To characterize the contribution of autophagy on the turnover of presynaptic proteins I generated temporally controlled Atg5-deficient mice. Tamoxifen addition to DIV0 cultured hippocampal neurons isolated from newborn mice carrying floxed alleles of ATG5 and expressing a tamoxifen-inducible Cre recombinase resulted in strongly reduced formation of autophagosomes. Conditional loss of ATG5 in hippocampal neurons in culture resulted in a reduced neuronal complexity. Further investigation showed that the transport of autophagosomes in neurons promotes neuronal complexity by transporting TrkB signals (published in Kononenko et al. Nat Commun 2017).
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).
Dissemination:
Papers
- 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).
Conferences
- 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)
