Assessing average somatic CAG repeat instability at the protein level

Hubert Aviolat,Hannah E Richey,Vanessa C Wheeler,Ricardo Mouro Pinto,Petr Vodicka,Ellen Sapp,Ryan Murtha,Marian Difiglia,Elizabeth Godschall,Kimberly B Kegel-Gleason

doi:10.1038/s41598-019-55202-x

Hubert Aviolat, Hannah E Richey + Show 8 more

Open Access

https://doi.org/10.1038/s41598-019-55202-x

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Abstract

Sandwich ELISA-based methods use Abs that target the expanded polyglutamine (polyQ) tract to quantify mutant huntingtin (mHTT). Using Meso Scale Discovery (MSD) assay, the mHTT signal detected with MW1 Ab correlated with polyQ length and doubled with a difference of only 7 glutamine residues between equivalent amounts of purified mHTTexon1 proteins. Similar polyQ length-dependent effects on MSD signals were confirmed using endogenous full length mHTT from brains of Huntington’s disease (HD) knock-in (KI) mice. We used this avidity bias to devise a method to assess average CAG repeat instability at the protein level in a mixed population of HTT proteins present in tissues. Signal detected for average polyQ length quantification at the protein level by our method exhibited a strong correlation with average CAG repeat length at the genomic DNA level determined by PCR method in striatal tissue homogenates from HdhQ140 KI mice and in human HD postmortem cortex. This work establishes that CAG repeat instability in mutant HTT is reflected at the protein level.

Highlights

Huntington’s disease (HD) is a neurodegenerative disease due to a CAG trinucleotide repeat expansion[1], ranging from 36 to 250 repeats[2] and resulting in an extended polyglutamine tract within huntingtin (HTT) protein
Meso Scale Discovery (MSD) is a method similar to ELISA except that electrochemiluminescence is used as detection readout: electricity is applied to the plate electrodes leading to light emission by electrochemiluminescent labels that are conjugated to detection antibodies
Lowering mutant HTT (mHTT) is a major therapeutic strategy under investigation in many laboratories and in clinical trials for HD patients[43,44], accurate quantification using ultra-sensitive immunodetection methods is vital. mHTT can be preferentially distinguished from WT by polyQ targeting Abs[23,24,25] sensitive to expanded polyQ repeats containing more epitopes than normal polyQ tracts

Summary

Introduction

Huntington’s disease (HD) is a neurodegenerative disease due to a CAG trinucleotide repeat expansion[1], ranging from 36 to 250 repeats[2] and resulting in an extended polyglutamine (polyQ) tract within huntingtin (HTT) protein. Genomic DNA (gDNA) from postmortem brain samples from two HD individuals, who died of other causes and with no microscopic evidence of pathological cell loss in the striatum (inherited CAG repeat length of 41 and 51 and an age at death of 40 and 27 years respectively), showed dramatic mutation length increases in striatum (up to >1,000 CAG repeats) and in the cortex, though to a lesser extent[11] These observations suggest that somatic instability could precede and influence the onset of symptoms. While MW1 and other polyQ targeting Abs (1C2 and 3B5H10) were initially proposed to recognize a specific mutant conformation[23,24] or a specific toxic monomeric conformation[25], recent studies have contradicted these hypotheses, suggesting a linear lattice model[26,27,28,29,30] These Abs bind a small polyQ epitope in similar linear and extended conformations, with a higher avidity for expanded polyQ tracts due to the Ab’s bivalence. This method could become a new benchmark to complement the PCR method, for detection of somatic expansion of unstable CAG repeats at the protein level

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