Abstract 1777 Loss of photoreceptor ankyrin repeat proteins Panky and Panky-like disturbs the photoreceptor cell membrane integrity and dampens the photoreceptor cell function in the mouse retina

Our vision begins with the retinal photoreceptor cells sensing ambient light changes. Visual information is transmitted in the form of analog electrical signals from photoreceptor to bipolar cells through chemical synapses, processed by the retinal circuitry, and finally output as digital signals to the brain via retinal ganglion cells. Our microarray and scRNA-seq analyses of conditional Otx2 transcription factor knockout (KO) mouse retinas where photoreceptor cells fail to differentiate revealed a variety of genes encoding factors important for photoreceptor development and functions. A pair of ankyrin repeat-containing proteins encoded by Ankrd33 and Ankrd33b, respectively, were found robustly and exclusively expressed in photoreceptor cells and hereafter termed as Panky (photoreceptor ankyrin repeat protein) and Panky-like, each containing 5 ankyrin repeats. The ankyrin repeats, composed of ∼33 highly conserved amino acid residues, have been suggested to mediate protein-protein interactions and are shown involved in a variety of cellular functions including transcription, signal transduction, and cytoskeleton structure. In the current study, we first generated knockout mice of Panky and Panky-like, aiming to elucidate their cellular functions in mature photoreceptor cells in vivo. All animal studies were conducted in accordance with the Animal Care and Use Committee guidelines of the Institute for Protein Research, Osaka University. Suggested by their highly homologous amino acid sequences to each other and the protein-protein interactions shown by co-immunoprecipitation in vitro, in photoreceptor cells Panky and Panky-like are likely to be functionally redundant as we found no significant difference in Panky or Panky-like single KO mice by immunohistochemistry and electroretinogram (ERG) examinations. In contrast, ERG recording from Panky/Panky-like double KO (DKO) mice showed reduced amplitudes of a-waves (i.e., photoreceptor cell activities) and b-waves (i.e., synaptic transmission from photoreceptor to bipolar cell) together with delayed b-wave implicit times under light-adapted conditions. These results indicate that light sensitivity and synaptic connections of cone photoreceptors are impaired in DKO mice. Morphological abnormalities of photoreceptor cells were revealed by immunostaining and transmission electron microscopy observation, especially in the outer segment compartment. The outer segment is the cellular compartment responsible for photo-perception and photo-transduction of photoreceptor cells and is known to be highly enriched in lipids. We therefore conducted the lipidomics analysis and found the amount of a variety of gangliosides and glycerophospholipids increased in DKO retinas compared to that of littermate controls. The altered composition of gangliosides due to the loss of Panky/Panky-like may disturb the membrane rigidity and integrity, resulting in the functional defect of cone photoreceptors in DKO mice. This project was funded by Grant-in-Aid for Scientific Research (B) (A21H026570), Grant-in-Aid for Scientific Research (C) (23K06303) from the Japan Society for the Promotion of Science, The Takeda Science Foundation and The Uehara Memorial Foundation.