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. 2021 Nov 24;10(12):3286. doi: 10.3390/cells10123286

Figure 8.

Figure 8

Exogenous expression of neuron-specific menin restores menin protein in α7 KD hippocampal neurons (in vitro). (A) IHC characterization of α7 KD hippocampal slices (Ai–vi) 12-week-old mice (n = 20 images, 5 independent samples, representative image). MEN1 encoding AAV labelled GFP-positive neurons (Aii,v) labelled with neurofilament NFL (neuronal marker) (Ai,iv) and merged (Aiii,vi) exhibiting a strong degree of colocalization between GFP and NFL. (B) ICC characterization of α7 KD hippocampal neuronal cultures (Bv–viii), co-transduced with GFP-labelled MEN1 encoding AAV (Bv) compared to scrambled controls, (Bi–iv) co-transduced with GFP only AAV, 4 weeks post-surgery (n = 35 images, six independent samples, representative image). tdTomato-positive neurons (Bii,vi) and GFP-positive neurons (Bi,v) labelled with α-C-terminal menin (Biii,vii) and merged (Biii,vi), Bvii exhibits significantly increased expression of C-menin in tdTomato GFP-positive hippocampal-pyramidal neurons compared to α7 KD+GFP only control (Biii). (C) Summary data, normalized menin expression in neurons from α7 nAChR scrambled AAV, α7 nAChR shRNA AAV, α7 nAChR scrambled AAV+MEN1, α7 nAChR AAV+MEN1AAV and α7 nAChR AAV+GFP AAV neuronal cultures compared to relevant controls (n ≥ 25 images, ≥7 independent samples) DIV 3, 7, 10, 14 and 20. (D) Summary data, normalized menin expression in neurons from α7 nAChR scrambled AAV, α7 nAChR shRNA AAV, α7 nAChR scrambled AAV+MEN1, α7 nAChR AAV+MEN1AAV and α7 nAChR AAV+GFP AAV compared to relative controls (n ≥ 30 images, ≥6 independent samples, 8 weeks old). Scale bars, (Ai–iii) 10, (Aiv–vi) 10, (Bi–iv) 5 and (Bv–viii) 8 μm. White dotted circles indicate GFP-positive pyramidal neurons. White asterisks on slices indicate colocalization of NFl and GFP. White asterisks on neurites indicate menin expression in tdTomato+ GFP-positive neurites. Statistical significance (one-way ANOVA followed by Tukey’s multiple comparison test) (one sample t-test) **** p < 0.0001, *** p < 0.001, ** p < 0.01, ns p > 0.9999. See Supplementary Tables S12–S14.