Novel methods and therapeutic approaches of diagnosis and treatment of Huntington's disease

Huntington’s Disease is a neurodegenerative disorder characterized by motor dysfunction, emotional disturbance, dementia and weight loss. The disorder is caused by an autosomal dominant expansion of a CAG repeat encoding for a polyglutamine stretch in exonof the huntingtin gene. Mutated huntingtin gains a neurotoxic function, leading to the onset of clinical symptoms mostly in mid-life. The progression of Huntington’s Disease is characterized by a marked degeneration of gray and white brain matter. A loss of vulnerable neurons, most notably striatal medium-sized spiny neurons, is observed, while resistant populations are spared. No cure for Huntington’s Disease exists and the disorder progresses relentlessly with a lethal outcome about two decades after diagnosis. In my thesis I explored four main projects. As the reported cellular dysfunctions in Huntington’s Disease are numerous, I generated an inducible, neuronal model to investigate the effects of mutant huntingtin expression at the cellular level (Chapter 4.1). This inducible model allowed for adjustable expression levels of different wild-type and mutant huntingtin fragments in proliferating or differentiated HN10 neuroblastoma cells, thus providing the ability to examine huntingtin protein effects under different cellular conditions. I was able to show that this model displays key major characteristics found in Huntington’s Disease patients like transcriptional dysregulation, mutant huntingtin aggregation and decrease in cell viability. Subsequently, I made use of this newly designed cellular model to develop huntingtin detection methods to further investigate the biological role of soluble or aggregated mutant huntingtin for Huntington’s Disease development and progression. Since the role of huntingtin aggregate formation in Huntington’s Disease is still under debate, I designed a simple method based on agarose gel electrophoresis for qualitative and quantitative characterization of huntingtin aggregates in my second project. Using this method, I proceeded to analyze samples of cellular and animal Huntington’s Disease models and was able to show that in the brain of transgenic R6/2 mice aggregates became larger as a function of age and disease progression. Importantly, I showed that in primary striatal neurons and in brains of two Huntington’s Disease mouse models (transgenic R6/2 and HdhQ150 knock-in mice), aggregate formation preceded detection of any functional deficits, supporting the theory that aggregates play an important pathogenic role in Huntington’s Disease (Chapter 4.2). In the third project, I developed a method for the detection of intracellular mutant huntingtin, the causative agent of Huntington’s Disease. I generated a small recombinant protein tag which is recognized by a pair of readily available, high affinity monoclonal antibodies, thus
making this method generally applicable for detection of other recombinant proteins. Using
this tag I was able to establish a time resolved fluorescence resonance energy transfer (time
resolved FRET) based assay which allows for rapid, sensitive and robust detection of cellular
mutant huntingtin levels. I miniaturized this assay to a homogeneous 1536 well microplate
format and demonstrated that the assay system is suitable for the identification of compounds
that increase or decrease the levels of huntingtin protein (Chapter 4.3).
In the fourth project, by using antibodies specific against endogenous huntingtin epitopes, I
expanded this time resolved FRET detection method to monitor the levels of endogenous
soluble mutant huntingtin in cellular, animal and human samples. I showed that the soluble
mutant huntingtin levels inversely correlate with the amount of mutant huntingtin aggregates
in the brains of aging R6/2 mice. Importantly, I was able to quantify mutant huntingtin
concentrations in blood fractions from Huntington’s Disease patients, providing for the first
time a bioassay to assess the relevance of mutant huntingtin levels as a marker for disease
progression. This biomarker could help to monitor the efficacy of drug treatments aimed at
lowering mutant huntingtin levels in preclinical and clinical trials (Chapter 4.4).