Degeneration of proprioceptive sensory nerve endings in mice harboring amyotrophic lateral sclerosis-causing mutations

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily targets the motor system. Although much is known about the effects of ALS on motor neurons and glial cells, little is known about its effect on proprioceptive sensory neurons. This study examines proprioceptive sensory neurons in mice harboring mutations associated with ALS, in SOD1(G93A) and TDP43(A315T) transgenic mice. In both transgenic lines, we found fewer proprioceptive sensory neurons containing fluorescently tagged cholera toxin in their soma five days after injecting this retrograde tracer into the tibialis anterior muscle. We asked whether this is due to neuronal loss or selective degeneration of peripheral nerve endings. We found no difference in the total number and size of proprioceptive sensory neuron soma between symptomatic SOD1(G93A) and control mice. However, analysis of proprioceptive nerve endings in muscles revealed early and significant alterations at Ia/II proprioceptive nerve endings in muscle spindles before the symptomatic phase of the disease. Although these changes occur alongside those at α-motor axons in SOD1(G93A) mice, Ia/II sensory nerve endings degenerate in the absence of obvious alterations in α-motor axons in TDP43(A315T) transgenic mice. We next asked whether proprioceptive nerve endings are similarly affected in the spinal cord and found that nerve endings terminating on α-motor neurons are affected during the symptomatic phase and after peripheral nerve endings begin to degenerate. Overall, we show that Ia/II proprioceptive sensory neurons are affected by ALS-causing mutations, with pathological changes starting at their peripheral nerve endings.

Keywords: Amyotrophic Lateral Sclerosis (ALS); Motor neuron; Neuromuscular junction (NMJ); Proprioceptive sensory neuron; SOD1g93A; TDP43A315T; VAChT; VGLUT1.

Figure 1

Fewer proprioceptive sensory neurons innervate the tibialis anterior muscle in SOD1^G93A and TDP43^A315T. We generated mice expressing YFP selectively in proprioceptive sensory neurons within DRGs (A–B,D) and then crossed this new mouse line with SOD1^G93A transgenic mice (C). Proprioceptive sensory neurons were identified in TDP43^A315T mice with NeuN and based on their large soma (see Fig. 2). Five days after focally injecting fCTB into the TA muscle, fewer sensory neurons were found in L3 DRGs of SOD1^G93A (n = 8) and TDP43^A315T (n = 4) compared with control (n = 5) mice (B–E) at 90 days of age. Fewer proprioceptive sensory neurons were also found in SOD1^G93A mice injected with fCTB into multiple locations of the TA (F). Despite these differences, the same numbers of YFP-positive neurons were found in L3 DRGs from SOD1^G93A mice as in control mice (G). Only male mice were used for this analysis. Error bar = standard error (STE). *P < 0.05 (E–F), ***P < 0.001 (E). Scale bars = 50 μm in C1 (applies to B–C1), 25 μm in D1 (applies to D,D1).

Figure 2

Sensory somata appear normal in SOD1^G93A and TDP43^A315T mice. Sensory somata labeled with fCTB were separated into three categories according to size (A–C). There is no difference in the average soma size for small (A,D), medium-sized (B,E), or large (C,F) sensory neurons among controls (n = 5), SOD1^G93A (n = 8), and TDP43^A315T (n = 4) mice at 90 days of age. Only male mice were used for this analysis. Error bar = standard error (STE). Scale bar = 25 μm.

Figure 3

Visualizing sensory and motor axons in the EDL muscle. Most nerve endings can be fully visualized in whole-mounted EDL muscles from mice expressing YFP (A). Sensory (A1) and motor (A2) branches can be traced from their nerve endings and counted. The nerve endings are shown in control (B) and SOD1^G93A (C) animals. α-MA = α-motor neuron axon; γ-MA = γ-motor neuron axon; Ia/II SA = Ia/II sensory afferent; NMJ, neuromuscular junction.

Figure 4

Peripheral proprioceptive nerve endings degenerate in SOD1^G93A and TDP43^A315T mice. In control, Ia proprioceptive sensory annulospiral endings are characterized by regular spacing of their spirals (A; arrow in C), and at least 10 spirals are found wrapping around the equatorial region of the muscle (A,D). In SOD1^G93A, the distance between spirals increases (B–C), and fewer spirals are found around the equatorial region of the muscle (B,D). Fewer spirals were also found in TDP43 mutants at 90 days of age (D). Proprioceptive sensory axons begin to degenerate alongside α-motor neuron axons during the early phase of the disease (E). At least six mice were used at each age. All animals were male; control n = 12, SOD1^G93A n = 16, TDP43^A315T n = 6. Error bar = STE. ***P < 0.001. Scale bar = 50 μm.

Figure 5

NMJs are affected in early symptomatic SOD1^G93A but not TDP43^A315T mice. NMJs were examined in 90-day-old male control (A1) and SOD1^G93A (B1) mice expressing YFP (A–B). AChR clusters (red) were labeled with fBTX (A1–B1). NMJs of male control (C1) and TDP43^A315T (D1) mice were also examined at 90 days of age by labeling the presynapse with synaptotagmin (C–D). NMJs are spared in TDP43^A315T (C–D1,E) but affected in SOD1^G93A mice (A–B1,E). At least 50 NMJs were counted per mouse, and five animals were examined per group. Error bar = STE. ***P < 0.001. Scale bars = 20 μm in B1 (applies to A–B1), 20 μm in D1 (applies to C–D1).

Figure 6

Analysis of γ-motor axons in SOD1^G93A mice. Representative images of γ-motor axons (green) innervating AChR clusters (red) located at the polar region of intrafusal muscle fibers (outlined by the dashed lines) in 90-day-old control (A–A2) and SOD1^G93A (B–B2) mice. The apposition of γ-motor axon with AChR clusters (stars) is unchanged in SOD1^G93A compared with control mice (A2,B2) even though α-motor axons are clearly retracting from NMJs (arrows). Scale bar = 20 μm.

Figure 7

Analysis of Ib proprioceptive axons in SOD1^G93A mice. Representative images of Ib proprioceptive axons (Ib afferent; A–A1,B–B1) alongside Ia/II proprioceptive sensory axons (Ia/II afferent; A–A2,B–B2) in the EDL muscle of 110-day-old control (A) and SOD1^G93A (B) mice. Ib nerve endings are readily found innervating Golgi tendons (B–B1) in muscles where Ia/II nerve endings have mostly degenerated (B,B2). Scale bars = 50 μm in A,B; 50 μm in B2 (applies to A1–B2).

Figure 8

Loss of VGluT1-positive synapses in the spinal cord of symptomatic SOD1^G93A and TDP43^A315T mice. Spinal cords at lumbar region 1 from male 90-day-old control (A), SOD1^G93A (B), and TDP43^A315T (C) mice were stained with VAChT (red) to label cholinergic synapses and VGluT1 (blue) to label proprioceptive synapses. The soma of α-motor neurons were marked by YFP in control and SOD1^G93A mice (A2–A3, B2–B3; green). The soma of neurons in TDP43^A315T mice were labeled with NeuN (C2–C3; green). When normalized to the perimeter of the motor neuron soma, there is a significant decrease in cholinergic synapses in SOD1^G93A mice (B,D) but not TDP43^A315T (C–D) compared with control mice (A,D). VGluT1-positive synapses, however, were reduced in both SOD1^G93A (B1,E) and TDP43^A315T (C1,D), mice. At least three animals were examined per group. Error bar = STE. *P < 0.05, **P < 0.01. Scale bar = 100 μm in C2 (applies to A–C2); 20 μm for A3–C3.

Figure 9

Numbers of VGluT1- and VAChT-positive synapses are unchanged in the spinal cord of 60-day-old SOD1^G93A mice. Spinal cords at lumbar region 1 from male 60-day-old control (A–A2) and SOD1^G93A (B–B2) animals expressing YFP in motor neurons were stained with VAChT (red) and VGluT1 (blue). (C–D) The number of VAChT and VGluT1 puncta on α-motor neuron somata is the same between 60-day-old control and SOD1^G93A mice. At least three animals were examined per group. Error bar = STE. Scale bar = 20 μm.