Masamitsu Yamauchi

Decreased cytochrome c oxidase activity but unchanged superoxide dismutase and glutathione peroxidase activities in the spinal cords of patients with amyotrophic lateral sclerosis

The cause of selective degeneration of motor neurons in the ventral horn of the spinal cord associated with amyotrophic lateral sclerosis (ALS) has still not been elucidated. Recently, so‐called oxidative stress has been suggested to be a significant factor in the pathogenesis of this disease. We measured the antioxidant actions of superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), and cytochrome c oxidase (CO) of the human spinal cord in patients with ALS in comparison with those in control patients. Total SOD activity in spinal cord transections from patients with sporadic ALS was not significantly different from the controls in ventral, lateral, or dorsal regions, although enzymic activity was relatively higher in the ventral compared with the dorsal region. GSH‐Px activity in the spinal cord of ALS patients was not very different from that in the control tissue. In contrast, CO activity was significantly reduced in all three regions of the spinal cord in patients with ALS, although the reduction was more marked in the ventral region. These results suggest that reactive oxygen species may attack the mitochondrial respiratory chain, leading eventually to the degeneration of vulnerable motor neurons in the spinal cord, even though no obvious changes in the activity of antioxidant enzymes are detectable.

Alteration of Transglutaminase Activity in Rat and Human Spinal Cord after Neuronal Degeneration

We measured the activity of transglutaminase (TG), a Ca2+-dependent enzyme and a biochemical marker of cell degeneration, in the adult rat spinal cord after unilateral occlusion of a branch of the dorsal spinal artery, and compared it to the enzyme activity in the tissue on the contralateral side without ischemic damage. The affected half of the spinal cord showed a significant rise in intrinsic (endogenous) TG activity one day after ischemic insult while no apparent morphological changes were observed in the tissue. However, the enzymic activity on the affected side gradually decreased to reach the level in the non-affected tissue, accompanying severe degeneration of neuronal cells at 7 days after the surgery, then it declined to nearly half the level in the intact tissue 30 days after the operation. We also determined the TG activity in transverse sections of the human spinal cord obtained at autopsy from 5 amyotrophic lateral sclerosis (ALS) and 9 non-ALS patients. TG activity in thoracic and lumbar cords was markedly low in ALS patients not only in ventral and lateral regions but also in the dorsal portion. These findings imply that the reduced TG activity in the ALS spinal cord is one of the characteristic features of the disease reflecting exhaustion of the enzyme in the tissue resulting from degeneration of the spinal neurons through cross-linkage of soluble intraneuronal cytoplasmic proteins.

Increase of glial fibrillary acidic protein fragments in the spinal cord of motor neuron degeneration mutant mouse

We analyzed protein fractions extracted from the spinal cord of the motor neuron degeneration (Mnd) mouse, a mutant that exhibits progressive degeneration of lower spinal motor neurons, by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) after solubilization of the tissue with medium containing sodium dodecyl sulfate (SDS)-urea during growth of the animal, in comparison with those of age-matched controls (C57BL/6). Several protein spots were detected around a region of pI 5.6-6.0 and molecular mass of 35-50 kDa in Mnd spinal cord tissue on the two-dimensional PAGE separation profile with Coomassie brilliant blue staining, while only a few spots around the same region were found in the control spinal cord. These spots were all immunoreactive with an antibody against glial fibrillary acidic protein (GFAP), a cytoskeleton filamentous protein specific to astroglial cells. The protein spot with molecular mass of 50 kDa showed immunoreactivity with anti-GFAP antibody, had a blocked amino-terminus, and is assumed to be intact GFAP. Several protein spots with slightly smaller molecular masses of 35 to 48 kDa lacked the head domain of the GFAP molecule as a result of cleavage at the 29th and 56th residues from the amino terminus. In Mnd spinal cord tissue, the densities of the immunoreactive GFAP bands with smaller molecular masses increased with development, and became dominant at the time of the appearance of behavioral paralytic gait around 6 to 7 months of age. These results suggest that the increased GFAPs devoid of head domains are related to the degenerative loss of motor neurons in the Mnd spinal cord. Histopathological and GFAP immunohistochemical examination of Mnd spinal cord preparation demonstrated progressive degenerative loss of motor neurons, and considerable increases in number of GFAP-stained astrocytes in the ventral horn at 7 to 9 months of age. These processes of degenerative loss of motor neurons and proliferation of reactive astrocytes with increased levels of fragmented GFAP in the Mnd spinal cord during development seem to be characteristic and preceded the deterioration of motor activities in this animal model of amyotrophic lateral sclerosis.

Improved fluorometric enzymatic sorbitol assay in human blood.

Samples for use in the fluorometric enzymatic assay of sorbitol in erythrocytes are normally prepared using HClO4 and K2CO3. We have replaced these reagents with NaOH and ZnSO4. Human whole blood, erythrocyte and plasma samples prepared with NaOH and ZnSO4 are colorless and clear, while erythrocyte samples prepared with HClO4 and K2CO3 are a pale yellow-brown color. The sorbitol dehydrogenase reaction in the supernatant of the mixture of NaOH and ZnSO4 is inhibited, but ethylenediaminetetraacetate completely eliminates this effect. The sorbitol assay in erythrocytes prepared with NaOH and ZnSO4 shows higher sensitivity and reproducibility than did that with HClO4 and K2CO3. Recovery of sorbitol added to erythrocytes is similar in both assay methods. Concentrations of whole blood and erythrocyte sorbitol assayed by the present method are significantly higher in diabetics than in normals. Poorly controlled diabetics had higher whole blood and erythrocyte sorbitol than well-controlled diabetics. Whole blood sorbitol concentrations differed more between diabetic and normal subjects than did erythrocyte sorbitol concentrations.

Increases in Fragmented Glial Fibrillary Acidic Protein Levels in the Spinal Cords of Patients with Amyotrophic Lateral Sclerosis

Using one-dimensional polyacrylamide gel electrophoresis, we analyzed protein fractions extracted from the spinal cords of patients with amyotrophic lateral sclerosis (ALS). Several protein bands with molecular weights of 35–55 kDa were stained with Coomassie brilliant blue much more intensely in the ALS than in the non-ALS spinal cord. Glial fibrillary acidic protein (GFAP) immunoreactivity showed a significant decrease of 50 and 45 kDa band and increase in fragmented 36 and 37 kDa bands, which represented GFAP fragments devoid of 59 and 40 residues from the N-terminal, respectively, as determined by protein sequence analysis. Immunohistochemical examination of ALS spinal cord transections demonstrated increased GFAP-stained astrocytes in the shrunken ventral horn with massive degeneration of motoneurons. These results will provide new insight into the possible role of astrocytes in the pathophysiology and/or pathogenesis of ALS.

Alteration of Enzymatic Activities Implicating Neuronal Degeneration in the Spinal Cord of the Motor Neuron Degeneration Mouse During Postnatal Development

Oxidative stress is suggested as a significant causative factor forpathogenesis of neuronal degeneration on spinal cord of human ALS. Wemeasured some enzymic activities implicating neuronal degenerationprocess, such as cytochrome c oxidase (CO), superoxidedismutase (SOD), and transglutaminase (TG) in spinalcord of an animal model of ALS, motor neuron degeneration(Mnd) mouse, a mutant that exhibits progressivedegeneration of lower spinal neurons during developmental growth, andcompared them with age-matched control C57BL/6 mice. CO activity inMnd spinal cord decreased during early postnatal period, whileSOD activity reduced in later stage. In Mnd tissue, TG activityin lumbar cord was increasing during early stage, but tended to declinein later period gradually. These biochemical alterations became evidentprior to the appearance of clinical motor dysfunction which wereobserved in later stages of development in Mnd spinal cord.

Neurochemical changes in the spinal cord in degenerative motor neuron diseases

Human amyotrophic lateral sclerosis (ALS), a typical motor neuron disease, is characterized pathologically by selective degenerative loss of motoneurons in the CNS. We have demonstrated significant reductions of neurotransmitter-related factors, such as acetylcholine-(ACh)-synthesizing enzyme activity and glutamate and aspartate contents in the ALS, compared to the non-ALS spinal cord obtained at autopsy. We have also shown considerable reductions in activities of cytochrome-c oxidase (CO), an enzyme contributing to aerobic energy production, and transglutaminase (TG), a Ca(2+)-dependent marker enzyme for tissue degeneration, in the ALS spinal cord. We found marked increases in fragmented glial fibrillary acidic protein (GFAP), a filamentous protein specifically associated with reactive astrocytes, in the ALS spinal cord relative to non-ALS tissue. These biochemical results corresponded well to pathomor-phological neuronal degenerative loss and reactive proliferation of astroglial components in the ALS spinal cord tissue. However, these results only indicate the final pathological and biochemical outcomes of ALS, and it is difficult to follow up cause and process in the ALS spinal cord during progression of the disease. Therefore, we used an animal model closely resembling human ALS, motor neuron degeneration (Mnd) mutant mice, a subline of C57BL/6 that shows late-onset progressive degeneration of lower motor neurons with paralytic gait beginning around 6.5 mo of age, to follow the biochemical and pathological alterations during postnatal development. We detected significant decreases in CO activity during early development and in activity of superoxide dismutase (SOD), an antioxidant enzyme, in later stages in Mnd mutant spinal cord tissue. TG activity in the Mnd spinal cord showed gradual increases during early development reaching a maximum at 5 mo, and then tending to decrease thereafter. Amounts of fragmented GFAPs increased continuously during postnatal development in Mnd spinal cord. These biochemical changes were observed prior to the appearance of clinical motor dysfunctions in the Mnd mutant mice. Such biochemical analyses using appropriate animal models will be useful for inferring the origin and progression of human ALS.

induction of tissue transglutaminase in rat superior cervical sympathetic ganglia following in vitro stimulation of retinoic acid

The addition of retinoic acid (RA, 50 nM) to Dulbeccos modifed Eagles medium containing 1.0 percent bovine serum albumin and 50 mu g/l of gentamicin markedly increased the activity of a Ca(2+) -dependent tissue transglutaminase (TGase) (ca. 3.2-fold), which stabilizes newly formed protein assemblies at the sites of synapses, in isolated rat superior cervical sympathetic ganglia (SCG), which is abundant in synapses, following in vitro aerobic incubation for 3 h at 37 degrees C. An isomer of RA, 13-cis-RA (50 nM), showed the same tendency but rather lesser magnitude (ca. 1.7-fold) in ganglionic TGase activation. Also, retinal (50 nM), a precursor of RA, had a little effect on TGase stimulation (ca. 1.5-fold) in SCG. The RA-induced enhancement of ganglionic TGase activity was completely eliminated in the presence of either actinomycin D (1.0 mu g/ml), a depressant of molecular transcriptional activity, or a potent inhibitor of protein synthesis, cycloheximide (10 mu g/ml). Kinetic analyses show that the stimulation of ganglionic TGase activity evoked by RA addition was associated with only an increase in V max value (ca. 3.3-fold) without change in Km value. Thus, the enzyme protein of TGase might be synthesized de novo in the ganglia in response to RA. The RA-induced activation effect of ganglionic TGase almost disappeared (ca. 1.3-fold) 1 week following denervation, by which time preganglionic cholinergic nerve terminals were degradated. In axotomized SCG, where sympathetic neurons were degenerated and reactive proliferation of glial cells was in progress, the RA-evoked increase in ganglionic TGase activity was attenuated (ca. 1.3-fold). These findings imply that some retinoids, especially RA effectively participate in the cholinergic potentiation of synaptic activity.

Connexin-subtype immunoreactivity in gap-junction channels of several vertebrate retinae

Gap junctions (GJs) are intercellular channels which permit small molecules to pass. These intercellular junctions also subserve electrotonic transmission between connected neurons in the nervous system. GJ channels are aggregates of connexons, each of which is a hexameric structure composed of subunit proteins, called connexins (CXs). The CXs represent a family of proteins composed of a variety of members (at least 14 members are known to exist in animal species). Studies on the expression of the known CXs in the central nervous system showed that CX 26, CX 32 and CX 43 are found in GJs between glial cells. All of the major cell types in the vertebrate retina exhibit GJ connections between homotypic neurons. To investigate CX subtypes expressed in retinal ccl1 types, Western blot analysis and immunohistochcmical examination were performed in retinae from rat, frog, carp and goldfish by using antisera directed against rat liver CXs 26 and 32, and rat heart CX 43. CX 43 immunoreactivity was identified in GJs between pigment epithelium cells in rat retina and GJs between Miiller glial cells in frog, carp and goldfish retina. Very weak labcling with anti-CX 32 antibody was obsemed at the outermost row of the inner nuclear layer where horizontal cell somata arc located. CX 26 immunorcactivity was obscncd in GJs labeled with anti-CX 43 serum. The immunoblots, probed with antibodies directed against CXs demonstrated that molecular weight of a CX subtype in GJs is consistent among animal species examined in our study.

1340 Biochemical analysis of glial fibrillary acidic protein (GFAP) in spinal cord tissue from patients with amyotrophic lateral sclerosis (ALS) and its model animal (mnd mouse)

We examined the effects of FK506, an inhibitor of Ca2+/caImodulin-dependent phosphatase 2B(calcineurin), on apoptotic neuronal death m gerbil hippocampus after ischemlc insults. Transient brain ischemia was induced m male Mongolian gerbils (60~90g) by occluding bilateral common carotid arteries for 4 minutes. Immediately after the clips were removed, FK506 (1 mg /kg), rapamycin (1 mg /kg) or vehicle were injected mtravenously. Apoptotic changes occurring CA1 pyramidal neurons were investigated using in siru DNA nick-end labeling(TUNEL). Neuronal damage was determined by counting pyramidal neurons within CA1 subfield of the hippocampus. Four days after Ischemia, TUNEL showed DNA fragmentation in situ in CA1 pyramidal neurons in vehicle-treated gerbils, while no TUNEL was detected m FK506-treated gerbils. After 7 days, hippocampal CA1 pyramidal neurons survived in gerbils treated with FK506, while the neurons were lost in rapamycinor vehicle-treated animals. These results suggest hat IX506 can prevent ischemia-induced apoptotic cell death in gerbil hippocampus and that the prevention may be involved in the inhibition of calcineurin.