Fig. 6: SMN deficiency is upstream to neuronal GABAergic signal and metabolism impairment in motor cortical areas.

a Representative IF images showing the RNAi decreased SMN expression (red) and related GABA signal intensity (green) in siRNA primary cortical neurons (labelled from MAP2, magenta) from WT mice compared to SCR controls. Scale bar: 30 um. The results of SMN silencing in DIV15 cortical neurons are then shown in (b) descriptive plot of the distribution of the number of siRNA and SCR neurons over a heterogeneous range of SMN signal intensity values. c Fold change histograms showing the average signal intensity values of GABA and SMN in ratio with those of the SCR neurons: the individual points show distributional differences of the neurons among samples (data are expressed as mean % ± SEM and normalized to SCR mean, referred as control; Student’s t-test, ****p < 0.0001;at least 100 neurons for each culture were analysed from n = 3 mice primary neuron cultures per group). Representative densitometries and protein levels quantifications of SNAT5 (d) and of the transporters GAT1 and GAT3 (e) in SMA P12 SM CRTX samples compared to WT controls [Data are expressed as mean ± SEM and normalized to VINC (referred as loading control) and to WT mean. Student’s t-test, *p < 0.05; for SNAT5 analysis: WT and SMA mice: n = 6; for GAT1 analysis: WT mice: n = 6; SMA mice: n = 6; for GAT3 analysis: WT mice: n = 4; SMA mice: n = 5]. f Representative widefield (left) and confocal (right) immunofluorescence images of WT and SMA primary cortical astrocytes (labelled by anti-GFAP antibody, in blue) cultures. Scale bar: 200 and 30 µm. g Quantification of GABA mean intensity in GFAP+ astrocytes among DIV15 WT and SMA cortical cells. Red dashed line in the graph indicates WT relative GABA mean signal intensity. Analyses included at least 100 cells from six independent cultures for each group. Each dot on the graph represents an individual cell (Mann-Whitney test, ***p < 0.005; WT and SMA mice: n = 6 primary cell co-cultures).