Fig. 5

Mef2c-KD induces mitochondrial dysfunction and motor neuronal damage, and alters motor neuron excitability in the cortical layer V of mice. (A) The pAAV-hSyn(pro)-shControl-GFP (shControl) or pAAV-hSyn(pro)-shMef2c-GFP (shMef2c) viruses was delivered into cortical layer V by bilateral stereotaxic injection method. (B) Immunostaining of ND4 and DAPI in shControl and shMef2c mice. Scale bars (white): 5 μm. Right: densitometry analysis of ND4 immunoreactivity level in GFP⁺ cells. A total of 30 cells/group were counted (6 cells/mouse) from N = 5 mice/group (shControl and shMef2c). Statistics were calculated using Student’s t-test (*, P  < 0.05). (C) Immunostaining of DRP1 and TOMM20 in shControl and shMef2c mice. Scale bars (white): 5 μm. Right: skeletonized images of mitochondria morphological structure using TOMM20 signals, and densitometry analysis of DRP1 immunoreactivity level in mitochondria of GFP⁺ cells. A total of 30 cells/group were counted(6 cells/mouse) from N = 5 mice/group (shControl and shMef2c). Statistics were calculated using Student’s t-test (***, P < 0.001). (D) Line measurement analysis for DRP1 and TOMM20 colocalization signals in shControl and shMef2c-transduced cortical pyramidal neuron. (E) Results of mitochondria network size analysis performed by MiNA plugin in Fiji ImageJ software. A total of 10 cells/group were counted (2 cells/mouse) from N = 5 mice/group (shControl and shMef2c). Statistics were calculated using Student’s t-test (**, P = 0.003). (F) Representative TEM images showed the presence of immunogold particles (red head arrows) in mitochondria of the pyramidal neurons layer V in shControl mice. Scale bars: 200 nm. (G) Representative TEM images showed mitochondria fission in shMef2c group. Scale bars: 2 μm. Right: a normalized curve for quantitative analysis of mitochondria size in the cortical pyramidal neurons of shControl and shMef2c mice. A total of n = 200 mitochondria/20 neurons/group were counted from N = 5 mice/group (shControl and shMef2c). (H) Immunostaining of CTIP2 in shControl and shMef2c mice. Scale bars: 5 μm. (I) Quantification analysis on the cell size of cortical layer V pyramidal neurons in GFP⁺ cells. A total of 15 cells/group were counted (3 cells/mouse) from N = 5 mice/group (shControl and shMef2c). Statistics were calculated using Student’s t-test (*, P  < 0.05). (J) A scheme illustrating performing whole-cell patch clamp on cortical layer V pyramidal neurons for shControl and shMef2c mice. (K) Representative recordings of action potentials from motor cortex layer V pyramidal neurons induced by step current injection, ranging from 0 to 330 pA (Scale bar: 20 mV, 200 ms). (L) shMef2c increased the number of action potentials triggered by a series of current injection (30-pA increment, 12 steps). shControl, n = 13 neurons/4 mice; shMef2c, n = 11 neurons/4 mice). Statistics were calculated using Student’s t-test (60(pA), P = 0.438; 90(pA), P = 0.088; 120(pA), *, P = 0.013; 150(pA), **, P = 0.009; 180(pA), **, P = 0.01; 210(pA), **, P = 0.001; 240(pA), **, P = 0.001; 270(pA), **, P = 0.002; 300(pA), **, P = 0.002; 330(pA), *, P = 0.03). (M) Representative traces of individual action potential (Scale bar: 20 mV, 50 ms). (N) The threshold to initiate action potentials is not altered in shMef2c group (shControl, n = 13 neurons/4 mice; shMef2c, n = 11 neurons/4 mice). Statistics were calculated using Student’s t-test, P = 0.828. Error bars represent means ± SEM