De membraanvervormende activiteit van Hsc70-4 bevordert de afbraak van synaptische proteïnen door endosomale microautofagie

Abstract

Synapses are often far from their cell bodies and must cope largely independently with dysfunctional proteins resulting from synaptic activity and stress. Protein quality control pathways are involved in the clearance of these dysfunctional proteins, however for some of these pathways, not much is known about their role at synapses. Hence, the main goal of this doctoral thesis is to further inquire into protein quality control at nerve terminals. In this work, we found that endosomal microautophagy, a protein degradation pathway, is active at nerve terminals in Drosophila. In endosomal microautophagy, Hsc70-4, a molecular chaperon, targets substrates with a recognition motif to late endosomes where the substrates are engulfed by the endosomal membrane. Moreover, we found that this pathway requires a novel function of Hsc70-4, namely membrane deformation. In an in vitro tubulation screen, performed in our lab, Hsc70-4 was identified as a membrane deforming protein. Since it was shown that this chaperone is involved endosomal microautophagy that requires reshaping of the endosomal membrane to take up substrate proteins, we hypothesize that this novel function of Hsc70-4 could be implicated in endosomal microautophagy at the synapse. Since this pathway has never been studied in neurons before, we designed a microautophagy sensor, which allowed us to observe the presence of this pathway at Drosophila neuromuscular junctions (NMJs). We found that this pathway requires Hsc70-4’s membrane deformation activity, but not its chaperone activity. Additionally, we showed that Sgt, a known co-chaperone of Hsc70-4, promotes Hsc70-4’s protein refolding activity and blocks synaptic microautophagy. This suggests a regulatory role for Sgt on these two functions of Hsc70-4. We observed that synaptic proteins with a specific recognition motif can be targeted by Hsc70-4 for degradation by microautophagy, while Sgt on the other hand seems to block their turnover via this pathway. Finally, we observe that loss of microautophagy reduces neurotransmission while increasing microautophagic activity increases neurotransmission. We suggest that increased microautophagy rejuvenates the synaptic protein pool, resulting in more competent machinery for neurotransmitter release. With this work, we unraveled a new protein quality control pathway at the synapse that can specifically target proteins for degradation.status: publishe