Reduction of lower motor neuron degeneration in wobbler mice by N-acetylcysteine

Jeffrey T. Henderson, Mohammed Javaheri, Susan Kopko and John C. Roder

Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
Program in Molecular Biology and Cancer, 600 University Ave., Toronto, Ontario M5G-1X5

Discussion

Stimuli which elevate intracellular levels of reactive oxygen species have been shown to enhance the rate of programmed cell death in a variety of neural cells in vitro (Sheen and Macklis, 1994; Kroemer et al., 1995; Greenlund et al., 1995; Verity et al., 1995). In addition, reactive oxygen species have been suggested to play an important role in several human neurodegenerative diseases; including amyotrophic lateral sclerosis ( Wakai et al., 1994; Abe et al., 1995; Beal, 1995, Busciglio and Yanker, 1995; Eisen, 1995). Consistent with this, agents which inhibit ROS have been shown to be neuroprotective both in vitro and in vivo; under conditions of acute neural injury (Pan and Perez-Pollo, 1993; Sampath et al., 1994, Sato et al., 1995).

NAC has previously been shown in several of these paradigms to be particularly effective in enhancing cell survival and reducing free radical damage in neural cells (Mayer and Noble, 1994; Rothstein et al., 1994; Ferrari et al., 1995; Knuckey et al., 1995; Talley et al., 1995). In order to explore the potential benefits of NAC under conditions of chronic neural injury in vivo, we have examined the murine mutant wobbler, a model of lower motor neuron degeneration similar to in some respects to human amyotrophic lateral sclerosis (Bird et al., 1971; Andrews, 1975; Bulac et al., 1983). Our present results demonstrate that daily oral administration of NAC significantly reduces the degeneration of lower motor neurons in wobbler mice. For motor axons of the facial nerve, application of NAC resulted in a generalized increase in axon number and caliber. Within the cervical spinal cord, NAC reduced losses of choline acetyltransferase positive neurons. Examination of both proximal (triceps) and distal (flexor carpi ulnaris) forelimb muscles also revealed significant increases in both muscle mass and mean fiber area. While these increases were nominal, they were sufficient to promote a substantial increase in the functional activity of wobbler forelimbs, littermates which received D-glucose or L-alanine supplementation.

The mechanism by which lower motor neurons degenerate in wobbler mice is presently unknown. The ability of NAC to reduce the loss of these neurons, together with the ability of this treatment to elevate levels of glutathione peroxidase activity in the cervical spinal cord, suggests a means by which NAC may act to reduce local ROS levels. Previous work also demonstrates that NAC directly supports glutathione synthesis in neural cells; thus reducing intracellular ROS levels (Mayer and Noble, 1994; Rattan et al., 1994; Rothstein et al., 1994). However, it is important to note that Yan et al. have demonstrated both the D and L isomers of NAC to be capable of promoting the survival of PC12 cells in culture. In addition, NAC has been shown induce genes such as NF-kappa b (Brennan and O'Neil, 1995, Staal et al., 1995). These data suggest that NAC may exert survival promoting effects in a manner independent of its actions as a direct antioxidant.

In humans, NAC has recently been used in a limited trial over a 12 month period on ALS patients (bulbar and limb isoforms, 50 mg/kg) (de Jong et al., 1987; Louwerse et al., 1995). While no net clinical improvement was observed in patients treated with NAC, segregation of ALS cases into bulbar and limb onset subgroups is informative. In ALS cases of bulbar onset, NAC clearly did not improve survival. However in patients with the limb onset form, NAC does appear to improve survival (NAC 74% 28/38, vehicle 51% 22/43). However, these results were not quite significant (p<0.06) due to the relatively small trial size and heterogeneous state of presenting patients and disease progression. It is interesting to speculate that the differential availability of NAC to these two neural populations may underlie the differences in their response. For comparison, one current therapy which shows promise for ALS patients are studies with the glutamate antagonist riluzole (100 mg/day). Given over a 12 month period, there are presently some indications of clinical improvements in ALS patients; suggesting that aberrant glutamate metabolism (and perhaps ROS) may play a role in this disorder (Bensimon et al., 1994).

The neuropathy present in wobbler mice has previously been treated with a combination of CNTF (1 mg/kg) and BDNF (5 mg/kg) by subcutaneous injection three times/week over a four weeks period (postnatal weeks 4-8) (Mistumoto et al., 1994). Treated animals exhibited substantial improvements in the number of ChAT positive neurons within the cervical spinal cord, together with improvements in muscle mass, fiber size and forelimb function. While the measures used in these studies are differ somewhat from our own, these results demonstrate that wr mice treated with neurotrophic factors retain greater function over s similar time-frame than those treated with NAC. These differences may reflect differences in the efficiency of the treatment agent to efficiently act on the affected population, as discussed below.

The comparisons given above demonstrate that while application of NAC does lead to significant improvements in motor function, these improvements have not been as great as those observed using the best current therapies. Thus NAC cannot completely compensate for the pathogenic aspects of these neuropathies. There are several possibilities for this. To be accessible to motor neurons, NAC must be taken up via contact with somatic tissue, through the motor (and sensory) innervation. Oral administration of NAC does result in enhanced glutathione peroxidase activity in the cervical spinal cord, suggesting that there is significant accumulation from the periphery. However, motor neurons which have already begun to degenerate at their terminals or show impaired axonal transport may be impaired in their ability to accumulate NAC and its metabolites, thus limiting its potential therapeutic effects. Because of this, this form of antioxidant treatment is likely to be more beneficial in the early stages of motor degeneration, rather than latter stages in which axon transport has already been substantially impaired. Clearly this issue can be addressed by altering the administration route. In addition, it is important to realize that NAC may be affecting only a portion of the components involved in the control of programmed cell death. The coordinated support of some or all of these components may be necessary to achieve optimal results with respect to cell viability. Despite this, the use of agents such as NAC would seem to be a practical approach to enhancing cell survival for several reasons. Unlike current neurotrophic therapies which involve the subcutaneous or intrathecal implantation of proteins which are quite labile in vivo, NAC can be given orally over dispersed intervals. NAC is also an inexpensive drug, possessing few side effects and with extensive clinical experience; in contrast to current neurotrophic therapies (Berkow and Fletcher, 1992; Brennan and O'Neil, 1995). Given the similarities in the neuropathy observed in wobbler to that of several human neurodegenerative disorders, application of NAC may prove useful in treating other mammalian neurodegenerative diseases.

Abstract

Introduction

Materials and Methods

Results

Discussion

References