V. PETROVA, R.EVA, J.W. FAWCETT
Numerous extracellular and intracellular processes contribute to the failure of long-range regeneration in the adult central nervous system (CNS) after injury. One reason why adult CNS axons have poor regenerative capabilities is that a developmental change occurs where essential growth molecules such as integrins and growth factor receptors become excluded from axons. These growth-promoting molecules are normally transported along axons in Rab11-positive recycling endosomes by motor proteins/adaptor complexes. However, this transport declines with maturation leading to a decline in regenerative capacity. Protrudin, a member of the ZFYVE family of zinc-binding proteins is a membrane-associated protein involved in neurite outgrowth and directional membrane trafficking in HeLa, PC12 and primary hippocampal cells (Shirane et al., 2006). Phosphorylated protrudin preferentially binds to Rab11-GDP, an association which is required for neurite outgrowth and for anterograde movement of this complex.
We hypothesised that increasing the phospho-protrudin/Rab11 interaction would result in anterograde transport of growth-promoting molecules to the tip of injured axons enabling regeneration of primary cortical neurons after laser axotomy. To test this, two phosphomimetic forms of protrudin were created at phosphorylation sites known to play an important role for its association with Rab11. We found that both constitutively phosphorylated protrudin forms (64%, 70%) and also wild-type protrudin (60%) increased the proportion of regenerating axons compared to control (27%). Live-cell imaging experiments are currently being performed to investigate whether the increase in regenerative capacity is indeed due to its association with Rab11 and increased anterograde transport of integrins. Protrudin is a complex molecule with numerous cellular functions such as ER shaping, vesicular transport, interactions with spastin – a microtubule-severing protein and many more. In order to unpick the mechanisms of action of protrudin on regeneration, five mutants were created – each targeting a specific region of the protein important for its involvement in various molecular pathways (ΔFYVE, ΔRab11-binding-domain, ΔKIF5, ΔFFAT, ΔSpastin). These mutants were overexpressed in primary cortical neurons and their effects on regeneration were tested. Protrudin was found to promote regeneration through multiple mechanisms, some of which, such as ER function have not previously been associated with the process of axon regeneration. Protrudin’s ability to promote regeneration is currently being tested in vivo in models of acute and chronic injury.
*V. PETROVA1, R. EVA1, J. W. FAWCETT1,2;
1Cambridge Univ., Cambridge, United Kingdom; 2Inst. of Exptl. Med., Ctr. of Reconstructive Neurosci., Prague, Czech Republic. Overexpression of protrudin in primary cortical neurons enhances regeneration after laser axotomy through multiple mechanisms. Program No. 115.05. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.
Grant Support: MRC Grant R004463, MRC Grant G1000864, Gates Cambridge Studentship, Gates Cambridge Academic Development Award,
International Foundation for Research in Paraplegia P172