Primary lateral sclerosis

Effects of glial cell line-derived neurotrophic factor (GDNF) were studied in transgenic (Tg) mice model for amyotrophic lateral sclerosis. GDNF protein or vehicle was injected three times a week from 35 weeks of age into the right gastrocnemius muscle of Tg mice carrying mutant human Cu/Zn superoxide dismutase gene, and histological analysis was performed at 46 weeks. Clinical data showed a tendency of improvement, but was not significantly different between the two animal groups. In contrast, total number of and phospho-Akt (p-Akt) positive large motor neurons in the treated side was significantly more preserved in GDNF-treated group than in vehicle group (p

Key words: GDNF; amyotrophic lateral sclerosis; therapy

INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective loss of motor neurons, causing muscular atrophy, complete paralysis and death. A variety of mutations in Cu/Zn superoxide dismutase (SODI) gene are present in about 20% of cases with familial ALS (FALS)'. The toxicity of mutant SOD has been proposed to involve one or more of the following; an increase in peroxynitrite formation2-4, an increase in peroxidase activity5-7, and aggregation of the enzyme8. A slowly degenerative death of selective neuronal population has been reported to occur under a disequilibrium between death and survival signals such as caspase-3 and Akt respectively9. Although several lines of transgenic (Tg) mice have been established that express a mutant SOD1 gene and provide valuable models for human ALS', the primary pathogenic mechanisms of ALS remain to be elucidated.

In vivo and in vitro experiments have suggested that motor neuron diseases might benefit from neurotrophic factor administration11. For example, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), and glial cell linederived neurotrophic factor (GDNF) protected motor neurons from acute death induced by peripheral nerve axotomy in neonate rodents12,13. GDNF is the most potent protector for motor neuron, and is present at a high level in embryonic limb and muscle at the time of innervation 14. GDNF prevents motor neuron degeneration in a mouse line called progressive motoneuropathy pmn15. However, the effects of GDNF on motor neuron degeneration in a genetic mouse model of FALS have not been reported.

Since the neuropathology of motor neurons in these mice develops over several months, consistant delivery of GDNF may be necessary to show an evident effectiveness. In this study, we treated Tg mice carrying an ALS-linked mutant SOD1 gene with intramuscular injection of GDNF protein, and observed a histological improvement.

MATERIALS AND METHODS

Tg mice expressing a mutant human SOM with a Gly 93 Ala (G93A-Tg) substitution were used in this study10,16 and the G93A-Tg progeny was identified by polymerase chain reaction (PCR) amplification according to our previous reports16,17. GDNF (3 g kg^sup -1^) or vehicle solution was injected into the right gastrocnemius muscle of the mice three times a week from 35 (pre-symptomatic, n = 5) weeks of age. At around 39-40 weeks of age, the Tg mice develop progressive paralysis beginning with a posterior limb leading to death in additional eight weeks. Therefore, the GDNF therapy started at 35 weeks for applying it to human in the future. To evaluate clinical effects of GDNF, body weight, clinical grade (CG, normal, 1; slightly symptomatic, 2; symptomatic, 3; heavy symptomatic, 4; death), rolling number of circular cage (CCR), and grasping on a rolling column (RC) were measured at 35, 40, 42, and 46 weeks. The CCR was measured as a voluntary movement of mice in an unilateral direction (forward or back) for 30 min in the narrow circular cage, which represents a locomotor activity of the mice. For RC, the rate of rolling of the transverse column (rota rod, diameter 3.5 cm, Acceler Rota-Rod 7650, UGO BASILE, Varese, Italy) was accelerated from 0 to 10 min18. The total rolling number before they fall down was recorded as an indicator of grasping power of the mice. All experimental protocols and procedures were approved by the Animal Committee of the Okayama University Graduate School of Medicine and Dentistry, Japan.

For measurement of free-GDNF protein content, serum samples were examined for GDNF enzymelinked immunoassay with a chicken polyclonal antibody in the 96-well immunoassay plate (Nunc MaxiSorp Nalge Nunc Int., Rochester, NY, USA), which was precoated with the anti-GDNF monoclonal antibody, using the kit (GDNF E^sub max^ Immunoassay System, Promega, Madison, WI, USA). After overnight incubation at 4 deg C, reaction of a second antibody-HRP conjugate (kit component) for chicken IgY was performed, followed by color development with TMB solution (kit component). After the stop reaction with 1 mol l^sup -1^ phosphoric acid, OD^sub 450^ was measured by a 96 well plate reader, and GDNF protein content of each sample was calculated. Using this system, immunoreactive GDNF in the sample can be quantitated in the range of 16 to 1,000 pg ml^sup -1^(19).

For histological study, mice were decapitated with a deep anesthesia by ether at the end of the experiment (46 weeks), and the lumbar spinal cord was removed and quickly frozen in a powdered dry ice. Transverse sections of 10 (mu)m thickness were cut on a cryostat at -20 deg C, and were stained with hematoxylin and eosin (HE). For immunohistochemistry, the sections were treated with a rabbit polyclonal antibody specific for phospho-Akt (p-Akt, 1:50, #9277, Cell Signaling Technology, MA, USA), phospho-ERK (p-ERK, 1 :100, #9101, Cell Signaling Technology), mouse anti-cleaved caspase-3 polyclonal antibody (1:50, #9661, Cell Signaling Technology), or rabbit anti-cleaved caspase-9 polyclonal antibody (11 :50, #9501, Cell Signaling Technology) in 10% normal rabbit serum and 0.3% Triton X-100 at 4 deg C for 16 h, and developed by the standard avidin-biotin complex method as our previous report17. The number of large ventral horn neurons were measured on the spinal cord sections (10 (mu)m thick). The sections were examined by light microscope. The diameter of each large motor neuron was determined as 20 (mu)m or more. For quantification of immunohistochemistry, the average optical densities were measured using a computer-based system (NIH Image Ver. 1.62, NIH, USA). The background density was subtracted from the positive signal. The species specificity of the secondary antibody was verified by omitting the primary antibodies.

The number of large ventral horn neurons was measured on the spinal cord sections. At least five transverse sections from each lumbar cord were examined by light microscope. The number in anterior horn areas in each section was combined and averaged in order to provide the number of neurons in unilateral anterior horns per section. Values are expressed as mean +/- SE. Comparisons of vehicle and drug-treated groups were made with a nonparametrical test such as the Wilcoxon rank-sum (Wilcoxon's U) test for a part of clinical data (CG, body weight, CCR, and RC) and biological data.

DISCUSSION

In the present study, the number of motor neuron and immunoreactivity of p-Akt were significantly preserved in the GDNF-treated group than in the vehicle group (Figures 3-5). The intramuscular injection of GDNF to Tg mice resulted in the long-term expression of GDNF protein with elevated serum level (Figure 2). The GDNF treatment prevented motor neuron loss with preserving p-Akt staining (Figure 5A) and without affecting p-ERK and caspases-3 and -9 (Figures 58, 6). Because the contralateral side (Lt) did not show such a protective effect (Figures 3-5), the effect of GDNF was not simply due to serum elevation of GDNF (Figure 2), but to the direct effect on innervated motor neuron of the injected side. The present results are consistent with such previous findings as early and progressive loss of survival signals phosphatidylinositol-3 kinase (PI3K) and Akt, and as late activation of caspase-3 in Tg mice9.

Recent studies showed that BDNF, NT-3, CNTF, and cardiotrophin-1 supported survival of embryonic motor neurons in cultured11,15,20. CNTF, NT-3, and BDNF, as well as GDNF, prevented motor neuron death in mouse models of motor neuron disease, such as wobbler and pmn mice15. GDNF was protective against motor neuron injury after axotomy of peripheral nerves in neonatal rats with adenoviral gene transfer. Grafting of GDNF-secreting myoblasts into two hindlimb muscles of a mouse model for ALS protected the innervating motor neurons from degeneration and delayed the onset of the disease symptoms21. This is the first report of a therapeutic effect of the intramuscular injection of GDNF in Tg model mice for ALS.

Both P13K and its key downstream molecule Akt play a critical role in neuronal survival, and are supported by neurotrophic factors such as nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1)22. Akt prevents apoptosis of many cells under pathological conditions23,24. The signaling through P13K/Akt may be the most important pathway for neuronal survival. Our study indicates that the GDNF treatment prevented motor neuron loss through preserving survival signaling pathway Akt, but not affecting p-ERK and active caspases-3 and -9 immunostainings. Although clinical data in CG, CCR, and RC showed a tendency of improvement, the difference was not significant between vehicle- and GDNF-treated groups (Figure 1). Because 35 weeks of age, when the GDNF therapy started, is just before the disease development, clinical improvement should become significant when the therapy started from an earlier stage. Alternatively, the present results indicate that supporting survival Akt signal of motor neurons by GDNF might not be sufficient to successfully treat clinical symptoms of motor neuron disease. In fact, the maintenance of motor neurons also depends on many other different neurotrophic factors25.

Because the present GDNF therapy started just before or at around the disease beginning for aiming to apply it to human ALS patients, clinical effectiveness was not strong and significant. However, GDNF therapy significantly prevented motor neuron loss in the Tg mice, still suggesting a possibility of GDNF therapy to ameliorate the progression of human ALS. Treatment with suitable combinations of neurotrophic factors might be much more beneficial than treatment with one factor alone.

ACKNOWLEDGEMENTS

This work was partly supported by Grant-in-Aid for Scientific Research (B) 12470141, (C) 13670649 and (Hoga) 12877211 from the Ministry of Education, Science, Culture and Sports of Japan, Kobayashi Magobe Memorial Medical Foundation, and by grants (Tashiro K, Itoyama Y, and Tsuji S), and Comprehensive Research on Aging and Health (H 11 Choju-010, No.207, Koizumi A) from the Ministry of Health and Welfare of Japan.

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Y. Manabe, I. Nagano, M.S.A. Gazi, T. Murakami, M. Shiote, M. Shoji, H. Kitagawa* and K. Abe

Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, Okayama

*Tokushima New Drug Research Institute, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan

Correspondence and reprint requests to: Yasuhiro Manabe, Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. [manabe@cc.okayama-u.ac.jpl Accepted for publication October 2002.

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