Huntingtin polyglutamine expansions impair the transport of signalling and degradative cargoes.

Abstract

Huntingtin scaffolds motor proteins and adaptors to vesicular cargoes. Through interactions with kinesin, dynein, and myosin-VI, huntingtin regulates the direction of transport along microtubules and switching between microtubules and actin filaments. Huntington's disease (HD) is caused by polyglutamine (polyQhtt) expansion of length >35Q at Huntington's N-terminus, with longer repeats leading to more severe neurodegeneration. Defects in transport of brain-derived neurotrophic factor (BDNF) and lysosomes have been reported in cells expressing polyQhtt. We track signalling BDNF vesicles and degradative lysosomes in induced neurons from isogenic human stem cell lines with repeat lengths of 18, 30, 45, 65, and 81Q to understand how transport of signalling and degradative cargoes are affected in HD. Preliminary data indicate that lysosomes have lower processivity and similar run lengths with pathological polyQhtt. Interestingly, the 45Q cells have lower processivity and run length compared to all other conditions, indicating divergent mechanisms might contribute to mild and severe disease phenotypes. Lysosomes are typically transported towards the soma. However, with polyQhtt we observe increased outward motility, suggesting huntingtin's ability to regulate transport direction is impaired. To examine how polyQ huntingtin alters the activity of kinesin and dynein, we use optical tweezers to measure the forces exerted on endocytosed BDNF-coated nanodiamonds in control compared to 81Q neurons. BDNF-coated nanodiamonds (∼100 nm) are readily endocytosed by neurons and enable reliable force measurements due to their high refractive index. In parallel, we developed methods to isolate BDNF-phagosomes from neurons and reconstitute their motility along microtubules. Together, live-cell imaging, optical trapping, and in vitro reconstitution reveal how HD mutations impact the transport of signalling and degradative cargoes by misregulating the recruitment and activity of kinesin and dynein.