Research ArticleGenetic Inactivation of Cholinergic C Bouton Output Improves Motor Performance but not Survival in a Mouse Model of Amyotrophic Lateral Sclerosis
Introduction
Motor Neuron Disease (MND) is a progressive and fatal neurodegenerative disease that affects motor neurons and can present with various phenotypes (Swinnen and Robberecht, 2014, Grad et al., 2017). The most common MND subtype is Amyotrophic Lateral Sclerosis (ALS), which is defined by combined upper and lower motor neuron degeneration. 90% of ALS patients suffer from the sporadic form of the disease (sALS), while 10% suffer from familial ALS (fALS). Survival in ALS typically varies between two and five years from symptom onset, albeit a small percentage of patients survive much longer (Swinnen and Robberecht, 2014). The most common cause of mortality is respiratory failure due to decreased respiratory muscle strength and difficulty in swallowing. To date, there is no effective treatment for the disease; the neuroprotective agents riluzole and edaravone may extend lifespan for a few months if administered at early disease stages (Bensimon et al., 1994, Lacomblez et al., 1996, The Writing Group on behalf of the Edaravone (MCI-186) ALS 19, 2017, Jaiswal, 2019).
Over the recent decades a number of different ALS mouse models have been generated based on identified mutations underlying certain familial and sporadic cases of ALS (De Giorgio et al., 2019). Mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene were the first to be identified (Rosen et al., 1993) and account for approximately 20% of fALS and 5% of sALS cases. Based on this discovery, the transgenic human SOD1G93A gain of toxic function mouse was generated (Gurney et al., 1994) and it has been the most widely used ALS mouse model (Vinsant et al., 2013, Mina et al., 2018, De Giorgio et al., 2019).
In the last decade a question that has been raised regarding neurodegenerative diseases, in both humans and animal models, is whether alterations in synaptic function occur before or during disease onset and whether they have detrimental or beneficial roles in disease progression (Fogarty, 2018). For instance, there is increasing evidence for a state of hyperexcitability in both upper and lower motor neurons revealed by transcranial magnetic stimulation and electrophysiology in ALS patients (Vucic et al., 2008, Menon et al., 2015) and in motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients harboring select ALS-causative mutations (Wainger et al., 2014, Devlin et al., 2015). There is also evidence for decreased glycinergic transmission in SOD1G93A spinal motor neurons (Chang and Martin, 2009). Importantly, hyperexcitability may confer a survival advantage to still-surviving SOD1G93A mutant motor neurons experiencing stress (Saxena et al., 2013). Consistent with studies revealing presynaptic structural and functional alterations in ALS, studies in spinal muscular atrophy (SMA), a genetic disorder characterized by progressive loss of lower motor neurons, have demonstrated that abolition of afferent input to α-motor neurons precedes motor neuron loss (Mentis et al., 2011). In ALS, deletion of sensory endings on motor neurons leads to better survival (Lalancette-Hebert et al., 2016). These findings raise the hypothesis that apart from intrinsic motor neuron excitability changes, synaptic alterations precede and may even precipitate motor neuron dysfunction and possibly degeneration.
We set out to define the role of an abundant modulatory cholinergic synapse, known as C bouton, on motor neurons in ALS. C boutons are large cholinergic synaptic terminals of V0C Pitx2 expressing interneurons (Zagoraiou et al., 2009, Rozani et al., 2019) apposed to postsynaptic clusters containing M2 muscarinic receptors (Hellström et al., 2003, Witts et al., 2014), voltage-gated potassium channels Kv2.1 (Deardorff et al., 2013), calcium-activated potassium channels (SK) (Deardorff et al., 2013) and the sigma-1 receptor (Mavlyutov et al., 2010). Besides C boutons, Pitx2 interneurons also form cholinergic synapses on Ia inhibitory interneurons (Siembab et al., 2010). C boutons increase motor neuron excitability in demanding motor tasks (Miles et al., 2007, Zagoraiou et al., 2009). The integrity, stability and downstream signaling of these cholinergic terminals critically depends on postsynaptic receptor clusters (Deardorff et al., 2014, Romer et al., 2019) and on proteins, such as neuregulin-1, that are associated with these receptors and may play important roles in MNDs (Gallart-Palau et al., 2014, Lasiene et al., 2016, Salvany et al., 2019).
Several studies have investigated C boutons in the SOD1G93A mouse model of ALS with conflicting results. There is evidence for increased surface occupancy by C boutons on surviving motor neurons during disease progression (Pullen and Athanasiou, 2009, Herron and Miles, 2012). This has been suggested to serve as a homeostatic mechanism in progressive motor neuron dysfunction (Landoni et al., 2019). A recent study tried to elucidate the precise changes of C boutons at different disease stages and showed that C boutons may be differentially regulated during the disease course, exhibiting an increase in their density at early time points, followed by a decrease starting between P75 and P100 (Milan et al., 2015). Together, these findings prompted us to delve into the role of this neuromodulatory synapse in ALS.
Based on the evidence presented above, we speculate that C boutons may aggravate or ameliorate disease progression. On the one hand, as motor neurons degenerate, there may be a compensatory increase in excitability to maintain a proper motor neuron output, which could be mediated by C boutons. However, this lasting excitatory overload could generate excitotoxic insults and lead to motor neuron death (Brownstone and Lancelin, 2018). On the other hand, based on evidence of synaptic alterations during disease progression, we speculate that C boutons are activated in order to compensate for the loss of excitatory synapses (Romer et al., 2017; Salamatina et al. 2020) and may have beneficial roles in motor neuron function. To explore if C boutons exert an aggravating or beneficial role in the disease we followed a genetic strategy to abolish the cholinergic output of C boutons on motor neurons in SOD1G93A transgenic mice and assessed the survival and the motor performance in the rotarod motor task of these mice and their control littermates at different stages of disease progression. This is one of very few attempts to elucidate the role of specific synapses in the SOD1G93A mouse by the implementation of a genetic approach (Lalancette-Hebert et al., 2016, Landoni et al., 2019; Salamatina et al. 2020). Our data reveal that inactivation of cholinergic output from C boutons does not alter survival, whereas it has a beneficial role on motor performance as mice lacking cholinergic transmission mediated by C boutons performed better on the rotarod task than control littermates at the early symptomatic stages of ALS. These findings suggest that C boutons have a negative impact at early symptomatic stages of the disease.
Section snippets
Mouse lines
All mice were housed in the facility of the Center for Experimental Surgery of the Biomedical Research Foundation of the Academy of Athens (BRFAA). The BRFAA facility is registered as breeding and experimental facility according to the Presidential Decree of the Greek Democracy 160/91, which harmonizes the Greek national legislation with the European Council Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes. All experiments were performed in
Genetic inactivation of the cholinergic C bouton output in control mice
C boutons are specialized cholinergic synaptic terminals of Dbx1 derived Pitx2 + V0C interneurons. To achieve their inactivation, we followed a genetic strategy for removing choline acetyltransferase (ChAT), the enzyme catalyzing the biosynthesis of acetylcholine (ACh), in these interneurons (Zagoraiou et al. 2009; Fig. 1A). We conditionally deleted ChAT with a Dbx1::Cre driver (Buffelli et al., 2003) by crossing Dbx1::Cre heterozygous mice with mice carrying floxed ChAT alleles (ChATfl/fl).
Discussion
This study implements genetic tools to investigate the role of C bouton cholinergic transmission in motor neuron function during the course of ALS in the SOD1G93A ALS mouse model. Previous studies that have shown synaptic alterations in ALS mouse models raise two issues: (i) they provide disparate and ambiguous findings on synapse alterations in ALS mutant motor neurons (Dukkipati et al., 2017) and (ii) they raise questions as to whether such synaptic changes underlie disease pathogenesis or
Funding
Eleni Konsolaki’s research is co-financed by Greece and the European Union (European Social Fund-ESF) through the Operational Programme «Human Resources Development, Education and Lifelong Learning» in the context of the project "Reinforcement of Postdoctoral Researchers" (MIS-5001552), implemented by the State Scholarships Foundation (ΙΚΥ). Laskaro Zagoraiou, Konstantinos Pothakos and Eirini Tsape were supported by Fondation Santé.
Acknowledgments
We are grateful to Gareth B. Miles, Robert M. Brownstone and Lora B. Sweeney for providing helpful comments on the manuscript. We thank the director of BRFAA mouse facility Nikolaos Kostomitsopoulos for his support.
References (59)
- et al.
Presynaptic activity and CaMKII modulate retrograde semaphorin signaling and synaptic refinement
Neuron
(2010) - et al.
Glycinergic innervation of motoneurons is deficient in amyotrophic lateral sclerosis mice: a quantitative confocal analysis
Am J Pathol
(2009) Driven to decay: Excitability and synaptic abnormalities in amyotrophic lateral sclerosis
Brain Res Bull
(2018)- et al.
Time course of preferential motor unit loss in the SOD1 G93A mouse model of amyotrophic lateral sclerosis
Neurobiol Dis
(2007) - et al.
Gender-specific perturbations in modulatory inputs to motoneurons in a mouse model of amyotrophic lateral sclerosis
Neuroscience
(2012) - et al.
Neuronal matrix metalloproteinase-9 is a determinant of selective neurodegeneration
Neuron
(2014) - et al.
Excitotoxicity in ALS: overstimulation, or overreaction ?
Exp Neurol
(2016) - et al.
Semaphorins and the dynamic regulation of synapse assembly, refinement, and function
Curr Opin Neurobiol
(2014) - et al.
Cholinergic modulation of motor neurons through the C-boutons are necessary for the locomotor compensation for severe motor neuron loss during amyotrophic lateral sclerosis disease progression
Behav Brain Res
(2019) - et al.
The sigma-1 receptor is enriched in postsynaptic sites of C-terminals in mouse motoneurons. An anatomical and behavioral study
Neuroscience
(2010)
Cortical hyperexcitability precedes lower motor neuron dysfunction in ALS
Clin Neurophysiol
Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy
Neuron
Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase
Neuron
Accessory respiratory muscles enhance ventilation in ALS model mice and are activated by excitatory V2a neurons
Exp Neurol
Neuroprotection through excitability and mTOR required in ALS motoneurons to delay disease and extend survival
Neuron
Detecting and avoiding problems when using the Cre/lox system
Trends Genet
The role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis
Biochim Biophys Acta
Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons
Cell Rep
A cluster of cholinergic premotor interneurons modulates mouse locomotor activity
Neuron
A controlled trial of riluzole in amyotrophic lateral sclerosis. ALS/Riluzole Study Group
N Engl J Med.
Multiple origins of Cajal-Retzius cells at the borders of the developing pallium
Nat Neurosci.
Onset and Progression in Inherited ALS determined by motor neurons and microglia
Science
Tests to assess motor phenotype in mice: a user’s guide
Nat Rev Neurosci
Escape from homeostasis: spinal microcircuits and progression of amyotrophic lateral sclerosis
J Neurophysiol
Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition
Nature
Transgenic and physiological mouse models give insights into different aspects of amyotrophic lateral sclerosis
Dis Model Mech
Expression of postsynaptic Ca2+-activated K+ (SK) channels at C-bouton synapses in mammalian lumbar α-motoneurons
J Physiol
Swimming against the tide: investigations of the C-bouton synapse
Front Neural Circuits
Human iPSC-derived motoneurons harbouring TARDBP or C9ORF72 ALS mutations are dysfunctional despite maintaining viability
Nat Commun
Cited by (11)
Beyond neuromuscular activity: botulinum toxin type A exerts direct central action on spinal control of movement
2024, European Journal of PharmacologyCytoplasmic TDP-43 accumulation drives changes in C-bouton number and size in a mouse model of sporadic Amyotrophic Lateral Sclerosis
2023, Molecular and Cellular NeuroscienceProprioception revisited: where do we stand?
2021, Current Opinion in PhysiologyCitation Excerpt :As removing pSN input on αMNs improves motor neuron survival, and reducing pSN activity through the elimination of γMNs delays disease onset [55•], these studies suggest αMN hypoexcitability may result from overtaxing proprioceptive input. In addition to changes in proprioceptive input, other alterations in premotor input have been reported in ALS [56–60]. Taken together, these studies suggest that pSN activity and synaptic excitation contribute to αMN degeneration in neurodegenerative disease and that restructuring of premotor contacts may play an important compensatory role during disease progression.