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Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells.

Massai L, Petricca L, Magnoni L, Rovetini L, Haider S, Andre R, Tabrizi SJ, Süssmuth SD, Landwehrmeyer BG, Caricasole A, Pollio G, Bernocco S - BMC Biochem. (2013)

Bottom Line: Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD.An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples.The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharmacology Department, Siena Biotech SpA, Strada del Petriccio e Belriguardo, 35, 53100 Siena, Italy. lmassai@sienabiotech.it.

ABSTRACT

Background: Huntington's disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD.

Results: An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course.

Conclusions: The method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials.

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Pharmacological assay validation. Panel A and B, Effect of HSP90 overexpression on HTT protein (by ELISA) and gene (by RT-PCR) respectively. Panel C and D, Effect of HSP90 inhibition by NVP-AUY922 on HTT protein (by ELISA) and gene (by RT-PCR) respectively. Readings are normalized against Ctrl or DMSO as appropriate; statistical analyses are performed with two-way ANOVA followed by Bonferroni test for multiple comparisons (* p < 0.05, ** p < 0.01 wrt Ctrl or DMSO).
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Figure 3: Pharmacological assay validation. Panel A and B, Effect of HSP90 overexpression on HTT protein (by ELISA) and gene (by RT-PCR) respectively. Panel C and D, Effect of HSP90 inhibition by NVP-AUY922 on HTT protein (by ELISA) and gene (by RT-PCR) respectively. Readings are normalized against Ctrl or DMSO as appropriate; statistical analyses are performed with two-way ANOVA followed by Bonferroni test for multiple comparisons (* p < 0.05, ** p < 0.01 wrt Ctrl or DMSO).

Mentions: As inhibitors of HSP90 have been demonstrated to modulate mHTT steady state levels in cellular systems [29], we decided to validate our assay by assessing the detection of soluble HTT in complex matrices following pharmacological modulation. Firstly we verified that co-expression of HSP90 with wild-type and mutant HTT significantly increased the levels of HTT detected by the assay in total cell lysates (Figure 3A). This effect is exerted at protein level, as no increase in either HTT-Q138 or HTT-Q17 mRNA was observed by real-time qPCR and paradoxically, HTT-Q138 mRNA was reduced (Figure 3B). For pharmacological modulation, cells were treated for 24 hours with NVP-AUY922, a small molecule known to be a potent HSP90 inhibitor [30]. Upon modulation of HSP90 activity, we observed a significant reduction in soluble HTT protein irrespective of the presence of the expanded polyQ stretch (Figure 3C). Interestingly this treatment not only reduced the soluble protein, but also induced the expression of its mRNA as shown by RT-PCR analysis (Figure 3D). In summary, the pharmacological validation of the assay demonstrated its capacity to detect, in a significant manner, small variations in soluble HTT levels induced by inhibition of an enzyme modulating protein degradation.


Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells.

Massai L, Petricca L, Magnoni L, Rovetini L, Haider S, Andre R, Tabrizi SJ, Süssmuth SD, Landwehrmeyer BG, Caricasole A, Pollio G, Bernocco S - BMC Biochem. (2013)

Pharmacological assay validation. Panel A and B, Effect of HSP90 overexpression on HTT protein (by ELISA) and gene (by RT-PCR) respectively. Panel C and D, Effect of HSP90 inhibition by NVP-AUY922 on HTT protein (by ELISA) and gene (by RT-PCR) respectively. Readings are normalized against Ctrl or DMSO as appropriate; statistical analyses are performed with two-way ANOVA followed by Bonferroni test for multiple comparisons (* p < 0.05, ** p < 0.01 wrt Ctrl or DMSO).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221641&req=5

Figure 3: Pharmacological assay validation. Panel A and B, Effect of HSP90 overexpression on HTT protein (by ELISA) and gene (by RT-PCR) respectively. Panel C and D, Effect of HSP90 inhibition by NVP-AUY922 on HTT protein (by ELISA) and gene (by RT-PCR) respectively. Readings are normalized against Ctrl or DMSO as appropriate; statistical analyses are performed with two-way ANOVA followed by Bonferroni test for multiple comparisons (* p < 0.05, ** p < 0.01 wrt Ctrl or DMSO).
Mentions: As inhibitors of HSP90 have been demonstrated to modulate mHTT steady state levels in cellular systems [29], we decided to validate our assay by assessing the detection of soluble HTT in complex matrices following pharmacological modulation. Firstly we verified that co-expression of HSP90 with wild-type and mutant HTT significantly increased the levels of HTT detected by the assay in total cell lysates (Figure 3A). This effect is exerted at protein level, as no increase in either HTT-Q138 or HTT-Q17 mRNA was observed by real-time qPCR and paradoxically, HTT-Q138 mRNA was reduced (Figure 3B). For pharmacological modulation, cells were treated for 24 hours with NVP-AUY922, a small molecule known to be a potent HSP90 inhibitor [30]. Upon modulation of HSP90 activity, we observed a significant reduction in soluble HTT protein irrespective of the presence of the expanded polyQ stretch (Figure 3C). Interestingly this treatment not only reduced the soluble protein, but also induced the expression of its mRNA as shown by RT-PCR analysis (Figure 3D). In summary, the pharmacological validation of the assay demonstrated its capacity to detect, in a significant manner, small variations in soluble HTT levels induced by inhibition of an enzyme modulating protein degradation.

Bottom Line: Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD.An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples.The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharmacology Department, Siena Biotech SpA, Strada del Petriccio e Belriguardo, 35, 53100 Siena, Italy. lmassai@sienabiotech.it.

ABSTRACT

Background: Huntington's disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD.

Results: An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course.

Conclusions: The method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials.

Show MeSH
Related in: MedlinePlus