Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man
Introduction
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive weakness leading to paralysis and death. Approximately 2% of cases are due to mutations in the superoxide dismutase (SOD1) gene. Transgenic mice carrying human SOD1 mutations develop progressive weakness similar to patients with ALS, making them useful models for understanding the pathogenesis of disease and testing new therapies. A widely used transgenic mouse model is the high-expressing SOD1G93A mutant that develops clinical disease at 80–90 days and dies at about 130 days (Chiu et al., 1995). This model has proven useful for examining the cellular pathology of motor neuron degeneration in ALS, and has provided a tool for developing preclinical data on drugs that may work to slow the course of ALS.
There is a general acceptance that weakness and death in the SOD1G93A mutant mouse, and in humans with ALS, occur as a direct consequence of motor neuron death. There are studies, however, that have demonstrated dysfunction and/or degeneration of the neuromuscular junction (NMJ) at times earlier than are reported for the loss of motor neurons. Using immunocytochemical methods, Frey et al. (2000) showed selective loss of fast-firing neuromuscular synapses as early as day 50, and Kennel et al. (1996) reported progressive loss of motor unit numbers by physiologic measures beginning at day 40. Motor neurons were not counted in either of these studies, but reports from other investigators do not suggest a significant loss of motor neurons in the G93A mouse until after 80–90 days (Chiu et al., 1995).
These data raise the question of where motor neuron dysfunction begins: within the motor neuron cell body, within the motor axon, or even at the level of the neuromuscular junction. Recent reports of abnormalities in retrograde axonal transport leading to motor neuron death support a peripheral localization for factors initiating the onset of this disease Hafezparast et al., 2003, LaMonte et al., 2002, Puls et al., 2003. Human studies of ALS do not provide an answer to this question, and no data are available correlating weakness or death with loss of spinal motor neurons.
In order to better understand the progression of disease in the SOD1G93A mutant mouse, we undertook a systematic pathological study of these animals at multiple time points along the course of disease. In the same animals, we quantified the numbers of spinal motor neurons, axons in the nerve roots, and the degree of denervation at neuromuscular junctions, providing a sequential view of motor neuron pathology in these animals. Our findings demonstrate that before any loss of motor neurons, there is a severe loss of ventral root motor axons and significant denervation at corresponding neuromuscular junctions. Progression of abnormalities was from distal to proximal, indicating a “dying back” pathophysiology. In addition, we had the opportunity to study at autopsy a patient with sporadic ALS who died early and unexpectedly. His pathology also suggested a pattern of disease similar to our findings in the mouse.
Section snippets
Animal breeding and behavioral analysis
All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee. Animals were housed in microisolator cages on a 12-h light–dark cycle and given free access to food and water. Breeding pairs of G93A high-expressing mice were obtained from Jackson Laboratories (Bar Harbor, ME), and identification of mutant mice was by standard PCR analysis on tail snip DNA.
Beginning at age 50 days, SOD-1 mutant mice (12 males and 12 females) were tested weekly for their
SOD1 clinical evaluation
The first indication of clinical disease was decreased performance on the accelerating Rotarod at day 78. Performance on the constant Rotarod began to decline at day 85. Weight was not a sensitive measure of disease onset, and did not deviate from baseline until after 120 days (Fig. 1).
Neuropathology
Initial evaluation of end-plates and ventral roots in 80-day-old presymptomatic animals showed significant abnormalities in both, prompting us to look for the initial neuropathologic changes at earlier time
Discussion
We examined the cell body, axon, and NMJ at multiple time points to illustrate the spatiotemporal progression of motor neuron pathology in the high-expressing SOD1G93A mutant mouse and related these findings to the clinical onset of weakness in these animals. As in previous studies, we first recognized signs of clinical disease at about 80 days, but there were significant pathological changes at much earlier time points. Quantitative analysis demonstrated denervation at the NMJ by day 47,
Acknowledgements
This work was supported by NIH grant P01 NS40405(JDG) and by a grant from the Robert Packard Center for ALS Research at Johns Hopkins. We thank Dr. Mark Rich for helpful discussion and Raphael James, Karen Carney, and Dayna McDermott for technical assistance.
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