Although the genetic origin of a multitude of neurodegenerative disease has been identified for decades, the underlying mechanism of protein aggregation remains poorly understood. With the advance and distribution of EM-methods, emphasis has been given to structural insights of protein aggregates and mutant substructure characterisation, thereby losing the temporal context of actual dynamics. Quantitative light microscopy methods can incorporate these insights to unveil structure-function relationships relevant to identify molecular interactions in the aggregation processes. Intracellular phase transitions, as a paradigm of misregulated protein self-assembly and increasing reports of proteins physiologically undergoing liquid-liquid phase separation, are a promising framework to investigate these neuropathologies.
Huntingtin (Htt) is our aggregation protein model system, which is linked to Huntington’s disease, a neurodegenerative disorder characterized by a progressive loss of striatal and cortical neurons. The pathology is thought to be caused by an expanded, unstable trinucleotide (CAG) repeat in the first exon (HttEx1) of the Htt gene, which translates to a polyglutamine (polyQ) repeat in the protein product. A multitude of potential and partially reversible aggregation pathways via soluble dimers and oligomers to protofibrils and amyloid fibers culminating in μm-scale inclusion bodies (IBs) have been proposed, in addition to various potential cytotoxic effects on neurons. The actual mechanism leading to aggregation, e.g. via chaperones, intrinsic aggregation due to misfolding and PTMs, or cellular modulators remains to be determined. Steady progress has been made in uncovering disease mechanisms downstream of mHtt expression. For example, there is some consensus that polyglutamine-expanded mHtt elicits gain-of-function proteinopathy that may affect both nuclear and cytoplasmic cellular function.
Htt mutant pathogenicity and disease onset have been show to correlate with its polyglutamine repeat length as soon as it passed the critical length of 36 CAG repeats. Further side domain (Nt17) and point mutation (S13 and S16, lysine 444), mutant construct length (Htt105-171), and post-translational modifications (proteolysis at D586, phosphorylation at S13 and S16) effects on aggregation propensity and nucleocytosolic location have to this end been reported.
It has further been shown that aggregation of Htt-polyQ-GFP fusion proteins express dramatically different aggregation kinetics and ultrastructural compositions of IBs compared to their native Htt-polyQ construct, underscoring the potential limitations of using fusion proteins to investigate the molecular, biochemical, and cellular determinants of Htt inclusion formation and mechanisms of toxicity. Hence, the need for label-free imaging and transient labelling strategies emerged to further investigate physiological intracellular aggregation dynamics.
The goal of our research is to develop and deploy a multifaceted nanoscale imaging approach to uncover various dynamics throughout the aggregation process of defined mHtt constructs, focusing on
- the structural polymorphism and pathway heterogeneity in amyloid-fiber formation,
- sub-diffraction limit protein aggregate structures and dynamics both nuclear and cytosolic,
- the nucleocytosolic shuttling via the nuclear pore and the involvement of ER (and cytoskeleton) in this transport,
- the actual pathology emerging from this aggregation process to elucidate a relative time point or aggregation maturity or distribution that predicts cellular decline.
