Zuoshang Xu
Departments of Pharmacology and Molecular Toxicology, Cell Biology and Neuroscience Program,
University of Massachusetts Medical School, Worcester, MA, USA
Correspondence:
Zuoshang Xu, MD, PhD.
Departments of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School, 55 Lake Avenue North,
Worcester, MA 01655, USA
Tel: (+1) 508 856 3309
Fax: (+1) 508 856 5080
E-mail: zuoshang.xu@umassmed.edu
Received 29 March 2000
Revised 10 July 2000
Accepted 17 July 2000
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes motoneuron degeneration, skeletal muscle atrophy, paralysis and death. The identification of mutations in Cu,Zn superoxide dismutase (SOD1) as a genetic cause of this disease has led to the creation of a number of in-vitro and in-vivo models. Experiments have been carried out in these model systems to address fundamental questions related to the disease: (1) what is the nature of toxicity of the mutated SOD1? (2) what are the cellular targets and pathways that lead to neuronal degeneration? (3) what makes motoneurons particularly vulnerable to the toxicity of the mutant enzyme? and (4) are there effective treatments for ALS based on current hypotheses regarding the disease mechanism? Current research on these questions is reviewed. (ALS 2000; 1: 225-234)
Keywords: neurodegenerative disease - superoxide dismutase - spinal cord - paralysis - degeneration
Amyotrophic lateral sclerosis (ALS) was first described by Charcot more than a century ago. It is an adult-onset, progressive and fatal neurodegenerative disease. Ninety percent of the cases are sporadic and 10% are familial. Although the primary cause of this disease is heterogeneous, the shared common end-point is motoneuron degeneration, skeletal muscle atrophy, paralysis and death.1,2 For a long time, studies of this disease were limited to pathological descriptions of autopsy specimens. Direct experimental study on the mechanism of motoneuron degeneration was not possible because suitable models for the disease were lacking. A breakthrough occurred in 1993 when mutations in the Cu,Zn superoxide dismutase (SOD1) gene were discovered to cause approximately 25% of familial ALS.3,4 At present, nearly 70 different mutations have been identified as causes of this disease.5 By expressing the disease-causing mutant SOD1 in different systems, numerous in-vitro and in-vivo models have been created to address the mechanism and the treatment of the disease. Here I briefly review the major hypotheses on the mechanisms by which the mutant SOD1 causes motoneuron degeneration, with a brief summary of the treatments that have been tested thus far on the transgenic mouse models.
SOD1 is a 17 KD protein that contains one copper and one zinc atom. It is present in cytoplasm as a homodimer. Copper is required for the enzymatic activity of SOD1 while zinc stabilizes the protein structure.6 Although SOD1 is known to have several different catalytic activities,7 its main natural function is believed to be superoxide dismutation, in which superoxide (O2-) is converted to hydrogen peroxide (H2O2) and oxygen:
The hydrogen peroxide produced by this reaction is further converted to water and oxygen by catalase or glutathion peroxidase. Superoxide is mainly a by-product of mitochondrial respiration and is an oxidative free radical toxic to cells. Thus, by acting concertedly with the downstream anti-oxidative enzymes, SOD1 detoxifies the toxicity of superoxide.
Because of this function, it was thought at first that mutations in the enzyme caused a reduction in enzyme activity, thereby leaving inside the cells an increased level of superoxide, which eventually kills motoneurons.8,9 Despite its logic and simplicity, and some early supportive evidence,10-12 this hypothesis has lost its validity, as experiments subsequently carried out in several laboratories overwhelmingly demonstrated that mutant SOD1 caused motoneuron degeneration by a gain of toxic property, rather than a loss of superoxide dismutation function. First, some disease-causing mutant enzymes retain normal levels of superoxide dismutation activity and the presence of mutant enzyme does not affect the activity and stability of the normal enzyme.13,14 Second, transgenic mice expressing the mutant SOD1 develop motoneuron degeneration and ALS without lowering the level of superoxide dismutation activity.15-18 Third, neither overexpression nor knockout of the normal SOD1 in transgenic mice causes the disease, indicating that neither an increase nor a decrease of normal SOD1 activity are causes.15,17,19 Finally, neither overexpression nor knockout of the normal SOD1 influence the course of the disease caused by the mutant SOD1 in transgenic mice, indicating that neither an increase nor a decrease of the normal SOD1 activity influence the progression of the disease.20 The collective power of these experiments has left little doubt that a gain of toxic property is the culprit which causes the death of motoneurons.
Two major hypotheses have been proposed. First, Beckman, Crow, Estevez and their colleagues suggest that the mutated SOD1 has an enhanced activity to catalyze nitration of tyrosine using peroxynitrite (ONOO-), which is generated by the reaction of superoxide with nitric oxide.21 They also propose that this enhanced activity may be brought about by a reduced Zn affinity as a consequence of mutations in SOD1.21,22 This could lead to loss of Zn from SOD1 in vivo. Loss of Zn causes a structural change, so that the Cu++ can be readily reduced to Cu+ by cellular antioxidants, resulting in the following reactions:23,24
Thus, Zn-deficient SOD1 could generate its own peroxynitrite in the presence of oxygen and nitric oxide, and nitrate tyrosine residues.
In support of this hypothesis, Beckman, Crow and colleagues showed that the affinity to Zn in various ALS-causing SOD1 mutants was reduced by 18-30 times compared with the wild-type enzyme.22 The Cu++ in Zn-deficient SOD1 was reduced much more rapidly and could generate peroxynitrite at higher rates than the normal enzyme.24 When Zn-deficient SOD1 was delivered into cultured motoneurons, it induced apoptosis, and this induction depended on the activity of nitric oxide synthetase.24 Consistent with this hypothesis are the observations that spinal cords from both patients and transgenic mice expressing the mutant SOD1 exhibit elevated levels of free 3-nitrotyrosine, and induction of iNOS (inducible nitric oxide synthase) that is correlated with motoneuron degeneration.25-27
But it remains to be demonstrated that this mechanism occurs in vivo. In particular, evidence is needed to show that the mutant SOD1 exists in a Zn-deficient form and that it kills motoneurons in a NO-dependent manner in the spinal cord. Recently, evidence against this latter suggestion has been presented.28,29 In addition, no protein-bound nitrotyrosine was detectable in transgenic mice expressing the mutant SOD1.26 Furthermore, it was proposed that neurofilament subunit L (NF-L) might exacerbate the toxicity of the mutant enzyme because of its high affinity to Zn, which could remove Zn from the mutant SOD1.22,24 However, a recent experiment showed that increased levels of NF-L prolonged the survival of the transgenic mice which expressed a mutant SOD1 G93A,30 suggesting that such a mechanism may not play a significant role in vivo.
The second hypothesis regarding the toxic property is that the mutant SOD1 has an enhanced peroxidase activity. This property was first discovered in the wild-type SOD1 by Yim and Stadtman.31,32 They found that SOD1 could catalyze the oxidation of a spin trap compound DMPO (5,5'-dimethyl-1-pyrroline N-oxide) in the presence of H2O2. The following mechanism has been proposed for this reaction:
Using the same assay, two groups have demonstrated that this activity was elevated in the mutant SOD1 in vitro.33-35 In support of this hypothesis, levels of products derived from oxidative damage of nucleic acids, proteins and lipids are found to be elevated in cases of human ALS and in transgenic mice expressing mutant SOD1.36-38 A particularly interesting observation was that in the presence of H2O2, mutant SOD1 could selectively inactivate a glial glutamate transporter GLT-1 (EAAT-2) expressed in xenopus oocytes.39 Although the exact mechanism of this inactivation was unclear, it could be reversed by Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), a SOD-mimicking compound, suggesting that reactive oxidative species were involved. Together with previous observations that oxidative modification of GLT-1 occurs in the spinal cords of ALS patients40 and that GLT-1 quantity and activity are reduced in mice expressing SOD1 mutants,18,41 this evidence provides an interesting link between SOD1 mutation and the excitotoxicity hypothesis for motoneuron death in ALS.42,43
Although these data support this hypothesis, several problems remain. For example, it has not been shown whether this activity is capable of directly damaging biologically relevant molecules. The elevated peroxidase activity has not been reproduced by other investigators. At the least, it appears that some of the mutants do not have an elevated peroxidase activity.44,45
Whichever of the above two hypotheses is true, a common central element in both is the Cu. Therefore a crucial test for both hypotheses is to determine whether Cu is required for the toxicity. Recent understanding of the mechanism by which SOD1 acquires its Cu in cells has provided an experimental paradigm for this test. It was initially discovered in yeast that Cu was acquired by SOD1 from a delivery protein called Lys7.46 Without Lys7, SOD1 cannot incorporate its Cu and remains an apo-enzyme. A mammalian homologue called CCS (copper chaperone for SOD) was also identified.46 The mammalian CCS has the same tissue distribution as SOD1 and also delivers Cu to SOD1.47-49 Thus, an obvious experiment to test whether Cu plays a role in toxicity is to generate CCS knockout mice and then breed the transgenic mice expressing mutant SOD1 onto the CCS knockout background. If this lowers the Cu content in the mutant SOD1 and alleviates the disease, it will provide strong evidence that Cu plays a central role in the toxicity. On the other hand, if this lowers the Cu content in the mutant SOD1 but does not slow the course of the disease, it will suggest that Cu does not play an important role in the mutant SOD1 toxicity.
Aside from the question of the nature of the toxicity, another important question is, what are the cellular targets for this toxicity, or what is the cellular pathway that leads to the eventual cell death? The significance of this relates not only to our understanding of the disease mechanism, but also to the designing of therapeutic strategies. By understanding the pathway, one may design ways to block or slow the progression of the disease at various steps in the degeneration pathway.
To begin to understand the pathway, it is important to know the sequence of pathological events that lead to the eventual motoneuron degeneration. The events that are associated with the end-stage of ALS have been studied for many years in human autopsy specimens and it is clear that there is massive death of motoneurons and their axons. Various inclusion bodies, including neurofilament aggregation and Lewy bodies, are observed in some of the surviving neurons.18,50 Several studies in transgenic mice expressing mutant SOD1 and in some human cases suggest that motoneurons die by the mechanism of apoptosis. This conclusion is largely based on immunoblotting and immunohistochemical staining for molecules known to be in apoptotic pathways. Kostic and colleagues detected an increase in c-Jun expression in the spinal cord.51 Martin found an increase in Bax and Bak, and a decrease in Bcl-2, in mitochondrial fractions from ALS spinal cords.52 Li and colleagues showed an increase in immunostaining for activated caspase 1 and 3 in motoneurons.53 The involvement of apoptosis was further supported by the response of anti-apoptotic treatment in mutant SOD1 transgenic mice (see below). However, it is still uncertain whether the typical apoptotic pathway is involved. Migheli and colleagues found that c-Jun expression is elevated mostly in astrocytes; and they failed to detect activated caspase-3 in motoneurons.54 Most importantly, there have been no reports of typical diagnostic changes, such as chromatin condensation and marginalization, or nuclear fragmentation, in ALS.54 Extensive search by the present author through electron micrographs taken from the spinal cord of mutant SOD1 mice at different stages of disease also failed to detect this type of change in motoneurons.
Perhaps more relevant to the understanding of the disease mechanism and development of treatments are the early events. There are two important questions: what is happening in the pre-symptomatic stage and what is happening in the early clinical stage? These questions were difficult to address without animal models, but with the transgenic mice that express mutant SOD1 and develop ALS, several groups have shown pathological changes beginning at asymptomatic stages, including vacuolation, fragmentation of the Golgi apparatus, astrogliosis, SOD1 aggregation and neurofilament accumulation in proximal axons.17,18,20,50,55-57
To obtain an overall picture of the sequence of pathological events correlating with the clinical progression of the disease, detailed quantitative studies of pathological events were conducted in transgenic mice expressing mutated SOD1 G93A at different disease stages.58,59 Two significant events come to light. First, massive motoneuron death does not occur after the onset of the disease, but occurs at the end-stage. If this is also the case in humans, then there should be a significant time window in which therapy could rescue motoneurons after diagnosis of the disease. Second, there are widespread mitochondrial abnormalities at the pre-symptomatic stage of the disease. At the onset, a massive mitochondrial vacuolation occurs, and the number of these vacuolations gradually declines as the disease progresses; i.e., mitochondrial vacuolation peaks at the onset of the disease. This suggests that mitochondria are an important target for mutant SOD1 toxicity and raises the question as to how mutant SOD1 damage mitochondria.
It may not be surprising that mitochondria should emerge as one of the earliest targets to be damaged by mutant SOD1. Mitochondria are a major, and probably the most predominant, source of oxidative free radicals in cells. If either of the two principal hypotheses regarding the nature of mutant SOD1 toxicity is correct, mitochondria could provide abundant substrates for the mutant SOD1 to generate more toxic free radicals, which in turn damage mitochondria in their vicinity. This possibility is consistent with results from experiments in cultured neuronal cells. When mutant SOD1 was introduced into these cells, mitochondrial damage and dysfunction were observed.60,61 Mitochondrial dysfunction may also play an important role in sporadic ALS.62 The downstream effect of mitochondrial damage could be considerable, including impairment in energy metabolism,63 elevated oxidative stress (see above), increased sensitivity to excitotoxicity,64 deficient axonal transport65 and apoptosis (see above).
One puzzling problem in this scenario is that in two transgenic mice expressing G85R or G86R, massive mitochondrial vacuolation has not been observed;16,18 nor is mitochondrial vacuolation common in human ALS cases. One possible explanation for this is that mitochondrial vacuolation is an early, transient phenomenon, which could be missed if the observation is not made in the time window that is correlated with the onset of the disease. Another possibility is that in some cases mitochondria are functionally defective but do not display vacuolation.
One of the major hypotheses on the mechanism of motoneuron degeneration in ALS is excitotoxicity.42 Although this was initially thought to be associated with sporadic ALS, some recent experiments have shown that mutant SOD1 could impair the function of glial glutamate transporter GLT-1,39 which could increase the extracellular concentration of glutamate. Motoneurons express both NMDA (N-methyl-D-aspartate) and non-NMDA glutamate receptors66-68 and are susceptible to high concentrations of glutamate receptor agonists.69-72 The observed efficacy (although mild) of riluzole,73 a compound thought to antagonize excitotoxicity, on a mutant SOD1 transgenic mouse also suggests that excitotoxicity contributes to motoneuron death. Although no direct link between SOD1 mutation and excitotoxicity has been established, the following scenario is conceivable. As discussed above, mutant SOD1 could directly or indirectly damage mitochondria, which would lead to energy deficiency. This energy deficiency sensitizes neurons to glutamate toxicity.74 At the same time, a potential decrease in glial glutamate transport activity could cause the extracellular glutamate level to rise, which would chronically act on the sensitized motoneurons and eventually cause motoneuron death.
The pathways discussed above suggest that the critical damage is initiated in motoneurons. A different idea is that early damage might be initiated in glial cells.17,18 Evidence for this mechanism is that focal astrogliosis is detected at pre-symptomatic stages in mice expressing mutant SOD1 G37R or G85R,17,18 and that mutant SOD1 is capable of inducing damage to glial glutamate transporter GLT-1.39-41 The latter could lead to a decrease in glial cell uptake of glutamate and consequently an increased extracellular glutamate concentration, and eventually cause excitotoxicity that damages motoneurons.42 However, the abnormalities of GLT-1 have only been associated with the late stage of the disease,18,40,41,75 consistent with the possibility that they are a consequence of motoneuron degeneration rather than a cause. Further, the astrogliosis observed at early stages is rather minor. Careful measurement of levels of astrogliosis at different disease stages indicate that massive astrogliosis develops gradually in parallel with ongoing motoneuron degeneration in the spinal cord, and mutant SOD1 is expressed at much higher levels in motoneurons than in astrocytes,59 suggesting that the initial damage occurs in motoneurons and that astrogliosis is a reaction to motoneuron degeneration. Consistent with this view, a recent experiment in which a disease-causing mutant SOD1 was specifically expressed in astrocytes showed no motoneuron degeneration.76
The paradox between selective cell vulnerability and widespread expression of mutated genes is a general phenomenon in several major neurodegenerative diseases and ALS is no exception. It presents one of most mysterious aspects of the disease mechanism. SOD1 is a ubiquitously expressed protein, yet motoneurons are most susceptible. How do we account for this selective vulnerability?
There is no definitive answer to this question at present. Nevertheless, several possibilities have been proposed. One is that motoneurons express higher levels of SOD1 than other neuronal cells.36 However, there are other neurons besides motoneurons that express high levels of SOD1. Some of these neurons, including pyramidal neurons in the hippocampus and Purkinje neurons in the cerebellum, are not affected by SOD1 mutations.49,77 Careful comparison of the levels of SOD1 in different neuronal groups needs to be conducted in order to resolve this question.
Another possibility is that the susceptibility is due to the large size of the neurons. Motoneurons are the largest neurons, with the longest axons, in mammals. Maintaining the large size may require high metabolic activity, which could result in high levels of oxidative free radicals. This idea was supported by a recent experiment in which sciatic nerve axons in mice expressing G93A mutation were transected and prevented from regeneration.78 This operation induced motoneuron atrophy, leading to an increase in the number of small neurons and axons.78-81 When measured at the end-stage of the disease, the increased number of small axons in the ventral root were found to have been spared from degeneration, while large axons degenerated to the same degree as non-transected controls, suggesting that large motoneuron size contributes to the susceptibility.78
One significant contributing factor for motoneuron vulnerability may be associated with its heavy dependence on intracellular transport of cellular organelles. As they are the largest type of cell, with elaborate dendritic trees and the longest axons, motoneurons probably depend on intracellular transport more than do any other cells. The toxicity of the mutant SOD1 might affect the intracellular transport indirectly: for example, by compromising mitochondria function or by damaging the components (such as neurofilaments) that are being transported.23,60 Consistent with this possibility, several studies have shown that axonal transport in transgenic mice expressing mutant SOD1 was slower:82-84 in particular, two studies showed that this abnormality could be detected at early stages of the disease.83,84 Despite these data, it is worth keeping in mind that the role of neurofilaments is probably more complicated than merely affecting the burden of axonal transport (see below).
Regulation of calcium levels plays an important role in excitotoxicity and cell death in many types of neurons.85-87 It has been proposed that selective motoneuron vulnerability may be due to the low capacity of motoneurons to control Ca++ homeostasis. Several studies have shown that the vulnerable neuronal population in ventral horn contains abundant neurofilaments but low levels of calbidin and parvalbumin.88,89 Calbindin and parvalbumin are Ca++ binding proteins capable of buffering Ca++ levels.90 In mice expressing SOD1 mutants, the neurons rich in calbindin and parvalbumin in ocular motor nuclei and ventral horn are spared from degeneration, but the neurons which are poor in calbindin and parvalbumin die at the terminal disease stage.88,89 Appel and colleagues recently suggested that there might be differences in the regulation of Ca++ levels between the spinal motoneurons and the ocular motoneurons, and these differences might explain the different vulnerability of these two neuronal populations in ALS.89
Based on the excitotoxicity hypothesis, it has been suggested that motoneurons might possess high levels, or special classes, of glutamate receptors that render them particularly vulnerable. The distribution of both NMDA and non-NMDA receptors in the CNS has been investigated extensively.66-68,91-94 The general consensus from these studies is that these receptors are broadly distributed and confer no selectivity in motoneurons. Only one of the AMPA (a-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid) receptor subunits, GluR1, is lower in motoneurons than are other types of neurons in the spinal cord.
Several groups examined whether another AMPA receptor subunit GluR2 expression was low or absent in motoneurons, since receptors with the GluR2 subunit are not permeable to Ca++.95 Two groups found that GluR2 was selectively decreased or absent in ventral horn motoneurons, suggesting that this may confer selective motoneuron vulnerability.96,97 This observation, however, has not been substantiated by other workers, who have found no selective decrease in GluR2 levels in motoneurons;98-100 nor any preferential survival of motoneurons expressing GluR2 in mice expressing a mutant SOD1 G86R.100 These results cast doubts on the possibility that a lack of GluR2 in motoneurons confers selective vulnerability, and indicate that other mechanisms, such as hypersensitivity to glutamate induced by energy deficiency, should be considered.
Based on the current hypotheses and understanding of the disease mechanism, several therapeutic treatments have been tried on the transgenic mouse models. Some of the trials showed modest effects (Table 1). Riluzole has been used to treat human ALS and it did prolong survival somewhat.101,102 Similar results were obtained in mice expressing SOD1 mutant G93A (Table 1). Its mechanism of action is thought to be inhibition of glutamate release, and therefore, blocking the excitotoxicity.103 However, this is by no means certain, and opinions remain divided.104
| Agent | Expected action | Mice treated | Disease progression | Survival | Reference no. |
| Riluzole | Lowering glutamate | G93A1Gur | Slowed | Ext.c (13 days, 10%) | 73,107 |
|
Vitamin E/selenium |
Anti-oxidant | G93A1Gur | DOa (14 days, 15%) | No effect | 107 |
| Carboxyfullerene | Anti-oxidant | G93A1Gur | NAb | Ext.c (8 days, 7%) | e |
| Putrescine-catalase | Anti-oxidant | G93A1Gur | DOa (21 days, 26%) | No effect | f |
| G93A1Gurdl | DOa (30 days, 15%) | No effect | f | ||
| Lysine acetylsalicylate | Anti-oxidant, anti-inflammation | G93A1Gur | Slowed | No effect | g |
| d-penicillamine | Cu chelater | G93A1Gur | DOa (10 days, 8%) | Ext.c (11 days, 8%) | h |
| Trientine/ascorbate | Cu chelater, anti-oxidant | G93A1Gurdl | DOa (25 days, 10%) | No effect | i |
| 2% Creatine | Energy balance, MTP inhibition | G93A1Gur | Slowed | Ext.c (26 days, 18%) | 106 |
| Genistein | Estrogenic, anti-oxidative stress | G93A1Gur/G93A1Gurdl | Slowed in males | Ext.c in males | j |
| NOS inhibitors | NO reduction | G93A1Gur/G93A1Gurdl | NAb | No effectd | 28,29 |
| GDNF | Promoting motoneuron survival | G93A1Gur | Slowed | NAb | 114 |
| bFGF | Promoting motoneuron survival | G93A1Gur | NAb | No effect | 117 |
| ZVAD-fmk | Inhibiting caspases | G93A1Gur | DOa (20 days, 20%) | Ext.c (27 days, 22%) | 53 |
As discussed earlier, the toxicity of the mutated SOD1 may be associated with its abnormal catalytic properties in cellular redox reactions. Oxidative free radicals are therefore thought to be a main contributor of damage to cellular organelles and motoneuron degeneration.105 Several trials using anti-oxidants have been attempted, and modest effects on disease onset or survival have been observed (Table 1). These results are encouraging, as they suggest that anti-oxidative treatment is a viable concept, although important issues such as the mechanism of action and optimal method of administration need to be further investigated, and new, more effective compounds remain to be found.
At the center of the abnormal catalytic property of the mutated SOD1, Cu is considered essential. In two studies, the transgenic mice were treated with Cu chelators. Treatment using d-penicillamine delayed the onset and prolonged survival to a small extent (Table 1). Treatment with trientine combined with ascorbate slowed the clinical progression but did not prolong survival (Table 1). As a potential target of the abnormal catalytic property of the mutated SOD1, mitochondria may become damaged and dysfunctional at early disease stages, triggering the beginning of massive motoneuron degeneration.58 Because creatine can enhance the energy storage capacity and inhibit the opening of mitochondrial transition pore (MTP) (which is thought to play an important role in mitochondrial degeneration), transgenic mice expressing a mutant SOD1 were treated with a creatine-supplemented diet.106 Significant improvement in clinical symptom and extension of survival were found (Table 1).
Several trials of other therapeutic interventions showed minimal or no effect. These include gabapentin, an anticonvulsant compound,107 diet restriction108 and several nitric oxide synthetase (NOS) inhibitors.28,29 In one experiment using a specific neuronal NOS (nNOS) inhibitor AR-R 17477, a modest extension of survival was observed.28 However, treatment using either non-selective or other nNOS inhibitors in other experiments did not show significant effects.28,29 Furthermore, crossing mice expressing mutant SOD1 with nNOS knockout mice did not affect the disease progression.28 Thus, whether NOS and its product NO play a role in the pathogenesis of disease mediated by SOD1 mutation remains uncertain.
Neurotrophic factors as potential therapeutic agents have been tested widely in other motoneuron disease models and in some cases displayed good therapeutic potential.109-113 Two neurotrophic factors have been tested in mice expressing mutated SOD1. GDNF, a potent neurotrophic factor that prevents axotomy-induced motoneuron death and slows motoneuron degeneration in a mouse line with progressive motoneuropathy (pmn), was used to treat mice expressing mutant SOD1 G93A. It was delivered by transplanting myoblasts infected with modified retrovirus carrying GDNF gene into gastrocnemius muscle. This treatment appeared to have slowed motoneuron degeneration and the decline of muscle strength.114 In another experiment, mice expressing G93A mutant were treated with basic fibroblast growth factor (bFGF). Despite previously reported efficacy in reducing axotomy-induced motoneuron death and slowing motoneuron degeneration in the wobbler mice,115,116 this treatment did not prolong the survival of mice expressing G93A mutation.117
One test for the therapeutic potential of different strategies has been to cross mice expressing mutant SOD1 with transgenic mice that overexpress, or lack, molecules related to certain neuronal functions. Using this approach, two groups tested anti-apoptotic strategies in the treatment of ALS. In one experiment, SOD1 mutant mice were crossed with mice overexpressing Bcl-2, an inhibitor of apoptosis.51 In the other experiment, SOD1 mutant mice were crossed with mice expressing a dominant negative form of ICE, which is one of the caspases involved in apoptosis.118 In both cases, modest extension of survival was observed in doubly transgenic mice. The most dramatic effect of the anti-apoptotic treatment has been observed using intraventricular infusion of N-benzyloxycarbonyl-Val-Asp-fluoromethylketone (zVAD-fmk), a broad caspase inhibitor. This treatment slowed the disease progression and prolonged survival by 22%.53 These results suggest that anti-apoptotic treatment could be effective in treating human ALS.
Because it has been suspected that the pathogenic pathway of motoneuron death plays a part in abnormal regulation in neurofilament economy (see above and also Bruijn and Cleveland119), several groups crossed mutant SOD1 mice with mice either lacking or overexpressing one of the neurofilament subunits. Based on the hypothesis that the heavy neurofilament burden contributes to the vulnerability of motoneurons to SOD1 mutation, the initial expectation was that the progression of the disease would be alleviated in neurofilament knockout mice but exacerbated in mice overexpressing neurofilaments. Indeed, when mice expressing the mutant SOD1 G85R were bred into the neurofilament subunit L (NF-L) knockout background, their survival was prolonged by about 11%.120 Surprisingly, however, when other mice expressing a different SOD1 mutation, G37R, were bred with transgenic mice overexpressing human neurofilament subunit H (NF-H), the doubly transgenic mice survived much longer (65%) than the G37R singly transgenic mice.121 This apparently contradicted the neurofilament burden hypothesis. However, a close examination of motoneurons revealed that overexpression of NF-H caused neurofilaments to accumulate in cell bodies but to decrease in axons, which suggests that overexpression of NF-H causes sequestration of neurofilaments in cell bodies, resulting in fewer neurofilaments being transported into axons. Therefore, it is possible that the burden of neurofilament transport in axons is reduced, consequently reducing the vulnerability of motoneurons.121
This result was confirmed recently by an experiment in which mice expressing mutant SOD1 G93A were crossed with mice overexpressing the mouse NF-H.30 However, in that same experiment, G93A was also crossed with mice overexpressing NF-L. In that transgenic line, the axonal neurofilaments were increased,122,123 yet the survival in doubly transgenic animals was prolonged to the same extent as mice overexpressing NF-H.30 These results indicate that the role of neurofilaments in motoneuron degeneration is not as simple as initially thought. Because alteration of the neurofilament economy has so far had the greatest effects on the survival of mice expressing mutant SOD1, further investigation of the role of neurofilaments in pathogenesis may yield valuable insights into the question of motoneuron vulnerability.
The discovery that SOD1 mutations cause ALS has moved the field of ALS research from a descriptive phase to an experimental one. With various models established by expressing the mutant SOD1, it is now well established that SOD1 mutations cause ALS by a gain of a toxic property. There are two hypotheses about the nature of this toxic property but neither has been proven. Both hypotheses involve Cu. Therefore, experiments that manipulate the Cu level in mutant SOD1 (e.g. by knocking out CCS) should help to establish whether these hypotheses are valid.
Several experiments have pointed out that the oxidative stress levels are increased in nervous tissue in mice expressing mutant SOD1 and in ALS patients, but whether these increases represent downstream consequences of neuronal degeneration, or reflect the upstream events intimately related to the property of mutated enzyme, remains to be resolved.
Currently, mitochondrial damage is perhaps the best-established early event associated with the preclinical, and the onset of the clinical, stages of the disease. The mechanism that leads to this damage is not known and needs to be further investigated. The most important event downstream of mitochondrial damage could possibly be energy deficiency, which could induce hypersensitivity to excitotoxicity and ultimately neuronal degeneration. Other
possible early events include fragmentation of the Golgi apparatus and damage to the astroglial cells.
The answer to the question regarding the selective vulnerability is uncertain, although characteristics such as large size, richness in neurofilaments and relative deficiency in Ca++ binding proteins have been suggested as contributing factors.
Several treatment strategies based on our current understanding of the disease mechanism have been tested, but more effective ones are yet to be found. With many studies currently directed to SOD1 mutant models, an important question is how representative this model is of ALS in general. The answer will not be known until the mechanism of mutant SOD1 toxicity and other causes are understood, although the similarity in clinical symptoms, pathology and response to treatment with riluzole in all ALS cases leads us to hope that this mechanism will be applicable to ALS in general.
I thank Drs Leslie Shinobu and Cynthia Higgins for their discussion and critical reading of the manuscript. This work is supported by grants from the National Institute of Neurological Disorders and Stroke and ALS Association.
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Table 1
Therapeutic approaches tested in mutant SOD1 transgenic mice
Agent Expected action Mice treated Disease progression Survival Reference no.
Riluzole Lowering glutamate G93A1Gur Slowed Ext.c (13 days, 10%) 73,107
Vitamin E/selenium Anti-oxidant G93A1Gur DOa (14 days, 15%) No effect 107
Carboxyfullerene Anti-oxidant G93A1Gur NAb Ext.c (8 days, 7%) e
Putrescine-catalase Anti-oxidant G93A1Gur DOa (21 days, 26%) No effect f
G93A1Gurdl DOa (30 days, 15%) No effect f
Lysine acetylsalicylate Anti-oxidant, anti-inflammation G93A1Gur Slowed No effect g
d
-penicillamine Cu chelater G93A1Gur DOa (10 days, 8%) Ext.c (11 days, 8%) hTrientine/ascorbate Cu chelater, anti-oxidant G93A1Gurdl DOa (25 days, 10%) No effect i
2% Creatine Energy balance, MTP inhibition G93A1Gur Slowed Ext.c (26 days, 18%) 106
Genistein Estrogenic, anti-oxidative stress G93A1Gur/G93A1Gurdl Slowed in males Ext.c in males j
NOS inhibitors NO reduction G93A1Gur/G93A1Gurdl NAb No effectd 28,29
GDNF Promoting motoneuron survival G93A1Gur Slowed NAb 114
bFGF Promoting motoneuron survival G93A1Gur NAb No effect 117
ZVAD-fmk Inhibiting caspases G93A1Gur DOa (20 days, 20%) Ext.c (27 days, 22%) 53
aDO, delayed onset. bNA, data not available. cExt., extended. dIn one experiment, treatment with a nNOS inhibitor extended survival. But this result is not replicated in other experiments (see text). eDugan et al, Proc Natl Acad Sci USA 1997; 94: 9434-9. fReinholz et al, Exp Neurol 1999; 159: 204-16. gBarneoud et al, Exp Neurol 1999; 155: 243-51. hHottinger et al, Eur J Neurosci 1997; 9: 1548-51. iNagano et al, Neurosci Lett 1999; 265: 159-62. jTrieu et al, Biophys Res Comm 1999; 258: 685-8.
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