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Daniel Elefant, Ignacio Hernandez Morato, Doreen Badheka, Jaliya Kumaratilake, Sansar C. Sharma, Stuart A. Newman; Endogenous alkaline phosphatase in the optic chiasm suggests a novel mechanism of retinal axonal guidance. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1579.
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© ARVO (1962-2015); The Authors (2016-present)
At the developing optic chiasm, numerous repulsive cues guide the contralateral and ipsilateral navigation for developing retinal ganglion cell (RGC) axons, although to date, no attractant molecules have been identified. We have localized a high concentration of enzymatically active intestinal-type alkaline phosphatase (IAP) closely associated with the developing avian chiasm. We tested and confirmed a hypothesis that this creates an adenosine (AD) gradient from the breakdown of extracellular ATP, providing an attractant cue for optic axons.
(1) Sections (15 μm) from 3d-10d chick embryos were stained immunohistochemically (IHC) with anti-IAP or -CD81 (a marker for exosomes), or with BCIP/NBT+levamisole with no 1o or 2o antibodies. (2) Retinal and chiasm explants isolated from 5-6 d embryos were cultured in native collagen gels (NCG) with AD+/- DMPX, ATP, CTP or in the presence of Elvax pellets with 1 mM AD. Images were analyzed with ImageJ software
(1) In 3-10 d embryos, endogenous levamisole resistant AP was detected at the optic chiasm (maximally at 7d). IHC confirmed the AP as the intestinal subtype. EM localized chiasmatic AP to the outer surface of exosome-like particles. Labeling with anti-CD81 with 3D imaging confirmed a similar distribution of exosomes to IAP. (2) Retinal explants cultured in NCG alone showed no significant neurite outgrowth. In the presence of AD, explants showed extensive nondirectional neurite growth compared to cytosine, ATP, or AMP. Elvax pellets infused with 1 mM AD induced directional neurite extension from explants towards higher concentrations of AD. Adding DMPX, an AD receptor blocker, decreased neurite outgrowth, indicating a role for the AD receptor. Retinal and chiasmatic tissue explants were co-cultured in the same NCG, with 1 mM ATP and neurite extension from the retinal towards the chiasm explant was seen. The same culture set-up, in the absence of ATP or the presence of CTP did not lead to any neurite outgrowth.
IAP is associated with high concentrations of exosomes in the midline of the developing diencephalon. Using NCG cultures, we obtained evidence that the localized AP, acting on ambient extracellular ATP, potentially generates an AD gradient with its high point at the midline, serving as a guidance cue for growing RGC axons. This finding has great potential impact on future axon regeneration studies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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