Detection of Endogenous Retinoids in the Molluscan CNS and Characterization of the Trophic and Tropic Actions of 9-cisRetinoic Acid on Isolated Neurons

Abstract

Retinoic acid (RA) is an active metabolite of Vitamin A that plays an important role in the growth and differentiation of many cell types. All-trans RA (atRA) is the retinoic acid isomer that has been most widely studied in the nervous system, and can induce and direct neurite outgrowth from both vertebrate and invertebrate preparations. The presence and role of the 9-cis-RA isomer in the nervous system is far less well defined. Here, we used high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) to show for the first time, the presence of both atRA and 9-cis-RA in the CNS of an invertebrate. We then demonstrated that 9-cis-RA was capable of exerting the same neurotrophic and chemotropic effects on cultured neurons as atRA. In this study, significantly more cells showed neurite outgrowth in 9-cis-RA versus the EtOH vehicle control, and 9-cis-RA significantly increased the number and length of neurites from identified neurons after 4 d in culture. 9-cis-RA also extended the duration of time that cells remained electrically excitable in culture. Furthermore, we showed for the first time in any species, that exogenous application of 9-cis-RA induced positive growth cone turning of cultured neurons. This study provides the first evidence for the presence of both atRA and 9-cis-RA in an invertebrate CNS and also provides the first direct evidence for a potential physiological role for 9-cis-RA in neuronal regeneration and axon pathfinding.

From carrot to clinic: an overview of the retinoic acid signaling pathway

“…However, the biological significance of 9-cis RA as the in vivo RXR ligand remains controversial as no significant levels of 9-cis RA could be detected in vivo in different vertebrates [130]. However, 9-cis RA was shown to be functional in other organisms, such as in the CNS of the mollusk Lymnaea stagnalis, where the presence of both all-trans RA and 9-cis RA has been shown [131]. Similarly, 9-cis RA was detected along with all-trans RA in locust embryos suggesting a functional role for retinoids in insects [132].…”

A Novel, Nongenomic Mechanism Underlies Retinoic Acid-Induced Growth Cone Turning

“…There are no specific Ca 2ϩ channel blockers that are effective in Lymnaea neurons, so to this end, we used the general Ca 2ϩ channel blocker, cadmium, which has previously been shown to be effective in Lymnaea neurons (Feng et al, 2000;Dmetrichuk et al, 2008). Again, we monitored neurite outgrowth for 1 h after the addition of cadmium to the bath (10 Ϫ5 M, final concentration) and found that it had no adverse effect on the rate of neurite outgrowth (0.682 Ϯ 0.369 m/min; p Ͼ 0.05).…”

Presence of Ribeiroia ondatrae in the developing anuran limb disrupts retinoic acid levels

“…Retinoid extraction was performed as described previously (Dmetrichuk et al 2008). Briefly, tissue was thawed and homogenised in 500 μl of ice cold homogenisation buffer (phosphate buffered saline, pH 7.3 containing 0.5% ascorbic acid and 0.5% EDTA).…”

“…High-pressure liquid chromatography Techniques used were performed as described previously (Dmetrichuk et al 2008). Briefly, chromatographic separation was performed using a Waters Spherisorb S5ODS2 C-18 Column.…”

“…This was connected online to an Agilent 1100 LC system with the conditions described above. As described previously (Dmetrichuk et al 2008), the mass spectrometer was set up to detect 255 m/z, which is a fragment observed in MS2 when the parent ion (299 m/z) (corresponding to either atRA or 9-cis RA) is fragmented according to the following scheme: molecular anion (299)→(255)+CO 2 . This specific fragmentation behaviour has been previously documented in our earlier work (unrelated to current study) and has been shown to correspond specifically to both isomers of RA.…”