Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

Macdonald, Douglas; Tessari, Michela; Boogaard, Ivette; Smith, Melanie; Pulli, Kristiina; Szynol, Agnieszka; Albertus, Faywell; Lamers, Marieke; Dijkstra, Sipke; Kordt, Daniel; Reindl, Wolfgang; Herrmann, Frank; McAllister, George; Fischer, David F.; Muñoz-Sanjuán, Ignacio

doi:10.1371/journal.pone.0096854

Cited by 56 publications

(97 citation statements)

References 42 publications

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“…However, the TR-FRET assay did not detect HTT in knock-in mice harboring a humanized HTT exon 1 with a CAG 7 repeat (Q7/Q7) 41 , as expected and previously reported 23 . Although we had no access to alternative mouse models expressing a wild type HTT with a polyQ length larger than Q7 and a corresponding mutant HTT protein, samples were available from a characterized Drosophila HD model expressing a large Amino-terminal (N469) fragment of human HTT with either Q25 or Q120 9 .…”

Section: Resultssupporting

confidence: 87%

Polyglutamine expansion affects huntingtin conformation in multiple Huntington’s disease models

Daldin

Fodale

Cariulo

et al. 2017

Sci Rep

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Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington’s disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine- and temperature-dependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperature- and polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients.

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Section: Resultssupporting

confidence: 87%

Polyglutamine expansion affects huntingtin conformation in multiple Huntington’s disease models

Daldin

Fodale

Cariulo

et al. 2017

Sci Rep

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“…We found marked selectivity for mutant HTT over WT HTT (Figure 1, A and B). This specificity is conferred by the MW1 anti-HTT polyQ antibody, as previously demonstrated in other assay platforms (11,12). The 2B7 antibody, meanwhile, binds the N-terminal 17 amino acids of mutant and WT HTT (13).…”

Section: Resultsmentioning

confidence: 55%

Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington’s disease patients

Wild¹,

Boggio²,

Langbehn³

et al. 2015

J. Clin. Invest.

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221

236

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BACKGROUND:Quantification of disease-associated proteins in the cerebrospinal fluid (CSF) has been critical for the study and treatment of several neurodegenerative disorders; however, mutant huntingtin protein (mHTT), the cause of Huntington's disease (HD), is at very low levels in CSF and, to our knowledge, has never been measured previously. METHODS:We developed an ultrasensitive single-molecule counting (SMC) mHTT immunoassay that was used to quantify mHTT levels in CSF samples from individuals bearing the HD mutation and from control individuals in 2 independent cohorts. RESULTS:This SMC mHTT immunoassay demonstrated high specificity for mHTT, high sensitivity with a femtomolar detection threshold, and a broad dynamic range. Analysis of the CSF samples showed that mHTT was undetectable in CSF from all controls but quantifiable in nearly all mutation carriers. The mHTT concentration in CSF was approximately 3-fold higher in patients with manifest HD than in premanifest mutation carriers. Moreover, mHTT levels increased as the disease progressed and were associated with 5-year onset probability. The mHTT concentration independently predicted cognitive and motor dysfunction. Furthermore, the level of mHTT was associated with the concentrations of tau and neurofilament light chain in the CSF, suggesting a neuronal origin for the detected mHTT. CONCLUSIONS:We have demonstrated that mHTT can be quantified in CSF from HD patients using the described SMC mHTT immunoassay. Moreover, the level of mHTT detected is associated with proximity to disease onset and diminished cognitive and motor function. The ability to quantify CSF mHTT will facilitate the study of HD, and mHTT quantification could potentially serve as a biomarker for the development and testing of experimental mHTT-lowering therapies for HD.

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“…In patients with Huntington's disease, polyQ-expanded HTT exon 1-encoded protein fragments accumulate in compact inclusion bodies inside the neurons (42). Macdonald et al (39) have provided a global value of the concentration of HTT averaged over the whole brain: 0.15 µM. According to this calculation at this concentration, the aggregation free energy profile would be very uphill, even in fulllength constructs containing longer repeats: NT17-Q40-P10 (Fig.…”

Section: Discussionmentioning

confidence: 98%

“…Within the cytosol, the concentration has been estimated to be around 0.15 µM. We can also estimate the concentration of HTT exon 1-encoded fragments enriched inside an inclusion body in brain tissue from multiple experiments (as we describe in SI Appendix) (3,38,39), suggesting that the concentration is around 10 µM inside an inclusion body. Extrapolating our predicted free energy curves to 10 µM, the NT17-Q40-P10 construct exhibits a downhill profile, whereas the predicted 1D aggregation free energy curves for NT17-Q20-P10 and NT17-Q30-P10 at this physiological concentration remain uphill.…”

Section: Aggregation Free Energy Landscapes Of Long Polyq Repeatmentioning

confidence: 96%

Aggregation landscapes of Huntingtin exon 1 protein fragments and the critical repeat length for the onset of Huntington’s disease

Chen

Wolynes

2017

Proc. Natl. Acad. Sci. U.S.A.

104

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Huntington's disease (HD) is a neurodegenerative disease caused by an abnormal expansion in the polyglutamine (polyQ) track of the Huntingtin (HTT) protein. The severity of the disease depends on the polyQ repeat length, arising only in patients with proteins having 36 repeats or more. Previous studies have shown that the aggregation of N-terminal fragments (encoded by HTT exon 1) underlies the disease pathology in mouse models and that the HTT exon 1 gene product can self-assemble into amyloid structures. Here, we provide detailed structural mechanisms for aggregation of several protein fragments encoded by HTT exon 1 by using the associative memory, water-mediated, structure and energy model (AWSEM) to construct their free energy landscapes. We find that the addition of the N-terminal 17-residue sequence (NT 17 ) facilitates polyQ aggregation by encouraging the formation of prefibrillar oligomers, whereas adding the C-terminal polyproline sequence (P 10 ) inhibits aggregation. The combination of both terminal additions in HTT exon 1 fragment leads to a complex aggregation mechanism with a basic core that resembles that found for the aggregation of pure polyQ repeats using AWSEM. At the extrapolated physiological concentration, although the grand canonical free energy profiles are uphill for HTT exon 1 fragments having 20 or 30 glutamines, the aggregation landscape for fragments with 40 repeats has become downhill. This computational prediction agrees with the critical length found for the onset of HD and suggests potential therapies based on blocking early binding events involving the terminal additions to the polyQ repeats.Huntington's disease | aggregation | solubility | aggregation free energy landscape | critical length H untingtin (HTT) is a huge protein (3,100 amino acid residues) that has been implicated in a variety of physiological functions (1, 2). Having an expanded polyglutamine (polyQ) region of 36 or more glutamine residues in the N terminus of HTT leads to Huntington's disease as witnessed by the deposition of large intracellular protein aggregates or inclusion bodies, which are comprised primarily of N-terminal fragments coded by the exon 1 sequence of the mutant HTT (2-5). These N-terminal fragments, each composed of an N-terminal 17-residue sequence (NT17), a polyQ sequence that has expanded in length, and a proline-rich region afterward, originate from proteolysis of HTT (2, 3). The aggregation of the HTT exon 1 product is, therefore, believed to cause Huntington's disease. Clinical studies have shown an inverse correlation between polyQ length and age of disease onset (5). By implicating polymer physics in the disease process, this regularity has intrigued biophysicists for decades.Expanded polyQ sequences, more generally, are associated with nine known neurodegenerative diseases (4). Biophysical chemists have, therefore, focused on first understanding the aggregation of pure polyQ peptides. The rate of aggregation of polyQ peptides is length-dependent, and primary nucleation is rate-limiti...

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Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

Cited by 56 publications

References 42 publications

Polyglutamine expansion affects huntingtin conformation in multiple Huntington’s disease models

Polyglutamine expansion affects huntingtin conformation in multiple Huntington’s disease models

Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington’s disease patients

Aggregation landscapes of Huntingtin exon 1 protein fragments and the critical repeat length for the onset of Huntington’s disease

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