Figure 2.

MHCI requires MEF2 to limit synapse density. A, Representative images of dendrites from 8 DIV cortical neurons transfected with the indicated constructs for 48 h. Excitatory glutamatergic synapses were quantified as the density of postsynaptic PSD-95 puncta (green) colocalized (yellow) with presynaptic VGluT1 puncta (red). Scale bar, 5 μm. Each representative image shown includes an adjacent “mask” panel displaying the regions obtained following thresholding that were used for quantification. The intensity of the images shown was increased for publication to enable visualization of the faint puncta, in addition to the bright ones, because both are detected by our image analysis, as shown in the mask panels. The brighter puncta are saturated in those images by this increase in intensity. B, MEF2A+MEF2D knockdown alone does not alter synapse density, but completely prevents the MHCI-mediated decrease in synapse density. Expression of shRNAs against MEF2A and D (shA+D) together with MHCI OE rescued the decrease in synapse density observed in neurons expressing a scrambled shRNA (shSCR) with MHCI OE. Results are normalized to shSCR control; n ≥ 61 dendrites, 21 cells per condition; four experiments. C, MEF2C+MEF2D knockdown and MEF2A+MEF2C knockdown (D) had similar effects as MEF2A+MEF2D knockdown shown in B. E, Similarly, expression of MEF2-En, a dominant negative form of MEF2, does not alter synapse density alone, but partially rescues the MHCI-mediated decrease in synapse density; n ≥ 51 dendrites (17 cells) per condition, two experiments. *p < 0.05, **p < 0.01, ***p < 0.001.