Axonal TDP-43 aggregates in sporadic amyotrophic lateral sclerosis.

AIMS: Axonal aggregates of phosphorylated (p-) transactive response DNA-binding protein 43 kDa (TDP-43) in sporadic amyotrophic lateral sclerosis (sALS) were examined in relation to propagation of the protein in the nervous system. METHODS: Brains and spinal cords of Japanese patients with sALS and control subjects were examined immunohistochemically using formalin-fixed paraffin-embedded specimens with special reference to the topographical distribution, microscopic features, presynaptic aggregates, and correlation between the aggregates in axons and the clinical course. RESULTS: (i) Aggregates of p-TDP-43 were frequently present in axons of the hypoglossal and facial nerve fibres and the spinal anterior horn cells. (ii) Aggregates of p-TDP-43 in the axons showed two characteristic microscopic features - dash-like granuloreticular aggregates (GRAs) and massive aggregates (MAs). (iii) MAs were surrounded by p-neurofilaments, but p-neurofilament immunnoreactivity decreased at the inside of axons with GRAs. (iv) Patients showing MAs and GRAs had a relatively shorter clinical course than patients without the aggregates. (v) Some neurones in the red nucleus in patients were surrounded by synapses containing p- and p-independent (i)-TDP-43, and almost all neurones had lost their nuclear TDP-43 immunoreactivity; 17% of those neurones in the red nucleus also had TDP-43-immunopositive neuronal cytoplasmic inclusions, but no postsynaptic p-TDP-43 deposition was evident. CONCLUSIONS: There are two types of axonal p-TDP-43 aggregates, MAs and GRAs, located predominantly in the facial and hypoglossal nuclei and anterior horn cells. These aggregates may influence the function of neurones, and presynaptic aggregates of the protein induce loss of p-i-TDP-43 in the nuclei of postsynaptic neurones.

[A case of progressive hemifacial atrophy associated with immunological abnormalities].

We reported a case of progressive hemifacial atrophy associated with immunological abnormalities. This patient, a 35-year-old woman, had a history of high fever of unknown etiology associated with anemia and leukocytopenia at the age of 19. Eight months later, right hemifacial atrophy, alopecia and partial lipodystrophy of the right upper arm were developed. And the progression had stopped in the latter twenties. At the age of 35, she visited our hospital because of the hemifacial atrophy. Physical examination revealed right hemifacial atrophy, large alopecia at parietal and right temporal region, and partial lipodystrophy of the right upper arm. CT, MRI and ultrasound tomography of the head and the arm showed skin and scalp atrophy, temporal muscle atrophy and decrease of subcutaneous soft tissues. There was no autonomic dysfunction. Serum IgG was increased, and RA factor, anti-nuclear antibody, anti-DNA antibody and LE cell were positive. In this case, immunological abnormalities were considered to be related to the pathogenesis of hemifacial atrophy.

Effects of 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol) and nerve growth factor on DNA, RNA and protein syntheses in neonatal rat superior cervical ganglia.

To investigate molecular mechanisms involved in the neurotoxicity of clioquinol (5-chloro-7-iodo-8-hydroxyquinoline), the inhibitory effects of this drug on DNA, RNA and protein syntheses were examined, in relation to the action of nerve growth factor (2.5S NGF). We used an organ culture of neonatal rat superior cervical ganglion (SCG). Ten microM clioquinol inhibited completely DNA and protein syntheses and abolished the stimulatory effect of NGF on RNA synthesis. With regard to the chemical structure of clioquinol, hydroxylation at the 8th carbon of quinoline is essential for the inhibition of DNA, RNA and protein syntheses, and the hydrophobicity of the 8-HQ derivatives is a required property for potent inhibition. Compared with effects of EDTA, alizarine, sodium alizarine sulfate, o-phenanthroline and alpha,alpha'-dipyridyl, the loss of the NGF-induced stimulation of RNA synthesis by clioquinol does not seem to be primarily caused by its metal-chelating property. Clioquinol did not significantly alter the uptake rate of thymidine, uridine and leucine, thereby suggesting that the primary action of clioquinol on inhibition of DNA, RNA and protein syntheses does not relate to uptake of the precursor into SCG. Clioquinol did not significantly alter the degradation of 3H-uridine-labeled RNA. NGF suppressed the degradation of RNA and this suppression was overcome by clioquinol. The release of free uridine from SCG into the culture medium was enhanced by clioquinol and was partially suppressed by NGF. The inhibitory effects of clioquinol were completely prevented by bovine serum albumin (BSA), but not by NGF even at a 5-fold concentration of clioquinol.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical analysis of decreased ornithine transport activity in the liver mitochondria from patients with hyperornithinemia, hyperammonemia and homocitrullinuria.

Hyperornithinemia, hyperammonemia and homocitrullinuria (HHH disorder) is an inherited metabolic disorder which shows peculiar amino acid changes in the serum and urine. The primary defect is considered to be the transport of ornithine across the mitochondrial membrane, but there is no direct evidence for this so far. We have analyzed ornithine transport activities in the liver mitochondria from three patients with HHH disorder. In coupled liver mitochondria we demonstrated low activities of citrulline synthesis and low rates of ornithine uptake. However, there were no abnormalities in carbamoyl-phosphate synthetase activity, ornithine carbamoyltransferase activity, N-acetylglutamate levels or O2 uptake with succinate. We also performed a kinetic study of citrulline synthesis as a function of ornithine concentration. We found increased Km values for ornithine and varied Vmax values of citrulline synthesis, which suggested the presence of a mutant transport protein. From these results we conclude that the defect of hyperornithinemia, hyperammonemia and homocitrullinuria lies in the transport of ornithine across the mitochondrial membrane.

5-Chloro-7-iodo-8-hydroxyquinoline (clioquinol) inhibits the nerve growth factor-induced stimulation of RNA synthesis in neonatal rat superior cervical ganglion, in vitro--comparison with effects of methylmercuric chloride and 4-hydroxyaminoquinoline-N-oxide.

The inhibitory effects of 5-chloro-7-iodo-8-hydroxy-quinoline (clioquinol), methylmercuric chloride and 4-hydroxyaminoquinoline-N-oxide(4-HAQO) on DNA, RNA and protein syntheses in the neonatal rat superior cervical ganglion (SCG) were studied in relation to the action of mouse 2.5S nerve growth factor (NGF), using organ cultures. RNA and protein syntheses in SCG were stimulated approximately 3- and 2-fold, respectively, by NGF (1 microgram/ml), but the DNA synthesis was only slightly or not at all stimulated. Methylmercuric chloride and 4-HAQO dose-dependently inhibited DNA, RNA and protein syntheses, either in the presence or in the absence of NGF. On the other hand, clioquinol (up to 100 microM) slightly or not at all inhibited RNA synthesis in the absence of NGF; however, it did abolish the NGF-induced stimulation of RNA synthesis in the presence of NGF. The DNA and protein syntheses were dose-dependently inhibited by clioquinol, either in the presence or in the absence of NGF. We conclude from this study that the interaction between clioquinol and the functions of NGF raises the question of a possible toxicity of the drug on specific neurons.

Fine structure of the synaptic endings between sympathetic axons and skeletal muscle cells and of the varicosities in the bundles of neurites in tissue culture.

In cocultures of skeletal muscle and sympathetic ganglia from chick embryos, synaptic boutons on skeletal muscle cells and varicosities in the neuritic bundles were observed electron microscopically. Synaptic endings on skeletal muscles were bulbous. Most synaptic boutons simply made contact with muscle fibers, but some boutons appeared as concave invaginations into the sarcolemma. In the neuritic bundles, numerous varicosities were observed. Close approximations were found between the synaptic boutons and skeletal muscles and between the varicosities and neurites (dendrites or axons), but no membrane thickening nor subsynaptic infolding was observed in either synapse; in our cultures the contacts were characteristic of the autonomic nervous system. In both synapses, three variations were demonstrated by KMnO4 fixation after 5-hydroxydopamine incubation: (i) containing predominantly small dense-core vesicles (noradrenergic type), (ii) predominantly small clear vesicles (cholinergic types), and (iii) a mixture of both small dense-core and small clear vesicles (mixed type). Cytochemically, the varicosities in the neuritic bundles were predominantly noradrenergic at 1 week in culture, and both the synaptic boutons on skeletal muscles and the varicosities in the neuritic bundles contained a mixed population of small dense-core and small clear vesicles at intermediate times, with a gradual shift to cholinergic characteristics. These findings strongly suggest that in this culture system some sympathetic neurons or fibers become cholinergic (neuronal plasticity).