Structural divergence of essential triad ribbon synapse proteins among placental mammals - Implications for preclinical trials in photoreceptor transplantation therapy.

As photoreceptor transplantation rapidly moves closer to the clinic, verifying graft efficacy in animal models may have unforeseen xenogeneic barriers. Although photoreceptor transplants have most convincingly exhibited functional synaptogenesis in conspecific studies, such evidence (while ruling out false-positives due to: viral graft labeling, fusion/cytosolic transfer, or neuroprotection) has not yet been shown for discordant xenografts. From this, a fundamental question should be raised: is useful xenosynaptogenesis likely between human photoreceptors and mouse retina? The triad ribbon synapse (TRS) that would normally form is unique and contains trans-synaptic proteins essential to its formation and function. Thus, could interspecific structural divergence be present that may inhibit this trans-synaptic bridge in discordant xenografts? In an effort to address this question computationally, we compared eight recently confirmed (including subcellular location) TRS specific (or predominantly expressed at the TRS) proteins among placental mammals (1-to-1 orthologs) using HyPhy selection analysis (a predictive measure of structural divergence) and by using Phyre2 tertiary structural modeling. Here, selection analysis revealed strong positive (diversifying) selection acting on a particularly important TRS protein: pikachurin. This positive selection was localized to its second Laminin-G (LG)-like domain and on its N-terminal domain - a putative region of trans-synaptic interaction. Localization of structural divergence to the N-terminus of each putative post-translational cleavage (PTC) product may suggest neofunctionalization from ancestral uncleaved pikachurin - this would be consistent with a recent counter-paradigm report of pikachurin cleavage predominating at the TRS. From this, we suggest a dual role after cleavage where the N-terminal fragment can still mediate the trans-synaptic bridge, while the C-terminal fragment may act as a diffusible trophic or "homing" factor for bipolar cell dendrite migration. Tertiary structural models mirrored the conformational divergence predicted by selection analysis. With human and mouse pikachurin (as well as other TRS proteins) likely to diverge considerably in structure among placental mammals - alongside known inter-mammalian variation in TRS phenotype and protein repertoire, high levels of diversifying selection acting on genes involving sensation, considerable timespans allowing for genetic drift that can create xenogeneic epistasis, and uncertainty surrounding the extent of xenosynaptogenesis in PPC transplant studies to date - use of distantly related hosts to test human photoreceptor graft therapeutic efficacy should be considered with caution.