Tubulin Polymerization-promoting Protein (TPPP/p25α) Promotes Unconventional Secretion of α-Synuclein through Exophagy by Impairing Autophagosome-Lysosome Fusion

Aggregation of alpha-synuclein can be promoted by the tubulin polymerization-promoting protein/p25 alpha, which we have used here as a tool to study the role of autophagy in the clearance of alpha-synuclein. In NGF-differentiated PC12 catecholaminergic nerve cells, we show that de novo expressed p25 alpha co-localizes with alpha-synuclein and causes its aggregation and distribution into autophagosomes. However, p25 alpha also lowered the mobility of autophagosomes and hindered the final maturation of autophagosomes by preventing their fusion with lysosomes for the final degradation of alpha-synuclein. Instead, p25 alpha caused a 4-fold increase in the basal level of alpha-synuclein secreted into the medium. Secretion was strictly dependent on autophagy and could be up-regulated (trehalose and Rab1A) or down-regulated (3-methyladenine and ATG5 shRNA) by enhancers or inhibitors of autophagy or by modulating minus-end-directed (HDAC6 shRNA) or plus-end-directed (Rab8) trafficking of autophagosomes along microtubules. Finally, we show in the absence of tubulin polymerization-promoting protein/p25 alpha that alpha-synuclein release was modulated by dominant mutants of Rab27A, known to regulate exocytosis of late endosomal (and amphisomal) elements, and that both lysosomal fusion block and secretion of alpha-synuclein could be replicated by knockdown of the p25 alpha target, HDAC6, the predominant cytosolic deacetylase in neurons. Our data indicate that unconventional secretion of alpha-synuclein can be mediated through exophagy and that factors, which increase the pool of autophagosomes/amphisomes (e.g. lysosomal disturbance) or alter the polarity of vesicular transport of autophagosomes on microtubules, can result in an increased release of alpha-synuclein monomer and aggregates to the surroundings.