During the course of cellular life, macromolecules, proteins and organelles become damaged or otherwise unwanted. Proper disposal of these unwanted cellular components is essential for cell functioning. While most cells are capable of diluting out cellular waste by division...
During the course of cellular life, macromolecules, proteins and organelles become damaged or otherwise unwanted. Proper disposal of these unwanted cellular components is essential for cell functioning. While most cells are capable of diluting out cellular waste by division, non-dividing neurons are extremely prone to accumulate damaged components over time. Therefore, proper waste management is extremely important for healthy neuron functioning. One of the systems responsible for the removal of proteins and organelles is autophagy. During autophagy a defined set of proteins (Atg, or autophagy-related proteins) coordinate the orderly degradation and recycling of cellular components. A membrane compartment termed the isolation-membrane is formed, expands in size and eventually closes to form a double-membrane vesicle called the autophagosome. Finally, the resulting autophagosome fuses with the lysosome where its cargo is degraded. This pathway has been implicated in many cellular processes such as neuronal development and ageing. Dysfunctional autophagy has been shown to contribute to neurodegeneration and is linked to neurodegenerative diseases such as Alzheimerâ€™s and Parkinsonâ€™s disease, the two neurodegenerative disorders of highest prevalence in our society. Moreover, autophagic activity declines during aging and enhancing expression of autophagy genes has been proven effective in prolonging life span. Consequently, understanding the mechanisms underlying autophagic degradation and finding the substrates targeted by this pathway will provide essential knowledge to fight age-related disorders and improve health span in our aging population.
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 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).
- 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 â€œProtein Trafficking in Health and Diseaseâ€, 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)
My data showed that autophagy inhibition does not affect presynaptic protein degradation but mainly affects the degradation of ER-membrane proteins and leads to the accumulation of ER. For that reason the project is focused more on autophagy and ER. The ER plays a crucial role in protein and lipid synthesis and modification, transport, quality control and calcium level regulation. The ER is a highly dynamic organelle and a constant turnover and modulation is needed for different cellular processes such as cell division, development and neuronal activity. Neurons may rely on the ER for Ca2+ needed for signalling as well as lipid synthesis required for neurite outgrowth. Moreover, alterations in ER shape are linked to neurologic disorders such as amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegias, indicating the importance of ER morphology in neurons. The hereditary spastic paraplegias (HSP) are disorders characterized by muscle weakness and axon degeneration. Many HSP-associated genes encode for so-called ER-shaping proteins, suggesting that dysregulation of ER homeostasis leads to neurodegeneration. While endoplasmic reticulum malfunctions play an important role in neuropathology, little is known about ER architecture and dynamics. My project will contribute to a better understanding of neurological disorders in which ER homeostasis is affected and might lead to future therapeutic approaches.