Promoting regeneration while blocking cell death preserves motor neuron function in a model of ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disease of motor neurons with very few treatment options. We had previously found that motor neuron degeneration in a mouse model of ALS can be delayed by deleting the axon damage sensor MAP3K12 or dual leucine zipper kinase (DLK). However, DLK is also involved in axon regeneration, prompting us to ask whether combining DLK deletion with a way to promote axon regeneration would result in greater motor neuron protection. To achieve this, we used a mouse line that constitutively expresses ATF3, a master regulator of regeneration in neurons. Although there is precedence for each individual strategy in the SOD1G93A mouse model of ALS, these have not previously been combined. By several lines of evidence including motor neuron electrophysiology, histology and behaviour, we observed a powerful synergy when combining DLK deletion with ATF3 expression. The combinatorial strategy resulted in significant protection of motor neurons with fewer undergoing cell death, reduced axon degeneration and preservation of motor function and connectivity to muscle. This study provides a demonstration of the power of combinatorial therapy to treat neurodegenerative disease.

Keywords: ALS; SOD1; axon regeneration; combinatorial therapy; motor neuron; neuronal death.

Figure 1

Motor neuron-to-muscle connectivity is preserved by constitutive expression of ATF3 in neurons of SOD1^G93A mice—and this is further enhanced by DLK deletion. (A) CMAP recorded bimonthly from the left TA muscle of each mouse from 7 to 19 weeks of age, n = 12–18 mice per SOD1^G93A genotype group and n = 7–16 for controls. See Table 2 for P-values of all pairwise comparisons by Tukey’s test. All data-points for this dataset are shown in Supplementary Fig. 1. (B) Overview of experimental time course. Two separate cohorts were used: one for survival and behaviour and another for CMAP and hindlimb grip strength measurements. Additional cohorts were generated for each of the three histology time points. IG = inverted grid; GS = grip strength; SC = spinal cord; SN = sciatic nerve. (C) Quantification of TA NMJ innervation in 9-week-old transgenic mice, n = 8–9 per genotype, n = 3 for wild-type (WT). (D) Hindlimb grip strength from transgenic mice at 15–19 weeks of age, n = 8–10 per genotype. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns (not significant) by repeated-measures ANOVA (standard least squares) in A or one-way ANOVA for C and D, for comparisons relative to the SOD1^G93A group (filled diamonds).

Figure 2

Combining ATF3 expression with DLK deletion results in better preservation of large myelinated axons in SOD1^G93A mice. (A) Representative images of myelin staining from the tibial branch of the sciatic nerve from 17-week-old mice. Scale bars = 50 µm (low magnification), 25 µm (high magnification). (B–E) Quantification of axon calibres within the tibial branch of the sciatic nerve: n = 7–11 per genotype. (B) Percentage of axons smaller than 6 µm². (C) Percentage of axons that are 6–12 µm². (D) Percentage of axons larger than 12 µm². (E) Parts of whole analysis including small axons in white (<6 µm²), medium axons in pink (6–12 µm²) and large axons in blue (>12 µm²). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA.

Figure 3

Combining DLK deletion with ATF3 overexpression preserves muscle mass and function. (A) Representative images of left TA muscle from male (M) and female (F) mice from each genotype group. Scale bar = 200 µm. (B) TA muscle weights from 19-week-old male and female mice, n = 3 for wild-type (WT) and n = 7–13 per SOD1^G93A carrier genotype group. Each point is the average weight of left and right TA muscles per animal. (C) Correlation of CMAP amplitude with TA muscle weight at 19 weeks of age, R² = 0.5532, slope = 0.568 and n = 3–13. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA.

Figure 4

Constitutive ATF3 expression combined with DLK deletion improves motor neuron viability in SOD1^G93A mice. Representative images of solochrome (A) and ChAT (B) staining of spinal cords from 14-week-old mice. Hemi-spinal cord scale bar = 200 µm, higher magnification view of ventral horn scale bar = 100 µm, applies to A and B. (C) Motor neuron (MN) counts evenly sampled across 3.6 mm in lumbar spinal cord (L2–L6), n = 10 per genotype group except n = 11 for SOD1^G93A; ATF3^OE. Each point is the average count from five lumbar sections per animal, *P < 0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns (not significant) by one-way ANOVA.

Figure 5

ATF3 expression combined with DLK deletion reduces neurodegeneration and microglial recruitment in spinal cord of SOD1^G93A mice. Representative images of amino cupric silver staining (A) and Iba1 staining (B) of spinal cords from 14-week-old mice. Hemi-spinal cord scale bar = 200 µm, higher magnification view of ventral horn scale bar = 50 µm for A and 100 µm for B. Quantification of the per cent area positive for silver stain (C) and Iba1 (D) in the ventral horn region of the spinal cord, n = 10 per genotype group, except n = 11 for SOD1^G93A; ATF3^OE. Each point in (C) and (D) is an average of five sections per animal over 3.6 mm of lumbar spinal cord (L2–L6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns (not significant) by one-way ANOVA.

Figure 6

Modest effects of ATF3 expression and/or DLK deletion on survival and motor behaviours of SOD1^G93A mice. (A) Survival curve of SOD1^G93A mice. Median survival times for SOD1^G93A, SOD1^G93A; ATF3^OE, SOD1^G93A; DLK^cKO and SOD1^G93A; ATF3^OE; DLK^cKO are 156, 152, 161 and 158.5 days, respectively, n = 21–29 mice per genotype group. (B) Graph of time until onset of a tail suspension (TS) score of 2. Median time of onset for SOD1^G93A, SOD1^G93A; ATF3^OE, SOD1^G93A; DLK^cKO and SOD1^G93A; ATF3^OE; DLK^cKO are 135, 137, 140 and 149 days, respectively, n = 21–27 mice per genotype group. (C) Inverted grid latency of male and female mice from 7 weeks of age until 24 weeks of age, n = 23–30 per genotype. (D) Forelimb grip strength of male and female mice from 7 weeks of age until 18 weeks of age, n = 21–30 per genotype group. ^#Indicates significance, see Supplementary Fig. 6 for more detailed statistics.