2024 Theses Doctoral

Mechanisms Regulating Axonal Transport of ESCRT Machinery

Kirwan, Konner

Turnover of synaptic vesicle (SV) proteins is vital for the maintenance of healthy and functional synapses. I recently showed that SV protein turnover is driven by neuronal activity in an endosomal sorting complex required for transport (ESCRT)-dependent manner. The ESCRT pathway comprises a series of protein complexes (ESCRT-0, I, II, III) that capture cargo and catalyze its sorting into multivesicular bodies (MVBs) for delivery to lysosomes. ESCRT-mediated protein degradation faces spatiotemporal challenges in neurons, as ESCRT components must undergo long-distance anterograde transport from soma to synapses in order to capture and sort cargo into MVBs.

Moreover, MVBs and MVB-associated ESCRT-III proteins undergo retrograde transport back to the soma, where degradative lysosomes primarily reside. How ESCRT machinery is transported to and from synapses in morphologically complex neurons remains poorly understood. Here, I use live imaging approaches to characterize the axonal transport of ESCRT-0 protein Hrs and ESCRT-III protein CHMP2b, representing the initial and final components of the ESCRT pathway. In addition, I investigate the consequences of frontotemporal dementia (FTD)-causative mutant CHMP2b‸(intron5) on CHMP2b axonal transport and synaptic localization. I find that Hrs is transported on a subset of Rab5⁺ early endosomes, and that neuronal activity stimulates the motility and synaptic delivery of these Hrs⁺ vesicles.

Furthermore, I identify kinesin motor protein KIF13A as essential for the activity-dependent anterograde transport of Hrs to presynaptic boutons and the degradation of SV membrane proteins. While CHMP2b also undergoes activity-dependent transport to presynaptic sites, this transport is typically retrograde and associated with Rab7, suggesting that a large fraction of CHMP2b⁺ vesicles represent late endosomes undergoing transport from presynaptic terminals to the soma.

In contrast, vesicles carrying the CHMP2b‸(intron5) mutant exhibit aberrant oscillatory behavior reminiscent of a tug-of-war between motor proteins that disrupts their transport to presynaptic sites and contributes to defects in their maturation and activity-dependent transport. I demonstrate that these phenotypes are due in part to deficient binding of CHMP2b‸(intron5) to kinesin binding protein (KBP), which I identify as a key regulator of CHMP2b axonal transport.

Together, these data demonstrate a novel activity- and KIF13A-dependent mechanism for mobilizing axonal transport of ESCRT-0 machinery to initiate the degradation of SV membrane proteins, and shed light on the mechanisms of CHMP2b/MVB transport and the etiology of CHMP2b‸(intron5)-induced FTD.