Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation

Abstract

Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix–like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.

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Figure 1: Molecular analysis and expression of Pikachurin.
Figure 2: Pikachurin localizes to the synaptic cleft of photoreceptor ribbon synapse in the OPL.
Figure 3: Generation of Pikachurin null mouse by targeted gene disruption.
Figure 4: Pikachurin is required for proper apposition of bipolar dendritic tips to the photoreceptor synaptic terminus.
Figure 5: Electrophysiological and OKR analyses of wild-type and Pikachurin null mice.
Figure 6: Interaction and colocalization of pikachurin with dystroglycan.

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Acknowledgements

We thank Y. Kambara, M. Murai, T. Tsujii, E. Oiki, S. Takiuchi and K. Sone for technical assistance; Y. Saijoh, M. Uehara and H. Hamada for advice on the production of a knockout mouse; and N. Maeda for statistical analysis. This work was supported by Molecular Brain Science, Grant-in-Aid for Scientific Research on Priority Areas and Grant-in-Aid for Scientific Research (B), Grand-in-Aid for Exploratory Research, Specially Designated Research Promotion, Takeda Science Foundation, Senri Life Science Foundation, The Uehara Memorial Foundation and Mochida Memorial Foundation for Medical and Pharmaceutical Research. A part of this work was supported by the Nanotechnology Network Project of the Ministry of Education, Culture, Sports, Science and Technology Japan at the Research Center for Ultrahigh-Voltage Electron Microscopy, Osaka University (Handai Multi-functional Nano-Foundry).

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Authors

Contributions

S.S. and T. Furukawa designed the project. S.S., Y.O. and K. Katoh carried out the molecular, immunocytochemistry and electron microscopy experiments. S.S., M.K., K.M. and T.K. carried out the ERG experiments. S.S., A.T. and T. Furukawa produced the knockout mice. S.S. and N.K. performed the electron tomography analysis. J.U. carried out the immuno–electron microscopy experiments. S.S. and K.F. performed the OKR experiments. T.M. and H.S. carried out the VEP experiments. S.S., Y.O., M.K., K. Kobayashi and T.T. conducted the pull-down experiments. S.S., Y.O. and T. Furukawa wrote the manuscript. Y.T., T. Fujikado. and T. Furukawa supervised the project.

Corresponding author

Correspondence to Takashi Fujikado.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Methods (PDF 753 kb)

41593_2008_BFnn2160_MOESM216_ESM.mpg

Rod photoreceptor synapse terminal from wild-type retina displayed as serial slices from a tomogram. Graphic representation of rod photoreceptor synapse termini based on electron tomography using UHVEM. Wild-type (ac) and pikachurin−/− retina (df) are displayed as serial slices from a tomogram (a,d), serial slices with colored segmentation (b,e), and as a surface-rendered three-dimensional model (c,f). In the wild-type retinas, the synaptic ribbon of a rod photoreceptor (green) is adjacent to the horizontal cell processes (dark blue) and bipolar cell dendrites. (MPG 3177 kb)

Supplementary Movie 1

Rod photoreceptor synapse terminal from wild-type retina displayed as serial slices from a tomogram. Graphic representation of rod photoreceptor synapse termini based on electron tomography using UHVEM. Wild-type (ac) and pikachurin−/− retina (df) are displayed as serial slices from a tomogram (a,d), serial slices with colored segmentation (b,e), and as a surface-rendered three-dimensional model (c,f). In the wild-type retinas, the synaptic ribbon of a rod photoreceptor (green) is adjacent to the horizontal cell processes (dark blue) and bipolar cell dendrites. (MPG 3177 kb)

41593_2008_BFnn2160_MOESM217_ESM.mpg

Rod photoreceptor synapse terminal from wild-type retina displayed as serial slices with colored segmentation. (MPG 6338 kb)

Supplementary Movie 2

Rod photoreceptor synapse terminal from wild-type retina displayed as serial slices with colored segmentation. (MPG 6338 kb)

41593_2008_BFnn2160_MOESM218_ESM.mpg

Rod photoreceptor synapse terminal from wild-type retina displayed as a surface-rendered three-dimensional model. (MPG 6620 kb)

Supplementary Movie 3

Rod photoreceptor synapse terminal from wild-type retina displayed as a surface-rendered three-dimensional model. (MPG 6620 kb)

41593_2008_BFnn2160_MOESM219_ESM.mpg

Rod photoreceptor synapse terminal from pikachurin−/− retina displayed as serial slices from a tomogram. (MPG 6649 kb)

Supplementary Movie 4

Rod photoreceptor synapse terminal from pikachurin−/− retina displayed as serial slices from a tomogram. (MPG 6649 kb)

41593_2008_BFnn2160_MOESM220_ESM.mpg

Rod photoreceptor synapse terminal from pikachurin−/− retina displayed as serial slices with colored segmentation. (MPG 6672 kb)

Supplementary Movie 5

Rod photoreceptor synapse terminal from pikachurin−/− retina displayed as serial slices with colored segmentation. (MPG 6672 kb)

41593_2008_BFnn2160_MOESM221_ESM.mpg

Rod photoreceptor synapse terminal from pikachurin−/− retina displayed as a surface-rendered three-dimensional model. (MPG 6624 kb)

Supplementary Movie 6

Rod photoreceptor synapse terminal from pikachurin−/− retina displayed as a surface-rendered three-dimensional model. (MPG 6624 kb)

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Sato, S., Omori, Y., Katoh, K. et al. Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation. Nat Neurosci 11, 923–931 (2008). https://doi.org/10.1038/nn.2160

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