Synaptojanin1 is involved in endolysosomal trafficking in cone photoreceptors

Thesis (Ph.D.)--University of Washington, 2015Cells require the ability to properly sort and traffic proteins to their correct subcellular destination. The production of new proteins must be balanced by the turnover of old and damaged proteins. A breakdown in this process is detrimental to any cell; however highly polarized, non-proliferating cells, such as neurons, are particularly vulnerable to defects in protein turnover. Photoreceptors are highly polarized, specialized neurons that have high protein trafficking demands. At one end of the photoreceptor is the outer segment; the site of photon detection and phototransduction. The outer segment requires a constant supply of new proteins and membranes to maintain proper function. At the opposite end of the cell is the site of synaptic transmission. The synapse also undergoes large amounts of membrane trafficking and turnover due to the release of neurotransmitter and subsequent synaptic vesicle recycling. Although both ends of the photoreceptor require efficient membrane trafficking, the majority of studies of protein trafficking in photoreceptors have focused on the outer segment. In addition, photoreceptors do not need to degrade old and damaged proteins and membranes from the outer segment; the retinal pigment epithelium phagocytoses outer segment discs. Therefore the focus on outer segment trafficking has resulted in a deficit in our understanding of the process of protein degradation in photoreceptor cells. In this study we establish that the zebrafish nrca14 mutant has a specific defect in endolysosomal and autophagic trafficking and can therefore be used as a model to understand these processes in cone photoreceptors. The trafficking defects in the nrca14 cones begin early in photoreceptor development. The accumulation of autophagosomes in the nrca14 mutant is due, at least in part, to impaired autophagosome maturation that is not caused by a decrease in autophagosome mobility. The causative mutation in the nrca14mutant is in the gene encoding a polyphosphoinositide phosphatase Synaptojanin1; highlighting the importance of the phosphoinositide lipids in protein degradation pathways. We analyzed the distribution of the phosphoinositides PI(3)P, PI(4)P, PI(3,5)P2 and PI(4,5)P2 in wild type and nrca14 photoreceptors. We found that the distribution of these lipids in wild type photoreceptors is the same as in non-neuronal cell types; PI(3)P on endosomes, PI(4)P on the Golgi, PI(3,5)P2 on late endosomes/lysosomes and PI(4,5)P2 in the plasma membrane. We found no gross change in the distribution of these phosphoinositides in nrca14 mutant photoreceptors. Further, we use the nrca14 mutant phenotypes to discover that the Synaptojanin1 dephosphorylates a PIP species with a 5'phosphate to regulate autophagy and endolysosomal trafficking. Collectively, my work has generated a large tool set for studying membrane trafficking in zebrafish cone photoreceptors and has defined a specific role for Synaptojanin1 in regulating autophagy and degradative trafficking pathways in cones