Selective disruption of acetylcholine synthesis in subsets of motor neurons: A new model of late-onset motor neuron disease

doi:10.1016/j.nbd.2014.01.014

Neurobiology of Disease

Volume 65, May 2014, Pages 102-111

https://doi.org/10.1016/j.nbd.2014.01.014 Get rights and content

Highlights

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We generated a mouse model bearing selective dysfunction of a subset of motor neurons.
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Mutant mice were obtained by conditional knockout of the ChAT gene.
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Mutants display a progressing phenotype characteristic of neurogenic diseases.
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These alterations could only be detected several months after reduction in ChAT level.
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Mice exhibit hallmarks of late-onset motor neuron diseases such as post-polio defects.

Abstract

Motor neuron diseases are characterized by the selective chronic dysfunction of a subset of motor neurons and the subsequent impairment of neuromuscular function. To reproduce in the mouse these hallmarks of diseases affecting motor neurons, we generated a mouse line in which ~ 40% of motor neurons in the spinal cord and the brainstem become unable to sustain neuromuscular transmission. These mice were obtained by conditional knockout of the gene encoding choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine. The mutant mice are viable and spontaneously display abnormal phenotypes that worsen with age including hunched back, reduced lifespan, weight loss, as well as striking deficits in muscle strength and motor function. This slowly progressive neuromuscular dysfunction is accompanied by muscle fiber histopathological features characteristic of neurogenic diseases. Unexpectedly, most changes appeared with a 6-month delay relative to the onset of reduction in ChAT levels, suggesting that compensatory mechanisms preserve muscular function for several months and then are overwhelmed. Deterioration of mouse phenotype after ChAT gene disruption is a specific aging process reminiscent of human pathological situations, particularly among survivors of paralytic poliomyelitis. These mutant mice may represent an invaluable tool to determine the sequence of events that follow the loss of function of a motor neuron subset as the disease progresses, and to evaluate therapeutic strategies. They also offer the opportunity to explore fundamental issues of motor neuron biology.

Introduction

Motor neuron diseases are etiologically heterogeneous disorders resulting from the progressive degeneration of motor neurons in spinal cord and brainstem and the consequent disconnection between neurons and muscle fibers. In response to the loss of motor neurons, compensatory mechanisms occur but progressively fail leading to the development of weakness. This phenomenon is particularly relevant to the late progression of neuromuscular deficits that may affect patients with anterior acute paralytic poliomyelitis (Trojan and Cashman, 2005). During the recovery period following poliomyelitis, surviving motor neurons compensate for the loss of neighboring motor neurons by reinnervating orphaned muscle fibers, thereby providing gradual improvement and prolonged stability of neuromuscular function. Several decades after the initial acute attack, these neurons become overused, unable to support the increased metabolic demands to sustain many muscle fibers, and hence degenerate. The resulting muscle fiber denervation and motor unit dysfunction lead to progressive muscle weakness and eventually muscular atrophy. Superimposition of normal aging during which a gradual loss in muscle innervation normally occurs (Deschenes et al., 2010) accentuates this process. To date, no animal model and no specific treatment are available for this type of disorder.

An animal model in which a subset of motor neurons is not functional should provide novel in vivo information about the cascade of events triggered by motor neuron dysfunction. One way to achieve this goal is to interrupt the synaptic transmission between nerve-endings and muscle in a subset of motor neurons. Here we report the generation and the phenotypic characterization of a new mouse line in which about half of the spinal cord and brainstem motor neurons do not produce choline acetyltransferase (ChAT), the biosynthetic enzyme of acetylcholine, and thus cannot sustain cholinergic neurotransmission at their neuromuscular junctions (NMJs).

Section snippets

Animals

All experiments were conducted in accordance with the European Community Council Directive (86/809/EEC) regarding the care and use of animals for experimental procedures. Mice were weaned at 4 weeks and housed two to four per cage by sex and litter regardless of the genotype under standard conditions, with food and water available ad libitum and a 12 h light/dark schedule (lights on at 07:30 a.m.).

Chat^lox/lox mice

A 90 kb genomic clone was isolated by screening a 129/SvJ strain mouse Bacterial Artificial Chromosome

Southern blotting and genotype determination

Southern blot analysis of HindIII-digested genomic DNA extracted from ES cells was performed using a [α-³²P]-labeled fragment (1079 bp). This probe was derived from genomic DNA outside the region of homology between the targeting vector and endogenous sequences (see Fig. 1A for the position of the probe).

Mice were genotyped by routine PCR amplification of tail genomic DNA using the following primer sets. Primers P1: 5′-GGTTCTACAGGATCAATAAGG-3′ (forward) and P2: 5′-TAGTGGTCACAGCTACTCTC-3′

Targeted disruption of the ChAT gene in subsets of motor neurons

To conditionally inactivate the ChAT gene, we first generated a mouse line with floxed ChAT alleles (ChAT^lox/lox mice) by homologous recombination (Fig. 1). We chose to target the 8th exon, which corresponds to the 5th coding exon of the gene (Misawa et al., 1992), because its deletion introduces a frame-shift leading to the synthesis of a truncated protein unable to acetylate choline (not shown). Next, ChAT gene expression was selectively abolished in motor neurons by crossing ChAT^lox/lox mice

Discussion

In this study, we generated a new mouse line, ChAT^lox/lox; Cre^+/− mice, in which cholinergic transmission is abolished selectively in a subset (~ 40%) of motor neurons of brainstem and spinal cord by conditional knockout of the ChAT gene. These mice thus reproduce the loss of functional motor units characteristic of motor neuron diseases. They display a late-onset and progressive impairment of motor activity and muscular strength, associated to overt phenotypic abnormalities and

Conclusion

The mouse line described here constitutes a new model of chronic dysfunction of a motor neuron subset which occurs naturally, is not lethal and leads to a late-onset and age-related progressive phenotype characteristic of motor neuron diseases. It provides a potential means not only to better understand both motor neuron biology and neuromuscular pathology during the course of diseases, but also to evaluate the efficacy of therapeutic strategies.

The following are the supplementary data related

Acknowledgments

The authors thank Y. Clément, J. Gallego, E. Lepicard, B. Matrot, A. Privat and G. Vodjdani for helpful discussions, J. Forsayeth for critical reading of the manuscript, G. Grannec for his help in the setting of the figures, M. Holzenberger for kindly providing the MeuCre40 transgenic mice, D. Metzger for kindly providing the floxed PGK-neo cassette, E. Pellé for his technical support in radiograph realization, C. Guimpied for animal housing and care, and B. Zalc for support. This work was

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¹: Present address: Endocells, ICM, Paris, France.

²: Present address: Takeda Cambridge Ltd, Cambridge CB4 OPZ, United Kingdom.

³: Present address: IPSEN Innovation, Les Ulis, France.

⁴: Present address: UMR 1141, Hôpital Robert Debré, Paris, France.

⁵: Present address: UMR-S 839, Institut du Fer à Moulin, Paris, France.

View full text

Selective disruption of acetylcholine synthesis in subsets of motor neurons: A new model of late-onset motor neuron disease

Highlights

Abstract

Introduction

Section snippets

Animals

Chatlox/lox mice

Southern blotting and genotype determination

Targeted disruption of the ChAT gene in subsets of motor neurons

Discussion

Conclusion

Acknowledgments

Physiol. Behav.

Exp. Gerontol.

J. Biol. Chem.

Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype expression

J. Neurosci. Res.

Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition

Nature

Creation of a rare cutter multiple cloning site

Biotechniques

Skeletal muscle mass and distribution in 468 men and women aged 18–88 yr

J. Appl. Physiol.

Severely impaired neuromuscular synaptic transmission causes muscle weakness in the Cacna1a-mutant mouse rolling Nagoya

Eur. J. Neurosci.

Neuromuscular function impairment is not caused by motor neurone loss in FALS mice: an electromyographic study

Neuroreport

Impaired synaptic vesicle release and immaturity of neuromuscular junctions in spinal muscular atrophy mice

J. Neurosci.

Cre-mediated germline mosaicism: a new transgenic mouse for the selective removal of residual markers from tri-lox conditional alleles

Nucleic Acids Res.

Chat^lox/lox mice