Inactivation of JAK2/STAT3 signaling axis and downregulation of M1 mAChR cause cognitive impairment in klotho mutant mice, a genetic model of aging

Abstract

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice.

Figure 1

Changes in protein expression of p-JAK2, p-STAT3 (a) and cholinergic receptors (b), and AG antagonism against McN-induced effects on reduced M1 mAChR expression (c) and [³H]pirenzepine-binding density (d) in the hippocampus of klotho mutant mice. Veh=Vehicle (50% DMSO in saline, the solvent for AG 490); Sal=Saline; AG=AG490 (15 mg/kg/2 ml, i.p.); McN=McN-A-343 (1.0 μg/μl in saline, i.c.v.). Each value is the mean±SEM of six animals. *P<0.01 vs corresponding wild-type mice; ^#P<0.01 vs klotho mutant mice treated with Veh and Sal; ^&P<0.05, ^&&P<0.01 vs klotho mutant mice treated with Veh and McN (one-way ANOVA (a, b) or three-way ANOVA followed by multiple pairwise comparisons with Bonferroni's correction (c, d)).

Figure 2

AG antagonism against McN-induced changes in acetylcholine level (a), and choline acetyltransferase (ChAT) protein expression (b), ChAT activity (b), AChE protein expression (c), AChE activity (c), p-JAK2 (d), p-STAT3 expression (e), and NMDAR-dependent LTP (f, g) in the hippocampus of klotho mutant mice. (f) Field EPSP slope (% of baseline) was plotted against time. LTP was induced by application of one TBS episode. Insets: representative traces recorded before (thin line), 60 min (thick line) after one TBS episode. A: Wild-type/Sal+Veh (n=11), B: klotho mutant/Sal+Veh (n=11), C: klotho mutant/McN+Veh (n=10), D: klotho mutant/McN+AG490 (n=11), E: wild-type/McN+Veh (n=8). (g) Comparison of average early-LTP magnitude 58–60 min after TBS application. AG490 blocked the reversal effects of McN on NMDAR-dependent LTP. Veh=Vehicle (50% DMSO in saline, the solvent for AG490); Sal=Saline; AG=AG490 (15 mg/kg/2 ml, i.p.); McN=McN-A-343 (1.0 μg/μl in saline, i.c.v.). Each value is the mean±SEM of six animals (a–e) or 8–11 animals (f, g). *P<0.05, ^**P<0.01 vs corresponding wild-type mice; ^#P<0.05, ^##P<0.01 vs klotho mutant mice treated with Veh and Sal; ^&P<0.05, ^&&P<0.01 vs klotho mutant mice treated with Veh and McN (three-way ANOVA followed by multiple pairwise comparisons with Bonferroni's correction).

Figure 3

AG490 antagonism against McN-induced effects on PKCβII (a), p-ERK (b), p-CREB (c), and BDNF (d) expression in the hippocampus of klotho mutant mice. Veh=Vehicle (50% DMSO in saline, the solvent for AG490); Sal=Saline; AG=AG490 (15 mg/kg/2 ml, i.p.); McN=McN-A-343 (1.0 μg/μl in saline, i.c.v.). Each value is the mean±SEM of six animals. *P<0.05, ^**P<0.01 vs corresponding wild-type mice; ^#P<0.01 vs klotho mutant mice treated with Veh and Sal; ^&P<0.05, ^&&P<0.01 vs klotho mutant mice treated with Veh and McN (three-way ANOVA followed by multiple pairwise comparisons with Bonferroni's correction).

Figure 4

K252a antagonism against McN-induced effects on p-JAK2 (a), p-STAT3 (b), choline acetyltransferase (c), AChE (d), and M1 mAChR (f), and the level of acetylcholine (e) in the hippocampus of klotho mutant mice. Veh=Vehicle (5% DMSO in saline, the solvent for k252a); Sal=Saline; k252a=k252a (0.3 mg/kg/2 ml, i.p.); McN=McN-A-343 (1.0 μg/μl in saline, i.c.v.). Each value is the mean±SEM of six animals. ^#P<0.05, ^##P<0.01 vs klotho mutant mice treated with Veh and Sal; ^&P<0.05, ^&&P<0.01 vs klotho mutant mice treated with Veh and McN (two-way ANOVA followed by multiple pairwise comparisons with Bonferroni's correction).

Figure 5

Antagonism by AG490 or k252a against McN-induced effects on novel object recognition (a) and conditioned-fear learning performance (b) of klotho mutant mice. Veh=Vehicle (50% DMSO in saline, the solvent for AG490); Sal=Saline; AG=AG490 (15 mg/kg/2 ml, i.p.); k252a=k252a (0.3 mg/kg/2 ml, i.p.); McN=McN-A-343 (1.0 μg/μl in saline, i.c.v.). Neither 50% DMSO nor 5% DMSO solution affect significantly memory performance; thus, shown as Veh for AG490. Each value is the mean±SEM of 15 animals. *P<0.01 vs corresponding wild-type mice; ^#P<0.01 vs klotho mutant mice treated with Veh and Sal; ^&P<0.05, ^&&P<0.01 vs klotho mutant mice treated with Veh and McN (three-way ANOVA followed by multiple pairwise comparisons with Bonferroni's correction).