Regulation of axonal arbor and synaptic dynamics by the apoptotic pathway: Interactions with the cytoskeleton, downstream substrates and upstream regulators
Final Report Abstract
The cell biology underlying the addition and retraction of axonal branches and synapse formation involves the dynamic modulation of the neuronal cytoskeleton and membrane trafficking events. However, how these processes are coordinated remains little studied. Since apoptotic cells undergo dramatic cytoskeletal and membrane trafficking events, the intrinsic pathway of which we, and others have shown local roles for in neurones, we are examining possible coordination roles using the zebrafish visual system as a model for in vivo cell biology. Very little is known about roles for the extrinsic pathways of apotosis in the nervous system in non-apoptoptic contexts and nothing in the role of arborisation and synapse formation. We found that loss of caspase-8a led to increased retinal ganglion cell (RGC) axonal arbor size, presynaptic sites and arbor dynamics were more stable compared to control arbors. Surprisingly, we were able to rescue both the increased arbor size and reduced dynamics of the caspase-knockdown phenotype with a catalytically-inactive caspase-8 mutant. To begin to examine membrane trafficking events in RGC arborisation we imaged axon arbors under conditions where key early endosome function was manipulated by activating or inhibiting Rab5c, a key early endosomal protein. Loss of Rab5c function lead to much larger arbors, similarly to the loss of Caspase-8a, identifying a novel role for early endosomes in arbor growth. We tested for and observed a genetic interaction between caspase-a8 and rab5c in restricting arbor growth and presynaptic sites. Combined with our previous study our results suggest that axonal arborisation is under the coordinated regulation of the intrinsic apoptotic pathway in a catalytic activity-dependent-manner and here we identify a novel catalytic activity-independent role for the extrinsic apoptotic pathway interacting with early endosomal signaling to restrict arbor and presynaptic growth, by promoting their dynamics As caspases are proteases that are usually thought to function through their catalytic activity, our observation that Caspase-8 could function in a noncatalytic manner in neurones is both surprising and exciting. We are now trying to understand how it functions and its upstream regulators. We hypothesize that the apoptotic pathway may act as a central coordinator of the cytoskeletal and membrane trafficking events underlying axonal arborisation which will from a focus of ongoing and future experiments. In addition to the above we have generated tools for labelling the cytoskseleton and membrane trafficking, such as exocytosis, autophagy and lysosomes which will be used in further studies.
Publications
- (2017) Connecting the Eye to the Brain: Visualizing lysosome Trafficking in Zebrafish Neurons. Amgen Scholar Cambridge Symposium, UK
McDermott, M. Kotov, I., Campbell, D. and Misgeld, T.
- (2018) A catalytic activity-independent role for the extrinsic apoptotic pathway interacting with early endosomes to restrict axonal arbor growth and synaptogenesis in vivo. Latest Advances in Development and Function of Neuronal Circuits, Cold Spring Harbor Asia Conference, Awaji island Japan
Kotov, I. N. and Campbell, D. S.
- (2018) A catalytic activity-independent role for the extrinsic apoptotic pathway interacting with early endosomes to restrict axonal arbor growth and synaptogenesis in vivo. The 24th Japanese Medaka and Zebrafish meeting, Nagoya, Japan
Kotov, I. N. and Campbell, D. S.
- (2019) Connecting the eye to the brain: A model system for studying cell biology and neuronal connectivity. 5th Core-to-Core International Symposium "3D Lab-Exchange Program" Okinawa Institute of Science and Technology (OIST), Onna-son, Okinawa, Japan
Campbell, D.S.
