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Nursing & Allied Health Premium</collection><collection>Advanced Technologies & Aerospace Database</collection><collection>ProQuest Advanced Technologies & Aerospace Collection</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>Materials Science Collection</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Engineering Collection</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Reindl, Wolfgang</au><au>Baldo, Barbara</au><au>Schulz, Jana</au><au>Janack, Isabell</au><au>Lindner, Ilka</au><au>Kleinschmidt, Markus</au><au>Sedaghat, Yalda</au><au>Thiede, Christina</au><au>Tillack, Karsten</au><au>Schmidt, Christina</au><au>Cardaun, Isabell</au><au>Schwagarus, Tom</au><au>Herrmann, Frank</au><au>Hotze, Madlen</au><au>Osborne, Georgina F</au><au>Herrmann, Simone</au><au>Weiss, Andreas</au><au>Zerbinatti, Celina</au><au>Bates, Gillian P</au><au>Bard, Jonathan</au><au>Munoz-Sanjuan, Ignacio</au><au>Macdonald, Douglas</au><au>Li, Xiao-Jiang</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Meso scale discovery-based assays for the detection of aggregated huntingtin</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2019-03-26</date><risdate>2019</risdate><volume>14</volume><issue>3</issue><spage>e0213521</spage><epage>e0213521</epage><pages>e0213521-e0213521</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Huntington's disease (HD) is a monogenic neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin (HTT) gene, leading to an expanded poly-glutamine (polyQ) stretch in the HTT protein. This mutant HTT (mHTT) protein is highly prone to intracellular aggregation, causing significant damage and cellular loss in the striatal, cortical, and other regions of the brain. Therefore, modulation of mHTT levels in these brain regions in order to reduce intracellular mHTT and aggregate levels represents a direct approach in the development of HD therapeutics. To this end, assays that can be used to detect changes in HTT levels in biological samples are invaluable tools to assess target engagement and guide dose selection in clinical trials. The Meso Scale Discovery (MSD) ELISA-based assay platform is a robust and sensitive method previously employed for the quantification of HTT. However, the currently available MSD assays for HTT are primarily detecting the monomeric soluble form of the protein, but not aggregated species. In this study, we describe the development of novel MSD assays preferentially detecting mHTT in an aggregated form. Recombinant monomeric HTT(1-97)-Q46, which forms aggregates in a time-dependent manner, was used to characterize the ability of each established assay to distinguish between HTT monomers and HTT in a higher assembly state. Further validation of these assays was performed using brain lysates from R6/2, zQ175 knock-in, and BACHD mouse models, to replicate a previously well-characterized age-dependent increase in brain aggregate signals, as well as a significant reduction of aggregate levels in the striatum following mHTT knockdown with a CAG-directed allele-specific zinc-finger repressor protein (ZFP). Lastly, size exclusion chromatography was used to separate and characterize HTT species from brain tissue lysates to demonstrate specificity of the assays for the fractions containing aggregated HTT. In summary, we demonstrate that the newly developed assays preferentially detect aggregated HTT with improved performance in comparison to previous assay technologies. These assays complement the existing MSD platform assays specific for soluble HTT monomers, allowing for a more comprehensive analysis of disease-relevant HTT species in preclinical models of HD.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>30913220</pmid><doi>10.1371/journal.pone.0213521</doi><tpages>e0213521</tpages><orcidid>https://orcid.org/0000-0001-8308-3179</orcidid><orcidid>https://orcid.org/0000-0001-5592-5594</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2019-03, Vol.14 (3), p.e0213521-e0213521 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2199462975 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS); PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Age Alzheimer's disease Alzheimers disease Amino acids Animal models Animals Assaying Biological Assay Biological properties Biological samples Biology and Life Sciences Brain Brain - metabolism Change detection Chromatography Clinical trials Cortex Dementia Diagnosis Discovery and exploration Disease Models, Animal Drug development Economic models Enzyme-linked immunosorbent assay Genes Glutamine Humans Huntingtin Huntingtin Protein - genetics Huntingtin Protein - metabolism Huntington Disease - genetics Huntington Disease - metabolism Huntington Disease - pathology Huntington's disease Huntingtons disease Intracellular Lysates Medical research Medicine and Health Sciences Mice Mice, Transgenic Monomers Neostriatum Nervous system diseases Neurodegenerative diseases Neuropathology Peptides - genetics Peptides - metabolism Physical Sciences Polyglutamine Protein Aggregation, Pathological - genetics Protein Aggregation, Pathological - metabolism Protein Aggregation, Pathological - pathology Proteins Research and Analysis Methods Size exclusion chromatography Species Supervision Technology Therapeutics Time dependence Trinucleotide repeats University colleges Zinc Zinc finger proteins |
| title | Meso scale discovery-based assays for the detection of aggregated huntingtin |
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