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Maintaining synaptic function for a healthy brain: Membrane trafficking and autophagy in neurodegeneration

Periodic Reporting for period 5 - RobustSynapses (Maintaining synaptic function for a healthy brain: Membrane trafficking and autophagy in neurodegeneration)

Período documentado: 2022-01-01 hasta 2022-06-30

Our brain operates by transmitting electrical signals between different areas. This is mediated by specialized neuronal connections, synapses, that are sometimes located far from the neuronal cell body where DNA is located and RNA is produced. For example, the axonal arbor of dopaminergic neurons that degenerate in Parkinson’s disease are >5,5m long. Synapses can be very active, including those of dopaminergic neurons, releasing signals at rates of >800/s, causing local damage. It was not known how neurons deal with this logistic problem. In this project, we uncovered new mechanisms that synapses use to turn-over dysfunctional proteins and maintain synapse health. We found new connections between proteins involved in endocytosis and the formation of synaptic autophagosomes, including that this process is induced by neuronal activity, thus coupling the recycling of dysfunctional proteins to neuronal activity. We also pursued large scale protein-based screens and genetic screens and found that chaperones not only re-fold damaged proteins but that they also control the function of multivesicular bodies that are degradative organelles. We found that hsc70 is required for the invagination of the endosomal membrane to encapsulate damaged cargo, while hsp90 is required for multivesicular body-to-plasma membrane fusion. Our findings are medically relevant as several of the proteins we uncovered and studied are mutated in Parkinson’s disease. Indeed, we show that key mechanisms we found in the course of our work are recapitulated in induced human dopaminergic neurons derived from patients, indicating they are cause dysfunction in the context of disease, and future work will define how these protein turn-over processes tie in to the formation of toxic protein aggregates that are so prominent in neurodegeneration.
Neurodegeneration is characterized by synaptic dysfunction. eg, different proteins implicated in Parkinson's disease are linked to mechanisms of protein turnover but how this relates to synaptic defects was not resolved. We have studied the connections between neurodegeneration (including Parkinson's disease and Tauopathies) and protein turnover at synapses. We took two approaches: one where we defined the function of proteins known to be mutated in disease and another where we conducted innovative screens to identify novel components of these processes. We found an unanticipated connection between different genes mutated in Parkinson's disease: we found that EndophilinA, a protein exclusively studied for its role in endocytosis, is required for synaptic autophagy, and that pathogenic mutations in the protein block the process. We found that neuronal stimulation and LRRK2-dependent phosphorylation induce EndophilinA-dependent autophagy and we uncovered the molecular and structural mechanism. This is interesting because EndophilinA is emerging as a central hub in Parkinson's disease: the protein also binds to Synaptojanin1 and is ubiquitinated by Parkin, and we show both these proteins also control autophagy at synapses. This work has been published in different papers, and it has been presented at venues across the world. In future work we are testing is other proteins mutated in Parkinson are also affecting synaptic autophagy.

In related work we also looked at how synaptic turnover mechanisms affect the accumulation of protein aggregates. Here we uncovered a novel function of the protein Tau that is associated with Parkinson's disease and also mutated in forms of dementia. We found that Tau binds to synaptic vesicles by interacting with the vesicle associated protein synaptogyrin-3. Removing synaptogyrin-3 rescues Tau-induced defects, indicating that the abnormal accumulation and association of toxic proteins associated with neurodegeneration is critical to the development of synaptic dysfunction. The significance of these findings are profound and we have filed several patents as well as obtained separate funding to target synaptogyrin-3 in a therapeutic setting to undo the toxic effects of Tau. This work has also been published in several papers and it has been presented at international meetings and schools.

Protein turnover by autophagy or EndoA-lysosomal sorting requires extensive membrane shaping as to engulf cytoplasm for degradation and we deployed a novel in vitro screen approach aimed at identifying proteins involved in these processes. We expressed all fly proteins in E. coli, cracked open the cells, applied the supernatant to labeled giant unilamellar vesicles (GUVs), and used fluorescence-microscopy-screening to determine if the membranes of the GUVs are deformed. There was no need to purify the proteins as protein lysate from wild-type E. coli does not display GUV membrane deformation activity. We have prepared special protein expression libraries and found 204 novel membrane deforming proteins. Interestingly, we find several proteins linked to protein homeostasis and autophagy, including a significant enrichment of 7 chaperones. A role for chaperones in membrane deformation is new and we showed that two chaperones are critically involved in orchestrating multivesiclular body function. We found that Hsc70, one of the most abundant synaptic chaperones, is involved in the invagination of the endosomal membrane to mediate endosomal microautophagy. This is exciting as we were able to show that more than 50% of the synaptic proteins have the capacity of being turned over by this process, including major disease released proteins such as alpha-synuclein and Tau. We also studied the most abundant chaperone, hsp90, that we show can deform membranes using a newly discovered amphiphatic alpha helix to mediate the fusion of multivesicular bodies with the plasma membrane. These new functions for these chaperones indicate that these proteins are not only (re-)folding dysfunctional proteins but that they are also regulating protein turnover by packaging them in luminal vesicles that are release from cells (and synapses) as exosomes or microvesicles. This work has been published in several impactful papers and it has been presented at seminars and international meetings.
We have significantly advanced our understanding of the in vivo mechanisms of synaptic demise in the context of neurodegenerative disease, including Parkinson's disease and Tauopathies. By studying the mechanisms of protein turnover in the context of these diseases we also revealed mechanisms healthy synapses use to remain functional during bouts of intense and long-lasting activity. Our work revealed novel components and mechanisms of synaptic protein turn-over and rejuvenation with a link to synapse robustness.
-We were the first to find a new pathway of autophagy that operates specifically at synapses and that is under control of different proteins mutated in Parkinson's disease: LRRK2, EndophilinA and Synaptojanin.
-We show that neuronal activity drives synaptic autophagy in an Endophilin1-dependent manner and in parallel to LRRK2 phosphorylation
-We found a new function for some of the most abundant proteins in our bodies: Hsc70 and Hsp90; they both control multi vesicular body function
-We show that Tau, a protein that aggregates in disease, binds to vesicles at synapses, causing synaptic dysfunction
-We identified Synaptogyrin-3 as the Tau receptor at synapses and removing the protein rescues Tau-induced synaptic dysfunction