Yoichiro Abe

Curcumin and genistein additively potentiate G551D-CFTR

BACKGROUND The G551D mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is a common cause of cystic fibrosis (CF). G551D-CFTR is characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Numerous small molecules have been shown to increase the activity of G551D-CFTR presumably by binding to the CFTR protein. METHODS We investigated the effect of curcumin, genistein and their combined application on G551D-CFTR activity using the patch clamp technique. RESULTS Curcumin increased G551D-CFTR whole-cell and single-channel currents less than genistein did at their maximally effective concentrations. However, curcumin further increased the channel activity of G551D-CFTR that had been already maximally potentiated by genistein, up to ~50% of the WT-CFTR level. In addition, the combined application of genistein and curcumin over a lower concentration range synergistically rescued the gating defect of G551D-CFTR. CONCLUSIONS The additive effects between curcumin and genistein not only support the hypothesis that multiple mechanisms are involved in the action of CFTR potentiators, but also pose pharmaceutical implications in the development of drugs for CF pharmacotherapy.

Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol uptake/transporter 1, acts as a negative regulator for N-terminal palmitoylation of Sonic hedgehog.

Mammalian glycerol uptake/transporter 1 (Gup1), a homolog of Saccharomyces cerevisiae Gup1, is predicted to be a member of the membrane‐bound O‐acyltransferase family and is highly homologous to mammalian hedgehog acyltransferase, known as Skn, the homolog of the Drosophila skinny hedgehog gene product. Although mammalian Gup1 has a sequence conserved among the membrane‐bound O‐acyltransferase family, the histidine residue in the motif that is indispensable to the acyltransferase activity of the family has been replaced with leucine. In this study, we cloned Gup1 cDNA from adult mouse lung and examined whether Gup1 is involved in the regulation of N‐terminal palmitoylation of Sonic hedgehog (Shh). Subcellular localization of mouse Gup1 was indistinguishable from that of mouse Skn detected using the fluorescence of enhanced green fluorescent protein that was fused to each C terminus of these proteins. Gup1 and Skn were co‐localized with an endoplasmic reticulum marker, 78 kDa glucose‐regulated protein, suggesting that these two molecules interact with overlapped targets, including Shh. In fact, full‐length Shh coprecipitated with FLAG‐tagged Gup1 by immunoprecipitation using anti‐FLAG IgG. Ectopic expression of Gup1 with full‐length Shh in cells lacking endogenous Skn showed no hedgehog acyltransferase activity as determined using the monoclonal antibody 5E1, which was found to recognize the palmitoylated N‐terminal signaling domain of Shh under denaturing conditions. On the other hand, Gup1 interfered with the palmitoylation of Shh catalyzed by endogenous Skn in COS7 and NSC34. These results suggest that Gup1 is a negative regulator of N‐terminal palmitoylation of Shh and may contribute to the variety of biological actions of Shh.

Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [(32)P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition.

Immunological function of aquaporin-4 in stab-wounded mouse brain in concert with a pro-inflammatory cytokine inducer, osteopontin.

During injury to the central nervous system (CNS), astrocytes and microglia proliferate and migrate around the lesion sites. Recently, it has been reported that one of the water channels, aquaporin-4 (AQP4) is seemed to have a role in astroglial migration and glial scar formation caused by brain injury, although its molecular mechanism is largely unknown. In the present study, we examined the expression profiles in wild-type (WT) and AQP4-deficient (AQP4/KO) mice after a stab wound to the cerebral cortex. Three days after the stab wound, AQP4 expression was observed in activated microglia around the lesion site as well as in astrocytes. A microarray analysis revealed that 444 genes around the lesion site were upregulated 3 days after the wounding in WT mice. Surprisingly, most of these up-regulations were significantly attenuated in AQP4/KO mice. Real-time RT-PCR and immunofluorescence showed that osteopontin (OPN) expression around the lesion site was much lower in AQP4/KO mice than in WT mice. Moreover, the up-regulation of pro-inflammatory cytokines was significantly attenuated in AQP4/KO mice. Taken together, these results suggest that AQP4 plays an important role in immunological function in concert with OPN under pathological conditions in the CNS.

Dynamic subcellular localization of aquaporin-7 in white adipocytes

Aquaporin‐7 (AQP7) is expressed in adipose tissue, permeated by water and glycerol, and is involved in lipid metabolism. AQP7‐null mice develop obesity, insulin resistance, and adipocyte hypertrophy. Here, we show that AQP7 is expressed in adipocyte plasma membranes, and is re‐localized to intracellular membranes in response to catecholamine in mouse white adipose tissue. We found that internalization of AQP7 was induced by PKA activation and comparative gene identification 58 (CGI‐58). This relocation was confirmed by functional studies in 3T3‐L1 adipocytes. Collectively, these results suggest that AQP7 makes several contributions to adipocyte metabolism, in both cortical and intracellular membranes.

SUMO3 Modification Accelerates the Aggregation of ALS-Linked SOD1 Mutants

Mutations in superoxide dismutase 1 (SOD1) are a major cause of familial amyotrophic lateral sclerosis (ALS), whereby the mutant proteins misfold and aggregate to form intracellular inclusions. We report that both small ubiquitin-like modifier (SUMO) 1 and SUMO2/3 modify ALS-linked SOD1 mutant proteins at lysine 75 in a motoneuronal cell line, the cell type affected in ALS. In these cells, SUMO1 modification occurred on both lysine 75 and lysine 9 of SOD1, and modification of ALS-linked SOD1 mutant proteins by SUMO3, rather than by SUMO1, significantly increased the stability of the proteins and accelerated intracellular aggregate formation. These findings suggest the contribution of sumoylation, particularly by SUMO3, to the protein aggregation process underlying the pathogenesis of ALS.

Sustained Down-regulation of β-Dystroglycan and Associated Dysfunctions of Astrocytic Endfeet in Epileptic Cerebral Cortex

Background: The molecular and cellular changes of astrocytes in epileptic cerebral cortex are poorly understood. Results: In the epileptic cortex, β-dystroglycan is down-regulated in a sustained manner with a concomitant impairment in the integrity of the astrocyte endfeet. Conclusion: Astrocyte endfeet undergo long lasting changes in the epileptic cortex. Significance: The pathological changes of astrocyte endfeet could be a new therapeutic target for epilepsy. Epilepsy is characterized by the abnormal activation of neurons in the cerebral cortex, but the molecular and cellular mechanisms contributing to the development of recurrent seizures are largely unknown. Recently, the critical involvement of astrocytes in the pathophysiology of epilepsy has been proposed. However, the nature of plastic modulations of astrocytic proteins in the epileptic cortex remains poorly understood. In this study, we utilized the zero magnesium in vitro model of epilepsy and examined the potential molecular changes of cortical astrocytes, focusing specifically on endfeet, where specialized biochemical compartments exist. We find that the continuous epileptic activation of neurons for 1 h decreases the expression level of β-dystroglycan (βDG) in acute cortical brain slices prepared from mice. This change is completely abolished by the pharmacological blockade of NMDA-type glutamate receptors as well as by matrix metalloproteinase inhibitors. Consistent with the highly specialized localization of βDG at astrocytic endfeet, where it plays a pivotal role in anchoring endfeet-enriched proteins in astrocytes, the down-regulation of βDG is accompanied by a decrease in the expression of AQP4 but not laminin. Importantly, this down-regulation of βDG persists for at least 1 h, even after the apparent recovery of neuronal activation. Finally, we show that the down-regulation of βDG is associated with the dysfunction of the endfeet at the blood-brain interface as a diffusion barrier. These results suggest that the sustained down-regulation of βDG leads to dysfunctions of astrocytic endfeet in the epileptic cerebral cortex and may contribute to the pathogenesis of epilepsy.

Expression and localization of aquaporin-4 in sensory ganglia

Aquaporin-4 (AQP4) is a water channel protein that is predominantly expressed in astrocytes in the CNS. The rapid water flux through AQP4 may contribute to electrolyte/water homeostasis and may support neuronal activities in the CNS. On the other hand, little is known about the expression of AQP4 in the peripheral nervous system (PNS). Using AQP4(-/-) mice as a negative control, we demonstrated that AQP4 is also expressed in sensory ganglia, such as trigeminal ganglia and dorsal root ganglia in the PNS. Immunohistochemistry revealed that AQP4 is exclusively localized to satellite glial cells (SGCs) surrounding the cell bodies of the primary afferent sensory neurons in the sensory ganglia. Biochemical analyses revealed that the expression levels of AQP4 in sensory ganglia were considerably lower than those in astrocytes in the CNS. Consistently, behavioral analyses did not show any significant difference in terms of mechanical and cold sensitivity between wild type and AQP4(-/-) mice. Overall, although the pathophysiological relevance of AQP4 in somatosensory perception remains unclear, our findings provide new insight into the involvement of water homeostasis in the peripheral sensory system.

An astrocyte-specific enhacer of the aquaporin-4 gene functions through a consensus sequence of POU transcription factors in concert with multiple upstream elements

J. Neurochem. (2012) 120, 899–912.

Cytotoxic mechanisms by M239V presenilin 2, a little-analyzed Alzheimer's disease-causative mutant

Although neurotoxic functions are well characterized in familial Alzheimers disease (FAD)‐linked N141I mutant of presenilin (PS)2, little has been known about M239V‐PS2, another established FAD‐causative mutant. We found that expression of M239V‐PS2 caused neuronal cytotoxicity. M239V‐PS2 exerted three forms of cytotoxicity: one was sensitive to both an antioxidant glutathione‐ethyl‐ester (GEE) and a caspase inhibitor Ac‐DEVD‐CHO (DEVD); the second was sensitive to GEE but resistant to DEVD; and the third was resistant to both. The GEE/DEVD‐sensitive cytotoxicity by M239V‐PS2 was likely through NADPH oxidase and the GEE‐sensitive/DEVD‐resistant cytotoxicity through xanthine oxidase (XO). Both mechanisms by M239V‐PS2 were suppressed by pertussis toxin (PTX) and were mediated by Gαo, but not by Gαi. Although Aβ1–43 itself induced no cytotoxicity, Aβ1–43 potentiated all three components of M239V‐PS2 cytotoxicity. As these cytotoxic mechanisms by M239V‐PS2 are fully shared with N141I‐PS2, they are most likely implicated in the pathomechanism of FAD by PS2 mutations. Notably, cytotoxicity by M239V‐PS2 could be inhibited by the combination of two clinically usable inhibitors of superoxide‐generating enzymes, apocynin and oxypurinol.