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

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.

Characterization of early pathogenesis in the SOD1(G93A) mouse model of ALS: part II, results and discussion.

Pathological events are well characterized in amyotrophic lateral sclerosis (ALS) mouse models, but review of the literature fails to identify a specific initiating event that precipitates disease pathology. There is now growing consensus in the field that axon and synapses are first cellular sites of degeneration, but controversy exists over whether axon and synapse loss is initiated autonomously at those sites or by pathology in the cell body, in nonneuronal cells or even in nonmotoneurons (MNs). Previous studies have identified pathological events in the mutant superoxide dismutase 1 (SOD1) models involving spinal cord, peripheral axons, neuromuscular junctions (NMJs), or muscle; however, few studies have systematically examined pathogenesis at multiple sites in the same study. We have performed ultrastructural examination of both central and peripheral components of the neuromuscular system in the SOD1(G93A) mouse model of ALS. Twenty percent of MNs undergo degeneration by P60, but NMJ innervation in fast fatigable muscles is reduced by 40% by P30. Gait alterations and muscle weakness were also found at P30. There was no change in axonal transport prior to initial NMJ denervation. Mitochondrial morphological changes are observed at P7 and become more prominent with disease progression. At P30 there was a significant decrease in excitatory axo-dendritic and axo-somatic synapses with an increase in C-type axo-somatic synapses. Our study examined early pathology in both peripheral and central neuromuscular system. The muscle denervation is associated with functional motor deficits and begins during the first postnatal month in SOD1(G93A) mice. Physiological dysfunction and pathology in the mitochondria of synapses and MN soma and dendrites occur, and disease onset in these animals begins more than 2 months earlier than originally thought. This information may be valuable for designing preclinical trials that are more likely to impact disease onset and progression.

Sarcoplasmic reticulum Ca2+ depletion in adult skeletal muscle fibres measured with the biosensor D1ER.

The endoplasmic/sarcoplasmic reticulum (ER/SR) plays a crucial role in cytoplasmic signalling in a variety of cells. It is particularly relevant to skeletal muscle fibres, where this organelle constitutes the main Ca2+ store for essential functions, such as contraction. In this work, we expressed the cameleon biosensor D1ER by in vivo electroporation in the mouse flexor digitorum brevis (FDB) muscle to directly assess SR Ca2+ depletion in response to electrical and pharmacological stimulation. The main conclusions are: (1) D1ER is expressed in the SR of FDB fibres according to both di-8-(amino naphthyl ethenyl pyridinium) staining experiments and reductions in the Förster resonance energy transfer signal consequent to SR Ca2+ release; (2) the amplitude of D1ER citrine/cyan fluorescent protein (CFP) ratio evoked by either 4-chloro-m-cresol (4-CmC) or electrical stimulation is directly proportional to the basal citrine/CFP ratio, which indicates that SR Ca2+ modulates ryanodine-receptor-isoform-1-mediated SR Ca2+ release in the intact muscle fibre; (3) SR Ca2+ release, measured as D1ER citrine/CFP signal, is voltage-dependent and follows a Boltzmann function; and (4) average SR Ca2+ depletion is 20% in response to 4-CmC and 6.4% in response to prolonged sarcolemmal depolarization. These results indicate that significantly depleting SR Ca2+ content under physiological conditions is difficult.

Role of Ca2+, membrane excitability, and Ca2+ stores in failing muscle contraction with aging.

Excitation-contraction (EC) coupling in a population of skeletal muscle fibers of aged mice becomes dependent on the presence of external Ca(2+) ions (Payne, A.M., Zheng, Z., Gonzalez, E., Wang, Z.M., Messi, M.L., Delbono, O., 2004b. External Ca(2+)-dependent excitation - contraction coupling in a population of aging mouse skeletal muscle fibers. J. Physiol. 560, 137-155.). However, the mechanism(s) underlying this process remain unknown. In this work, we examined the role of (1) extracellular Ca(2+); (2) voltage-induced influx of external Ca(2+) ions; (3) sarcoplasmic reticulum (SR) Ca(2+) depletion during repeated contractions; (4) store-operated Ca(2+) entry (SOCE); (5) SR ultrastructure; (6) SR subdomain localization of the ryanodine receptor; and (7) sarcolemmal excitability in muscle force decline with aging. These experiments show that external Ca(2+), but not Ca(2+) influx, is needed to maintain force upon repetitive fiber electrical stimulation. Decline in fiber force is associated with depressed SR Ca(2+) release. SR Ca(2+) depletion, SOCE, and the putative segregated Ca(2+) release store do not play a significant role in external Ca(2+)-dependent contraction. More importantly, a significant number of action potentials fail in senescent mouse muscle fibers subjected to a stimulation frequency. These results indicate that failure to generate action potentials accounts for decreased intracellular Ca(2+) mobilization and tetanic force in aging muscle exposed to a Ca(2+)-free medium.

Sarcoplasmic reticulum Ca2+ release declines in muscle fibers from aging mice.

This study hypothesized that decline in sarcoplasmic reticulum (SR) Ca(2+) release and maximal SR-releasable Ca(2+) contributes to decreased specific force with aging. To test it, we recorded electrically evoked maximal isometric specific force followed by 4-chloro-m-cresol (4-CmC)-evoked maximal contracture force in single intact fibers from the mouse flexor digitorum brevis muscle. Significant differences in tetanic, but not in 4-CmC-evoked, contracture forces were recorded in fibers from aging mice as compared to younger mice. Peak intracellular Ca(2+) in response to 4-CmC did not differ significantly. SR Ca(2+) release was recorded in whole-cell patch-clamped fibers in the linescan mode of confocal microscopy using a low-affinity Ca(2+) indicator (Oregon green bapta-5N) with high-intracellular ethylene glycol-bis(alpha-aminoethyl ether)-N,N,N'N'-tetraacetic acid (20 mM). Maximal SR Ca(2+) release, but not voltage dependence, was significantly changed in fibers from old compared to young mice. Increasing the duration of fiber depolarization did not increase the maximal rate of SR Ca(2+) release in fibers from old compared to young mice. Voltage-dependent inactivation of SR Ca(2+) release did not differ significantly between fibers from young and old mice. These findings indicate that alterations in excitation-contraction coupling, but not in maximal SR-releasable Ca(2+), account for the age-dependent decline in intracellular Ca(2+) mobilization and specific force.

Loss of skeletal muscle strength by ablation of the sarcoplasmic reticulum protein JP45.

Skeletal muscle constitutes approximately 40% of the human body mass, and alterations in muscle mass and strength may result in physical disability. Therefore, the elucidation of the factors responsible for muscle force development is of paramount importance. Excitation-contraction coupling (ECC) is a process during which the skeletal muscle surface membrane is depolarized, causing a transient release of calcium from the sarcoplasmic reticulum that activates the contractile proteins. The ECC machinery is complex, and the functional role of many of its protein components remains elusive. This study demonstrates that deletion of the gene encoding the sarcoplasmic reticulum protein JP45 results in decreased muscle strength in young mice. Specifically, this loss of muscle strength in JP45 knockout mice is caused by decreased functional expression of the voltage-dependent Ca(2+) channel Ca(v)1.1, which is the molecule that couples membrane depolarization and calcium release from the sarcoplasmic reticulum. These results point to JP45 as one of the molecules involved in the development or maintenance of skeletal muscle strength.