Shigenobu Nakamura

Tissue-specific expression of three types of β-protein precursor mRNA: Enhancement of protease inhibitor-harboring types in Alzheimer's disease brain

Abstract Expression of three types of mRNA encoding amyloid β-protein precursor (APP) in various tissues was analysed, using a ribonuclease protection assay, with special reference to Alzheimers disease (AD). The total content and the proportion of APP mRNAs were specific to each tissue. Among eight tissues examined, the brain was distinct in that the expression level was highest and APP695 mRNA was expressed in abundance. The ratio of APP770/APP751/APP695 mRNAs was approximately 1:10:20 in the cerebral cortex of control brain. The proportions of APP770 mRNA and APP770-plus-APP751 mRNAs increased up to 2.6- and 1.4-fold, respectively, in various regions of AD brain compared with control. The enhanced expression of protease inhibitor-haboring types (APP770 and APP751) may disturb the balance between biosynthesis and degradation of APPs and ultimately lead to accumulation of β-protein as amyloid.

Tryptophan-5-Hydroxylase in Mammalian Brain*

Publisher Summary This chapter describes a procedure to explore the properties and function of tryptophan-5-hydroxylase in mammalian brain. The biosynthesis of 5-HT in the brain was reported recently following direct administration of radioactive tryptophan into the brain of rats and pigeons. The animals were treated with a monoamine oxidase (MAO) inhibitor prior to injection to prevent the breakdown of 5-HT. Subsequent reports from several laboratories, described the hydroxylation of tryptophan in vitro with extracts of various mammalian tissues such as carcinoid tumors, brain, and neoplastic mouse mast cells. The detailed properties of tryptophan-5-hydroxylases as well as the reaction mechanism involved are still largely unexplored, mainly because of the relatively low activity of this enzyme and the lack of a sensitive and reliable assay for its activity. Assay methods so far available are based on the determination of radioactive 5-HT separated from a large excess of unreacted tryptophan by paper chromatography, paper electrophoresis, or ion-exchange resins. Mast cell tryptophan 5-hydroxylase is an exception, being active enough to accumulate 5-HTP.

The P300 event-related potentials in dementia of the Alzheimer type. Correlations between P300 and monoamine metabolites

We studied auditory event-related potentials (ERPs) in 40 patients with dementia of the Alzheimer type (DAT). Peak latencies and amplitudes of N100, P200 and P300 components were measured. To study the possible relationships between the ERPs and monoaminergic neurotransmitter functions, CSF neurotransmitter metabolites were also analyzed. In patients with DAT, the P300 amplitude was significantly reduced and the P300 latency was prolonged. CSF concentrations of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) were decreased. The P300 amplitude was significantly correlated with the 5-HIAA concentration. There were no correlations between the P300 components and the HVA concentration. These findings suggest that the serotonergic system modulates the P300 component of ERPs in patients with DAT.

Determination of amyloid β protein precursors harboring active form of proteinase inhibitor domains in cerebrospinal fluid of Alzheimer's disease patients by trypsin-antibody sandwich ELISA

beta-Amyloid protein precursors (APP) having proteinase inhibitor domains (APPI) were quantified by a new sandwich enzyme linked immunosorbent assay for detection of active (free) form of proteinase inhibitors by using trypsin in place of the first antibody and by denaturation of APPI-trypsin complex in the microtiterplate. The concentration of APPs having APPI in cerebrospinal fluid of Alzheimers disease patients was found, by this method, to be significantly elevated compared with those of multi-infarct dementia.

Increased manganese level in spinal cords of amyotrophic lateral sclerosis determined by radiochemical neutron activation analysis.

The manganese distribution in the cross-section of the cervical, thoracic and lumbar portion of the spinal cords from 7 autopsied cases with amyotrophic lateral sclerosis (ALS) and 6 control subjects were determined by radiochemical neutron activation analysis. It was possible to determine 1 ng levels of manganese content accurately in the small tissues about 1 mg of dried weight using wet ashing and chemical separation after neutron activation. The dried weight of spinal cord was about 1/3 of the wet weight. Manganese concentration in the wet tissue was calculated from the ratio of dried to wet weight. In the anterior horn of the cervical cords of ALS, manganese concentration was the highest; 1.75 +/- 0.39 ng/mg of dried weight and 0.59 +/- 0.09 ng/mg of wet weight, respectively. These were significantly higher (P less than 0.01) compared to those in controls; 1.02 +/- 0.12 ng/mg of dried weight and 0.35 +/- 0.04 ng/mg of wet weight, respectively. The elevation of manganese level in the spinal cords of ALS was more prominent in the anterior horn and lateral column than in the posterior column both in dried and wet tissues. Since manganese inhibits neuronal transmission, it is likely that neurological degenerative changes occur as a result of local disturbances of manganese metabolism in the spinal cord of ALS.

Acetylcholinesterase activity in cerebrospinal fluid of patients with Alzheimer's disease and senile dementia.

Acetylcholinesterase (AChE) in the cerebrospinal fluid (CSF) from patients with Alzheimer-type dementia and control subjects was analyzed by centrifugation on a sucrose density gradient, and by column chromatography on Sephadex G-200. The sedimentation coefficient and molecular weight of CSF AChE were calculated as 10S and 380,000, respectively, which corresponded to those of G4 isozyme in the brain. Other isozymes of AChE were not detected in the CSF of either patients with Alzheimer-type dementia or the controls. Sufficient activity of AChE was observed in the CSF of a patient with familial pseudocholinesterase deficiency, although the pseudocholinesterase activity was not found either in the serum or in the CSF. CSF AChE activity in control subjects increased with advancing age (P less than 0.02). AChE activity in the CSF was significantly lower in patients with presenile dementia (Alzheimers disease), compared with age-matched control subjects (P less than 0.001). However, AChE activity in the CSF showed a wide variation among patients of Alzheimer-type dementia with a late onset (senile dementia).

Heavy metal concentrations in blood cells in patients with amyotrophic lateral sclerosis

Manganese (Mn) and selenium (Se) concentrations in blood cells were measured by neutron activation analysis. Blood was obtained from patients with amyotrophic lateral sclerosis (ALS), patients with other neurological diseases and control subjects. Dried blood cells were activated by neutron irradiation. Mn was determined after chemical separation and Se was determined nondestructively. Mn concentrations in blood cells from ALS patients were significantly lower (P less than 0.01) than those from the other groups. The Mn concentrations were also significantly lower (P less than 0.01) in late than in earlier stages of ALS. Se concentrations in blood cells from ALS patients were significantly higher (P less than 0.01) than those from the other two groups. A generalized abnormal distribution of these metals may play a role in the pathogenesis of this disorder. Bromine, zinc, rubidium, and iron concentrations of erythrocytes were the same in all groups.

Regional and Subcellular Distribution in Mammalian Brain of the Enzymes Producing Adenosine

Abstract: The activities of 5′‐nucleotidase, 2′‐nucleotid‐ase, alkaline phosphatase, and acid phosphatase were measured in rat and autopsied human brains. The four phosphatases in the rat brain showed little change in activity after death. The activities of adenosine‐producing enzymes were compared in various parts of rat and human brains. When phosphatase activity was measured at pH 7.5, 5′‐nucleotidase showed the highest activity in the most parts of the brain. The activity of 2′‐nucleotidase and that of nonspecific phosphatase were almost the same at pH 7.5. However, higher phosphatase activity was observed in all parts of the brain when nonspecific phosphatase activity was measured at pH 10.0 or 5.5. High specific activity of 5′‐nucleotidase in the brain was detected in the membranous components, especially in the synaptic membranes. The activity of 2′‐nucleotidase was distributed in the soluble and synaptosomal fractions. The highest activity of both alkaline and acid phosphatases was recovered in the crude mitochondrial fraction, with the highest specific activity in the microsomal fraction. Phosphatase activity was distributed widely in the rat brain. The activity of 5′‐nucleotidase was high in the medulla oblongata, thalamus, and hippocampus, but low in the peripheral nerve, spinal cord, and occipital lobe. The activity of 2′‐nucleotidase was high in the vermis and frontal lobe. The highest acid and alkaline phosphatase activities were detected in the frontal lobe and in the olfactory bulb, respectively. The distribution of the four phosphatases in the autopsied human brain was similar to that in the rat brain. The highest 5′‐nucleotidase activity was observed in the temporal lobe and thalamus. High activity of 2′‐nucleotidase was found in the basal ganglia, substantia nigra, and cortical motor area. Alkaline phosphatase was enriched in the temporal cortex, parietal cortex, and amygdala, and low in the white matter. High acid phosphatase activity was detected in the insula and cerebellar cortex.

Reversible serotoninergic neurotoxicity ofN-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mouse striatum studied by neurochemical and immunohistochemical approaches

Effects of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on serotonin (5-HT) were studied in mouse striatum. The 5-HT level was significantly reduced at 1 week after MPTP, though returned at 4 weeks. The level of 5-hydroxyindoleacetic acid (5-HIAA) increased at both 1 and 4 weeks. The ratio of increase in 5-HT induced by pargyline administration was larger in MPTP-pretreated mice than in vehicle-pretreated controls at 1 week, although it was identical in both groups at 4 weeks. The 5-HT immunohistochemical study confirmed the biochemical change of 5-HT. These results suggest that MPTP functionally affects the 5-HT metabolism in mouse striatum.

Plasma glutamate decarboxylase activity in neuropsychiatry

Plasma glutamate decarboxylase (GAD) activity was measured in patients with endogenous psychoses and neurologic diseases. Unmedicated schizophrenic patients showed no difference in plasma GAD levels compared to controls. Administration of neuroleptics together with anticholinergic agents increased plasma GAD activity in schizophrenic patients. Compared to controls, patients with major depression and bipolar illness showed significantly lower GAD activity. No effect of antidepressants and minor tranquilizers on plasma GAD activity was found. Relatively lower GAD activity was shown in neurotic patients. The enzyme activity in plasma of patients with Huntingtons chorea (HC) was lower than control levels. The plasma GAD concentrations correlated with cerebrospinal fluid concentrations in five HC patients.