Hiroki Iwashita

Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma

Airway hyperresponsiveness (AHR), goblet cell metaplasia, and mucus overproduction are important features of bronchial asthma. To elucidate the molecular mechanisms behind these pulmonary pathologies, we examined for genes preferentially expressed in the lungs of a murine model of allergic asthma by using suppression subtractive hybridization (SSH). We identified a gene called gob-5 that had a selective expression pattern in the airway epithelium with AHR. Here, we show that gob-5, a member of the calcium-activated chloride channel family, is a key molecule in the induction of murine asthma. Intratracheal administration of adenovirus-expressing antisense gob-5 RNA into AHR-model mice efficiently suppressed the asthma phenotype, including AHR and mucus overproduction. In contrast, overexpression of gob-5 in airway epithelia by using an adenoviral vector exacerbated the asthma phenotype. Introduction of either gob-5 or hCLCA1, the human counterpart of gob-5, into the human mucoepidermoid cell line NCI-H292 induced mucus production as well as MUC5AC expression. Our results indicated that gob-5 may play a critical role in murine asthma, and its human counterpart hCLCA1 is therefore a potential target for asthma therapy.

2-{3-[4-(Alkylsulfinyl)phenyl]-1-benzofuran-5-yl}-5-methyl-1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-3beta with good brain permeability.

Glycogen synthase kinase 3beta (GSK-3beta) inhibition is expected to be a promising therapeutic approach for treating Alzheimers disease. Previously we reported a series of 1,3,4-oxadiazole derivatives as potent and highly selective GSK-3beta inhibitors, however, the representative compounds 1a,b showed poor pharmacokinetic profiles. Efforts were made to address this issue by reducing molecular weight and lipophilicity, leading to the identification of oxadiazole derivatives containing a sulfinyl group, (S)-9b and (S)-9c. These compounds exhibited not only highly selective and potent inhibitory activity against GSK-3beta but also showed good pharmacokinetic profiles including favorable BBB penetration. In addition, (S)-9b and (S)-9c given orally to mice significantly inhibited cold water stress-induced tau hyperphosphorylation in mouse brain.

A novel glycogen synthase kinase-3 inhibitor 2-methyl-5-(3-{4-[(S )-methylsulfinyl]phenyl}-1-benzofuran-5-yl)-1,3,4-oxadiazole decreases tau phosphorylation and ameliorates cognitive deficits in a transgenic model of Alzheimer’s disease

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07532.x

Efficacy of a novel, orally active GSK-3 inhibitor 6-Methyl-N-[3-[[3-(1-methylethoxy)propyl]carbamoyl]-1H-pyrazol-4-yl]pyridine-3-carboxamide in tau transgenic mice

Neurofibrillary tangles (NFTs) composed of hyperphosphorylated and aggregated tau are common pathological characteristics in Alzheimers disease (AD) and other tauopathies. Aberrant tau phosphorylation is an early and pivotal event in the pathogenesis of tauopathies, and since GSK-3 is a key factor implicated in aberrant tau phosphorylation, GSK-3 inhibition is expected to suppress tauopathy disease progression. In the present study, we report the efficacy of a newly discovered small molecule GSK-3 inhibitor, 6-methyl-N-[3-[[3-(1-methylethoxy)propyl]carbamoyl]-1H-pyrazol-4-yl]pyridine-3-carboxamide (compound A), to inhibit tau phosphorylation and to reduce the amount of pathological aggregated tau in JNPL3 mice that overexpress a mutant form of human tau. Compound A is a highly potent and selective inhibitor of GSK-3 with an IC(50) of 2 nM, with at least 230-fold lower potency against 27 other kinases. Oral administration of compound A resulted in a significant reduction of tau phosphorylation at several GSK-3 directed sites. Furthermore, chronic oral administration of compound A markedly reduced aggregated tau in old JNPL3 mice. These results suggest that a novel, orally active GSK-3 inhibitor, compound A, has potency in the prevention of tau pathology.

Increased human Ca2+-activated Cl- channel 1 expression and mucus overproduction in airway epithelia of smokers and chronic obstructive pulmonary disease patients

BackgroundThe mechanisms underlying the association between smoking and mucus overproduction remain unknown. Because of its involvement in other airway diseases, such as asthma, we hypothesized that Ca2+-activated Cl- channel 1 (CLCA1) was associated with overproduction of mucus in the airways of smokers and COPD patients.MethodsUsing real-time quantitative PCR analyses, we compared the CLCA1 mRNA expression levels in induced-sputum cells from COPD patients (n = 20), smokers without COPD (n = 5), and non-smokers (n =13). We also examined the relationship between CLCA1 protein expression and mucus production in lung airway epithelia of COPD patients (n = 6), smokers without COPD (n = 7), and non-smokers (n = 7).ResultsCLCA1 mRNA expression was significantly up-regulated in the induced-sputum cells of COPD patients compared with cells of non-smokers (p = 0.02), but there was no significant difference compared with cells of smokers without COPD. Using immunostaining with an anti-CLCA1 antibody, semi-quantitative image analyses of airway epithelium demonstrated significantly increased CLCA1 expression in smokers without COPD (p = 0.02) and in COPD patients (p = 0.002) compared with non-smokers. There were significant negative correlations between CLCA1 protein expression and FEV1/FVC (r = −0.57, p = 0.01) and %predicted FEV1 (r = −0.56, p = 0.01). PAS staining for mucus showed that there was a significant positive correlation between CLCA1 protein expression and mucus production (r = 0.67, p = 0.001). These markers were significantly increased in smokers without COPD (p = 0.04) and in COPD patients (p = 0.003) compared with non-smokers (non-smokers < smokers ≤ COPD).ConclusionsCLCA1 expression is significantly related to mucus production in the airway epithelia of smokers and COPD patients, and may contribute to the development and pathogenesis of COPD by inducing mucus production.

Phosphodiesterase 2A Inhibitor TAK-915 Ameliorates Cognitive Impairments and Social Withdrawal in N-Methyl-d-Aspartate Receptor Antagonist–Induced Rat Models of Schizophrenia

The pathophysiology of schizophrenia has been associated with glutamatergic dysfunction. Modulation of the glutamatergic signaling pathway, including N-methyl-d-aspartate (NMDA) receptors, can provide a new therapeutic target for schizophrenia. Phosphodiesterase 2A (PDE2A) is highly expressed in the forebrain, and is a dual substrate enzyme that hydrolyzes both cAMP and cGMP, which play pivotal roles as intracellular second messengers downstream of NMDA receptors. Here we characterize the in vivo pharmacological profile of a selective and brain-penetrant PDE2A inhibitor, (N-{(1S)-1-[3-fluoro-4-(trifluoromethoxy)phenyl]-2-methoxyethyl}-7-methoxy-2-oxo-2,3-dihydropyrido[2,3-b]pyrazine-4(1H)-carboxamide) (TAK-915) as a novel treatment of schizophrenia. Oral administration of TAK-915 at 3 and 10 mg/kg significantly increased cGMP levels in the frontal cortex, hippocampus, and striatum of rats. TAK-915 at 10 mg/kg significantly upregulated the phosphorylation of α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptor subunit GluR1 in the rat hippocampus. TAK-915 at 3 and 10 mg/kg significantly attenuated episodic memory deficits induced by the NMDA receptor antagonist (+)-MK-801 hydrogen maleate (MK-801) in the rat passive avoidance test. TAK-915 at 10 mg/kg significantly attenuated working memory deficits induced by MK-801 in the rat radial arm maze test. Additionally, TAK-915 at 10 mg/kg prevented subchronic phencyclidine-induced social withdrawal in social interaction in rats. In contrast, TAK-915 did not produce antipsychotic-like activity; TAK-915 had little effect on MK-801- or methamphetamine-induced hyperlocomotion in rats. These results suggest that TAK-915 has a potential to ameliorate cognitive impairments and social withdrawal in schizophrenia.

A novel glycogen synthase kinase-3 inhibitor 2-methyl-5-(3-{4-[(s)-methylsulfinyl]phenyl}-1-benzofuran-5-yl)-1,3,4-oxadiazole (mmbo) decreased tau phosphorylation and ameliorated cognitive deficits in a transgenic model of Alzheimer's disease

potential epitope specificity and safety of this promising therapeutic effect, we are examining several tau epitopes. Here we assessed the efficacy of using a pseudo-phosphorylated tau immunogen. Methods: Homozygous JNPL3 mice were immunized with Tau379-408[ESer396, E-Ser404] in alum adjuvant (n 1⁄4 13) or with adjuvant only (n 1⁄4 7), starting at 2 months. Mice were tested on various sensorimotor tasks (rotarod, traverse beam, locomotor activity and grip strength) at 5 and 8 months of age. Antibody titers were determined and at 8 months their brains were processed for tau biochemistry and histology. Results: The vaccine elicited a robust antibody response towards the immunogen, and its phosphorylated and non-phosphorylated analogs. Which is as expected since this region of the tau protein is highly immunogenic. The immunized mice had a 24% reduction in soluble PHF1/total tau ratio on western blots (p 1⁄4 0.04), and a 42% reduction in PHF1 immunostaining in the dentate gyrus (p < 0.03), compared to alum-treated mice. Levels of sarkosyl insoluble human and total tau were highly variable in both groups and not significantly different. Biochemical and histological analyses with other antibodies and of other brain regions is underway. Disappointingly, potential improvements in motor function of the immunized mice could not be assessed since the animals did not develop overt signs of such impairments at the ages tested, in contrast to our previous observation in the same homozygous model (Asuni A. et al., J. Neurosci., 2007). Unfortunately, such changes in phenotype are commonly observed in transgenic mice.Conclusions: These findings indicate that immunological targeting using a pseudo-phosphorylated tau epitope can reduce pathological tau within the brain, further supporting the feasibility of tau immunotherapy.

Efficacy of a novel, orally active GSK-3 Inhibitor 6-Methyl-N-[3-[[3-(1-methylethoxy)propyl]carbamoyl]-1H-pyrazol-4-yl]pyridine-3-carboxamide in tau transgenic mice

hibit phosphorylation of Tau on several AD-relevant Tau epitopes. 21,22 Specifically, at early stages (3 months of age), hyperphosphorylated Tau protein are visualized (AD2, AT270) in the hippocampus while abnormal Tau species (AT100, pS422) are faintly observed. Over the time, hippocampal pathology increases with strong AT100 and pS422 immuno reactivity’s at 8-10 months of age, ultimately correlated with prominent cognitive deterioration. Three months old THY-Tau22 mice were immunized with a peptide including the phosphoserine 422 residuewhile control mice received the adjuvant alone. After optimization, a protocol (time of immunization and peptide length) was set up and different parameters were analyzed. Results: A specific antibody response against the pSer422 epitope was observed. We noticed a decrease in insoluble tau species (AT100and pS422 immunoreactive) correlate with a significant memory improvement using the Y-maze spatial memory task. Conclusions: Overall, our data support that immunotherapy targeting pathological pSer422 epitope leads to a specific response against pathological Tau and may improve cognitive deficits promoted by Tau pathology.