2-4-dinitrophenylhydrazine and Amyotrophic-Lateral-Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, which has been linked to the generation of free radicals and oxidative stress. Oxidative damage to spinal cord proteins is suggested to be a contributory factor to neuronal death in ALS. Since proteins are the major targets for free radicals and the so-called "reactive species", therefore the objective of our study was to identify oxidatively damaged spinal cord proteins. The material consisted of spinal cords of 8 sporadic ALS cases and 5 controls. We estimated the level of protein carbonyl moieties, which react quantitatively with 2,4-dinitrophenylhydrazine (DNPH). Afterwards proteins were separated by SDS-polyacrylamide gel electrophoresis and the protein bound DNPH moieties were detected immunochemically. We also morphologically examined spinal cords after immune staining against DNPH. The protein carbonyl content of the ALS spinal cords significantly increased in all examined cases. In most ALS patients, proteins with 125 kDa, 70 kDa and 36kDa were highly oxidized. The 70-kDa protein was identified immunochemically to be neurofilament 68. The morphological examination of ALS spinal cords indicated a pronounced anti-DNPH immune reaction in neurones of the anterior horns; the reaction in the posterior horns was less intense. Microglia in the white matter was immunoreactive; astroglia was DNPH-negative. Although the exact mechanism by which reactive oxygen species induce motor neurones to die is not known yet, the presented data indicate that they affect spinal cord cellular proteins, including neurofilament 68. In this study, we successfully examined the neurochemical features accompanying motor neuron injury in ALS, and the results may help to develop a rationale anti-oxidative neuroprotective strategy.

Topics: Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Astrocytes; Blotting, Western; Cell Death; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immunohistochemistry; Male; Microglia; Middle Aged; Nerve Tissue Proteins; Neurofilament Proteins; Neurons; Oxidation-Reduction; Phenylhydrazines; Reactive Oxygen Species; Spinal Cord

Topics: Adult; Aged; Aged, 80 and over; Aldehydes; Amyotrophic Lateral Sclerosis; Cerebral Cortex; Female; Humans; Ketones; Male; Middle Aged; Muscular Atrophy, Spinal; Nerve Tissue Proteins; Oxidative Stress; Phenylhydrazines; Reactive Oxygen Species; Reproducibility of Results

The Gly93-->Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (approximately 50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100-120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.

Topics: Aging; Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Blotting, Western; Female; Humans; Lumbosacral Region; Male; Mice; Mice, Transgenic; Neck; Nerve Tissue Proteins; Oxidation-Reduction; Phenylhydrazines; Precipitin Tests; Spinal Cord; Superoxide Dismutase