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J. Han, J. Wang, F. Kung, E. Townes-Anderson; Localization of the Newly-Identified Synaptic Protein Pikachurin in Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4121.
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Pikachurin is a recently identified protein that plays a key role in the formation of ribbon synapses in the retina (Sato, et al. Nature Neuroscience, 2008). Unlike other presynaptic and postsynaptic molecules vital for the synaptogenesis of ribbon synapses, pikachurin only affects synapses between photoreceptors and bipolar cells and has been shown to reside in the synaptic cleft. Specific synaptogenesis during retina regeneration after injury and disease could be potentially enhanced by pikachurin. However, it is not clear where pikachurin is synthesized, how it is released, and whether retinal degeneration and regeneration affect its expression. To address these issues, the expression of pikachurin in salamander and porcine retinas were examined in this study.
Salamander and porcine retinas were dissociated by enzymatic treatment with papain followed by gentle mechanical trituration. Cells were fixed immediately after or cultured for up to 2 weeks before fixation. Fixed cells were immunostained with polyclonal against pikachurin raised in rabbit (gift of Dr. T. Furukawa, Osaka Bioscience Institute, Japan). Pikachurin expression was also examined in cell lysates of salamander and porcine whole retinas by Western blot.
In acutely isolated retinal cells, pikachurin immunofluorescence was visible in various cell types, including photoreceptors, Müller cells, and multipolar cells. Typically, the fluorescence was diffused throughout the cytoplasm, often appearing punctate. In photoreceptors, there was frequently strong immunostaining in the synaptic pedicles. When retinal cells were cultured for several days in vitro, pikachurin immunofluorescence remained prominent in photoreceptors and Müller cells. In addition, co-localization of pikachurin and synaptic vesicle markers (e.g. SV2) was observed in photoreceptor inner segments and newly formed synaptic varicosities. Pikachurin antibody bound to the expected 130 Kd band in Western blots as well as a 260 Kd and a 55 Kd band, interpreted as a dimer and a smaller isoform, respectively.
Pikachurin is expressed in both neural and glial retinal cells. With time in culture, it is co-localized with synaptic vesicles in neurites, suggesting its release is associated with synaptic vesicle exocytosis. Expression of pikachurin in Müller cells suggests a role for glia in synaptogenesis.
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