Neuromuscular denervation and deafferentation but not motor neuron death are disease features in the Smn2B/- mouse model of SMA

Abstract

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of motor neurons and skeletal muscle atrophy which is caused by ubiquitous deficiency in the survival motor neuron (SMN) protein. Several cellular defects contribute to sensory-motor circuit pathology in SMA mice, but the underlying mechanisms have often been studied in one mouse model without validation in other available models. Here, we used Smn2B/- mice to investigate specific behavioral, morphological, and functional aspects of SMA pathology that we previously characterized in the SMNΔ7 model. Smn2B/- SMA mice on a pure FVB/N background display deficits in body weight gain and muscle strength with onset in the second postnatal week and median survival of 19 days. Morphological analysis revealed severe loss of proprioceptive synapses on the soma of motor neurons and prominent denervation of neuromuscular junctions (NMJs) in axial but not distal muscles. In contrast, no evidence of cell death emerged from analysis of several distinct pools of lumbar motor neurons known to be lost in the disease. Moreover, SMA motor neurons from Smn2B/- mice showed robust nuclear accumulation of p53 but lack of phosphorylation of serine 18 at its amino-terminal, which selectively marks degenerating motor neurons in the SMNΔ7 mouse model. These results indicate that NMJ denervation and deafferentation, but not motor neuron death, are conserved features of SMA pathology in Smn2B/- mice.

Fig 1. Behavioral characterization of Smn^2B/- SMA mice.

(A) Body weight of control Smn^2B/+ (n = 32) mice and Smn^2B/- SMA mice (n = 31). Data represent mean and SEM. Statistics were performed with two-way ANOVA and Bonferroni’s multiple comparison test. ** P < 0.01; **** P < 0.0001. (B) Kaplan-Meier survival curves from the same experimental groups as in (A). Statistics were performed with Log-rank (Mantel-Cox) test. **** P < 0.0001. (C) Righting time from the same experimental groups shown in (A). Data represent mean and SEM. Statistics were performed with two-way ANOVA and Bonferroni’s multiple comparison test. Not Significant. (D) Time to fall in the hindlimb suspension test from the same experimental groups shown in (A). Data represent mean and SEM. Statistics were performed with two-way ANOVA and Bonferroni’s multiple comparison test. *** P < 0.001; **** P < 0.0001.

Fig 2. Smn ^2B/- SMA mice display severe loss of proprioceptive synapses on motor neurons.

(A) Immunostaining of VGluT1⁺ synapses (grayscale) and ChAT⁺ motor neurons (blue) in the L2 spinal cord from control (Smn ^2B/+) and SMA (Smn^2B/-) mice at P16. Scale bars: 25μm. (B) Number of VGluT1⁺ synapses on the soma of L2 motor neurons from the same groups as in (A) at P16. The box-and-whiskers graph shows the individual values, median, interquartile range, minimum and maximum from 5 mice per group. Statistics were performed with two-tailed unpaired Student’s t-test. **** P < 0.0001.

Fig 3. Selective loss of NMJ innervation in axial but not distal muscles in Smn^2B/- SMA mice.

(A and C) NMJ staining with bungarotoxin (BTX), Synaptophysin (SYP), and Neurofilament-M (NF-M) of the axial muscle quadratus lumborum (A) and the distal muscle tibialis anterior (C) from control (Smn^2B/+) and SMA (Smn^2B/-) mice at P16. Arrowheads indicate denervated NMJs. Scale bars: 25 μm. (B and D) Percentage of fully innervated, partially innervated, and denervated NMJs from the same groups as in (A) and (C). The box-and-whiskers graph shows the individual values, median, interquartile range, minimum and maximum from Smn^2B/+ (n = 4) and Smn^2B/- (n = 5) mice. Statistics were performed with two-tailed unpaired Student’s t-test. **** P < 0.0001, ns = not significant.

Fig 4. Death of lumbar motor neurons is not a disease feature of Smn^2B/- SMA mice.

(A) ChAT immunostaining of motor neurons in the L1 spinal segment from control (Smn ^2B/+) and SMA (Smn^2B/-) mice at P16. Scale bars: 50μm. (B) Total number of L1 motor neurons from the same groups as in (A). The box-and-whiskers graph shows the individual values, median, interquartile range, minimum and maximum from 5 mice per group. Statistics were performed with two-tailed unpaired Student’s t-test. ns = not significant. (C) ChAT immunostaining of motor neurons in the L2 spinal segment from control (Smn ^2B/+) and SMA (Smn^2B/-) mice at P16. Scale bars: 50μm. (D) Total number of L2 motor neurons from the same groups as in (C). The box-and-whiskers graph shows the individual values, median, interquartile range, minimum and maximum from 5 mice per group. Statistics were performed with two-tailed unpaired Student’s t-test. ns = not significant. (E) ChAT immunostaining of motor neurons in the L5 spinal segment from control (Smn ^2B/+) and SMA (Smn^2B/-) mice at P16. Scale bars: 50μm. (F and G) Total number of L5 LMC (F) and L5 MMC (G) motor neurons from the same groups as in (E). The box-and-whiskers graph shows the individual values, median, interquartile range, minimum and maximum from Smn^2B/+ (n = 4) and Smn^2B/- (n = 5) mice. Statistics were performed with two-tailed unpaired Student’s t-test. ns = not significant.

Fig 5. Smn deficiency induces p53 accumulation but not serine 18 phosphorylation in L1 motor neurons of Smn^2B/- SMA mice.

(A) ChAT and p53 immunostaining of the L1 spinal cord from control (Smn^2B/+) and SMA (Smn^2B/-) mice at P16. Scale bars: 100 μm. (B) ChAT and p53 immunostaining of L1 motor neurons from the same groups as in (A). Scale bars: 50 μm. (C) ChAT and phospho-p53^S18 immunostaining of L1 motor neurons from the same groups as in (A). Scale bars: 50 μm. (D) Percentage of p53⁺ L1 motor neurons from the same groups as in (A). (E) Percentage of phospho-p53^S18+ L1 motor neurons from the same groups as in (A). Data represents individual values, mean and SEM from 3 mice per group. Statistics were performed with two-tailed unpaired Student’s t-test. *** P < 0.001.