Opposite Synaptic Alterations at the Neuromuscular Junction in an ALS Mouse Model: When Motor Units Matter

Abstract

Denervation of the neuromuscular junction (NMJ) precedes the loss of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). ALS is characterized by a motor unit (MU)-dependent vulnerability where MNs with fast-fatigable (FF) characteristics are lost first, followed by fast fatigue-resistant (FR) and slow (S) MNs. However, changes in NMJ properties as a function of MU types remain debated. We hypothesized that NMJ synaptic functions would be altered precociously in an MU-specific manner, before structural alterations of the NMJ. Synaptic transmission and morphological changes of NMJs have been explored in two nerve-muscle preparations of male mice and their wild-type (WT) littermates: the soleus (S and FR MU); and the extensor digitorum longus (FF MU). S, FR, and FF NMJs of WT mice showed distinct synaptic properties from which we build an MU synaptic profile (MUSP) that reports MU-dependent NMJ synaptic properties. At postnatal day 180 (P180), FF and S NMJs of SOD1 already showed, respectively, lower and higher quantal content compared with WT mice, before signs of MN death and before NMJ morphological alterations. Changes persisted in both muscles until preonset (P380), while denervation was frequent in the mutant mouse. MN death was evident at this stage. Additional changes occurred at clinical disease onset (P450) for S and FR MU. As a whole, our results reveal a reversed MUSP in SOD1 mutants and highlight MU-specific synaptic changes occurring in a precise temporal sequence. Importantly, changes in synaptic properties appear to be good predictors of vulnerability to neurodegeneration. The inadequate excitability of motor neurons and their output, the neuromuscular junctions (NMJs), has been considered a key factor in the detrimental outcome of the motor function in amyotrophic lateral sclerosis. However, a conundrum persists at the NMJ whereby persistent but incoherent opposite neurotransmission changes have been reported to take place. This article untangles this conundrum by systematically analyzing the changes in synaptic properties over the course of the disease progression as a function of the motor unit type. This temporal analysis reveals that early synaptic alterations evolve with disease progression but precede NMJ neurodegeneration. These data provide a novel framework of analysis and comparison of synaptic transmission alterations in neurodegenerative disorders.

Sex-Specific Differences in Motor-Unit Remodeling in a Mouse Model of ALS

“…For NMJ innervation ( Fig. 3), the main effects of each variable were compared using a GLM with a logistic distribution and Holm-Sidak's correction was applied to all pairwise comparisons for the post hoc test (referred to as "GLM post hoc test") as previously described (Tremblay et al, 2017). Data are presented as mean 6 SEM in the histograms and in the text.…”

“…Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons. The early dysfunction and loss of neuromuscular junctions (NMJs) is a key event in patients and animal models of the disease (Fischer et al, 2004;Pun et al, 2006;Armstrong and Drapeau, 2013a,b;Clark et al, 2016;Tallon et al, 2016;Tremblay et al, 2017;Chand et al, 2018). A recent study has shown in SOD1 G37R mice that NMJ loss within single motor-units (MUs), defined as a motor neuron and the muscle fibers it innervates, is initially slow and asynchronous (Martineau et al, 2018).…”

Neuroleptics as therapeutic compounds stabilizing neuromuscular transmission in amyotrophic lateral sclerosis

“…We determined NMJ synaptic strength by performing intracellular recordings at NMJs from the extensor digitorum longus (EDL) muscle ( Figure 6, A and B), a fast-twitch muscle essentially composed of fast-fatigable motor units, which are known to be selectively vulnerable in ALS (30)(31)(32). We showed that the amplitude of evoked endplate potentials (EPPs) as well as the quantal content (QC) in mutant SOD1 mice were significantly reduced compared with WT mice and pimozide-treated NMJs at both symptomatic (P400) ( Figure 6, C and D, P < 0.05) and presymptomatic (P160) ( Figure 6, G and H) stages.…”

A robust TDP-43 knock-in mouse model of ALS

“…Since the paralysis happened in the hindlimb of N390D/+ mice, we guessed that the hindlimbs were affected earlier than the forelimbs. Also, we analyzed mainly the soleus muscle, which was one of the main muscles that control walking, running and jumping, following the study by Tremblay et al [62] on the denervation of neuromuscular junctions in SOD-1 mice. In contrast to young N390D/+ male mice, the 12-month and 24-month old N390D/+ male mice exhibited significant skeletal muscle atrophy when compared to +/+ or A315T/+ mice, as represented by decrease of the wet weight of soleus muscle and reduction of the calf muscle volume ( Fig.…”

“…The NMJ staining was carried out as the methods described by Tremblay et al, [62]. The fixed soleus muscle samples were teased apart to fibers by using forceps, and the soleus muscle fibers were permeabilized with 10% FBS, 1% Triton-X100 diluted in PBS for 1 h at room temperature.…”