秋葉 龍太朗

<jats:p> The retinal fovea in human and nonhuman primates is essential for high acuity and color vision. Within the fovea lies specialized circuitry in which signals from a single cone photoreceptor are largely conveyed to one ON and one OFF type midget bipolar cell (MBC), which in turn connect to a single ON or OFF midget ganglion cell (MGC), respectively. Restoring foveal vision requires not only photoreceptor replacement but also appropriate reconnection with surviving ON and OFF MBCs and MGCs. However, our current understanding of the effects of cone loss on the remaining foveal midget pathway is limited. We thus used serial block-face electron microscopy to determine the degree of plasticity and potential remodeling of this pathway in adult <jats:italic>Macaca fascicularis</jats:italic> several months after acute photoreceptor loss upon photocoagulation. We reconstructed MBC structure and connectivity within and adjacent to the region of cone loss. We found that MBC dendrites within the scotoma retracted and failed to reach surviving cones to form new connections. However, both surviving cones and ON and OFF MBC dendrites at the scotoma border exhibited remodeling, suggesting that these neurons can demonstrate plasticity and rewiring at maturity. At six months postlesion, disconnected OFF MBCs clearly lost output ribbon synapses with their postsynaptic partners, whereas the majority of ON MBCs maintained their axonal ribbon numbers, suggesting differential timing or extent in ON and OFF midget circuit remodeling after cone loss. Our findings raise rewiring considerations for cell replacement approaches in the restoration of foveal vision. </jats:p>

Retinitis pigmentosa (RP) is a group of hereditary diseases that involve loss of photoreceptors. There has been no established treatment for RP, and it is now the 2nd leading cause of blindness in Japan. Previous clinical researches using human fetal retina transplantation suggested some functional recovery in vision, but it did not become a standard therapy because of ethical concerns for using fetus tissues. Invention of induced pluripotent stem cells (iPSC) in 2006 and the establishment of retinal organoids induction protocol from ES/iPS cells have paved a way of cell therapy for RP without ethical concerns. Our team has shown that mouse iPSC derived retinas can survive and mature after subretinal transplantation to the end-stage retinal degeneration model mice. Further, human ESC derived retinas survived and matured in retinal degeneration monkey models. Recently, we have established a qualitative and quantitative evaluation tool for photoreceptor synapses, QUANTOS, and showed that photoreceptors in mouse iPSC derived retina can form photoreceptor synapses in a time dependent manner after transplantation. We are now moving toward 1st in human clinical trial using iPSC derived retina for RP.

Quantitative and qualitative evaluation of synapses is crucial to understand neural connectivity. This is particularly relevant now, in view of the recent advances in regenerative biology and medicine. There is an urgent need to evaluate synapses to access the extent and functionality of reconstructed neural network. Most of the currently used synapse evaluation methods provide only all-or-none assessments. However, very often synapses appear in a wide spectrum of transient states such as during synaptogenesis or neural degeneration. Robust evaluation of synapse quantity and quality is therefore highly sought after. In this paper we introduce QUANTOS, a new method that can evaluate the number, likelihood, and maturity of photoreceptor ribbon synapses based on graphical properties of immunohistochemistry images. QUANTOS is composed of ImageJ Fiji macros, and R scripts which are both open-source and free software. We used QUANTOS to evaluate synaptogenesis in developing and degenerating retinas, as well as de novo synaptogenesis of mouse iPSC-retinas after transplantation to a retinal degeneration mouse model. Our analysis shows that while mouse iPSC-retinas are largely incapable of forming synapses in vitro, they can form extensive synapses following transplantation. The de novo synapses detected after transplantation seem to be in an intermediate state between mature and immature compared to wildtype retina. Furthermore, using QUANTOS we tested whether environmental light can affect photoreceptor synaptogenesis. We found that the onset of synaptogenesis was earlier under cyclic light (LD) condition when compared to constant dark (DD), resulting in more synapses at earlier developmental stages. The effect of light was also supported by micro electroretinography showing larger responses under LD condition. The number of synapses was also increased after transplantation of mouse iPSC-retinas to rd1 mice under LD condition. Our new probabilistic assessment of synapses may prove to be a valuable tool to gain critical insights into neural-network reconstruction and help develop treatments for neurodegenerative disorders.