Kazuhiro Tashima

Overexpression of Ca2+/calmodulin-dependent protein kinase II inhibits neurite outgrowth of PC12 cells

Abstract: To investigate the physiological role of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the α subunit of mouse CaM kinase II (CaM kinase IIα) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase IIα gene. The expression of CaM kinase IIα was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the γ and δ subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase IIα through autophosphorylation and generation of the Ca2+/calmodulin‐independent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase IIα. The activity of cyclic AMP‐dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system.

Role of nitric oxide in the cerebellar degeneration during methylmercury intoxication.

To investigate the role of nitric oxide in the cerebellar degeneration during methylmercury intoxication, interaction of the change in nitric oxide synthase activity and degeneration of the granular layer neurons was examined in rats after methylmercury administration. Both reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and anti-nitric oxide synthase antibody staining, and measurement of glutamate, and nitrite and nitrate levels in the cerebrospinal fluid were performed after oral administration of 5 mg/kg of methylmercury for 12 days. Nitric oxide synthase activity in the cerebellum was also assayed by monitoring the conversion of arginine to citrulline. Methylmercury levels in the blood and the cerebellum gradually increased up to day 13 after the initial methylmercury administration, and neurological disturbances, such as hindleg crossing and abnormal gait, were observed from day 17 after administration. Although a significant decrease in the number of granular layer neurons was recognized at day 84, no such decrease either in NADPH-diaphorase or anti-nitric oxide synthase antibody positive neurons was seen. Glutamate levels in the cerebrospinal fluid transiently increased at day 9 and finally decreased at day 84. Also a transient increase in both nitrite and nitrate levels in the cerebrospinal fluid and nitric oxide synthase activity in the cerebellum was seen prior to the start of degeneration of the granular layer neurons. These results suggest that nitric oxide may play an important role in the degeneration process of the granular layer neurons during methylmercury intoxication.

Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) by proline-directed protein kinases and its dephosphorylation

Abstract: We identified two major substrates for the proline‐directed protein kinases—cdc2 kinase and tau protein kinase II (TPKII)—in the cytosol fraction from rat brains. The molecular masses of the proteins were 80 and 46 kDa. Because the 80‐kDa protein was phosphorylated by protein kinase C and was heat stable, we examined the possibility that the protein might be myristoylated alanine‐rich C kinase substrate (MARCKS). On the basis of a comparison between the properties of the 80‐kDa protein and purified MARCKS, we concluded that the 80‐kDa protein is indeed MARCKS. The amounts of phosphate incorporated into MARCKS by protein kinase C, cdc2 kinase, and TPKII were 1.7, 1.4, and 0.6 mol/mol of the protein, respectively. Two‐dimensional tryptic peptide mapping indicated that phosphorylation sites by protein kinase C and proline‐directed protein kinases completely differed. Only the seryl residue was phosphorylated by protein kinase C, whereas both seryl and threonyl residues were phosphorylated by cdc2 kinase and TPKII. Phosphorylation of MARCKS by protein kinase C inhibited the binding to calmodulin, whereas phosphorylation by cdc2 kinase and TPKII significantly increased the binding to calmodulin. The holoenzyme of protein phosphatase 2A dephosphorylated MARCKS that had been phosphorylated by protein kinase C, cdc2 kinase, or TPKII, whereas calcineurin was unable to dephosphorylate it. These results suggest that cdc2 kinase and TPKII regulate the functions of MARCKS in different ways from protein kinase C.

Dephosphorylation of abnormal sites of tau factor by protein phosphatases and its implication for Alzheimer's disease

The abnormally phosphorylated forms of tau factor are major constituents of neurofibrillary tangles in Alzheimers disease brain. In order to investigate protein phosphatases which are related to dephosphorylation of abnormal phosphorylation sites, we examined the dephosphorylation of tau factor phosphorylated by three proline-directed type protein kinases. Tau factor phosphorylated by cdc2 kinase and tau protein kinase II was dephosphorylated by the holoenzyme of protein phosphatase 2A and calcineurin, while either the catalytic subunit of protein phosphatase 2A or protein phosphatase 2C could not catalyze the dephosphorylation. From the kinetic analysis, we concluded that tau factors phosphorylated by the protein kinases serve as good substrates for protein phosphatase 2A and calcineurin. On the other hand, tau factor phosphorylated by glycogen synthase kinase 3 alpha was dephosphorylated by the catalytic subunit of protein phosphatases 2A as well as the holoenzyme of protein phosphatase 2A and calcineurin. It has been reported that serines 199, 202 and 396 according to the numbering of the longest human tau isoform are among the major abnormal phosphorylation sites of tau factor. We synthesized two phosphopeptides which contained phosphoserines 199 and 202 or phosphoserine 396 and prepared the polyclonal antibodies specific for the phosphopeptides. Using these antibodies, we confirmed that the holoenzyme of protein phosphatase 2A and calcineurin could dephosphorylate phosphoserines 199, 202 and 396 in tau factor. The catalytic subunit of protein phosphatase 2A could dephosphorylate phosphoserine 396 but not phosphoserines 199 and 202. Neurofibrillary tangles in Alzheimers disease brain were immunostained with both antibodies but the normal neurons in the normal aged brains were not. The results suggest that protein phosphatase 2A and calcineurin can be involved in the dephosphorylation of abnormal phosphorylation sites in tau factor and that the dephosphorylation of phosphoserine 396 is differently regulated from phosphoserines 199 and 202.

Gastrointestinal dysfunction in familial amyloidotic polyneuropathy (ATTR Val30Met)--comparison of Swedish and Japanese patients.

The aim of the present study was to compare the clinical symptoms of Swedish and Japanese patients with familial amyloidotic polyneuropathy (ATTR Val30Met), especially gastrointestinal disturbances, and to correlate the findings with survival. Seventy-three Swedish and 47 Japanese patients were available for the study. Thirty-two Swedish and 7 Japanese patients had undergone liver transplantation. The mean age at onset was 50 for Swedish and 35 for Japanese patients (P < 0.001; non-transplanted patients). Fifty-five percent of Japanese patients had gastrointestinal disturbances from onset, compared to 22% of Swedish patients (P < 0.05; whole population). The Swedish patients average survival after the onset was significantly longer than Japanese patients (P < 0.05; non-transplanted patients). An early onset of gastrointestinal symptoms was correlated to a decreased survival for Swedish, but not for Japanese patients. Age at onset was not correlated to survival neither for Swedish nor for Japanese patients. We conclude that the clinical expressions of familial amyloidotic polyneuropathy ATTR Val30Met are different in Swedish and Japanese patients. The survival after the onset was shorter for Japanese patients, who also had an earlier onset of gastrointestinal disturbances, especially diarrhea than Swedish patients.

Down regulation of a harmful variant protein by replacement of its normal protein.

To compensate for the hypoprotein and hypoalbuminemia of familial amyloidotic polyneuropathy (FAP) patients, 800 ml of fresh frozen plasma (FFP) was intravenously administered and change in total and variant transthyretin (TTR) levels were measured in the plasma. After injection of FFP, total plasma TTR levels were elevated and variant TTR levels decreased from 24 to 48 h, accompanied by an elevation of plasma total protein, albumin levels and TTR levels. To elucidate the mechanism of this phenomenon, a large amount of purified normal TTR from normal human plasma was intravenously injected in mice and FAP patients. By intravenous injection of 3 mg of the purified TTR to C57Black6, the expression of TTR mRNA decreased from 6 to 24 h post injection, and gradually increased up to 48 h post injection. After injecting 400 mg of normal TTR in each of 3 FAP patients, total plasma TTR levels were elevated and variant TTR levels decreased significantly from 24 to 48 h. These results suggested that down regulation of the harmful protein by replacement of its normal form of the protein occurred by this method. This phenomenon should be applied as the basis for one of the useful methods for decreasing the harmful proteins in the circulation.

Autonomic dysfunction and anemia in neurologic disorders

The effect of autonomic dysfunctions on anemia in various neurological disorders, such as familial amyloidotic polyneuropathy (FAP) Type I, pandysautonomia, and Shy-Drager syndrome was examined. As a control, hemograms of patients with amyotrophic lateral sclerosis (ALS), which is known to be free from autonomic dysfunction, was compared with patients with the above neurological disorders. FAP and pandysautonomia patients showed significant anemia comparable with the severity of the autonomic dysfunctions. Shy-Drager patients exhibited mild anemia. However, in ALS patients, no such anemia was recognized at all even in the end stage of this disease. In pandysautonomia patients, hypoplastic bone marrow was recognized, which was quite consistent with the data previously reported in FAP patients. Human recombinant erythropoietin improved orthostatic hypotension as well as anemia in 4 FAP patients. These results suggest that autonomic dysfunction may be deeply connected with erythropoiesis.

Heterogeneity of clinical symptoms in patients with familial amyloidotic polyneuropathy (FAP TTR Met30)

Patients with familial amyloidotic polyneuropathy (FAP TTR Met 30) manifest clinical jindings, such as auto-nomic dysfunction, sensori-motor polyneuropathy, and visceral organ impairment with the progression of the disease. to clarih the clinical features of each patient and to determine the accurate clinical stages of FAt patients were given clinical scores and divided into 4 different groups by their clinical symptoms. Radar charts of the scores revealed that the shapes of the plotted scores between 2 different time examinations were similar until the bedridden stage of FAc suggesting that the clinical type of FAP had been determined at the onset of the disease and did not change as the disease progressed Retrospective stu4 revealed that most of the patients were near death when the total score became about 70. This evaluating method for FAP TTR Met30 may be useful in systematically evaluating the heterogeneity and the stage of each FAP patient, and also in elucidating the effect of therapies on the pat...

Role of nitric oxide in the peripheral vessels of patients with familial amyloidotic polyneuropathy (FAP) type I

In order to establish the generation of endothelial derived relaxating factor (EDRF) in patients with familial amyloidotic polyneuropathy (FAP) type I, the muscle temperature of the lower limb was measured with a deep portion thermometer. The temperature of the gastrocunemius muscle in FAP patients was significantly decreased compared with control subjects. In order to investigate the generation of EDRF in FAP patients, we administered NG-monomethyl-L-arginine (L-NMMA), specific inhibitor of nitric oxide, from the brachial artery and measured the changes in blood flow. Although 61.7 +/- 18.2% of the blood flow was transiently decreased after administration of L-NMMA in control subjects, FAP patients showed poor responses. In contrast, in the same way as in control subjects, significant vasodilatation was seen in FAP patients after administration of L-arginine. The urinary secreted NO2-/NO3- levels per day, which reflect the synthesis of nitric oxide in the systemic circulation, was a great deal lower in FAP patients than in control subjects. These results suggest that, although peripheral vessels can be dilated when a large amount of the substrate for NO synthase, L-arginine, is supplied, production of nitric oxide may be suppressed, and, as the result of this phenomenon, blood flow is decreased in the peripheral tissues of FAP patients in a static state.

Identification of a New Transthyretin Variant (Ile49) in Familial Amyloidotic Polyneuropathy Using Electrospray Ionization Mass Spectrometry and Nonisotopic RNase Cleavage Assay

Mutation of the transthyretin (TTR) plasma protein and gene in a Japanese patient with amyloid polyneuropathy was investigated by electrospray ionization mass spectrometry (ESI-MS) and nonisotopic RNase cleavage assay (NIRCA), respectively. ESI-MS analysis showed normal TTR peaks and additionally a variant TTR with 12-dalton-higher molecular weight than normal TTR. NIRCA suggested that the mutation existed near either the 5′ or 3′ end of exon 3. Direct DNA sequencing revealed both a normal ACC (threonine) and a variant ATC (isoleucine) at codon 49, which was located near the 5′ end of exon 3. The molecular weight shift of this mutation was 12 D, consistent with the result of ESI-MS.