Takafumi Ichikawa

LRRK2 Phosphorylates Tubulin-Associated Tau but Not the Free Molecule: LRRK2-Mediated Regulation of the Tau-Tubulin Association and Neurite Outgrowth

Leucine-rich repeat kinase 2 (LRRK2), a large protein kinase containing multi-functional domains, has been identified as the causal molecule for autosomal-dominant Parkinsons disease (PD). In the present study, we demonstrated for the first time that (i) LRRK2 interacts with tau in a tubulin-dependent manner; (ii) LRRK2 directly phosphorylates tubulin-associated tau, but not free tau; (iii) LRRK2 phosphorylates tau at Thr181 as one of the target sites; and (iv) The PD-associated LRRK2 mutations, G2019S and I2020T, elevated the degree of tau-phosphorylation. These results provide direct proof that tau is a physiological substrate for LRRK2. Furthermore, we revealed that LRRK2-mediated phosphorylation of tau reduces its tubulin-binding ability. Our results suggest that LRRK2 plays an important role as a physiological regulator for phosphorylation-mediated dissociation of tau from microtubules, which is an integral aspect of microtubule dynamics essential for neurite outgrowth and axonal transport.

Distinct effects of tetragastrin, histamine, and CCh on rat gastric mucin synthesis and contribution of NO

Although gastrin, histamine, and carbachol (CCh) accelerate gastric mucin metabolism, information about their target cells of mucin production is lacking. To clarify this, we examined the effects of these stimulants, including the possible participation of nitric oxide (NO), on mucin biosynthesis in distinct sites and layers of rat gastric mucosa. Pieces of tissue obtained from the corpus and antrum were incubated in a medium containing radioactive precursors and each stimulant, with or without NO synthase (NOS) inhibitor. Distribution of NOS was compared with that of the specific mucins by immunostaining using specific antiserum and monoclonal antibodies. In the full-thickness corpus mucosa, tetragastrin enhanced [3H]glucosamine incorporation into mucin but had no effect on [14C]threonine incorporation. Both histamine and CCh dose dependently increased3H- and14C-labeled corpus mucin. Only CCh stimulated antral mucin biosynthesis. CCh stimulation was noted in the corpus mucosa after removal of surface mucous cells, but stimulation by tetragastrin or histamine disappeared as a result of this pretreatment. Only tetragastrin-induced activation was completely blocked by the NOS inhibitor. NOS immunoreactivity was limited to surface mucous cells. Mucus-producing cells present in the different sites and layers of the gastric mucosa have distinct mechanisms for regulation of mucin biosynthesis. Gastrin-stimulated mucin biosynthesis mediated by NO is limited to surface mucous cells of rat gastric oxyntic mucosa.Although gastrin, histamine, and carbachol (CCh) accelerate gastric mucin metabolism, information about their target cells of mucin production is lacking. To clarify this, we examined the effects of these stimulants, including the possible participation of nitric oxide (NO), on mucin biosynthesis in distinct sites and layers of rat gastric mucosa. Pieces of tissue obtained from the corpus and antrum were incubated in a medium containing radioactive precursors and each stimulant, with or without NO synthase (NOS) inhibitor. Distribution of NOS was compared with that of the specific mucins by immunostaining using specific antiserum and monoclonal antibodies. In the full-thickness corpus mucosa, tetragastrin enhanced [3H]glucosamine incorporation into mucin but had no effect on [14C]threonine incorporation. Both histamine and CCh dose dependently increased 3H- and 14C-labeled corpus mucin. Only CCh stimulated antral mucin biosynthesis. CCh stimulation was noted in the corpus mucosa after removal of surface mucous cells, but stimulation by tetragastrin or histamine disappeared as a result of this pretreatment. Only tetragastrin-induced activation was completely blocked by the NOS inhibitor. NOS immunoreactivity was limited to surface mucous cells. Mucus-producing cells present in the different sites and layers of the gastric mucosa have distinct mechanisms for regulation of mucin biosynthesis. Gastrin-stimulated mucin biosynthesis mediated by NO is limited to surface mucous cells of rat gastric oxyntic mucosa.

Stimulatory effect of α-synuclein on the tau-phosphorylation by GSK-3β

Hyperphosphorylation of tau protein (tau) causes neurodegenerative diseases such as Alzheimer’s disease (AD). Recent studies of the physiological correlation between tau and α‐synuclein (α‐SN) have demonstrated that: (a) phosphorylated tau is also present in Lewy bodies, which are cytoplasmic inclusions formed by abnormal aggregation of α‐SN; and (b) the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) increases the phosphorylation of tau as well as the protein level of α‐SN in cultured neuronal cells, and also in mice. However, the molecular mechanism responsible for the α‐SN‐mediated hyperphosphorylation of tau remains to be elucidated. In this in vitro study, we found that: (a) α‐SN directly stimulates the phosphorylation of tau by glycogen synthase kinase‐3β (GSK‐3β), (b) α‐SN forms a heterotrimeric complex with tau and GSK‐3β, and (c) the nonamyloid beta component (NAC) domain and an acidic region of α‐SN are responsible for the stimulation of GSK‐3β‐mediated tau phosphorylation. Thus, it is concluded that α‐SN functions as a connecting mediator for tau and GSK‐3β, resulting in GSK‐3β‐mediated tau phosphorylation. Because the expression of α‐SN is promoted by oxidative stress, the accumulation of α‐SN induced by such stress may directly induce the hyperphosphorylation of tau by GSK‐3β. Furthermore, we found that heat shock protein 70 (Hsp70) suppresses the α‐SN‐induced phosphorylation of tau by GSK‐3β through its direct binding to α‐SN, suggesting that Hsp70 acts as a physiological suppressor of α‐SN‐mediated tau hyperphosphorylation. These results suggest that the cellular level of Hsp70 may be a novel therapeutic target to counteract α‐SN‐mediated tau phosphorylation in the initial stage of neurodegenerative disease.

Effects of substance P and calcitonin gene-related peptide on axonal transport in isolated and cultured adult mouse dorsal root ganglion neurons

Substance P and calcitonin gene-related peptide (CGRP) released from primary sensory neurons are known to play important roles in nociception and nociceptive transmission. In the present study, we attempted to clarify the roles of these neuropeptides in the regulation of axonal transport in sensory neurons. Cells were isolated from adult mouse dorsal root ganglia and cultured in F-12 medium containing fetal bovine serum for 48 h until their neurites were grown. These isolated and cultured DRG cells were mostly (>98%) small (diameter <25 microm) and medium (diameter, 25-40 microm) in size, and were immunoreactive for substance P and CGRP (85.9 and 66. 0% of total cells, respectively). Video-enhanced microscopy was applied to observe particles transported within neurites. Application of substance P (100 nM) decreased the number of particles transported in both anterograde and retrograde directions in each of DRG neurons tested (n=5). The instantaneous velocities of individual particles transported in anterograde and retrograde directions were also reduced by substance P. In contrast, alpha-CGRP (100 nM) increased the number of particles transported in both directions in each of DRG neurons tested (n=5), and also increased the instantaneous velocities of particles transported bidirectionally. Application of beta-CGRP (100-1000 nM) did not elicit any effect on axonal transport. Therefore, axonal transport in sensory neurons seems to be modulated by substance P and alpha-CGRP, both of which can be derived from its own and adjacent sensory neurons.

Leucine-rich repeat kinase 2 regulates tau phosphorylation through direct activation of glycogen synthase kinase-3β.

Leucine‐rich repeat kinase 2 (LRRK2) has been identified as the causal molecule for autosomal‐dominant Parkinsons disease (PD). Experimental evidence indicates that LRRK2 may play an important role in the pathology induced by abnormal phosphorylation of tau. In the present study, we demonstrated that LRRK2 directly associates with GSK‐3β, and that this interaction enhances the kinase activity of GSK‐3β. Furthermore, we found that LRRK2‐mediated activation of GSK‐3β induces high phosphorylation of tau at Ser396 in SH‐SY5Y cells. From our present findings, we conclude that LRRK2 may function as a novel enhancer for GSK‐3β and as a physiological regulator of neurite outgrowth and axonal transport through regulation of the GSK‐3β‐mediated phosphorylation of tau at the cellular level. Since LRRK2 is detected in tau‐positive inclusions in brain tissue affected by various neurodegenerative disorders, including PD, LRRK2‐stimulated phosphorylation of tau by GSK‐3β may be involved in development of pathological features in the initial stage of PD.

Changes in the mucus barrier of the rat during 5-fluorouracil-induced gastrointestinal mucositis

Objective. A frequent complication of antineoplastic chemotherapy (CT) is gastrointestinal (GI) mucositis. Although clinically this mucositis can be treated, data on the effect of CT on the mucosal defense mechanisms are scant, so the effects of 5-fluorouracil (5-FU) on mucin, one of the principal defense factors of the GI mucosa, were investigated. Material and methods. 5-FU was administered orally to rats at a dose of 50 mg/kg once daily for 5 days. Using anti-mucin monoclonal antibodies, the immunoreactivity in different areas of the rats’ GI tracts was compared, as well as the mucin content. Changes in the GI mucin during the process of recovery from the injury were also investigated. Immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) was used to determine whether or not the effects of 5-FU on cell proliferation contributed to the changes in mucin. Results. 5-FU caused significant alterations of the immunoreactivity and content of mucin in the rat GI mucosa, especially in the jejunum. The jejunal mucin content was most markedly reduced on day 1 after drug withdrawal, and increased thereafter. By day 7, the content had transiently but significantly increased approximately 1.5-fold, and returned to the basal level by day 13. The number of PCNA-positive cells strikingly decreased at day 1, but by day 7 had increased approximately 2-fold, compared with the control. Conclusion. The activation of mucus cells in the jejunum, if appropriately manipulated, could lead to more effective prevention of CT-induced GI mucositis.

A monoclonal antibody, PGM34, against 6-sulfated blood-group H type 2 antigen, on the carbohydrate moiety of mucin

Mucin, a major component of mucus, is a highly O‐glycosylated, high‐molecular‐mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site‐specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide‐alditols obtained from pig gastric mucin were fractionated by successive gel‐filtration, ion‐exchange, and normal‐phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide‐alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucα1–2Galβ1–4GlcNAc(6SO3H)β1–6(Fucα1–2Galβ1–3)GalNAc‐ol and Fucα1–2Galβ1–4GlcNAc(6SO3H)β1–6(Galβ1–3)GalNAc‐ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucα1–2Galβ1–4GlcNAc(6SO3H)β‐ sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.

Lafutidine-induced stimulation of mucin biosynthesis mediated by nitric oxide is limited to the surface mucous cells of rat gastric oxyntic mucosa

Although the new histamine H2 receptor antagonist, lafutidine (FRG-8813), N-[4-[4-(piperidinylmethyl)pyridyl-2-oxy]-(Z)-2-butenyl]-2-(fur furylsulfinyl)acetamide accelerates mucin metabolism of rat gastric mucosa, the physiological mechanisms by which this drug stimulates the biosynthesis remain unclear. In this paper, we report the effect of lafutidine on mucin biosynthesis in distinct sites and layers of rat gastric mucosa, including the possible participation of nitric oxide (NO). Lafutidine enhanced [3H]glucosamine incorporation into the mucin in the full thickness corpus mucosa, but not in the antrum. This stimulation on mucin biosynthesis disappeared by the removal treatment of surface mucosal cells. The lafutidine-induced increase of [3H]-labeled mucin in the corpus was completely blocked by either NG-nitro-L-arginine (10[-5] M) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazolne-1-oxyl-3-oxide (10[-5] M). The inhibitory action of NG-nitro-L-arginine was totally reversed by L-arginine (5 x 10[-3] M). These results suggest that the lafutidine-induced stimulation of mucin biosynthesis mediated by NO is limited to the surface mucous cells of rat gastric oxyntic mucosa.

Effects of a novel histamine H2-receptor antagonist, lafutidine, on the mucus barrier of human gastric mucosa

Background and Aim:  Lafutidine is a novel histamine H2‐receptor antagonist used primarily as an antisecretory agent in Japan. Previous human studies have not assessed its gastroprotective effects. The purpose of the present study was to determine the effects of lafutidine on the human gastric mucus layer using both histological and biochemical methods.

Mechanisms for cytoprotection by vitamin U from ethanol-induced gastric mucosal damage in rats

A comparison was made of the effects of a nonsulfhydryl compound, vitamin U (methylme-thioninesulfonium chloride, MMSC), and a sulfhydryl compound, cysteine (Cys), with regard to the inducement of acute gastric mucosal damage in the presence and absence ofN-ethylmaleimide (NEM), a sulfhydryl-blocking reagent. The effects of MMSC, Cys, or NEM on gastric mucin content were examined using a newly developed biochemical method. MMSC and Cys inhibited mucosal damage due to 50% ethanol. The preinjection of NEM had no effect on cytoprotection of prostaglandins, but prevented the effects of Cys and MMSC. MMSC and Cys increased surface mucin content but lessened that of deep mucin. NEM decreased surface mucin and increased deep mucin. It thus follows that sulfhydryl compounds accelerate the secretion of deep mucin and accumulate surface mucin. The cytoprotective mechanism of MMSC may thus be mediated by sulfhydryl compounds, and the increase in surface mucosal mucin may possibly be related to cytoprotection.