Carol Milligan

Complete Dissociation of Motor Neuron Death from Motor Dysfunction by Bax Deletion in a Mouse Model of ALS

The death of cranial and spinal motoneurons (MNs) is believed to be an essential component of the pathogenesis of amyotrophic lateral sclerosis (ALS). We tested this hypothesis by crossing Bax-deficient mice with mice expressing mutant superoxide dismutase 1 (SOD1), a transgenic model of familial ALS. Although Bax deletion failed to prevent neuromuscular denervation and mitochondrial vacuolization, MNs were completely rescued from mutant SOD1-mediated death. However, Bax deficiency extended lifespan and delayed the onset of motor dysfunction of SOD1 mutants, suggesting that Bax acts via a mechanism distinct from cell death activation. Consistent with this idea, Bax elimination delayed the onset of neuromuscular denervation, which began long before the activation of cell death proteins in SOD1 mutants. Additionally, we show that denervation preceded accumulation of mutant SOD1 within MNs and astrogliosis in the spinal cord, which are also both delayed in Bax-deficient SOD1 mutants. Interestingly, MNs exhibited mitochondrial abnormalities at the innervated neuromuscular junction at the onset of neuromuscular denervation. Additionally, both MN presynaptic terminals and terminal Schwann cells expressed high levels of mutant SOD1 before MNs withdrew their axons. Together, these data support the idea that clinical symptoms in the SOD1 G93A model of ALS result specifically from damage to the distal motor axon and not from activation of the death pathway, and cast doubt on the utility of anti-apoptotic therapies to combat ALS. Furthermore, they suggest a novel, cell death-independent role for Bax in facilitating mutant SOD1-mediated motor denervation.

Extracellular Heat Shock Protein 70: A Critical Component for Motoneuron Survival

The dependence of developing spinal motoneuron survival on a soluble factor(s) from their target, muscle tissue is well established both in vivo and in vitro. Considering this apparent dependence, we examined whether a specific component of the stress response mediates motoneuron survival in trophic factor-deprived environments. We demonstrate that, although endogenous expression of heat shock protein 70 (HSP70) did not change during trophic factor deprivation, application of e-rhHsp70 (exogenous recombinant human Hsp70) promoted motoneuron survival. Conversely, depletion of HSP70 from chick muscle extract (MEx) potently reduces the survival-promoting activity of MEx. Additionally, exogenous treatment with or spinal cord overexpression of Hsp70 enhances motoneuron survival in vivo during the period of naturally occurring cell death [programmed cell death (PCD)]. Hindlimb muscle cells and lumbar spinal astrocytes readily secrete HSP70 in vitro, suggesting potential physiological sources of extracellular Hsp70 for motoneurons. However, in contrast to exogenous treatment with or overexpression of Hsp70 in vivo, muscle-targeted injections of this factor in an ex vivo preparation fail to attenuate motoneuron PCD. These data (1) suggest that motoneuron survival requirements may extend beyond classical trophic factors to include HSP70, (2) indicate that the source of this factor is instrumental in determining its trophic function, and (3) may therefore influence therapeutic strategies designed to increase motoneuron Hsp70 signaling during disease or injury.

Characterization of early pathogenesis in the SOD1G93A 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 SOD1G93A 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 SOD1G93A 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.

Characterization of early pathogenesis in the SOD1G93A 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 SOD1G93A 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.

Phosphorylation of c-Jun in Avian and Mammalian Motoneurons In Vivo during Programmed Cell Death: An Early Reversible Event in the Apoptotic Cascade

c-Jun is a transcription factor that is involved in various cellular events, including apoptotic cell death. For example, phosphorylation of c-Jun is one of the earliest biochemical changes detected in dying sympathetic neurons after NGF deprivation in vitro. However, currently, it is not known whether a similar molecular event is involved in the developmental programmed cell death (PCD) of neurons in vivo.We observed that only a subpopulation of motoneurons (MNs) exhibit c-Jun phosphorylation during the PCD period in chick [embryonic day 5 (E5)-E12] and mouse (E13-E18) embryos. Experimental perturbation of MN survival-promoting signals by limb bud removal (reduced signals) or by activity blockade (increased signals) in the chick embryo demonstrated that the presence of those signals is negatively correlated with the number of c-Jun-phosphorylated MNs. This suggests that insufficient survival signals (e.g., neurotrophic factors) may induce c-Jun phosphorylation of MNs in vivo. Consistent with the idea that c-Jun phosphorylation is a reversible event during normal PCD of MNs, we found that c-Jun phosphorylation was transiently observed in a subpopulation of mouse MNs rescued from PCD by deletion of the proapoptotic gene Bax. Inhibition of c-Jun signaling significantly reduced MN death in chick embryo, indicating that activation of c-Jun signaling is necessary for the PCD of MNs. Together, c-Jun phosphorylation appears to be required for the initiation of an early and reversible event in the intracellular PCD cascade in vivo after loss of survival-promoting signals such as neurotrophic factors.

A Single-Dose Resveratrol Treatment in a Mouse Model of Amyotrophic Lateral Sclerosis

The underlying causes of denervation of the neuromuscular junction and eventual motor neuron death in amyotrophic lateral sclerosis (ALS) have not been resolved. The superoxide dismutase 1 (SOD1)(G93A) mutant mouse is a frequently used animal model of ALS. We hypothesized that resveratrol (RSV), a polyphenolic molecule that enhances mammalian NAD(+)-dependent SIRT1 deacetylases and may increase life span, would improve motor function and survival in the SOD1 mouse model via modulation of p53 acetylation. Data were collected for mean survival times, neuromuscular performance on the ROTOR-ROD™ (San Diego Instruments, San Diego, CA, USA), body weight, and p53 acetylation. Mean survival times were not statistically different (P=.23) between control and experimental (RSV-fed) groups (mean +/- SD, control [n=11] 138 +/- 6 days vs. experimental [n=10] 135 +/- 8 days). Performance was not significantly different between groups at time points corresponding to 50%, 80%, and 90% mean life span (P=.46), nor did RSV treatment attenuate body weight loss. Thus although manipulation of SIRT1 deacetylase activity has effects at the protein level in healthy aging organisms, we conclude that RSV treatment does not lead to functional improvement or increased longevity in a mouse model of ALS. We speculate that RSV-mediated modulation of p53 acetylation is either incapable of increasing or insufficient to increase motor performance and longevity in this model of ALS.

Administration of Recombinant Heat Shock Protein 70 Delays Peripheral Muscle Denervation in the SOD1 G93A Mouse Model of Amyotrophic Lateral Sclerosis

A prominent clinical feature of ALS is muscle weakness due to dysfunction, denervation and degeneration of motoneurons (MNs). While MN degeneration is a late stage event in the ALS mouse model, muscle denervation occurs significantly earlier in the disease. Strategies to prevent this early denervation may improve quality of life by maintaining muscle control and slowing disease progression. The precise cause of MN dysfunction and denervation is not known, but several mechanisms have been proposed that involve potentially toxic intra- and extracellular changes. Many cells confront these changes by mounting a stress response that includes increased expression of heat shock protein 70 (Hsp70). MNs do not upregulate Hsp70, and this may result in a potentially increased vulnerability. We previously reported that recombinant human hsp70 (rhHsp70) injections delayed symptom onset and increased lifespan in SOD1G93A mice. The exogenous rhHsp70 was localized to the muscle and not to spinal cord or brain suggesting it modulates peripheral pathophysiology. In the current study, we focused on earlier administration of Hsp70 and its effect on initial muscle denervation. Injections of the protein appeared to arrest denervation with preserved large myelinated peripheral axons, and reduced glial activation.

In vitro methods to prepare astrocyte and motoneuron cultures for the investigation of potential in vivo interactions

This protocol details methods to isolate and purify astrocytes and motoneurons (MNs) from the chick lumbar spinal cord. In addition, an approach to study the influences of astrocyte secreted factors on MNs is provided. Astrocytes are isolated between embryonic days 10 and 12 (E10–12), propagated in serum (2–3 h) and differentiated in chemically defined medium (3–4 h). When prepared according to this protocol, astrocyte cultures are more than 98% pure when assessed using the astrocyte-specific markers glial fibrillary acidic protein (GFAP) and S100β. MNs are isolated between E5.5 and 6.0 (3–4 h) using a procedure that takes selective advantage of the large size of these cells. These cultures can be maintained using individual trophic factors, target-derived factors or astrocyte-derived factors, the preparation of which is also described (5–6 h). All or part of these techniques can be used to investigate a variety of processes that occur during nervous system development and disease or after injury.

Decreases in phosphoinositide‐3‐kinase/Akt and extracellular signal‐regulated kinase 1/2 signaling activate components of spinal motoneuron death

Motoneuron dependence on target‐derived trophic factors during development is well established, with loss of trophic support leading to the death of these cells. A complete understanding of the intracellular signal transduction machinery associated with extracellular survival signals requires the examination of individual pathways in various cellular and environmental contexts. In cells deprived of trophic support, and hence compromised for survival, phosphoinositide‐3‐kinase (PI3K) is decreased when compared with healthy cells supplied with trophic support. Extracellular signal‐regulated kinase 1/2 (ERK1/2) signaling is dramatically decreased in deprived cells. We have examined the role of these two pathways to understand how changes in their activity regulate motoneuron survival and death. Pharmacological inhibition of PI3K attenuated motoneuron survival and was important in the regulation of Bcl‐2 serine phosphorylation, limited release of cytochrome c into the cytoplasm and caspase activation. Bax translocation from cytoplasm to mitochondria was not altered when PI3K was inhibited. High levels of ERK1/2 inhibition robustly attenuated motoneuron survival in cells supplied with trophic support, whereas moderate inhibition of ERK1/2 activation had little effect. ERK1/2 inhibition in these cells decreased Bcl‐2 phosphorylation and resulted in release of cytochrome c from the mitochondria. Bax translocation and caspase activation were not affected by ERK1/2 inhibition. These data reveal that changes in PI3K and ERK1/2 signaling lead to individual and overlapping effects on the cell‐death machinery. Characterizing the role of these pathways is critical for a fundamental understanding of the development and degeneration of specific neuronal populations.

Fewer active motors per vesicle may explain slowed vesicle transport in chick motoneurons after three days in vitro.

Vesicle transport in cultured chick motoneurons was studied over a period of 3 days using motion-enhanced differential interference contrast (MEDIC) microscopy, an improved version of video-enhanced DIC. After 3 days in vitro (DIV), the average vesicle velocity was about 30% less than after 1 DIV. In observations at 1, 2 and 3 DIV, larger vesicles moved more slowly than small vesicles, and retrograde vesicles were larger than anterograde vesicles. The number of retrograde vesicles increased relative to anterograde vesicles after 3 DIV, but this fact alone could not explain the decrease in velocity, since the slowing of vesicle transport in maturing motoneurons was observed independently for both anterograde and retrograde vesicles. In order to better understand the slowing trend, the distance vs. time trajectories of individual vesicles were examined at a frame rate of 8.3/s. Qualitatively, these trajectories consisted of short (1-2 s) segments of constant velocity, and the changes in velocity between segments were abrupt (<0.2 s). The trajectories were therefore fit to a series of connected straight lines. Surprisingly, the slopes of theses lines, i.e. the vesicle velocities, were often found to be multiples of ~0.6 mum/s. The velocity histogram showed multiple peaks, which, when fit with Gaussians using a least squares minimization, yielded an average spacing of 0.57 mum/s (taken as the slope of a fit to peak position vs. peak number, R(2)=0.994). We propose that the abrupt velocity changes occur when 1 or 2 motors suddenly begin or cease actively participating in vesicle transport. Under this hypothesis, the decrease in average vesicle velocity observed for maturing motoneurons is due to a decrease in the average number of active motors per vesicle.