Characterization of early pathogenesis in the SOD1(G93A) mouse model of ALS: part I, background and methods

Abstract

Charcot first described amyotrophic lateral sclerosis (ALS) in 1869; however, its causes remain largely unknown and effective, long-term treatment strategies are not available. The first mouse model of ALS was developed after the identification of mutations in the superoxide dismutase 1 (SOD1) gene in 1993, and accordingly most of our knowledge of the etiology and pathogenesis of the disease comes from studies carried out using this animal model. Although numerous preclinical trials have been conducted in the mutant SOD1 mouse models, the results have been disappointing because they did not positively translate to clinical trials. One explanation may be that current understanding of when and where pathogenesis begins is insufficient to accurately guide preclinical trials. Further characterization of these early events may provide insight into disease onset, help in the discovery of presymptomatic diagnostic disease markers, and identify novel therapeutic targets. Here, we describe the rationale, approach, and methods for our extensive analysis of early changes that included an ultrastructural examination of central and peripheral components of the neuromuscular system in the SOD1(G93A) mouse and correlated these alterations with early muscle denervation, motor dysfunction, and motoneuron death. We also provide a discussion of published work to review what is known regarding early pathology in the SOD1 mouse model of ALS. The significance of this work is that we have examined early pathology simultaneously in both the spinal cord and peripheral neuromuscular system, and the results are presented in the companion paper (Part II, Results and Discussion). Our results provide evidence as to why a thorough characterization of animal models throughout the life span is critical for a strong foundation to design preclinical trials that may produce meaningful results.

Keywords: Axons; NMJs; cytoplasmic vacuoles; glia; mega-mitochondria; mitochondria; motoneurons; motor function.

Figure 1

Illustration of approaches used to identify MNs. (A and B) The TA and soleus motor pools exhibit rostral caudal overlap when identified via retrograde labeling after muscle injections of fluorescently labeled Cholera toxin B subunit (CTB; A), but can be distinctly identified when soleus is injected with Alexa-Fluor 488 CTB (green in B) and TA is injected with Alexa-Fluor 555 CTB (red in B). (C) Immunohistochemistry was used to confirm CTB in MNs of mice where the TA was injected with CTB, confirming motor pool location. (D) Arrows in 1 mm toluidine blue stained sections (areas similar to those shown in A–C) from animals prepared for EM indicate αMNs typically used in the evaluation of synaptic type on MNs. Dotted line outlines the VH boundary, and the box indicates location of thin section. X-Y coordinates on the electron microscope were used to map the adjacent sections and located specific MNs. MNs, motoneurons; TA, tibialis anterior; VH, ventral horn.

Figure 2

(A) Lateral view of a right hind paw during one stride depicting instances of time in swing and stance. Stance is comprised of braking and propulsion. (B) Paw Placement Angle is measured between the long axis through the hind paw and a line drawn through the center of the animal in its direction of motion. (C) Illustration of additional measurements for motor behavior. As described in the companion paper, the SOD1 mice exhibit a more closed hind paw placement angle, and less consistent step-to-step paw placement (increased paw placement angle variability) compared to WT mice. SOD1, superoxide dismutase; WT, wild type.

Figure 3

A wild-type (WT) mouse performing the loaded grid test is shown.

Figure 4

Chronology of pathophysiology in the SOD1^G93A mouse model of ALS. SOD1, superoxide dismutase; ALS, amyotrophic lateral sclerosis.

Figure 5

A summary diagram illustrating some of the pathological changes associated with mutant superoxide dismutase (SOD1) mouse models and putative patient disease progression. Research directed toward understanding how these events (puzzle pieces) are related and lead to clinical symptoms is critical to solving the puzzle for developing effective therapies.