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En Leh Samuel Tsai, Arturo Ortin-Martinez, Akshay Gurdita, Lacrimioara Comanita, Nicole Yan, Sheila Smiley, Philip Nickerson, Vianney Delplace, Molly Shoichet, Valerie Wallace; An in-vitro retina aggregate system to investigate the effects of environment on photoreceptor neurite outgrowth. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3312.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptor transplantation is being investigated as a potential therapy for retinal degeneration; however, recent studies have shown that very few donor cells make synaptic connections with the host retina. To address the role of environmental cues in photoreceptor neurite outgrowth we established an in vitro co-culture system to model the effect of retinal cells on immature photoreceptor neurite outgrowth.
GFP-labeled photoreceptor (donors) were transplanted into the subretinal space of wildtype (WT) or degenerative (CrxKO) animals. The recipients were then harvested 3 weeks post-transplant and analyzed via flatmount and cryosection. To form aggregates, WT or CrxKO retinas of P1 pups were harvested, dissociated and plated at high density to form aggregates after six days in culture. For co-cultures, FACS-purified donors were added to aggregates and cultured for an additional 3 days. We ablated Müller glia (MG) using α-aminoadipic acid (AAA) and inhibited Rho/ROCK signaling using an inhibitor (Y27632). The aggregates and co-cultures were fixed, stained and analyzed using Imaris 9.0.
We first characterized our aggregate assay and established that aggregate formation is reproducible in terms of size and cellular composition. Neurite extension in donors is significantly enhanced in the presence of WT aggregates relative to purified donors cultured alone. In vivo, transplanted photoreceptors in CrxKO recipients exhibit enhanced total neurite length (37.2±3.1 um) when compared with WT recipients (6.8±1.4 um). When we co-cultured donors with aggregates, we observe that the neurite enhancement effect of CrxKO is recapitulated: total neurite length for CrxKO co-cultures (37.1±1.0 um) was significantly increased compared to wildtype co-cultures (26.9±0.8 um). Total neurite length of photoreceptors is significantly decreased when MG are ablated by AAA treatment in both WT (-15%) and CrxKO (-38%). In contrast, Y27632 treatment significantly enhanced total neurite length in both WT (+32%) and CrxKO (+28%).
Collectively, these data show that photoreceptors require either non-retinal cell types or some level of retinal organization to extend neurites. Photoreceptor neurite extension partially requires Müller glia and is effected by Rho/ROCK signaling. Finally, we show that Crx-dependent photoreceptor differentiation is an inhibitor of neurite outgrowth.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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