Fig. 6: miR106sp-mediated MECP2 expression in RTT neurons improves the morphological and calcium transient deficits. | Nature Communications

Fig. 6: miR106sp-mediated MECP2 expression in RTT neurons improves the morphological and calcium transient deficits.

From: Targeting microRNA-dependent control of X chromosome inactivation improves the Rett Syndrome phenotype

Fig. 6

A qRT-PCR analysis monitoring the expression of Xi-linked MECP2 in 4- and 12-week-old RTT neurons infected with LTV-empty or LTV-miR106sp. Neurons derived from a clone expressing wild-type MECP2 from the Xa were used as a positive isogenic control (Control), which was set to 1. n = 3. For 4 weeks, LTV-empty vs. LTV-miR106sp, *p = 0.014; LTV-miR106sp vs. Control, **p = 0.0012. For 12 weeks, LTV-empty vs. LTV-miR106sp, *p = 0.018; LTV-miR106sp vs. Control, **p = 0.0068. B Quantification of 4- and 12-week-old LTV-empty or LTV-miR106sp-infected RTT-neurons and control neurons with nuclear MECP2 immunostaining. n = 200 cells per group in three independent experiments. For 4 weeks, LTV-empty vs. LTV-miR106sp, **p = 0.0075; LTV-miR106sp vs. Control, **p = 0.0046. For 12 weeks, LTV-empty vs. LTV-miR106sp, ***p = 4.77 × 10-05; LTV-miR106sp vs. Control, ***p = 0.00011. C Representative images of MAP2 (Green) and TubIII (Red) staining in ~4-week-old LTV-empty or LTV-miR106sp-infected RTT- and control neurons. DAPI stains the nucleus Blue (Top). Representative reconstructed neuronal images for the quantitative assessment of all orders of branches in each group by Sholl analysis are shown (Bottom). D Quantitative analysis of the soma cross-sectional area (Top) and the number of neuronal branch points (Bottom) in 4- and 12-week-old MAP2 + RTT neurons expressing empty or miR106sp and control neurons. n = 200 cells per group in three independent experiments. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Soma size: For 4 weeks, LTV-empty vs. LTV-miR106sp, *p = 0.024; LTV-miR106sp vs. Control, **p = 0.001. For 12 weeks, LTV-empty vs. LTV-miR106sp, ***p = 0.00039; LTV-miR106sp vs. Control, nsp = 0.425. Branches density: For 4 weeks, LTV-empty vs. LTV-miR106sp, *p = 0.015; LTV-miR106sp vs. Control, nsp = 0.491. For 12 weeks, LTV-empty vs. LTV-miR106sp, *p = 0.015; LTV-miR106sp vs. Control, nsp = 0.142. E, F Representative fluorescent intracellular calcium transient in RTT neurons expressing empty or miR106sp or control neurons over 5 min (n = 20; E). The quantitation of calcium spikes for each group is shown (F, Left). The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. For calcium event frequency, LTV-empty vs. LTV-miR106sp, ***p = 7.43 × 10-13; LTV-miR106sp vs. Control, ***p = 1.09 × 10-11. The percentage of GCaMP6-positive neurons in each group is shown (F, Right). (n = 60 per group) G ChIP analysis monitoring binding of MECP2 to the Actin, DLX (LTV-empty vs. LTV-miR106sp, *p = 0.049), JUNB (LTV-empty vs. LTV-miR106sp, *p = 0.040), and SNRPN (LTV-empty vs. LTV-miR106sp, *p = 0.026) promoter in RTT neurons expressing control or miR106sp. Data is expressed as mean ± SD (A, B, F, G). Unpaired, two-sided t-test with no multiple adjustments. Scale bars: 40 µm (D).

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