Fig. 5.
C1q deletion leads to a preferential loss of cholinergic C-bouton nerve terminals onto motor neurons in affected SOD1 mutant-expressing ALS mice. (A–P) Confocal images of triple-immunofluorescence stainings to assess cholinergic C-bouton nerve terminals on large lumbar (L4/5) ventral horn motor neurons during disease course in SOD1G37R ALS mice with (A–D and I–L) or without (E–H and M–P) C1q at presymptomatic (A–H) and end-stage (I–P) time points. C-boutons were identified by presynaptic VAChT (A, E, I, and M; green) and apposed postsynaptic Kv2.1 (B, F, J, and N; red) densities, whereas motor neurons were marked by fluorescent Nissl (FN, blue; D, H, L, and P). Apposition/colocalization of VAChT/Kv2.1 are shown as yellow overlap (C, G, K, O) with enlargements (Insets) showing an individual C-bouton. Although ALS mice with normal C1q content did not show major loss of C-boutons on motor neuron during disease (compare A–D vs. I–L), C1q-deleted ALS mice developed a loss of C-boutons at disease end stage (compare I–L vs. M–P). (Q and R) Analysis of large surviving motor neurons at three disease stages in SOD1G37R ALS mice revealed a slight shrinkage of diameter (Q) (independent of C1q), whereas perikaryal VAChT fluorescence intensity (designated perVAChT within the dotted white line; A, E, I, and M) stayed relatively constant (R). (S and T) Quantification of C-bouton synaptic density analysis on motor neurons indicated that C1q deletion led to a strong loss of presynaptic VAChT-positive input (designated synVAChT) and corresponding Kv2.1-positive postsynaptic densities on motor neurons [measured as relative fluorescence units (rfu) of synVAChT and Kv2.1 per micrometer of contour length] at end stage in SOD1G37R ALS mice (S). Detailed analysis indicated that C-boutons were not simply lost, but [as a result of greater synVAChT than Kv2.1 loss (S)] developed reduced VAChT/Kv2.1 apposition/colocalization (measured as the ratio of fully apposed to total VAChT/Kv2.1 synaptic densities) (T), consistent with reduced synaptic integrity. (**P < 0.01, Student t test; ns, nonsignificant; n = 4 mice per genotype and disease stage; error bars represent SEM). Gray bars in Q–T: 6-mo-old control, nontransgenic mice. ES, end stage (complete hindlimb paralysis, average of 6 mo of age); OS, onset (18 wk of age, average weight peak); PS, presymptomatic (at 8 wk of age). (Scale bar: P, 25 μm.)