Coordinated proteasome and lysosome retrograde axonal transport is regulated by kinesin-1 clustering at projection ends

OTERO MG; CROMBERG L; FALZONE T

Vancouver

Simposio; International Conference on Alzheimer Disease; 2012

Alzheimer Association

Dysfunction of intracellular protein degradation pathways have been implicated in the abnormal aggregates and axonal swellings observed in neurodegenerative disease such as Alzheimer Disease (AD). An interplay connection between protein degradation by Ubiquitin-Proteasome System (UPS) and Autophagy-Lysosome Pathways (ALP) implies the correct trafficking and delivery of lysosomal vesicles and the regulation of membrane recycling by proteasome function. Axonal transport by the coordinated function of motor proteins can regulate proteins and organelles positioning and delivery. Transport defects are implicated as early mechanisms that can lead to abnormal protein aggregation and axonoathies. Consequently, it is important to understand the role of motor proteins in the regulation of the relevant crosstalk between UPS and ALP clearance pathways involved in the progression of AD phenotypes. Methods To test whether proteasome activity mediates the regulation of lysosome retrograde axonal transport we performed live imaging experiments in primary hippocampal neurons to track the dynamics of fluorescent lysosome vesicles (lysotracker) under proteasome inhibition. Immunofluorescent and biochemical experiments were performed to unravel the accumulation and clustering of anterograde motor proteins after proteasome inhibition. shRNA against different motor proteins were transfected in primary hippocampal neurons for live imaging experiments of fluorescent lysosome vesicle tracking to study motor protein clustering and lysosome dynamics. Results In vivo imaging of fluorescent lysosomes after prteasome inhibition revealed a decrease in retrograde proportions and vesicle density in primary hippocampal axons. Immunofluorescent and biochemistry experiments showed poly-ubiquitin independent clustering of kinesin heavy chain motor proteins in growth cones and projection tips after proteasome inhibition. Sucrose density gradient on proteasome inhibited N2a cells showed impairments in endosome-lysosome membrane density. Motor protein reduction by shRNA-KIF5b transfection enhanced retrograde lysosome vesicle proportions and increased lysosome axonal density. Conclusions  Our experiments revealed an interesting neuronal pathway where local UPS impairment regulate the density, axonal loading and retrograde transport of lysosome vesicles by changing the confined concentration of motor proteins. Thus, we propose a new mechanism of crosstalk between UPS and ALP depending on motor protein load at neuronal ends mediating the abnormal protein degradation phenotype observed in many neurodegenerative diseases such as AD.