Synthesis and Evaluation of Two Novel All -trans-Retinoic Acid Conjugates: Biocompatible and Functional Tools for Retina Research

The vitamin A derivative all- trans-retinoic acid (ATRA) is an important biologically active metabolite that regulates a variety of essential biological processes in particular via gene-regulatory mechanisms. In the retina, ATRA is a light-dependent byproduct of the phototransduction cascade. Here, ATRA is not only needed for proper retinal development, but it also acts as a neuromodulator on horizontal cells, second-order inhibitory neurons in the outer retina, which reveal morphological and physiological changes when the retina is treated with ATRA. There is evidence that gene-regulatory mechanisms may only be partially involved in these neuromodulatory processes and the underlying nontranscriptional mechanisms are still elusive. This is, among other things, due to the lack of appropriately labeled ATRA, which would allow the tracking of ATRA in cells or a given tissue. To overcome this obstacle, we designed, synthesized, and evaluated two conjugates of ATRA, one conjugated with biotin (biotin-ATRA) and one conjugated with diaminoterephthalate fluorophore (DAT-ATRA), as molecular tools for different fields of application. The biocompatibility of both compounds was demonstrated via cell viability assays in cultured N2a-cells. N2a-cells exposed to the compounds showed no significant changes in the viability rate. The functionality of synthesized ATRA-conjugates was verified using retinal tissue derived from adult carp. The binding of ATRA-conjugates to distinct retinal cells was assessed in primary cultures of carp retina. Hereby, horizontal and Müller cells have been identified as specific target cells of the new ATRA compounds. Electron microscopy further confirmed that the new substances are still able to induce synaptic plasticity at horizontal cell dendrites resulting in formation of spine synapses, as it is shown for native ATRA. Taken together, the novel ATRA-conjugates represent biocompatible and functional molecular tools, which may further provide the possibility to track ATRA in neuronal cells and study its modulatory effects in different cell systems.