The etiology of amyotrophic lateral sclerosis (ALS) remains unknown although an existence of neurotoxic substances in cerebrospinal fluid (CSF) from ALS patients have been postulated. In order to investigate a possible effect of CSF from ALS patients on cellular signaling in spinal neurons, we compared Fos-like immunoreactivity (Fos-LI) in organotypic cultures of rat lumbar spinal cord after addition of CSF from ALS patients or another neurologic disease. Fos-LI was normally present predominantly in dorsal horn neurons, whereas only a few ventral horn neurons were positive for Fos-LI. The number of Fos-LI positive neurons significantly increased in dorsal horn with addition of CSF from ALS patients as well as glutamate at 100 pM. However, the increase was not observed with addition of CSF from other neurologic diseases. The increase in Fos-LI positive neurons in dorsal horn was reversed by a further supplement of MK801, an N-methyl-D-aspartate (NMDA) receptor antagonist, but not of CNQX, an alpha-amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA)/kainate antagonist. These results indicate that there may be substances in CSF from ALS patients that stimulate Fos expression in certain populations of spinal neurons via the NMDA receptors. [Neurol Res 1999; 21: 309-312]
Keywords: c-fos expression; cerebrospinal fluid; amyotrophic lateral sclerosis; spinal cord; slice culture
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease which affects the motor neurons of the spinal cord and brainstem as well as the pyramidal cells of the motor cortex. A clinical feature of ALS is its rapid course, compared with other neurodegenerative diseases. The etiology of the disease remains unknown. Experimental and clinical findings suggest that glutamate, an excitatory neurotransmitter and potent toxin to central nervous system, contributes to the pathogenesis of ALS1,2. Some investigators have reported generalized abnormalities in glutamate metabolism in ALS, as indicated by reduced activity of leukocyte glutamate dehydrogenase3 and increased glutamate concentrations in plasma and in cerebrospinal fluid (CSF)2. These observations in association with a finding of reduced glutamate levels in neural tissue, suggest an abnormal distribution of glutamate in intracellular and extracellular pools . Recent studies revealed that there may be a specific defect in splicing excitatory amino acid transporter type 2 (EAAT^sub 2^) mRNA in sporadic ALS tissue5, and that alternative splicing forms of GLT-1 mRNAs was present in ALS spinal cords6.
Glutamate is known to be neurotoxic through both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. The latter group includes alpha-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. Stimulation of any of these receptors leads to increased intra-cellular calcium accumulation via voltage-sensitive calcium channels and activation of intra-cellular protein kinases by calcium and cyclic AMP, resulting in effects on the Ca/cAMP-responsive binding (CREB) protein which has a rapid and profound influence on gene expression leading to induction of immediate early genes (IEG) such as c-fos7.
To evaluate and characterize a possible neurotoxicity of CSF from patients with ALS, we studied expression of Fos, a transcription product of c-fos, in organotypic cultures of rat lumbar spinal cord neurons, comparing the results with those using glutamate and CSF from agematched patients with spinocerebellar ataxia (SCA) or peripheral nerve disorders. In addition, we examined a possible blocking effect of subtype-specific glutamate receptor antagonists against the CSF-induced Fos expression.
MATERIALS AND METHODS
Eighteen patients admitted to our Department of Neurology between December 1995 and August 1997 gave their consent for a portion of the CSF obtained from diagnostic lumbar punctures to be used for the present research. Lumbar puncture was performed during each patient's first admission, and CSF was stored at -80oC. All CSF samples had normal conventional biochemical and cytologic profiles.
Six patients (three males and three females, age range 57-73 years, mean 64.0, SD 5.8 years) had the sporadic form of typical ALS by clinical and electromyographic criteria. Controls with and without other neurodegenerative disease were matched for age. The six neurodegenerative controls (two males and four females, age range 53-68 years, mean 62.0, SD 6.3 years) had SCA (four with olivopontocerebellar atrophy, one with Joseph's disease, and one with dentatorubral-pallidoluysian atrophy). The six controls with no history of degenerative neurologic disorders (three males and three females, age range 50-66 years, mean 60.2, SD 6.1 years) had peripheral nerve disorders (two with chronic inflammatory demyelinating polyneuropathy, two with peripheral neuropathy, one with radiculopathy, and one with hereditary motor and sensory neuropathy).
Organotypic cultures were prepared from the lumbar spinal cords of 11-day-old Sprague-Dawley rats weighing 18 to 23 g (Charles River Co., Yokohama, Japan). Lumbar spinal cords were removed and sliced into 5-mm thick transverse sections, and one slice was placed on a Millipore CM semipermeable membrane insert 30 mm in diameter (Falcon, Franklin Lakes, NJ, USA). The inserts were placed in 3 ml of culture medium in wells 35 mm in diameter. Culture medium consisted of 50% minimal essential medium and HEPES (25 mM; Gibco, Grand Island, NY, USA), 25% heat-inactivated horse serum (Gibco), and 25% Hanks' balanced salt solution (Gibco) supplemented with D-glucose (25.6 mg ml^sup -1^) and glutamine (2 mM), at a final pH of 7.2. Cultures were incubated at 37degC in a 5% CO^sub 2^containing humidified environment.
Three days after initiation of cultures, the spinal slices were exposed to glutamic acid (for 30 min, 1 h, 2 h, 100 #M of final concentration), CSF from ALS patients, or control CSF by adding 50 pL of CSF to the medium of each slice. Tissues were exposed for 1 h. In blocking experiments, either the NMDA receptor antagonist MK801 (final concentration, 10 HM) or the AMPA/ kainate receptor antagonist CNQX (20 HM) was added to media 5 min before adding CSF from ALS patients. After incubation with CSF, slices were fixed immediately with 4% paraformaldehyde in 0.1 M phosphate buffer for 1 h. Ten-micron sections of each spinal slice were cut using a cryostat. Sections were mounted on 3-aminopropyltriethoxysilane-coated glass slides. Sections on slides were incubated in primary antiserum (1:2500 dilution in phosphate-buffered saline containing 0.3% Triton X-100, 0.05% sodium azide and 2% normal rabbit serum) for 72 h at 40degC. The polyclonal antibody used (Cambridge Research Biochemicals, Cambridge, England) was raised in sheep against residues 2 to 16 of the N-terminal region of the Fos protein. Sections were visualized using a diaminobenzidine (DAB)-nickel method. Because this antibody recognizes Fos and some related antigens, positive staining was referred to as Foslike immunoreactivity (Fos-LI). Neuronal nuclei showing Fos-LI were counted unilaterally in five sections of lumbar spinal cord, resulting in five determinations per 5-mm slice, which were then averaged.
Data are presented as mean +/- SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Student's t-test. Significance was set at p
Neurons showing Fos-LI in untreated control were seen in dorsal (Figure la) and ventral horns. However, the number of Fos-LI positive neurons was much larger in dorsal horn, while only a few neurons were positive for Fos-LI in ventral horn (Table 1). Addition of glutamate (final concentration 100 HM) to culture medium did not affect the number of Fos-LI positive neurons at 30 min. However, it increased the number of Fos-LI at 1 h in dorsal horn (Figure lb) and thereafter there was a decrease at 2 h (Table 1). No change in the number of Fos-LI positive neurons was observed in ventral horn. Addition of CSF from ALS patients to culture medium significantly increased Fos-LI to a level of 215% of untreated control values in dorsal horn (p
In this study, CSF from ALS patients increased Fos-LI in the dorsal horn of primary cultured rat spinal neurons in acute phase. Glutamate induced Fos-LI in the same region, and MK801 but not CNQX prevented the induction (Table 1). These results suggest that a constituent of CSF from ALS patients induced Fos-LI in dorsal horn neurons probably through NMDA receptors. Only one previous study has explored relationships between IEG and ALS. Virgo et al. reported that c-jun mRNA level was greater in dorsal and ventral horns in human ALS spinal cord than in control specimens. Our report provides the first evidence of an association between expression of Fos protein in cultured normal rat spinal cords after an addition of CSF from ALS patients.
In motoneuron-enriched cultures, excitatory amino acids provide toxic effects following activation of either NMDA or non-NMDA glutamate receptors9. In this study, addition of glutamate to culture medium significantly increased Fos-LI in comparison to untreated control (p
Couratier et al.21 found that CSF from ALS patients was toxic to cortical neurons in primary brain cultures from rat embryos, apparently via activation of nonNMDA glutamate receptors. However, a possible effect of CSF on spinal neuronal cells was not mentioned. Several authors have reported that most subtypes of glutamate receptor are distributed intensely in the dorsal horn of rat spinal cord22,23. In fact, our results showed that Fos-LI was expressed most prominently in an area particularly rich in NMDA receptors. While CSF from ALS patients appears to contain substances that stimulate an IEG, no increase in Fos-LI was detected in the ventral horn. Dorsal horn neurons were not usually involved in ALS, but ventral horn neurons are most involved. One possible explanation of this discrepancy would be that IEG expression may not directly be involved in the neurodegenerative process in ALS patients. A second might be that the activated interneurons of dorsal horn expressing Fos-LI may influence motor neurons to progressively degenerate via prolonged cellular response. A third would be that rather than Fos, other proteins such as Jun may be induced by stimulation of NMDA receptors, which are abundant in motor neurons and may activate the degenerative process in the spinal cord. Smeyne et al.24 have provided evidence using a fos-lacZ transgenic mouse showing that the continuous expression of Fos appeared to be a hallmark of terminal differentiation and a harbinger of subsequent death. Anderson et al.25 reported that some neurons co-express Fos or Jun immunoreactivity with abnormally phosphorylated tau protein in degenerating neurons of Alzheimer's disease. The data suggest that Fos and Jun family may be involved in Alzheimer's disease and other chronic neurodegenerative conditions including ALS. A possible relationship between expression of such IEG and apoptosis related protein would be necessary in the future under similar experimental condition to the present study.
In conclusion, CSF from ALS patients may contain substances which stimulate c-Fos expression via the NMDA receptors in spinal cord neurons, and this signal may trigger or otherwise be involved in a neurodegenerative condition such as ALS.
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Department of Neurology, Okayama University Medical School, Okayama, Japan
Correspondence and reprint requests to: Yasuhiro Manabe, MD, Department of Neurology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Accepted for publication September 1998.
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