Synaptojanin1 is Involved in Trafficking Synaptic Proteins in Cone Photoreceptors

Abstract

Purpose: Photoreceptors are highly polarized cells that require efficient and accurate means of sorting and transporting proteins to different cellular locations. The zebrafish visual mutant nrc^a14 lacks the polyphosphoinositide phosphatase Synaptojanin 1 (SynJ1). Cone photoreceptors in nrc^a14 zebrafish larvae have normal outer segments, but altered synaptic morphology and function. In addition, synaptic proteins and large vesicular structures accumulate in nrc^a14 cone inner segments. The goal of this study is to characterize these membranous structures within the inner segment. This will allow us to understand the role of SynJ1 and phosphoinositides in regulating synaptic protein trafficking.

Methods: Transgenic zebrafish with GFP targeted to various subcellular compartments within cones were generated and crossed into the nrc^a14 zebrafish line. To determine the effects of altering synaptic activity on these subcellular compartments, WT and nrc^a14 larvae were incubated at 28°C on a normal 14/10hr light/dark cycle, or complete darkness for 24hrs. Cones of larvae at 5 days post fertilization were analyzed by confocal or transmission electron microscopy.

Results: We used Golgi, early and late endosomal, and synaptic markers to determine the identity of the aberrant vesicular structures within nrc^a14 cone inner segments. Cone inner segments of nrc^a14 larvae contain mislocalized synaptic proteins, fragmented Golgi and enlarged early endosomes. To determine the effects of altering synaptic activity on nrc^a14 cones we incubated larvae in the dark for 24hrs. Dark incubation dramatically increased the appearance of vesicular structures in nrc^a14, but not WT, cone inner segments. To correlate the vesicular structures to a specific subcellular membrane, we are currently analyzing the effect of dark adaptation on our subcellular markers.

Conclusions: SynJ1 is involved in multiple membrane trafficking events in cones. These trafficking events are modulated by synaptic activity as dark incubation increases the accumulation of vesicular structures in nrc^a14 cones. The appearance of both abnormal Golgi and early endosomes implicates SynJ1 in trafficking of newly synthesized synaptic proteins to the synapse and/or recycling of synaptic proteins from the synapse. The nrc^a14 zebrafish provides a tool for understanding how the trafficking of synaptic proteins is regulated in cones.

Keywords: 728 synapse • 648 photoreceptors • 583 lipids