C1q induction and global complement pathway activation do not contribute to ALS toxicity in mutant SOD1 mice

Abstract

Accumulating evidence from mice expressing ALS-causing mutations in superoxide dismutase (SOD1) has implicated pathological immune responses in motor neuron degeneration. This includes microglial activation, lymphocyte infiltration, and the induction of C1q, the initiating component of the classic complement system that is the protein-based arm of the innate immune response, in motor neurons of multiple ALS mouse models expressing dismutase active or inactive SOD1 mutants. Robust induction early in disease course is now identified for multiple complement components (including C1q, C4, and C3) in spinal cords of SOD1 mutant-expressing mice, consistent with initial intraneuronal C1q induction, followed by global activation of the complement pathway. We now test if this activation is a mechanistic contributor to disease. Deletion of the C1q gene in mice expressing an ALS-causing mutant in SOD1 to eliminate C1q induction, and complement cascade activation that follows from it, is demonstrated to produce changes in microglial morphology accompanied by enhanced loss, not retention, of synaptic densities during disease. C1q-dependent synaptic loss is shown to be especially prominent for cholinergic C-bouton nerve terminal input onto motor neurons in affected C1q-deleted SOD1 mutant mice. Nevertheless, overall onset and progression of disease are unaffected in C1q- and C3-deleted ALS mice, thus establishing that C1q induction and classic or alternative complement pathway activation do not contribute significantly to SOD1 mutant-mediated ALS pathogenesis in mice.

Keywords: amyotrophic lateral sclerosis; gender differences; motoneuron; neuroinflammation; synaptic density.

Fig. 1.

Early induction of C1q and classic complement components in spinal cords of ALS mice. (A and B) Complement components and glial activation markers were assessed in ALS mice (open bars), and their induction levels compared with ALS mice lacking C1q (the initiating component of the classic complement pathway; filled bars). By using quantitative RT-PCR, lumbar spinal cord mRNA levels of all three C1q genes (C1qa/b/c) (A) and downstream complement components (C4, C3) (B) were assessed in mutant SOD1^G37R mice (open bars), presymptomatically (PS; at 8 wk of age), at onset (OS; 18 wk of age, the average weight peak), and end stage (ES; complete hindlimb paralysis, average of 6 mo of age) and correlated to microglial (CD11b/Itgam) and astrocytic (Gfap) activation (B). Compared with control (6-mo-old) SOD1^WT samples, early induction of C1q and classic complement components were detected in mutant SOD1^G37R mice and paralleled glial activation (A and B). In C1q-deleted ALS mice (filled bars), loss of C1qa was confirmed (arrows, A). Mice deleted for C1qa are unable to form a functional C1q polypeptide, and induction levels of C1qb/c genes were strongly reduced. C1q deletion in ALS mice led to a transient delay in induction of downstream complement components (C4, C3) as well as glial activation markers (microglial CD11b/Itgam and astrocytic Gfap) (B). (**P < 0.01, Student t test; n = 4 mice per genotype and disease stage; error bars represent SEM).

Fig. 2.

Time course of global glial activation is unchanged in spinal cords of C1q-deleted ALS mice. (A–X) Immunofluorescence stainings for two microglial [Iba1/Aif (A–H), Mac2/Lgals3 (I–P)] and one astrocytic (GFAP) (Q–X) activation markers. No signs of glial activation were present in 6-mo-old control SOD1^WT mice with normal C1q content (D, L, and T) or in 6-mo-old control mice deleted in C1q (H, P, and X). In mutant SOD1^G37R ALS mice, overall glial activation was detectable at onset (B, J, and R) (at 18 wk of age, the average weight peak) and increased further at end stage (C, K, and S) (complete hindlimb paralysis,average of 6 mo of age); however, this was independent of whether C1q was present (A–C, I–K, and Q–S) or absent (E–G, M–O, and U–W) (n = 4 mice per genotype and disease stage). (Scale bar: D, 100 μm.)

Fig. 3.

C1q-dependent morphological changes of microglia during disease course of ALS mice. (A–J) Confocal images of immunofluorescence stainings for the microglial marker Iba1. (A and F) Control 6-mo-old SOD1^WT (A) or C1q-deleted mice (F) showed microglia with a resting phenotype and long branched processes. (B and C, G and H) In presymptomatic (at 8 wk of age) mutant SOD1^G37R ALS mice, C1q-deletion led to an increased number of bulbous termini (arrows, G’ and H’, Insets) on microglial processes (compare B′ and C′ for ALS mice with normal C1q-content vs. G′ and H′ for C1q-deleted ALS mice). (D, E, I, and J) In mutant SOD1^G37R ALS mice at onset (at 18 wk of age, the average weight peak) and end stage (complete hindlimb paralysis, average of 6 mo of age) time points, microglia had acquired a classic activated morphology with retracted processes, but overall morphology was independent of C1q deletion (n = 4 mice per genotype and disease stage). (Scale bars: J, 20 µm; H′, 10 µm.)

Fig. 4.

Motor neuron loss is unchanged but loss of presynaptic density onto motor neurons is more prounounced in C1q-deleted ALS mice. (A–C) Motor neuron loss. Shown are Nissl stainings with enlargements (Insets) of lumbar spinal cord ventral horns to identify motor neurons in mutant SOD1^G37R ALS mice presymptomatically (A) and at end stage (B). (C) Quantification indicated equal numbers of large lumbar (L4/5) motor neurons in (6-mo-old) control SOD1^WT and C1q-deleted mice whereas significant motor neuron loss was measured at onset and end stage in SOD1^G37R ALS mice, but independent of the prescence of C1q. (Scale bar: A, 100 µm.) (D–G) Loss of general presynaptic density onto large lumbar (L4/5) ventral horn motor neurons during disease course. Confocal images of double-immunofluorescence stainings for the general presynaptic marker Syt1 (green) and for a fluorescent Nissl stain (MN; red) indicated significant disease-linked reduction of presynaptic density (measured between the perikaryon and the dotted white line) on affected motor neurons, when comparing presymptomatic (D) vs. end-stage (E) mutant SOD1^G37R ALS mice. (F) Quantification of the loss in presynaptic density [measured as relative fluorescence units (rfu) of Syt1 per micrometer of motor neuron contour length] observed in D and E. (G) Quantification showing increased loss after onset in presynaptic density in the absence of C1q (**P < 0.01, Student t test; n = 4 mice per genotype and disease stage; error bars indicate SEM). ES, end stage (complete hindlimb paralysis, average of 6 mo of age); OS, onset (18 wk of age, average weight peak); PS, presymptomatic (8 wk of age). (Scale bar: E, 15 μm.)

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 SOD1^G37R 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 SOD1^G37R 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 SOD1^G37R 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.)

Fig. 6.

C1q induction and its activation of the classic complement pathway do not contribute to overall disease mechanism in SOD1^G37R mutant ALS mice. (A–C) Kaplan–Meier plots of ages (in days) at which onset (weight peak) (A), early disease (10% weight loss) (B), or end stage (complete hindlimb paralysis) (C) were reached in mutant SOD1^G37R mice with normal (C1q^+/+, ○), partially (C1q^+/−, □) or fully deleted (C1q^−/−, ●) C1q content. Average (±SEM) ages (in days) of the different disease stages are shown, and animal numbers are indicated in brackets (equally sex-mixed). No statistical significant difference could be detected among the three genotypes [P = 0.19/P = 0.26 (A), P = 0.12/P = 0.34 (B), and P = 0.06/P = 0.27 (C), log-rank and Student t test; Table S1].

Fig. 7.

Unaltered survival and motor neuron loss in mutant SOD1-expressing ALS mice deleted in C3 demonstrating no contribution of an alternative complement activation pathway to toxicity in this ALS model. (A) Kaplan–Meier plot of ages (in days) at which end stage (complete hindlimb paralysis) was reached in mutant SOD1^G93A mice with normal (C3^+/+, ○) or partially (C3^+/−, □) or fully deleted (C3^−/−, ●) C3 content. Average (±SEM) ages (in days) of the disease end stages are shown, and animal numbers are indicated in brackets (equally sex-mixed). No statistically significant difference was detected among the three genotypes [P = 0.50/P = 0.23 (A), log-rank and Student t test]. (B) Quantification of motor neuron loss indicated equal numbers of large lumbar (L4/5) motor neurons in (5 mo old) nontransgenic control and C3-deleted mice. Significant motor neuron loss measured at end stage was independent of the prescence of C3 (**P < 0.01, Student t test; n = 4 mice per genotype and disease stage; error bars indicate SEM). ES, end stage (complete hindlimb paralysis).