Periodic Reporting for period 1 - SYNPT (The role of autophagy in presynaptic protein turnover)
Reporting period: 2015-05-01 to 2017-04-30
Interneuronal communication primarily takes place at synapses. Over the years studies have shown that protein synthesis and degradation processes affect the properties of synapses by changing the abundance of particular synaptic proteins in a spatially confined manner. Studying protein turnover and autophagy in the presynapse will help to understand how plastic changes occur at the level of individual synapses and give insights into the role of autophagy in neurodegenerative diseases. To this end I characterized the role of autophagy in presynaptic protein degradation and synaptic functioning. Unexpectedly, the degradation of most proteins, including presynaptic proteins, was not affected upon autophagy inhibition.
To further investigate the role of autophagy in neurons I used dynamic SILAC (stable isotope labeling by amino acids in cell culture) and mass spectrometry to measure degradation rates of thousands of neuronal proteins (Figure 1A). I compared degradation rates of proteins in wildtype (WT) mouse neurons with ATG5 knockout (KO) neurons, where autophagosome formation is blocked. Unexpectedly, the degradation of most proteins, including presynaptic proteins, was not affected. Additional live cell microscopy, immunofluorescence and western blot studies also showed no accumulation of presynaptic proteins after autophagy inhibition. Interestingly, I observed a significant decrease in the degradation of endoplasmic reticulum (ER) membrane proteins. Immunofluorescence, western blot and electron microscopy data indeed showed accumulation of ER membranes and proteins in ATG5 KO neurons (Figure 1B).
- Kononenko NL, Classen GA, Kuijpers M, Puchkov D, Maritzen T, Tempes A, Malik AR, Skalecka A, Bera S, Jaworski J, et al.: Retrograde transport of TrkB-containing autophagosomes via the adaptor AP-2 mediates neuronal complexity and prevents neurodegeneration. Nat Commun 2017, 8.
- Kuijpers M, Haucke V: Autophagosome Formation by Endophilin Keeps Synapses in Shape. Neuron. 2016 Nov 23;92(4).
- 4th International Symposium “Protein Trafficking in Health and Disease”, 2017, Hamburg (poster presentation: Autophagy in neurons: implications for ER structure and function )
- MDC-FMP-BIH-Charite Postdoc Day 2017, Berlin (poster presentation: Autophagy in neurons: implications for ER structure and function )
- 2016 CSHL Meeting: Axon Guidance, Synapse Formation & Regeneration (talk: Autophagosome transport via the endocytic adaptor AP-2 mediates BDNF-TrkB signalling)