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 ADP/ATP ratios and hypophosphorylated huntingtin protein. We additionally observed dysregulated 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 TP53 function critical for huntingtin transcriptional regulation and genomic integrity.Canada for assistance with flow cytometry experiments.

N6-Furfuryladenine is protective in Huntington’s disease models by signaling huntingtin phosphorylation

“…Given the role of huntingtin as a scaffold for the ATM complex (5), dominant mutant huntingtin effects coupled with an age-related ROS increase could lead to the age-onset accumulation of DNA damage. As mutant huntingtin is hypophosphorylated at the N17 domain (13,19), and phosphorylation is beneficial in HD models (12,18), the effects we observed with N6FFA treatment connect DNA-damage repair, altered bioenergetics, and mutant huntingtin phosphorylation and inclusion load.…”

“…We therefore asked whether CK2 and huntingtin colocalize within the cell both at resting states and during active DNA-damage repair. We have previously reported that the anti-N17-S13pS16p antibody highlights insoluble, chromatin-dependent nuclear puncta (5,11,13), which we recently identified as nuclear speckles (19). Using antibodies against CK2 and N17-S13pS16p and superresolution structured illumination microscopy (SR-SIM), we observed colocalization of these endogenous proteins at nuclear speckles ( Fig.…”

“…We therefore set out to identify compounds that could modulate N17 phosphorylation in an attempt to restore the hypophosphorylation of N17 seen in HD cells (13,19). Using an extensively validated antibody against phosphorylated S13 and S16 (13), we conducted a high-content screen for the effects of diverse small-molecule natural products on N17 phosphorylation.…”

DNA Damage Repair in Huntington's Disease and Other Neurodegenerative Diseases

“…Huntingtin is a ROS sensor and will relocate from the outer leaf of the pervasive endoplasmic reticulum bilayer to the nucleus upon oxidation of a single methionine at position 8 [42]. Reactive oxygen species (ROS) also increases the number of nuclear speckles: liquid-liquid phase separated droplets at which huntingtin colocalizes with DNA repair factors including ATM [38,[42][43][44]. Huntingtin acts as a scaffold that can localize to DNA damage, and modulates its associated complex in the presence of ROS stress [38].…”

“…N17 comprises the first 17 amino acids that modulate the location of the massive 3144 amino acid protein as a "tail that wags the dog" [6,71]. This modification is deficient in HD [9,44] and its restoration is beneficial in HD model systems [9,43,72,73] making it an attractive therapeutic target.…”