Mutant huntingtin fragmentation in immune cells tracks Huntington's disease progression

Abstract

Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Therapeutic approaches to lower mutant HTT (mHTT) levels are expected to proceed to human trials, but noninvasive quantification of mHTT is not currently possible. The importance of the peripheral immune system in neurodegenerative disease is becoming increasingly recognized. Peripheral immune cells have been implicated in HD pathogenesis, but HTT levels in these cells have not been quantified before. A recently described time-resolved Förster resonance energy transfer (TR-FRET) immunoassay was used to quantify mutant and total HTT protein levels in leukocytes from patients with HD. Mean mHTT levels in monocytes, T cells, and B cells differed significantly between patients with HD and controls and between pre-manifest mutation carriers and those with clinical onset. Monocyte and T cell mHTT levels were significantly associated with disease burden scores and caudate atrophy rates in patients with HD. mHTT N-terminal fragments detected in HD PBMCs may explain the progressive increase in mHTT levels in these cells. These findings indicate that quantification of mHTT in peripheral immune cells by TR-FRET holds significant promise as a noninvasive disease biomarker.

Figure 1. Relationship between HTT levels in peripheral immune cells and disease stage.

Total HTT and mHTT protein levels were quantified by TR-FRET in monocytes, T cells, and B cells. Total HTT quantification relies on simultaneous binding of 2B7 and 2166 anti-HTT antibodies. mHTT is quantified using the 2B7 antibody and a polyglutamine-specific antibody, MW1. (A) Total HTT levels in leukocytes showed no significant differences between patients with HD and control subjects or between HD gene carriers at different disease stages. (B) mHTT protein was detected in samples from patients with HD and pre-manifest HD mutation carriers, as compared with that in controls. Differences in mean mHTT levels in leukocytes were observed between pre-manifest and manifest HD patients (P < 0.01) and between pre-manifest and early-stage HD subjects (P = 0.051 and P < 0.05 for monocytes and T cells/B cells, respectively). Colored circles indicate multiple samples from a single subject. White circles indicate samples from individual subjects. Horizontal bars indicate the mean. Em, emission; Ex, excitation; N, N-terminal; C, C-terminal; Tb, terbium cryptate; polyQ, polyglutamine tract; pre-HD, pre-manifest HD sample; Early HD, early-stage HD sample; Moderate HD, moderate-stage HD sample.

Figure 2. Associations among mHTT levels in peripheral immune cells and disease burden score and caudate atrophy rate.

(A) mHTT protein levels in leukocytes show a statistically significant positive association with HD disease burden score. Repeated measurements for a single subject are joined by a line. (B) mHTT levels in monocytes are significantly associated with caudate atrophy rates measured by serial volumetric MRI.

Figure 3. mHTT protein fragments are present in HD PBMCs.

(A) HTT in PBMCs from 2 subjects with HD and an age-matched control was immunoprecipitated with 2166, 2B7, or 4C9 anti-HTT antibodies, as compared with that precipitated by an IgG control. Immunoprecipitates were blotted with 4C9 and 2166 anti-HTT antibodies. Several HTT-specific bands were found in HD patient and control subject PBMC samples. (B) HTT protein in PBMCs from 2 patients with early-onset HD (HTT CAG repeat lengths of 76 and 59) and an age-matched control was immunoprecipitated with 2B7, 3B5H10, or 2166 anti-HTT antibodies. Immunoprecipitates were blotted with either the polyglutamine-specific MW1 antibody or the 2166 anti-HTT antibody. Several mHTT-specific bands were found in the lysates from the PBMCs from the patients with early-onset HD (colored boxes), including N-terminal HTT fragments (yellow boxes) immunoprecipitated with the 2B7 and 3B5H10 antibodies but not with the more C-terminal epitope-binding 2166. ID, immunodetection; IgG heavy chain, antibody heavy chain.