Fig 2: Mutant HTT is hypophosphorylated on residue T3 in Hdh Q111 knock-in HD mice cortex. (A) Western immunoblot of homogenates from Hdh knock-in mouse (6-mo cortex), analyzed using anti-pT3 (for HTT pT3), mAb 2166 (for total HTT), and MW1 (for expanded polyQ) antibodies, showing that mutant HTT is hypophosphorylated relative to its Q7 counterpart. (B) Relative HTT pT3 Singulex signal in brain cortex samples from WT (Q7/Q7) or heterozygous mutant (Q7/Q111) Hdh HD animals, normalized to total HTT levels measured either with the MW1/2B7 Singulex immunoassay (i) or with the MW1/2166 Singulex immunoassay (ii). Means and SD of four Q7/Q7 samples and six Q7/Q111 samples, unpaired t-test (two-tailed; **P < 0.01; ***P < 0.005). (C) Relative HTT pT3 (alternative anti-pT3 Ab from ref. 39) Singulex signal in brain cortex samples from WT (Q7/Q7) or heterozygous mutant (Q7/Q111) Hdh HD animals, normalized to total HTT levels measured either with the MW1/2B7 Singulex immunoassay (i) or with the MW1/2166 Singulex immunoassay (ii). Means and SD of four Q7/Q7 samples and six Q7/Q111 samples, unpaired t-test (two-tailed; **P < 0.01; ***P < 0.005).

Fig 3: Specificity of the MW1/pT3 and MW1/2B7 Singulex immunoassays. (A) Western immunoblot of HEK293T protein lysates demonstrating the effects of HTT mRNA knockdown using a specific HTT siRNA on endogenous and overexpressed full-length (FL) HTT for both pT3 HTT and total HTT protein levels. (B) Singulex analysis using MW1/pT3 or MW1/2B7antibody combinations showing the effects of HTT RNA knockdown on endogenous HTT levels (i) and on HTT protein levels obtained in cells transfected with a plasmid encoding a full-length HTT Q18 cDNA (ii). Data demonstrate that signals from both assays are sensitive to specific HTT expression knockdown. Means and SD of three independent experiments, paired t-test (two-tailed; *P < 0.05; **P < 0.01). (C) Western immunoblot of Phos-Tag SDS/PAGE, representing HEK293T cells transfected with each HTT EX1 plasmid construct bearing different CAG repeats with or without a T3A mutation and probed either with anti-pT3 pAb or with mAb 4C9. The HTT pT3 signal is sensitive to the T3A mutation. (D) Singulex immunoassay detecting effects of the T3A mutation on relative HTT pT3 levels in lysates from HEK293T cells expressing HTT EX1 constructs bearing different polyQ repeats. Means and SD of at least four independent experiments, paired t-test (two-tailed; *P < 0.05; **P = 0.01).

Fig 4: Mutant HTT is hypophosphorylated on residue T3 in HD iPSC-derived neuronal populations and in peripheral (blood) PBMCs isolated from HD individuals. (A) Relative HTT pT3 Singulex signal in iPSC-derived neuronal population protein lysate from WT and HD individuals, normalized to total HTT measured with the MW1/2B7 Singulex immunoassay. Means and SD of three CTRL iPSC lines (#CTRL21: CAG status 21/18; #CTRL28: CAG status 28/18; #CTRL33: CAG status 33/18) and two HD iPSC lines (#HD60: CAG status 60/18; #HD109: CAG status 109/19). Unpaired t-test (two-tailed; *P < 0.05). (B) HTT pT3 Singulex signal in PBMCs isolated from HD individuals, relative to PBMCs from normal individuals and normalized to total HTT measured with the MW1/2B7 Singulex immunoassay. Means and SD of three control and three HD samples, unpaired t-test (two-tailed; *P < 0.05).

Fig 5: T3 phosphorylation impacts mutant HTT N-terminal conformational rigidity and decreases HTT EX1 aggregation. (A, i) T3 phosphorylation has no effect on the conformation of soluble semisynthetic HTT EX1 Q23 protein as measured by the 2B7/MW1 TR-FRET conformational immunoassay. (A, ii) T3 phosphorylation decreases the conformational rigidity of HTT EX1 Q43 protein assessed with the same TR-FRET. Other relevant N17 PTMs, specifically acetylation on residues K6 and K9, do not affect the conformational rigidity of HTT EX1 Q43 protein as measured in parallel using the same assay. Summary of three independent experiments (one-way ANOVA test; ***P < 0.005). (B) The control 2B7/4C9 TR-FRET immunoassay, the signal of which on HTT protein is not temperature- and polyQ-dependent, is not affected by T3 phosphorylation. (C) T3 phosphorylation reduces the aggregation propensity of mutant HTT protein. (i) WB and corresponding densitometric analysis of equivalent nominal amounts of semisynthetic HTT EX1 Q43 protein and its T3 phosphorylated counterpart. Means and SD of three independent experiments, paired t-test (two-tailed; ns, not significant). (ii) Detergent-insoluble material was present in lanes loaded with HTT EX1 Q43 protein, the presence of which was invariably reduced if this protein was phosphorylated on residue T3. Means and SD of three independent experiments, paired t-test (two-tailed; **P < 0.01). (iii) Filter trap analysis of semisynthetic HTT EX1 Q43 protein aggregation in the presence/absence of T3 phosphorylation, confirming reduction of insoluble HTT EX1 Q43 aggregates if residue T3 is phosphorylated. Note that a small difference in soluble Q43 and pT3 Q43 semisynthetic protein levels is observed as measured by densitometry; however, a much larger difference in insoluble protein levels is observed. Means and SD of three independent experiments, paired t-test (two-tailed; *P < 0.05).