The novel cognitive enhancer ST101 induces neurite outgrowth in NG108-15 cells and hyperalgesia in mice through T-type Ca2+ channels

Sumire Ohno1, Daiki Kanaoka1, Hiroki Ide1, Fumiko Sekiguchi1, Shigeru Yoshida1, Kohji Fukunaga2, Atsufumi Kawabata1. 1Kinki University, Higashi-Osaka, Japan, 2Tohoku University, Sendai, Japan

ST101 (ZSET1446) is a possible therapeutic agent for Alzheimer’s disease. Recently, it has been reported that the effect of ST101 might be mediated by T-type Ca2+ channels (T-channels) (1). We have revealed that a donor of hydrogen sulphide, NaHS, causes neurite outgrowth in NG108-15 cells (2, 3) and hyperalgesia in mice (4) via Cav3.2 T-channels. In the present study, we thus investigated the effect of ST101 on neurite outgrowth and T-channel-dependent currents (T-currents) in NG108-15 cells and mechanical nociception in mice, and determined the possible involvement of T-channels.

Neurite outgrowth in NG108-15 cells was evaluated by counting cells with neurites that were longer than the cell body diameter. The cells were stimulated with ST101 at 0.001-1 nM or vehicle for 16 or 24 h. Inhibitors were applied 30 min before stimulation with ST101. Using a whole-cell patch clamp technique, T-currents in NG108-15 cells were measured in response to a test pulse at -20 mV from a holding potential at -80 mV. Mechanical nociceptive threshold was determined by the von Frey test in male ddY mice. Mice received intraplantar (i.pl.) administration of ST101 in a volume of 10 µL. RQ-00311651 (RQ), a T-channel inhibitor, or vehicle was administered orally 45 min before i.pl. ST101.

ST101caused neurite outgrowth in a concentration-dependent manner in the range of 0.001-0.1 nM (Fig. 1a). The ST101-induced neuritogenesis was suppressed by NNC 55-0396 (NNC), RQ and mibefradil, T-channel inhibitors, and ascorbic acid (AA) and ZnCl2, known to selectively inhibit Cav3.2 among three T-channel subtypes (Fig. 1b). T-currents in NG108-15 cells were significantly increased by stimulation with ST101 for 3 h, but not 10 min (Fig. 1c). In mice, i.pl. injection of ST101 in the range of 0.01-0.1 nmol/paw decreased nociceptive threshold in a dose-dependent manner (Fig. 1d), an effect abolished by oral administration of RQ at 40 mg/kg.

In summary, ST101 causes neurite outgrowth in NG108-15 cells and hyperalgesia through enhancement of T-channel functions.

Fig. 1 Effect of ST101 on neuritogenesis (n=8)(a, b) and T-currents (n=11-14 or 18-26)(c) in NG108-15 cells, and mechanical nociceptive threshold in mice (n=4-7)(d). Data show the mean ±SEM for 8 (a, b), 11-14 (c, left panel) or 18-26 (c, right panel) different experiments, or 4-7 (d) mice. Parametric analysis was performed by Student’s t-test or Tukey’s test, and non-parametric analysis was achieved by the Kruskal-Wallis H test / LSD-type test.

(1) Moriguchi et al, J Neurochem. 121:44, 2012; (2) Nagasawa, Kawabata et al, J Neurochem. 108:676, 2009; (3) Tarui, Kawabata et al, J Neurochem. 114:512, 2010;(4) Okubo, Kawabata et al, Br J Pharmacol. 166:1738, 2012.