fig3

Figure 3. Progressive degeneration of CSMNs in P56S KI mice. (A and B) Immunostaining of CTIP2 and NeuN in the motor cortex of 24-month-old WT and P56S KI mice. CSMNs of 2-, 12-, and 24-month-old WT and P56S KI mice were counted by unbiased stereology (n ≥ 3 at each time point); (C and D) Immunostaining of CTIP2 and NeuN (purple) in the motor cortex of 12-month-old WT/Thy1-YFP and P56S KI/Thy1-YFP mice. Unbiased stereological counting of the YFP⁺ CSMNs was performed (n = 5); (E and F) Immunostaining of ankyrin G in the motor cortex layer V of 12-month-old WT/Thy1-YFP and P56S KI/Thy1-YFP mice. The length and middle diameter of the CSMN’s AIS were measured (n = 5); (G-J) Coronal-section imaging of the YFP-labeled axons in the pyramidal tract at the medulla level (G) and the dorsal corticospinal tract at the cervical spinal cord level (I) of 12-month-old WT/Thy1-YFP and P56S KI/Thy1-YFP mice. The relative density of axons in the pyramidal tract at the medulla level (H) and the dorsal corticospinal tract at the cervical spinal cord level (J) was quantified (n ≥ 4 at each time point). Scale bar: 200 μm in (A) and (C); 20 μm in (E);100 μm in (G); 500 μm (upper panel) and 50 μm (lower panel) in (I). Two-way ANOVA: not significant (ns) P = 0.999936 (2M), ns P = 0.999673 (12M), and ^****P < 0.0001 (24M) in (B); ns P = 0.6026 (2M) and ^****P < 0.0001 (12M) in (H); ns P = 0.9439 (2M) and ^***P = 0.0001 (12M) in (J). Unpaired t test: ns P = 0.8251 in (D); ns P = 0.4301 (length of AIS) and ns P = 0.5211 (middle diameter of AIS) in (F). CSMN: corticospinal motor neuron; KI: knock-in; CTIP2: COUP-TF-interacting protein 2; NeuN: neuronal nuclei; WT: wild-type; AIS: axon initial segment.