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The dynamics of mutated huntingtin processing, oligomerization, aggregation and clearance

Huntington’s disease (HD) is a progressive and fatal neurological disorder associated with severe motor and psychiatric symptoms. HD is caused by a genomic expansion of a CAG repeat in the gene (HTT) that encodes for huntingtin (Htt), a large protein whose physiological functions are only partly understood. The HD-associated CAG expansion, which translates into an abnormally long glutamine repeat, increases the tendency of mutated huntingtin (mHtt) to oligomerize and form aggregates and nuclear inclusion bodies. Although much is still uncertain, it is clear that mHtt in the form of minute oligomers or large aggregates impairs brain cell viability and ultimately leads to cell death. This association between protein aggregation and cell death, a common theme in prevalent neurological diseases, has driven a disproportionally large amount of research on HD and mHtt biology, mostly because the unequivocal identification of its cause raises hopes that comprehensive understanding is attainable and potentially transferable to other major diseases.

Although much importance is attributed to mHtt oligomerization and aggregation, very few studies have actually followed these processes from start to end, as they happen. Thus, many questions regarding these processes remain unanswered: What are oligomerization and aggregation kinetics? What is the source of mHtt that gives rise to mHtt oligomers and aggregates? Is it co-translationally misfolded mHtt? Where does oligomerization and aggregation begin? What is the fate of newly formed oligomers and aggregates? Are they mobilized within the cell? Can aggregates, once formed, fall apart? If so, how is mHtt cleared? How are these processes affected by eliminating two HD-specific, N-terminal ubiquitination sites we recently described? What aspects of these processes are the strongest indicators of cell death?

Here we propose to address these questions by continuously following mHtt oligomerization and aggregation in live cortical neurons from the moment of mHtt expression, for durations of weeks and beyond. To that end, we will combine long-term imaging methodologies we pioneered and new labeling techniques, which provide means for resolving mHtt age (time from synthesis), oligomerization, aggregate formation kinetics, aggregate dynamics and fate. These approaches will be augmented with correlative electron microscopy to resolve aggregate ultrastructure and relationships with intracellular organelles as a function of aggregate age and history. These methodologies will then be used to examine how elimination of two, N-terminal HD specific ubiquitination sites we recently described, affects mHtt oligomerization and aggregation processes and how these processes relate to cell viability and cell death.

We expect that the program described here will provide a better understanding of mHtt processing and misprocessing, and their relationships with cell dysfunction and death. Moreover, it is our hope that the findings will provide general insights on life cycles of molecules whose size, solubility and folding significantly threat cell viability, as is the case in many of the most prevalent neurological disorders.

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