Toshiharu Nagatsu

Tumor necrosis factor-α (TNF-α) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients

Tumor necrosis factor-alpha (TNF-alpha), a glial-cell-related factor, was measured for the first time in the brain (striatum) and cerebrospinal fluid (CSF) from control and parkinsonian patients by a sensitive sandwich enzyme immunoassay. The concentrations of TNF-alpha in the brain and CSF were significantly higher in parkinsonian patients than those in controls. Since TNF-alpha is an important signal transducer of the immune system with cytotoxic and stimulator properties, these results suggest that an immune response may occur in the nigrostriatal dopaminergic regions in Parkinsons disease and that TNF-alpha may be related, at least in part, to the neuronal degeneration.

Interleukin-1β, interleukin-6, epidermal growth factor and transforming growth factor-α are elevated in the brain from parkinsonian patients

Interleukin (IL)-1 beta, IL-6, epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha) were measured for the first time in the brain (caudate nucleus, putamen and cerebral cortex) from control and parkinsonian patients by highly sensitive sandwich enzyme immunoassays. The concentrations of IL-1 beta, IL-6, EGF, and TGF-alpha in the dopaminergic, striatal regions were significantly higher in parkinsonian patients than those in controls, whereas those in the cerebral cortex did not show significant differences between parkinsonian and control subjects. Since these cytokines and growth factors may play important roles as neurotrophic factors in the brain, the present results suggest that they may be produced as compensatory responses in the nigrostriatal dopaminergic regions in Parkinsons disease, and may be related, at least in part, to the process of neurodegeneration in Parkinsons disease.

The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells

We reported that one of the isoquinolinesulfonamide derivatives, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M. and Hidaka, H. (1990) J. Biol. Chem. 265, 4315-4320). We have now investigated the inhibitory property of a newly synthesized methoxybenzenesulfonamide, KN-93, on CaMKII activity in situ and in vitro. KN-93 elicited potent inhibitory effects on CaMKII phosphorylating activity with an inhibition constant of 0.37 microM but this compound had no significant effects on the catalytic activity of cAMP-dependent protein kinase, Ca2+/phospholipid dependent protein kinase, myosin light chain kinase and Ca(2+)-phosphodiesterase. KN-93 also inhibited the autophosphorylation of both the alpha- and beta-subunits of CaMKII. Kinetic analysis indicated that KN-93 inhibits CaMKII, in a competitive fashion against calmodulin. To evaluate the regulatory role of CaMKII on catecholamine metabolism, we examined the effect of KN-93 on dopamine (DA) levels in PC12h cells. The DA levels decreased in the presence of KN-93. Further, the tyrosine hydroxylase (TH) phosphorylation induced by KCl or acetylcholine was significantly suppressed by KN-93 in PC12h cells while events induced by forskolin or 8-Br-cAMP were not affected. These results suggest that KN-93 inhibits DA formation by modulating the reaction rate of TH to reduce the Ca(2+)-mediated phosphorylation levels of the TH molecule.

Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Interleukin (IL)-1β, IL-2, IL-4, IL-6 and transforming growth factor-α levels are elevated in ventricular cerebrospinal fluid in juvenile parkinsonism and Parkinson's disease ☆

Interleukin (IL)-1β, IL-2, IL-4, IL-6, epidermal growth factor (EGF), and transforming growth factor (TGF)-α were measured for the first time in ventricular cerebrospinal fluid (VCSF) from control non-parkinsonian patients, patients with juvenile parkinsonism (JP) and patients with Parkinsons disease (PD) by highly sensitive sandwich enzyme immunoassays. All cytokines were detectable in VCSF from control and parkinsonian patients, and the concentrations were much higher than those in lumbar CFS. The concentrations of IL-1β, IL-2, IL-4 and TGF-α in VCSF were higher in JP than those in controls (P < 0.05). In contrast, the concentrations of IL-2 and IL-6 in VCSF from patients with PD were higher than those from control patients (P < 0.05). These results agree with our previous reports, in which the cytokine levels were elevated in the striatal dopaminergic region of the brain from patients with PD. Since VCSF is produced in the ventricles, the alteration of cytokines in VCSF may reflect the changes of cytokines in the brain. Because cytokines play an important role as mitogens and neurotrophic factors in the brain, the increases in cytokines as a compensatory response may occur in the brain of patients of JP or PD during the progress of neurodegeneration. Increase in cytokines may contribute not only as a compensatory response but as a primary initiating trigger for the neurodegeneration.

Distribution of major histocompatibility complex class II-positive microglia and cytokine profile of Parkinson's disease brains

There are numerous observations confirming that microglia expressing major histocompatibility complex (MHC) class II molecules are associated with the central nervous system (CNS) in aging and pathological conditions. In this study, we investigated the distribution of MHC class II-positive microglia in Parkinsons disease (PD) brains. The number of MHC class II-positive microglia in the substantia nigra (SN) and putamen increased as the neuronal degeneration of the SN proceeded. These cells were also ICAM-1 (CD54) and LFA-1 (CD11a) positive. The number of activated microglia not only in the SN and putamen but also in the hippocampus, transentorhinal cortex, cingulate cortex and temporal cortex in PD was significantly higher than that in the normal control. Most activated microglia persisted regardless of the presence or absence of Lewy bodies. They were frequently associated not only with α-synuclein-positive Lewy neurites, but also with TH-16-positive dopaminergic and WH-3-positive serotonergic neurites, as well as MAP-2- and SMI-32-positive neurites. These activated microglia were also positive for TNF-α and interleukin-6, which are known to have a neuroprotective function. We conclude that MHC class II-positive microglia are a sensitive index of neuropathological change and are actively associated with damaged neurons and neurites.

Brain-derived growth factor and nerve growth factor concentrations are decreased in the substantia nigra in Parkinson's disease.

Using highly sensitive sandwich enzyme-linked immunosorbent assays (ELISA), we measured for the first time the concentrations of brain-derived growth factor (BDNF) in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients. BDNF in the human brain (the order of ng/mg protein) was significantly lower specifically in the nigrostriatal dopamine (DA) regions from parkinsonian patients than in those from control patients. The concentration of nerve growth factor (NGF) was also significantly decreased in the substantia nigra of parkinsonian patients in comparison with that in the controls. Since BDNF and NGF may play important roles in survival and differentiation of neuronal cells, the present data indicate that the lack of neurotrophins, especially BDNF, may be involved in the pathogenesis of PD during progress of neurodegeneration of the nigrostriatal DA neurons.

New chromogenic substrates for X-prolyl dipeptidyl-aminopeptidase

We have synthesized several new chromogenic substrates, p-nitroanilides of the dipeptides, Gly-Pro, Ala-Pro, Lys-Pro, Arg-Pro, Glu-Pro, and Asp-Pro, for X-prolyl dipeptidyl-aminopeptidase. These have permitted the development of a simple assay of the enzyme in which p-nitroaniline liberated directly or after the Bratton-Marshall reaction is measured spectrophotometrically. The enzyme activity was measured in human serum or in homogeneous enzyme purified from human submaxillary gland. The homogeneous enzyme hydrolyzed each substrate to produce X-Pro and p-nitroaniline. The optimum pH was at 8.7, except with Arg-Pro p-nitroanilide (8.0). Serum enzyme hydrolyzed Gly-Pro p-nitroanilide to p-nitroaniline and Gly-Pro, which was further hydrolyzed to Gly and Pro by an imidodipeptidase in serum. Gly-Pro β-naphthylamide or Gly-Pro-Leu was a competitive inhibitor with each X-Pro p-nitroanilide as substrate. Gly-Pro p-nitroanilide had the highest activity among the substrates at pH 8.7, followed by p-nitroanilides of Ala, Lys, Arg, Glu, and Asp in a decreasing order of activity.

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.

Cytokines in Parkinson’s disease

We found that in Parkinsons disease (PD) the levels of various cytokines [tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-2, IL-4, IL-6, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, TGF-beta1] were significantly increased in the striatum (caudate and putamen) of the postmortem brain and in ventricular or spinal cerebrospinal fluid (VCSF, LCSF). Furthermore, the levels of the apoptosis-related proteins such as bcl-2 and soluble Fas (sFas) in the striatum were also elevated in PD. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonism mice, the levels of IL-1beta in the striatum were significantly increased, but those of nerve growth factor (NGF) were significantly decreased, compared with control mice. In hemiparkinsonism rats produced by injection of 6-hydroxydopamine (6-OHDA) into one side of the median forebrain bundle, the levels of TNF-alpha in the 6-OHDA-treated side were increased in the striatum and substantia nigra, but not in the cerebral cortex, compared with those in the control side. Repeated administration of L-DOPA in the 6-OHDA-treated rats did not change the TNF-alpha levels in the control side and in the 6-OHDA-treated side in the substantia nigra, striatum, and cerebral cortex. Our results suggest that the changes in the levels of cytokines, neurotrophins, and apoptosis-related proteins in the nigrostriatal regions of PD may be involved in apoptosis and degeneration of the nigrostriatal DA neurons.