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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.

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Decrease of soluble mutant HTT levels in R6/2 brain tissues is associated with increased age of the mice.(A) Homogenates from brains of R6/2 female mice obtained from The Jackson Laboratory (bearing an expanded polyglutamine tract of 120 CAG repeats on average) were analyzed for detection of soluble mutant human HTT at different ages (4, 8 and 12 weeks). Tissues analyzed showed significant signals in the expanded polyglutamine human HTT MSD assay (antibody pair pAb146/MW1) and a significant signal decrease between 4 and 8 weeks of age. Data are averages of n = 4 independent samples with correspondent standard deviations. B, assay background. ***, P<0.001. (B) SDS-PAGE and immunoblotting for HTT with the MW8 antibody reveals high molecular weight bands (presumably HTT aggregates) in R6/2 brain homogenates that increase with increasing age of the animals. Progressive decrease of soluble monomeric HTT fragments can be observed. WT, wild type (CBA×C57Bl/6) F1 (CBF) (B6CBAF1/OlaHsd, Harlan Olac) mice.
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pone-0096854-g004: Decrease of soluble mutant HTT levels in R6/2 brain tissues is associated with increased age of the mice.(A) Homogenates from brains of R6/2 female mice obtained from The Jackson Laboratory (bearing an expanded polyglutamine tract of 120 CAG repeats on average) were analyzed for detection of soluble mutant human HTT at different ages (4, 8 and 12 weeks). Tissues analyzed showed significant signals in the expanded polyglutamine human HTT MSD assay (antibody pair pAb146/MW1) and a significant signal decrease between 4 and 8 weeks of age. Data are averages of n = 4 independent samples with correspondent standard deviations. B, assay background. ***, P<0.001. (B) SDS-PAGE and immunoblotting for HTT with the MW8 antibody reveals high molecular weight bands (presumably HTT aggregates) in R6/2 brain homogenates that increase with increasing age of the animals. Progressive decrease of soluble monomeric HTT fragments can be observed. WT, wild type (CBA×C57Bl/6) F1 (CBF) (B6CBAF1/OlaHsd, Harlan Olac) mice.

Mentions: Brain tissue homogenates obtained from 4, 8 and 12 week-old R6/2 mice were analyzed using the expanded polyglutamine human HTT MSD assay. This transgenic mouse model contains a 1.9-kb fragment encompassing the human HTT promoter and exon-1 [22] bearing an expanded polyglutamine tract of 120 CAG repeats on average in the colony used. The R6/2 mice exhibit both early and severe behavioral and anatomical symptoms [19], [22]. Significant signals were observed for all transgenic R6/2 tissue samples analyzed (Figure 4A). In addition, tissue homogenates showed a decreased signal with increased age of the R6/2 mice. We measured a 2-fold signal decrease for 8 week-old brains compared with 4 week-old R6/2 tissues, whereas no additional signal reduction was seen for 12 week-old R6/2 samples. This signal decrease is most likely caused by a shift of mutant HTT exon-1 fragment protein from a soluble to aggregated form as a function of disease progression as previously reported [36] and was also observed in immunoblot analysis of these tissues (Figure 4B). Similar results were seen when micro-dissected cortex, striatum and brain stem tissue homogenates obtained from 4, 8, 12, and 15 week-old R6/2 mice of a different colony (bearing an expanded polyglutamine tract of 206 CAG repeats on average) were analyzed using the expanded polyglutamine human HTT MSD assay (Figure S5). The overall signals observed for cortex and striatum samples were comparable, whereas those detected for the brain stem samples were lower, especially for the 4 and 8 week time-points, suggesting lower levels of HTT exon-1 expression in this tissue. The most significant signal decrease was observed up to 12 weeks where we we measured a 2.7-, 3-, and 2-fold signal decrease respectively for 12 week-old cortex, striatum, and brain stem tissues compared with 4 week-old R6/2 tissues. The signals detected for 12 and 15 week time-points were comparable for all tissues tested. These data demonstrated that the pAb146-MW1 MSD HTT assay is specific for predominantly soluble forms of mutant HTT.


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)

Decrease of soluble mutant HTT levels in R6/2 brain tissues is associated with increased age of the mice.(A) Homogenates from brains of R6/2 female mice obtained from The Jackson Laboratory (bearing an expanded polyglutamine tract of 120 CAG repeats on average) were analyzed for detection of soluble mutant human HTT at different ages (4, 8 and 12 weeks). Tissues analyzed showed significant signals in the expanded polyglutamine human HTT MSD assay (antibody pair pAb146/MW1) and a significant signal decrease between 4 and 8 weeks of age. Data are averages of n = 4 independent samples with correspondent standard deviations. B, assay background. ***, P<0.001. (B) SDS-PAGE and immunoblotting for HTT with the MW8 antibody reveals high molecular weight bands (presumably HTT aggregates) in R6/2 brain homogenates that increase with increasing age of the animals. Progressive decrease of soluble monomeric HTT fragments can be observed. WT, wild type (CBA×C57Bl/6) F1 (CBF) (B6CBAF1/OlaHsd, Harlan Olac) mice.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096854-g004: Decrease of soluble mutant HTT levels in R6/2 brain tissues is associated with increased age of the mice.(A) Homogenates from brains of R6/2 female mice obtained from The Jackson Laboratory (bearing an expanded polyglutamine tract of 120 CAG repeats on average) were analyzed for detection of soluble mutant human HTT at different ages (4, 8 and 12 weeks). Tissues analyzed showed significant signals in the expanded polyglutamine human HTT MSD assay (antibody pair pAb146/MW1) and a significant signal decrease between 4 and 8 weeks of age. Data are averages of n = 4 independent samples with correspondent standard deviations. B, assay background. ***, P<0.001. (B) SDS-PAGE and immunoblotting for HTT with the MW8 antibody reveals high molecular weight bands (presumably HTT aggregates) in R6/2 brain homogenates that increase with increasing age of the animals. Progressive decrease of soluble monomeric HTT fragments can be observed. WT, wild type (CBA×C57Bl/6) F1 (CBF) (B6CBAF1/OlaHsd, Harlan Olac) mice.
Mentions: Brain tissue homogenates obtained from 4, 8 and 12 week-old R6/2 mice were analyzed using the expanded polyglutamine human HTT MSD assay. This transgenic mouse model contains a 1.9-kb fragment encompassing the human HTT promoter and exon-1 [22] bearing an expanded polyglutamine tract of 120 CAG repeats on average in the colony used. The R6/2 mice exhibit both early and severe behavioral and anatomical symptoms [19], [22]. Significant signals were observed for all transgenic R6/2 tissue samples analyzed (Figure 4A). In addition, tissue homogenates showed a decreased signal with increased age of the R6/2 mice. We measured a 2-fold signal decrease for 8 week-old brains compared with 4 week-old R6/2 tissues, whereas no additional signal reduction was seen for 12 week-old R6/2 samples. This signal decrease is most likely caused by a shift of mutant HTT exon-1 fragment protein from a soluble to aggregated form as a function of disease progression as previously reported [36] and was also observed in immunoblot analysis of these tissues (Figure 4B). Similar results were seen when micro-dissected cortex, striatum and brain stem tissue homogenates obtained from 4, 8, 12, and 15 week-old R6/2 mice of a different colony (bearing an expanded polyglutamine tract of 206 CAG repeats on average) were analyzed using the expanded polyglutamine human HTT MSD assay (Figure S5). The overall signals observed for cortex and striatum samples were comparable, whereas those detected for the brain stem samples were lower, especially for the 4 and 8 week time-points, suggesting lower levels of HTT exon-1 expression in this tissue. The most significant signal decrease was observed up to 12 weeks where we we measured a 2.7-, 3-, and 2-fold signal decrease respectively for 12 week-old cortex, striatum, and brain stem tissues compared with 4 week-old R6/2 tissues. The signals detected for 12 and 15 week time-points were comparable for all tissues tested. These data demonstrated that the pAb146-MW1 MSD HTT assay is specific for predominantly soluble forms of mutant HTT.

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