Limits...
Quantification assays for total and polyglutamine-expanded huntingtin proteins.

Macdonald D, Tessari MA, Boogaard I, Smith M, Pulli K, Szynol A, Albertus F, Lamers MB, Dijkstra S, Kordt D, Reindl W, Herrmann F, McAllister G, Fischer DF, Munoz-Sanjuan I - PLoS ONE (2014)

Bottom Line: In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions.We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples.Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.

View Article: PubMed Central - PubMed

Affiliation: CHDI Management/CHDI Foundation, Los Angeles, California, United States of America.

ABSTRACT
The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.

Show MeSH

Related in: MedlinePlus

Anti-HTT antibody epitopes and analysis by immunoblot.(A) Diagram representing antibody epitopes on human HTT protein (relative to GenBank accession CAD38447.1). A stretch of glutamine (Q) residues near the N-terminus is expanded in individuals affected by Huntington's disease. Amino-acid 1-92 encoded by exon-1 are shown. Blue, pAb147 antibody epitope on mouse HTT protein (GenBank accession NP_034544). (B) Immunoblot analysis of human large fragment recombinant HTT proteins detected by the indicated anti-huntingtin antibodies. 5 ng of both HTT (1–573) Q23 (lanes 1, 3, 5, 7, 9, 11, 13 and 15) and HTT (1–573) Q73 (lanes 2, 4, 6, 8, 10, 12, 14, and 16) purified large fragment proteins were analyzed by SDS-PAGE. M, molecular weight marker (kDa). (C) Immunoblot analysis of wild type littermate and transgenic BAC HD mouse whole brain extracts. Mouse endogenous wild type and human transgenic polyglutamine-expanded HTT proteins were detected using the indicated anti-huntingtin antibodies. 25 µg of normal (wt) and transgenic (tg) mouse brain extracts were analyzed by SDS-PAGE as indicated. M, molecular weight marker (kDa). Tg mHTT, transgenic human polyglutamine-expanded HTT. WT HTT, endogenous mouse wild type HTT.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4016121&req=5

pone-0096854-g001: Anti-HTT antibody epitopes and analysis by immunoblot.(A) Diagram representing antibody epitopes on human HTT protein (relative to GenBank accession CAD38447.1). A stretch of glutamine (Q) residues near the N-terminus is expanded in individuals affected by Huntington's disease. Amino-acid 1-92 encoded by exon-1 are shown. Blue, pAb147 antibody epitope on mouse HTT protein (GenBank accession NP_034544). (B) Immunoblot analysis of human large fragment recombinant HTT proteins detected by the indicated anti-huntingtin antibodies. 5 ng of both HTT (1–573) Q23 (lanes 1, 3, 5, 7, 9, 11, 13 and 15) and HTT (1–573) Q73 (lanes 2, 4, 6, 8, 10, 12, 14, and 16) purified large fragment proteins were analyzed by SDS-PAGE. M, molecular weight marker (kDa). (C) Immunoblot analysis of wild type littermate and transgenic BAC HD mouse whole brain extracts. Mouse endogenous wild type and human transgenic polyglutamine-expanded HTT proteins were detected using the indicated anti-huntingtin antibodies. 25 µg of normal (wt) and transgenic (tg) mouse brain extracts were analyzed by SDS-PAGE as indicated. M, molecular weight marker (kDa). Tg mHTT, transgenic human polyglutamine-expanded HTT. WT HTT, endogenous mouse wild type HTT.

Mentions: We characterized a panel of anti-HTT antibodies raised to different epitopes of the HTT protein (Figure 1A and Table S1 in Text S1) by immunoblot using purified large fragment (HTT 1–573) recombinant HTT proteins with different CAG repeat lengths (Figure 1B) as well as brain homogenates obtained from 3 month-old BAC HD mice or wild type littermates (Figure 1C). BAC HD mice express full-length human mutant HTT with 97 glutamine repeats under the control of the endogenous Htt regulatory machinery [23]. This animal model recapitulates several key phenotypic features of HD (e.g. late onset, atrophy of the cortex and striatum).


Quantification assays for total and polyglutamine-expanded huntingtin proteins.

Macdonald D, Tessari MA, Boogaard I, Smith M, Pulli K, Szynol A, Albertus F, Lamers MB, Dijkstra S, Kordt D, Reindl W, Herrmann F, McAllister G, Fischer DF, Munoz-Sanjuan I - PLoS ONE (2014)

Anti-HTT antibody epitopes and analysis by immunoblot.(A) Diagram representing antibody epitopes on human HTT protein (relative to GenBank accession CAD38447.1). A stretch of glutamine (Q) residues near the N-terminus is expanded in individuals affected by Huntington's disease. Amino-acid 1-92 encoded by exon-1 are shown. Blue, pAb147 antibody epitope on mouse HTT protein (GenBank accession NP_034544). (B) Immunoblot analysis of human large fragment recombinant HTT proteins detected by the indicated anti-huntingtin antibodies. 5 ng of both HTT (1–573) Q23 (lanes 1, 3, 5, 7, 9, 11, 13 and 15) and HTT (1–573) Q73 (lanes 2, 4, 6, 8, 10, 12, 14, and 16) purified large fragment proteins were analyzed by SDS-PAGE. M, molecular weight marker (kDa). (C) Immunoblot analysis of wild type littermate and transgenic BAC HD mouse whole brain extracts. Mouse endogenous wild type and human transgenic polyglutamine-expanded HTT proteins were detected using the indicated anti-huntingtin antibodies. 25 µg of normal (wt) and transgenic (tg) mouse brain extracts were analyzed by SDS-PAGE as indicated. M, molecular weight marker (kDa). Tg mHTT, transgenic human polyglutamine-expanded HTT. WT HTT, endogenous mouse wild type HTT.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016121&req=5

pone-0096854-g001: Anti-HTT antibody epitopes and analysis by immunoblot.(A) Diagram representing antibody epitopes on human HTT protein (relative to GenBank accession CAD38447.1). A stretch of glutamine (Q) residues near the N-terminus is expanded in individuals affected by Huntington's disease. Amino-acid 1-92 encoded by exon-1 are shown. Blue, pAb147 antibody epitope on mouse HTT protein (GenBank accession NP_034544). (B) Immunoblot analysis of human large fragment recombinant HTT proteins detected by the indicated anti-huntingtin antibodies. 5 ng of both HTT (1–573) Q23 (lanes 1, 3, 5, 7, 9, 11, 13 and 15) and HTT (1–573) Q73 (lanes 2, 4, 6, 8, 10, 12, 14, and 16) purified large fragment proteins were analyzed by SDS-PAGE. M, molecular weight marker (kDa). (C) Immunoblot analysis of wild type littermate and transgenic BAC HD mouse whole brain extracts. Mouse endogenous wild type and human transgenic polyglutamine-expanded HTT proteins were detected using the indicated anti-huntingtin antibodies. 25 µg of normal (wt) and transgenic (tg) mouse brain extracts were analyzed by SDS-PAGE as indicated. M, molecular weight marker (kDa). Tg mHTT, transgenic human polyglutamine-expanded HTT. WT HTT, endogenous mouse wild type HTT.
Mentions: We characterized a panel of anti-HTT antibodies raised to different epitopes of the HTT protein (Figure 1A and Table S1 in Text S1) by immunoblot using purified large fragment (HTT 1–573) recombinant HTT proteins with different CAG repeat lengths (Figure 1B) as well as brain homogenates obtained from 3 month-old BAC HD mice or wild type littermates (Figure 1C). BAC HD mice express full-length human mutant HTT with 97 glutamine repeats under the control of the endogenous Htt regulatory machinery [23]. This animal model recapitulates several key phenotypic features of HD (e.g. late onset, atrophy of the cortex and striatum).

Bottom Line: In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions.We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples.Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.

View Article: PubMed Central - PubMed

Affiliation: CHDI Management/CHDI Foundation, Los Angeles, California, United States of America.

ABSTRACT
The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.

Show MeSH
Related in: MedlinePlus