Defective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy

Abstract

Proximal spinal muscular atrophy (SMA) is a motoneuron disease for which there is currently no effective treatment. In animal models of SMA, spinal motoneurons exhibit reduced axon elongation and growth cone size. These defects correlate with reduced beta-actin messenger RNA and protein levels in distal axons. We show that survival motoneuron gene (Smn)-deficient motoneurons exhibit severe defects in clustering Cav2.2 channels in axonal growth cones. These defects also correlate with a reduced frequency of local Ca2+ transients. In contrast, global spontaneous excitability measured in cell bodies and proximal axons is not reduced. Stimulation of Smn production from the transgenic SMN2 gene by cyclic adenosine monophosphate restores Cav2.2 accumulation and excitability. This may lead to the development of new therapies for SMA that are not focused on enhancing motoneuron survival but instead investigate restoration of growth cone excitability and function.

Figure 1.

Reduced spontaneous Ca²⁺ influx into distal axons of Smn-deficient motoneurons cultured on laminin-111. (A) Spontaneous Ca²⁺ transients in isolated motoneurons were recorded in cell bodies, proximal axons, and axonal growth cones. These spontaneous Ca²⁺ transients can be inhibited by addition of 1 μM TTX or CTX, respectively. (B and C) The frequency of spontaneous generalized spikelike Ca²⁺ transients was determined on day 3 (n = 51/54), 4 (n = 91/84), 5 (n = 74/84), and 7 (n = 20/34) from control (Smn ^+/+; SMN2) and Smn ^−/− ; SMN2 motoneurons on laminin-111. No difference was observed in cell bodies and proximal axons (B) of Smn ^−/− ; SMN2 motoneurons, but a change was detected in distal axons and growth cones (C), first becoming detectable on day 4 in culture. (D and E–N) Axon length of cultured motoneurons from control and Smn-deficient embryos on laminin-111. Axon length was measured on day 3 (n = 129/106), 4 (n = 106/87), 5 (n = 162/206), 6 (n = 59/74), and 7 (n = 87/115). (D, G–I, and L–N) After day 4, the difference in axon elongation became significant. Axons of Smn-deficient motoneurons did not extend further after day 4 in comparison with control cells. Results represent the mean ± SEM of pooled data from three independent experiments. n, number of motoneurons that were scored in total from control or Smn ^−/− ; SMN2 embryos. *, P < 0.05; **, P < 0.001, tested by one-way ANOVA.

Figure 2.

Decreased frequency of local spontaneous Ca²⁺ transients in growth cones of Smn^−/−; SMN2 motoneurons on laminin-211/221. (A) Local Ca²⁺ transients in growth cones (black) were more frequent in Smn ^+/+; SMN2 motoneurons than in Smn ^−/−; SMN2 motoneurons cultured for 5 d. (B) The frequency of local spontaneous Ca²⁺ transients was significantly higher both for control (n = 34) and Smn-deficient motoneurons (n = 39) on laminin-211/221 in comparison with laminin-111 (n = 35/42). Despite the higher frequency on laminin-211/221, local transients on laminin-211/221 were significantly less frequent in Smn-deficient motoneurons in comparison with controls. Results represent the mean ± SEM of pooled data from three independent experiments. n, number of motoneurons that were scored in total from control or Smn ^−/− ; SMN2 embryos. *, P < 0.05 tested by one-way ANOVA.

Figure 3.

Expression and cellular distribution of Ca_v2.2 protein and mRNA in primary motoneurons of Smn^−/−; SMN2 and Smn^+/+; SMN2 embryos. (A–E) Distribution and quantitative analysis of Ca_v2.2 protein in 7-d-old primary motoneurons of Smn ^−/−; SMN2 and Smn ^+/+; SMN2 embryos on laminin-211/221. The signal intensity for Ca_v2.2 protein in the cell body of Smn ^−/−; SMN2 motoneurons (A and E; n = 30) is not reduced in comparison to control cells (B and E; n = 30). In contrast, the signal intensity for Ca_v2.2 is lower in axonal growth cones of Smn ^−/−; SMN2 motoneurons (D and E; n = 30) in comparison to controls (C and E; n = 30). (F–I) Ca_v2.2 immunoreactivity is highly enriched in growth cone protrusions in control motoneurons (F and G; n = 25) but not in Smn-deficient motoneurons (H and I; n = 23) that were fixed for short time periods and immunostained in the absence of detergent to enrich staining of extracellular versus intracellular Ca_v2.2. (J and K) Differences in Ca_v2.2 mRNA distribution are not detected in cell bodies (arrows) and growth cones (arrowheads and insets) of motoneurons from Smn ^−/−; SMN2 embryos (n = 35) in comparison to controls (n = 32). (L) The specificity of the antisense probe was tested with a sense Ca_v2.2 cDNA probe. (M and N) For high-resolution microscopy, STED microscopy was applied to reduce the focal spot area by one order of magnitude, thus allowing the distinction of small vesicle-like structures (arrowheads) from larger areas that probably (as discussed in the text) reflect clusters at the cell surface (arrows). (O) The ratio between vesicles and clusters is increased in Smn-deficient motoneurons, caused by a significant reduction of the cluster-like structures in Smn ^−/−; SMN2 growth cones (N and O) in contrast to growth cones of control motoneurons (M and O). Results represent the mean ± SEM of pooled data from three independent experiments. n, number of motoneurons that were scored in total from control or Smn ^−/− ; SMN2 embryos. *, P < 0.05; **, P < 0.001, tested by one-way ANOVA and t test, respectively.

Figure 4.

Axon growth and growth cone morphology of Smn^−/−; SMN2 motoneurons on laminin-211/221. (A–E) Axon length of control and Smn-deficient motoneurons on laminin-111 and laminin-211/221 after 7 d in culture. (A–C) Axons of Smn ^−/−; SMN2 motoneurons (n = 210) are shorter than those of control motoneurons (n = 93) on laminin-111. (A, D, and E) In contrast, axons of Smn ^−/−; SMN2 motoneurons (n = 141) extend significantly longer on laminin-211/221. (F–J) Growth cones of Smn-deficient motoneurons are significantly smaller both on laminin-111 (F–H; n = 32) and laminin-211/221 (F, I, and J; n = 30). Results represent the mean ± SEM of pooled data from three independent experiments. n, number of motoneurons that were scored in total from control or Smn ^−/−; SMN2 embryos. *, P < 0.05; **, P < 0.001, tested by one-way ANOVA.

Figure 5.

Axon growth after blockade of Ca_v2.2. with CTX in Smn^−/−; SMN2 and control motoneurons. (A and B) Axon length of 7-d-old isolated motoneurons from control and Smn-deficient embryos that were grown on laminin-111 (A) and laminin-211/221 (B) treated with 1 and 0.3 μM CTX, respectively. (A) On laminin-111, control neurons responded to 1 (n = 162) and 0.3 μM CTX (n = 186) with reduced axon growth, whereas Smn- deficient cells were unaffected in the presence of both concentrations (n = 151 for 1 μM and n = 82 for 0.3 μM CTX). (B) On laminin-211/221, CTX-treatment, both with 1 and 0.3 μM, increases axon extension in control motoneurons (n = 99 for 1 μM and n = 59 for 0.3 μM CTX) but not in Smn-deficient neurons (n = 102 for 1 μM and n = 82 for 0.3 μM CTX). Results represent the mean ± SEM of pooled data from three independent experiments. n, number of motoneurons that were scored in total from control or Smn ^−/−; SMN2 embryos. *, P < 0.05; **, P < 0.001, tested by one-way ANOVA.

Figure 6.

8-CPT-cAMP treatment restores local Ca²⁺ transients and N-type Ca²⁺ channel (Ca_v2.2) immunoreactivity in axonal growth cones of Smn^−/−; SMN2 motoneurons. (A) Local Ca²⁺ transients in growth cones of Smn- deficient motoneurons (n = 30) that were cultured for 5 d on laminin-211/221 are restored by 8-CPT-cAMP in comparison with controls (n = 30). (B) Quantitative analysis of immunoreactivity for Ca_v2.2 and β-actin in 5-d-old control (white bar; n = 30), Smn ^−/−; SMN2 (gray bar; n = 30), and 8-CPT-cAMP–treated Smn ^−/− ; SMN2 motoneurons (checkered bar; n = 30) cultured on laminin-211/221. (C–E) In control motoneurons, Ca_v2.2 (C and D) and β-actin (E) are enriched in axonal growth cones. (F–H) Smn-deficient motoneurons exhibit reduced Ca_v2.2 and β-actin accumulation in comparison to controls. (B [checkered bar] and I–K) The reduction of signal intensity for Ca_v2.2 and β-actin is restored by 100 μM 8-CPT-cAMP. Results represent the mean ± SEM of pooled data from three independent experiments. n, number of motoneurons that were scored in total from control or Smn ^−/−; SMN2 embryos. *, P < 0.05; **, P < 0.001, tested by one-way ANOVA.

Figure 7.

Smn protein distribution in control and Smn^−/−; SMN2 motoneurons after treatment with 8-CPT-cAMP. (A–I) Distribution of Smn protein in cell body (A–C), axon (D–F), and growth cones (G–I) of Smn ^+/+; SMN2 (A, D, and G), Smn ^−/−; SMN2 (B, E, and H), and 8-CPT-cAMP–treated Smn ^−/−; SMN2 (C, F, and I) primary motoneurons cultured for 7 d on laminin-211/221. 100 μM 8-CPT-cAMP increases Smn immunoreactivity in cell bodies, axons, and growth cones of Smn ^−/−; SMN2 motoneurons (n = 20 for controls, n = 20 for Smn ^−/−; SMN2, and n = 20 for Smn ^−/−; SMN2 treated with 100 μM 8-CPT-cAMP). (J) Quantitative analysis of Smn immunoreactivity revealed a significant Smn increase in 8-CPT-cAMP–treated motoneurons (checkered bars) in comparison to untreated Smn ^−/−; SMN2 motoneurons. Results represent the mean ± SEM of pooled data from three independent experiments. n, number of motoneurons that were scored in total from control, Smn ^−/−; SMN2, or Smn ^−/−; SMN2 embryos treated with 100 μM 8-CPT-cAMP. *, P < 0.05; **, P < 0.001, tested by one-way ANOVA.

Figure 8.

8-CPT-cAMP restores axon elongation, growth cone size, and responses to laminin-211/221 in Smn^−/−; SMN2 motoneurons. (A–E) The smaller growth cone size of 7-d-old Smn ^−/−; SMN2 motoneurons (A and C) is rescued by 100 μM 8-CPT-cAMP (A [gray checkered bar] and D) and reaches the size of control motoneurons (A, B, and D), also in the presence of CTX (A and E; n = 30 for each genotype with and without 100 μM 8-CPT-cAMP). (F) Ratio of β-actin content in the most distal part of the axon and the most proximal part of the axon. Smn ^−/−; SMN2 motor axons (n = 82) show reduced distal β-actin signal intensity in comparison to control cells (n = 78). 8-CPT-cAMP treatment of Smn ^−/−; SMN2 restores distal β-actin levels (checkered gray bar; n = 90). (G–N) Analysis of actin mRNA levels in axonal growth cones by in situ hybridization in motoneurons from Smn ^−/−; SMN2 (G, I, and L; n = 30), 8-CPT-cAMP–treated Smn ^−/−; SMN2 (G, J, and M; n = 32), and control embryos (G, H, and K; n = 30). (G, J, and M) 8-CPT-cAMP restores the actin mRNA deficit in axon terminals of Smn ^−/−; SMN2 motoneurons. (N) A sense actin probe was used as a negative control for the specificity of the actin probe. (O–R) 8-CPT-cAMP also restores the response of Smn ^−/−; SMN2 motor axons to laminin-211/221. Axon elongation after 7 d on laminin-211/221 in control (P; n = 141), Smn ^−/−; SMN2 (Q; n = 141) and 8-CPT-cAMP–treated Smn ^−/−; SMN2 (R; n = 60) motoneurons. (O) Quantitative analysis of data. Results represent the mean ± SEM of pooled data from three independent experiments. n, number of motoneurons that were scored in total from control, Smn ^−/−; SMN2, or Smn ^−/−; SMN2 embryos were treated with 100 μM 8-CPT-cAMP. *, P < 0.05; **, P < 0.001, tested by one-way ANOVA.