Bile Acids Induce Neurite Outgrowth in Nsc-34 Cells via TGR5 and a Distinct Transcriptional Profile

Abstract

Increasing evidence supports a neuroprotective role for bile acids in major neurodegenerative disorders. We studied major human bile acids as signaling molecules for their two cellular receptors, farnesoid X receptor (FXR or NR1H4) and G protein-coupled bile acid receptor 1 (GPBAR1 or TGR5), as potential neurotrophic agents. Using quantitative image analysis, we found that 20 μM deoxycholic acid (DCA) could induce neurite outgrowth in NSC-34 cells that was comparable to the neurotrophic effects of the culture control 1 μM retinoic acid (RA), with lesser effects observed for chenodexoycholic acid (CDCA) at 20 μM, and similar though less robust neurite outgrowth in SH-SY5Y cells. Using chemical agonists and antagonists of FXR, LXR, and TGR5, we found that TGR5 agonism was comparable to DCA stimulation and stronger than RA, and that neither FXR nor liver X receptor (LXR) inhibition could block bile acid-induced neurite growth. RNA sequencing identified a core set of genes whose expression was regulated by DCA, CDCA, and RA. Our data suggest that bile acid signaling through TGR5 may be a targetable pathway to stimulate neurite outgrowth.

Keywords: FXR; TGR5; bile acids; neurite outgrowth.

Figure 1

Neurite outgrowth measured as neurite length using NeuronCyto II. (A) NSC-34 exposed to basal medium (DM), 1 μM retinoic acid (RA), and 20 μM of the bile acid deoxycholic acid (DCA) for 6 days. Four microscopic fields (colored examples shown below the red/blue DAPI/immunostaining images) from each of ten 20X images (40 total fields) of each culture condition were processed, and neurite lengths calculated and plotted. RA and DCA resulted in increased neurite length in NSC-34 (Kruskal–Wallis statistic, * p < 0.0001). (B) SH-SY5Y cells NSC-34 exposed to basal medium (DM), 1 μM retinoic acid (RA), and 20 μM of the bile acid deoxycholic acid (DCA) for 6 days and analyzed as described in A for NSC-34 cells. RA and DCA resulted in increased neurite length in NSC-34 (Kruskal–Wallis statistic,** p = 0.0196).

Figure 2

Photomicrographs of NSC-34 cells. (A) Basal medium. (B) 1 μM RA. (C) 20 μM DCA. (D) 20 μM CDCA. (E) 1 μM GW4064 (FXR Activator). (F) 10 μM GW4064 (FXR Activator). (G) 1 μM WAY 252623 (LXR activator). (H) 10 μM WAY 252623 (LXR activator). (I) 1 μM INT 747 (FXR/TGR5 dual activator). (J) 10 μM INT 747 (FXR/TGR5 dual activator). (K) 1 μM RG-239 (TGR5 activator). (L) 10 μM RG-239 (TGR5 activator). (M) 5 μM Z-Guggulsterone (FXR inhibitor). (N) 5 μM Z-Guggulsterone (FXR inhibitor) + 20 μM DCA. (O) 5 μM GSK 2033 (LXR inhibitor). (P) 5 μM GSK 2033 (LXR inhibitor) + 20 μM DCA.

Figure 3

Neurite outgrowth measured as neurite length, as described in Figure 1, from cells in Figure 2. The horizontal line at ~8 μm is drawn through + 1 standard deviation of control neurite length measurements. The conditions in which 1 standard deviation did not overlap with control (i.e., above horizontal line) are colored: 1 μM RA in red, 20 μM DCA in dark blue, 20 μM CDCA in light blue, 1 μM INT 747 (FXR/TGR5 dual activator) in light gray, 10 μM INT 747 (FXR/TGR5 dual activator) in light gray, 10 μM RG-239 (TGR5 activator) in dark gray, 5 μM Z-Guggulsterone (FXR inhibitor) + 20 μM DCA in dark blue and 5 μM GSK 2033 (LXR inhibitor) + 20 μM DCA in dark blue. * p < 0.01, ** p < 0.001, ns = not significant.