Fig 2: M4 Synthesis Downstream of mGlu5 Is Mimicked and Occluded in the Fmr1-/y Hippocampus(A) Time course for DHPG stimulation experiments.(B) Analysis of transcripts encoding hippocampal mAChR subunits reveals a striking upregulation of Chrm4 mRNA in CA1-TRAP IP after mGlu1/5 stimulation (Veh = 1.00 ± 0.12, DHPG = 1.72 ± 0.23, *p = 0.0047, n = 15), with no changes seen in Chrm1 or Chrm3 (Chrm1: Veh = 1.00 ± 0.07, DHPG = 1.03 ± 0.06, p = 0.75, n = 16; Chrm3: Veh = 1.00 ± 0.12, DHPG = 0.82 ± 0.09, p = 0.209, n = 14).(C) DHPG stimulation of WT slices shows dramatic increase in Chrm4 mRNA in the TRAP IP fraction. In Fmr1-/y slices, Chrm4 mRNA is already significantly elevated in the TRAP IP and does not increase further with mGlu1/5 activation (WT vehicle = 1.00 ± 0.24, WT DHPG = 2.13 ± 0.41, KO vehicle = 2.28 ± 0.43, KO DHPG = 2.34 ± 0.44, ANOVA genotype *p = 0.03, treatment *p = 0.048, WT versus KO veh *p = 0.03, KO veh versus DHPG p > 0.999, n = 7). Chrm1 mRNA is significantly elevated in the Fmr1-/y CA1-TRAP IP, but DHPG does not increase Chrm1 in either WT or Fmr1-/y CA1-TRAP IPs (WT vehicle = 1.00 ± 0.09, WT DHPG = 1.13 ± 0.08, KO vehicle = 1.50 ± 0.13, KO DHPG = 1.47 ± 0.16, ANOVA genotype *p = 0.005, treatment p = 0.753, WT versus KO veh *p = 0.04, WT veh versus DHPG p = 0.944, n = 7). Chrm3 mRNA is neither increased in the Fmr1-/y CA1-TRAP IP nor elevated with DHPG (WT veh = 1.00 ± 0.14, WT DHPG, = 1.029 ± 0.18, KO veh = 0.946 ± 0.19, KO DHPG = 1.09 ± 0.19, ANOVA genotype p = 0.97, treatment p = 0.55, n = 7).(D) Immunoblotting shows a robust increase in M4 expression in WT slices after 5 min of DHPG stimulation, which is maintained at 30 min and 60 min post-stimulation. In contrast, the elevated expression of M4 in Fmr1-/y slices is not further increased with DHPG stimulation (WT vehicle = 100% ± 6.63%, WT DHPG 5 min = 159.59% ± 9.37%, WT DHPG 30 min = 131.09% ± 13.23%, WT DHPG 60 min = 141.66% ± 12.08%, KO vehicle = 132.70% ± 7.31%, KO DHPG 5 min = 146.95% ± 7.94% KO DHPG 30 min = 131.04% ± 13.01%, KO DHPG 60 min = 155.75% ± 10.28%, ANOVA treatment × genotype *p = 0.037, n = 7).(E) Time course for MTEP slice experiments.(F) Incubation with 10 µM MTEP reduces Chrm4 in the Fmr1-/y CA1-TRAP to WT levels (WT vehicle = 1.00 ± 0.112, WT MTEP = 1.48 ± 0.230, KO vehicle = 2.63 ± 0.352, KO MTEP = 1.63 ± 0.161, ANOVA genotype *p = 0.0014, treatment p = 0.1923, genotype × treatment *p = 0.0119, WT veh versus KO veh *p = 0.0024, WT veh versus WT MTEP p = 0.306, KO veh versus KO MTEP *p = 0.0289, WT MTEP versus KO MTEP p = 0.880, n = 8).(G) Immunoblotting shows a significant reduction in M4 expression in MTEP-treated Fmr1-/y slices (WT vehicle = 100% ± 9.7%, WT MTEP = 97% ± 5.4%, KO vehicle = 181% ± 32.6%, KO MTEP = 103% ± 14.1%, ANOVA genotype *p = 0.034, WT veh versus KO veh *p = 0.0181, KO veh versus KO MTEP *p = 0.0145, n = 6). n = number of littermate pairs. Error bars indicate SEM.

Fig 3: Effect of Rg3, ATXN8OS, and miR-424-5p on the target genes of miR-424-5p at the protein level. Western blot analysis of CHRM3 and DACH1 was performed after treating the MCF-7 cells with Rg3 (A) or deregulating ATXN8OS (40 µM siRNA) and miR-424-5p (40 µM for mimic and inhibitor) (B,C). Testing was done using siNC, control siRNA (40 µM); mimic NC, negative control mimic for miR-424-5p (40 µM); and inhibitor NC, negative control inhibitor for miR-424-5p (20 µM). The band intensity was measured with the Image Lab software and indicated by bar graphs. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: M1 and M4 Are Excessively Synthesized and Overexpressed in Fmr1-/y CA1 Pyramidal Neurons(A) Chrm4 and Chrm1, but not Chrm3, are enriched in the Fmr1-/y CA1-TRAP IP (Chrm4: WT = 1.00 ± 0.124, KO = 1.72 ± 0.195, *p = 0.0044, n = 14; Chrm1: WT = 1.00 ± 0.062, KO = 1.47 ± 0.079, *p < 0.0001, n = 14; Chrm3: WT = 1.00 ± 0.085, KO = 1.184 ± 0.073, p = 0.192, n = 10).(B) Total mRNA levels of Chrm4, Chrm1 and Chrm3 are unchanged in FACS-isolated Fmr1-/y CA1 pyramidal neurons (Chrm4: WT = 1.00 ± 0.209, KO = 0.98 ± 0.154, p = 0.926, n = 9; Chrm1: WT = 1.00 ± 0.150, KO = 0.87 ± 0.185, p = 0.602, n = 11; Chrm3: WT = 1.00 ± 0.232, KO = 1.14 ± 0.230, p = 0.668, n = 8).(C) Immunoblotting shows overexpression of M4 protein in hippocampal slice homogenates (WT = 100% ± 5.7%, KO = 121% ± 5.7%, *p = 0.0186, n = 12) and synaptoneurosomes (WT = 100% ± 7.10%, KO = 121% ± 6.89%, *p = 0.0203, n = 9).(D and E) Hippocampal slice homogenates show no difference in M1 (D) (WT = 100% ± 2.5%, KO = 105% ± 2.5%, p = 0.129, n = 12) or M3 (E) (WT = 100% ± 9.5%, KO = 98% ± 12.9%, p = 0.94, n = 9) expression.(F) Schematic shows steps for FACS immunostaining.(G) FACS-immunostaining reveals significant increase in the expression of M4 and M1 (M4: WT = 0.925 ± 0.045, KO = 1.075 ± 0.045, *p = 0.0389, n = 6; M1: WT = 0.920 ± 0.002, KO = 1.080 ± 0.046, *p = 0.0092, n = 6), but not M3 (WT = 0.962 ± 0.07, KO = 1.038 ± 0.1011 p = 0.547, n = 7), in Fmr1-/y CA1 pyramidal neurons. n = number of littermate pairs. Error bars indicate SEM.