A patient-derived cellular model for Huntington's disease reveals phenotypes at clinically relevant CAG lengths

Abstract

The huntingtin protein participates in several cellular processes that are disrupted when the polyglutamine tract is expanded beyond a threshold of 37 CAG DNA repeats in Huntington's disease (HD). Cellular biology approaches to understand these functional disruptions in HD have primarily focused on cell lines with synthetically long CAG length alleles that clinically represent outliers in this disease and a more severe form of HD that lacks age onset. Patient-derived fibroblasts are limited to a finite number of passages before succumbing to cellular senescence. We used human telomerase reverse transcriptase (hTERT) to immortalize fibroblasts taken from individuals of varying age, sex, disease onset, and CAG repeat length, which we have termed TruHD cells. TruHD cells display classic HD phenotypes of altered morphology, size and growth rate, increased sensitivity to oxidative stress, aberrant adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratios, and hypophosphorylated huntingtin protein. We additionally observed dysregulated reactive oxygen species (ROS)-dependent huntingtin localization to nuclear speckles in HD cells. We report the generation and characterization of a human, clinically relevant cellular model for investigating disease mechanisms in HD at the single-cell level, which, unlike transformed cell lines, maintains functions critical for huntingtin transcriptional regulation and genomic integrity.

FIGURE 1:

Generation of TruHD-immortalized cell lines. (A) hTERT mRNA levels normalized to β-actin mRNA levels in RPE1 cells (positive control), primary cells, and TruHD cells. hTERT levels in primary cells were not detectable (ND). n = 5. Error bars represent SEM. *p = 0.0369 comparing TruHD-Q21Q18F, TruHD-Q43Q17M, and TruHD-Q50Q40F by one-way analysis of variance (ANOVA). (B) Telomeric repeat amplification product (TRAP) assay. Amplification products run on 10% TBE gel after telomere extension reaction, showing telomeric repeats >50 base pairs in increments of 6 base pairs. Template strand is 36 base pairs. Negative control contains no Taq polymerase or template strand. (C) Representative karyotypes of TruHD-Q21Q18F, TruHD-Q43Q17M, and TruHD-Q50Q40F cells. “mar” denotes marker chromosomes, “+” are additional chromosomes and “?add(4)(p14)” denotes additional patterns observed on chromosome 4 at band p14. Results from full karyotype shown in Table 2.

FIGURE 2:

TruHD cell properties. (A) Immunofluorescence images of TruHD-Q21Q18F, TruHD-Q43Q17M, and TruHD-Q50Q40F. Scale bar = 10 μm. (B) PCA plot of images sorted with Phenoripper. (C) Cell surface area comparison in TruHD cells. n = 3, N > 200. Error bars represent SEM; *p < 0.0001. (D) Relative cell count measured every 24 h. n = 3, N > 200. Error bars represent SEM. ***p = 0.0003 at 48 h; ***p = 0.0001 at 72 h by one-way ANOVA. (E) Percentage cell viability of TruHD cells compared with STHdh cells. n = 3, N > 200. Error bars represent SEM. At 24 h, ****p < 0.0001 for STHdh^Q7/Q7 vs. STHdh^Q111/Q111 by two-ANOVA and ****p < 0.0001 for TruHD-Q21Q18F vs. TruHD-Q43Q17M and TruHD-Q50Q40F by two-way ANOVA. (F) Normalized ADP/ATP ratio in TruHD cells at ∼75% confluency 24 h after seeding. n = 3, N > 200. Error bars represent SEM. *p = 0.0371 and **p = 0.0048.

FIGURE 3:

Huntingtin protein levels in TruHD cells. (A) Densitometric analysis of total huntingtin levels using Western blot with EPR5526 antibody. Immunoblots were cut horizontally at the 75-kDa marker so that GAPDH loading control was probed for separately. Normalized to control TruHD-Q21Q18F cells. n = 6. Error bars represent SEM. **p = 0.0087 and *p = 0.0254 by unpaired Student’s t test. (B) Densitometric analysis of total huntingtin levels using Western blot with mAb2166 antibody. Immunoblots were cut horizontally at the 75-kDa marker so that GAPDH loading control was probed for separately. Normalized to control TruHD-Q21Q18F cells. n = 4. Error bars represent SEM. (C) Densitometric analysis of N17-phospho levels using Western blot. Immunoblots were cut horizontally at the 75-kDa marker so that GAPDH loading control was probed for separately. Normalized to control TruHD-Q21Q18F cells. n = 4. Error bars represent SEM. **p = 0.0020 by unpaired t test. (D) Mean fluorescence intensity analysis of N17-phospho using flow cytometry. Normalized to control TruHD-Q21Q18F. n = 4. Error bars represent SEM. ***p = 0.0005, *p = 0.0159 and ****p < 0.0001 by unpaired t test.

FIGURE 4:

Huntingtin localizes to nuclear speckles in response to stress. (A) N17-phospho localizes to SC35+ nuclear speckles. (B) Nuclear speckles are increased in TruHD-Q21Q18F control cells on treatment with 0.1 mM 3NP but not for TruHD-Q43Q17M or TruHD-Q50Q40F. Scale bar = 10 μm. (C) Quantification of nuclear speckles. n = 3, N = 180. Error bars represent SEM. ****p < 0.0001 by unpaired t test. Comparison of TruHD-Q21Q18F 3NP, TruHD-Q43Q17M CTRL, TruHD-Q43Q17M 3NP, TruHD-Q50Q40F CTRL, and TruHD-Q50Q40F 3NP by one-way ANOVA shows no significant difference (p = 0.8475).