Dynamic interaction between sigma-1 receptor and Kv1.2 shapes neuronal and behavioral responses to cocaine.

The sigma-1 receptor (Sig-1R), an endoplasmic reticulum (ER) chaperone protein, is an interorganelle signaling modulator that potentially plays a role in drug-seeking behaviors. However, the brain site of action and underlying cellular mechanisms remain unidentified. We found that cocaine exposure triggers a Sig-1R-dependent upregulation of D-type K(+) current in the nucleus accumbens (NAc) that results in neuronal hypoactivity and thereby enhances behavioral cocaine response. Combining ex vivo and in vitro studies, we demonstrated that this neuroadaptation is caused by a persistent protein-protein association between Sig-1Rs and Kv1.2 channels, a phenomenon that is associated to a redistribution of both proteins from intracellular compartments to the plasma membrane. In conclusion, the dynamic Sig-1R-Kv1.2 complex represents a mechanism that shapes neuronal and behavioral response to cocaine. Functional consequences of Sig-1R binding to K(+) channels may have implications for other chronic diseases where maladaptive intrinsic plasticity and Sig-1Rs are engaged.

Effects of a Psychoeducation Program for People with Schizophrenia Aimed at Increasing Subjective Well-Being and the Factors Influencing Those Effects: a Preliminary Study.

There have been no intervention studies of psychoeducation programs for schizophrenia that focus on improving subjective well-being or studies to determine the factors influencing such effects. This study aimed to examine the effects of a psychoeducation program combining traditional psychoeducation with a focus on providing knowledge and information and a new intervention to raise patients' subjective well-being and to clarity the factors affecting the program's efficacy. Subjects were 117 patients who participated in a psychoeducation program for schizophrenia between 2012 and 2018. In addition to comparing subjective well-being (Subjective Well-being under Neuroleptic Drug Treatment Short Form, Japanese version, SWNS-J) and attitudes towards drugs (Drug Attitude Inventory-10, DAI-10) before and after the program, basic information such as psychiatric symptoms was surveyed. The factors influencing the effects of the program were assessed with multiple regression analysis. Scores for SWNS-J subscales and total SWNS-J score increased significantly after the program. Higher total scores on subjective well-being after the program were significantly associated with having less severe negative symptoms and higher total subjective well-being before the program, and with more positive attitudes toward drugs after the program. These results suggest that intervention with a focus on improving subjective well-being can be an effective part of psychoeducation programs for patients with schizophrenia. The results also suggest that the level of improvement in subjective well-being gained from the program may be higher in patients with milder negative symptoms and patients with a better understanding of drug treatment.

Identification of Middle Chain Fatty Acyl-CoA Ligase Responsible for the Biosynthesis of 2-Alkylmalonyl-CoAs for Polyketide Extender Unit.

Understanding the biosynthetic mechanism of the atypical polyketide extender unit is important for the development of bioactive natural products. Reveromycin (RM) derivatives produced by Streptomyces sp. SN-593 possess several aliphatic extender units. Here, we studied the molecular basis of 2-alkylmalonyl-CoA formation by analyzing the revR and revS genes, which form a transcriptional unit with the revT gene, a crotonyl-CoA carboxylase/reductase homolog. We mainly focused on the uncharacterized adenylate-forming enzyme (RevS). revS gene disruption resulted in the reduction of all RM derivatives, whereas reintroduction of the gene restored the yield of RMs. Although RevS was classified in the fatty acyl-AMP ligase clade based on phylogenetic analysis, biochemical characterization revealed that the enzyme catalyzed the middle chain fatty acyl-CoA ligase (FACL) but not the fatty acyl-AMP ligase activity, suggesting the molecular evolution for acyl-CoA biosynthesis. Moreover, we examined the in vitro conversion of fatty acid into 2-alkylmalonyl-CoA using purified RevS and RevT. The coupling reaction showed efficient conversion of hexenoic acid into butylmalonyl-CoA. RevS efficiently catalyzed C8-C10 middle chain FACL activity; therefore, we speculated that the acyl-CoA precursor was truncated via ß-oxidation and converted into (E)-2-enoyl-CoA, a RevT substrate. To determine whether the ß-oxidation process is involved between the RevS and RevT reaction, we performed the feeding experiment using [1,2,3,4-(13)C]octanoic acid. (13)C NMR analysis clearly demonstrated incorporation of the [3,4-(13)C]octanoic acid moiety into the structure of RM-A. Our results provide insight into the role of uncharacterized RevS homologs that may catalyze middle chain FACL to produce a unique polyketide extender unit.

The non-apoptotic action of Bcl-xL: regulating Ca(2+) signaling and bioenergetics at the ER-mitochondrion interface.

Bcl-2 family proteins are known to competitively regulate Ca(2+); however, the specific inter-organelle signaling pathways and related cellular functions are not fully elucidated. In this study, a portion of Bcl-xL was detected at the ER-mitochondrion interface or MAM (mitochondria-associated ER membrane) in association with type 3 inositol 1,4,5-trisphosphate receptors (IP3R3); an association facilitated by the BH4 and transmembrane domains of Bcl-xL. Moreover, increasing Bcl-xL expression enhanced transient mitochondrial Ca(2+) levels upon ER Ca(2+) depletion induced by short-term, non-apoptotic incubation with thapsigargin (Tg), while concomitantly reducing cytosolic Ca(2+) release. These mitochondrial changes appear to be IP3R3-dependent and resulted in decreased NAD/NADH ratios and higher electron transport chain oxidase activity. Interestingly, extended Tg exposure stimulated ER stress, but not apoptosis, and further enhanced TCA cycling. Indeed, confocal analysis indicated that Bcl-xL translocated to the MAM and increased its interaction with IP3R3 following extended Tg treatment. Thus, the MAM is a critical cell-signaling junction whereby Bcl-xL dynamically interacts with IP3R3 to coordinate mitochondrial Ca(2+) transfer and alters cellular metabolism in order to increase the cells' bioenergetic capacity, particularly during periods of stress.

Sigma-1 receptor: the novel intracellular target of neuropsychotherapeutic drugs.

Sigma-1 receptor ligands have been long expected to serve as drugs for treatment of human diseases such as neurodegenerative disorders, depression, idiopathic pain, drug abuse, and cancer. Recent research exploring the molecular function of the sigma-1 receptor started unveiling underlying mechanisms of the therapeutic activity of those ligands. Via the molecular chaperone activity, the sigma-1 receptor regulates protein folding/degradation, ER/oxidative stress, and cell survival. The chaperone activity is activated or inhibited by synthetic sigma-1 receptor ligands in an agonist-antagonist manner. Sigma-1 receptors are localized at the endoplasmic reticulum (ER) membranes that are physically associated with the mitochondria (MAM: mitochondria-associated ER membrane). In specific types of neurons (e.g., those at the spinal cord), sigma-1 receptors are also clustered at ER membranes that juxtapose postsynaptic plasma membranes. Recent studies indicate that sigma-1 receptors, partly in sake of its unique subcellular localization, regulate the mitochondria function that involves bioenergetics and free radical generation. The sigma-1 receptor may thus provide an intracellular drug target that enables controlling ER stress and free radical generation under pathological conditions.

Conversion of psychological stress into cellular stress response: roles of the sigma-1 receptor in the process.

Psychiatrists empirically recognize that excessive or chronic psychological stress can result in long-lasting impairments of brain functions that partly involve neuronal cell damage. Recent studies begin to elucidate the molecular pathways activated/inhibited by psychological stress. Activation of the hypothalamic-pituitary-adrenal axis under psychological stress causes inflammatory oxidative stresses in the brain, in part due to elevation of cytokines. Psychological stress or neuropathological conditions (e.g., accumulation of ß-amyloids) trigger 'cellular stress responses', which promote upregulation of molecular chaperones to protect macromolecules from degradation. The unfolded protein response, the endoplasmic reticulum (ER)-specific cellular stress response, has been recently implicated in the pathophysiology of neuropsychiatric disorders and the pharmacology of certain clinically used drugs. The sigma-1 receptor is an ER protein whose ligands are shown to exert antidepressant-like and neuroprotective actions. Recent studies found that the sigma-1 receptor is a novel ligand-operated ER chaperone that regulates bioenergetics, free radical generation, oxidative stress, unfolded protein response and cytokine signaling. The sigma-1 receptor also regulates morphogenesis of neuronal cells, such as neurite outgrowth, synaptogenesis, and myelination, which can be perturbed by cellular stress. The sigma-1 receptor may thus contribute to a cellular defense system that protects nervous systems against chronic psychological stress. Findings from sigma receptor research imply that not only cell surface monoamine effectors but also intracellular molecules, especially those at the ER, may provide novel therapeutic targets for future drug developments.

First Japanese family with primary familial brain calcification due to a mutation in the PDGFB gene: an exome analysis study.

AIMS: Primary familial brain calcification (PFBC) is a rare disorder characterized by abnormal deposits of calcium in the basal ganglia and cerebellum. PFBC can present with a spectrum of neuropsychiatric symptoms resembling those seen in dementia and schizophrenia. Mutations in a few genes have been identified as causing PFBC: namely, the SLC20A2 gene that codes for the sodium-dependent phosphate transporter and the PDGFRB gene that codes for the platelet-derived growth factor receptor ß (PDGF-Rß). A recent study identified mutations in PDGFB coding for PDGF-B, the main ligand for PDGF-Rß, in six families with PFBC. Here we report the first Japanese family with PFBC carrying a mutation in PDGFB, which causes the substitution of an arginine with a stop codon at amino acid 149 of the PDGF-B protein (p. Arg149*). METHODS: Clinical histories and computed tomography scan images were provided. Sanger sequencing was performed for the exome analysis of SLC20A2 and PDGFB genes. RESULTS: One family member began to complain of auditory hallucination at 16 years of age and had been treated for schizophrenia. His father suffered from memory and gait disturbances in his late 60s. A computed tomography scan revealed a symmetrical area of calcification over the basal ganglia in both cases. A known mutation in PDGFB (c.445C>T, p.Arg149*) was consistently detected in both PFBC cases by Sanger sequencing. No mutations in SLC20A2 were detected. CONCLUSIONS: Our findings suggest that this mutation in PDGF-B is responsible for PFBC in this Japanese family and that abnormal PDGF signaling may be involved in the pathophysiology of certain psychiatric disorders.

Intractable hiccups (singultus) abolished by risperidone, but not by haloperidol.

Hiccups or singulata are rhythmic involuntary movements of the diaphragm, caused by a variety of conditions that interfere with the functions of the nerve nuclei in the medulla and supra-spinal hiccup center. Although neurotransmitters and receptors involved in the pathophysiology of hiccups are not defined well, dopamine has been considered to play an important role. In some cases, chlorpromazine or other antipsychotics are used for the treatment of intractable hiccups but their efficacy is often limited. This report involves an 18-year-old patient who experienced two episodes of intractable hiccups triggered by stress, which lasted for weeks or even months. In both episodes, haloperidol was initially used, but there was no significant effect. In contrast, risperidone, the second-generation antipsychotic that possesses a dopamine-serotonin antagonist property, completely abolished the hiccups 6 hours after administration. This is one of few case reports in which two antipsychotics were challenged for a single patient with hiccups, and the effects of the drugs were obviously different. Our finding suggests that, in addition to dopaminergic system, the serotonergic systems may be involved in the pathophysiology of some hiccup cases and that the serotonin-acting antipsychotics such as risperidone should be considered as a choice in the drug treatment of intractable hiccups.

Bcl-2 family in inter-organelle modulation of calcium signaling; roles in bioenergetics and cell survival.

Bcl-2 family proteins, known for their apoptosis functioning at the mitochondria, have been shown to localize to other cellular compartments to mediate calcium (Ca2+) signals. Since the proper supply of Ca2+ in cells serves as an important mechanism for cellular survival and bioenergetics, we propose an integrating role for Bcl-2 family proteins in modulating Ca2+ signaling. The endoplasmic reticulum (ER) is the main Ca2+ storage for the cell and Bcl-2 family proteins competitively regulate its Ca2+ concentration. Bcl-2 family proteins also regulate the flux of Ca2+ from the ER by physically interacting with inositol 1,4,5-trisphosphate receptors (IP3Rs) to mediate their opening. Type 1 IP3Rs reside at the bulk ER to coordinate cytosolic Ca2+ signals, while type 3 IP3Rs reside at mitochondria-associated ER membrane (MAM) to facilitate mitochondrial Ca2+ uptake. In healthy cells, mitochondrial Ca2+ drives pyruvate into the citric acid (TCA) cycle to facilitate ATP production, while a continuous accumulation of Ca2+ can trigger the release of cytochrome c, thus initiating apoptosis. Since multiple organelles and Bcl-2 family proteins are involved in Ca2+ signaling, we aim to clarify the role that Bcl-2 family proteins play in facilitating Ca2+ signaling and how mitochondrial Ca2+ is relevant in both bioenergetics and apoptosis. We also explore how these insights could be useful in controlling bioenergetics in apoptosis-resistant cell lines.

A feasibility study of provider-level implementation strategies to improve access to colorectal cancer screening for patients with schizophrenia: ACCESS2 (N-EQUITY 2104) trial.

BACKGROUND: People with schizophrenia have a lower colorectal screening rate than the general population. A previous study reported an intervention using case management to encourage colorectal cancer screening for patients with schizophrenia in psychiatric outpatient settings. In this feasibility study, we developed provider-level implementation strategies and evaluated the feasibility of conducting a definitive trial in terms of the penetration of the intervention assessed at the patient level. Additionally, we examined the fidelity of strategies to implement the intervention at the provider level in a routine clinical psychiatric setting. METHODS: This was a multicenter, single-arm feasibility study with medical staff at psychiatric hospitals in Japan. The provider-level implementation strategies developed in this study included three key elements (organizing an implementation team appointed by the facility director, interactive assistance using a clear guide that outlines who in the hospital should do what, and developing accessible educational materials) to overcome major barriers to implementation of the intervention and four additional elements (progress monitoring, joint meetings and information sharing among participating sites, adaptation of encouragement methods to specific contexts, and education of on-site staff), with reference to the elements identified in the Expert Recommendations for Implementing Change (ERIC). The feasibility of the strategies was evaluated by the proportion of patients who were eligible for encouragement (patients with schizophrenia aged 40, 50, or 60) who received encouragement. We set the goal of providing encouragement to at least 40% of eligible patients at each site. RESULTS: Two public and four private psychiatric hospitals in Okayama and Shimane prefectures participated in this study. Regarding fidelity, all elements of the strategies were conducted as planned. Following the procedures in the guide, each team prepared and provided encouragement according to their own facility and region. Penetration, defined as the proportion of eligible patients who received encouragement, ranged from 33.3 to 100%; five of the six facilities achieved the target proportion. CONCLUSIONS: The provider-level implementation strategies to implement encouragement were feasible in terms of penetration of the intervention assessed at the patient level. The results support the feasibility of proceeding with a future definitive trial of these strategies. TRIAL REGISTRATION: jRCT, jRCT1060220026 . Registered on 06/04/2022.

The lifetime of UDP-galactose:ceramide galactosyltransferase is controlled by a distinct endoplasmic reticulum-associated degradation (ERAD) regulated by sigma-1 receptor chaperones.

The glycosphingolipid biosynthesis is initiated by monoglycosylation of ceramides, the action of which is catalyzed either by UDP-glucose:ceramide glucosyltransferase or by UDP-galactose:ceramide galactosyltransferase (CGalT). CGalT is expressed predominantly at the endoplasmic reticulum (ER) of oligodendrocytes and is responsible for synthesizing galactosylceramides (GalCer) that play an important role in regulation of axon conductance. However, despite the importance of ceramide monoglycosylation enzymes in a spectrum of cellular functions, the mechanism that fine tunes activities of those enzymes is largely unknown. In the present study, we demonstrated that the sigma-1 receptor (Sig-1R) chaperone, the mammalian homologue of a yeast C8-C7 sterol isomerase, controls the protein level and activity of the CGalT enzyme via a distinct ER-associated degradation system involving Insig. The Sig-1R forms a complex with Insig via its transmembrane domain partly in a sterol-dependent manner and associates with CGalT at the ER. The knockdown of Sig-1Rs dramatically prolonged the lifetime of CGalT without affecting the trimming of N-linked oligosaccharides at CGalT. The increased lifetime leads to the up-regulation of CGalT protein as well as elevated enzymatic activity in CHO cells stably expressing CGalT. Knockdown of Sig-1Rs also decreased CGalT degradation endogenously expressed in D6P2T-schwannoma cells. Our data suggest that Sig-1Rs negatively regulate the activity of GalCer synthesis under physiological conditions by enhancing the degradation of CGalT through regulation of the dynamics of Insig in the lipid-activated ER-associated degradation system. The GalCer synthesis may thus be influenced by sterols at the ER.

Cocaine hijacks σ1 receptor to initiate induction of activated leukocyte cell adhesion molecule: implication for increased monocyte adhesion and migration in the CNS.

Human immunodeficiency virus (HIV)-associated increase in monocyte adhesion and trafficking is exacerbated by cocaine abuse. The underlying mechanisms involve cocaine-mediated upregulation of adhesion molecules with subsequent disruption of the blood-brain barrier (BBB). Recently, a novel activated leukocyte cell adhesion molecule (ALCAM) has been implicated in leukocyte transmigration across the endothelium. We now show that upregulation of ALCAM in the brain endothelium seen in HIV(+)/cocaine drug abusers paralleled increased CD68 immunostaining compared with HIV(+)/no cocaine or uninfected controls, suggesting the important role of ALCAM in promoting leukocyte infiltration across the BBB. Furthermore, ALCAM expression was increased in cocaine-treated mice with concomitant increase in monocyte adhesion and transmigration in vivo, which was ameliorated by pretreating with the neutralizing antibody to ALCAM, lending additional support to the role of ALCAM. This new concept was further confirmed by in vitro experiments. Cocaine-mediated induction of ALCAM in human brain microvascular endothelial cells through the translocation of σ receptor to the plasma membrane, followed by phosphorylation of PDGF-ß (platelet-derived growth factor-ß) receptor. Downstream activation of mitogen-activated protein kinases, Akt, and NF-κB (nuclear factor-κB) pathways resulted in induced expression of ALCAM. Functional implication of upregulated ALCAM was confirmed using cell adhesion and transmigration assays. Neutralizing antibody to ALCAM ameliorated this effect. Together, these findings implicate cocaine-mediated induction of ALCAM as a mediator of increased monocyte adhesion/transmigration into the CNS.

The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria.

The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP(3) receptor-mediated Ca(2+) influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-ß-cyclodextrin (MßC) significantly increases the association between MAMs and mitochondria, whereas MßC saturated with cholesterol does not change the association. (14)C-Serine pulse-labeling demonstrated that the treatment of living cells with MßC decreases the level of de novo synthesized (14)C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of (14)C-phosphatidylethanolamine (PtEt). Apparently, cholesterol depletion increased the PtSer transport from MAMs to mitochondria. Our findings suggest that cholesterol is an important substrate in regulating the association between MAMs of the ER and mitochondria.

Compromising σ-1 receptors at the endoplasmic reticulum render cytotoxicity to physiologically relevant concentrations of dopamine in a nuclear factor-κB/Bcl-2-dependent mechanism: potential relevance to Parkinson's disease.

The endoplasmic reticulum (ER) chaperone σ-1 receptor (Sig-1R) is cytoprotective against ER stress-induced apoptosis. The level of Sig-1Rs in the brain was reported to be lower in early parkinsonian patients. Because dopamine (DA) toxicity is well known to be involved in the etiology of Parkinson's disease, we tested in this study whether a relationship might exist between Sig-1Rs and DA-induced cytotoxicity in a cellular model by using Chinese hamster ovary (CHO) cells. DA in physiological concentrations (e.g., lower than 10 µM) does not cause apoptosis. However, the same concentrations of DA cause apoptosis in Sig-1R knockdown CHO cells. In search of a mechanistic explanation, we found that unfolded protein response is not involved. Rather, the level of protective protein Bcl-2 is critically involved in this DA/Sig-1R knockdown-induced apoptosis. Specifically, the DA/Sig-1R knockdown causes a synergistic proteasomal conversion of nuclear factor κB (NF-κB) p105 to the active form of p50, which is known to down-regulate the transcription of Bcl-2. It is noteworthy that the DA/Sig-1R knockdown-induced apoptosis is blocked by the overexpression of Bcl-2. Our results therefore indicate that DA is involved in the activation of NF-κB and suggest that endogenous Sig-1Rs are tonically inhibiting the proteasomal conversion/activation of NF-κB caused by physiologically relevant concentrations of DA that would otherwise cause apoptosis. Thus, Sig-1Rs and associated ligands may represent new therapeutic targets for the treatment of parkinsonism.

Sigma-1 receptor chaperones regulate the secretion of brain-derived neurotrophic factor.

The sigma-1 receptor (Sig-1R) is a novel endoplasmic reticulum (ER) molecular chaperone that regulates protein folding and degradation. The Sig-1R activation by agonists is known to improve memory, promote cell survival, and exert an antidepressant-like action in animals. Cutamesine (SA4503), a selective Sig-1R ligand, was shown to increase BDNF in the hippocampus of rats. How exactly the intracellular chaperone Sig-1R or associated ligand causes the increase of BDNF or any other neurotrophins is unknown. We examined here whether the action of Sig-1Rs may relate to the post-translational processing and release of BDNF in neuroblastoma cell lines. We used in vitro assays and confirmed that cutamesine possesses the bona fide Sig-1R agonist property by causing the dissociation of BiP from Sig-1Rs. The C-terminus of Sig-1Rs exerted robust chaperone activity by completely blocking the aggregation of BDNF and GDNF in vitro. Chronic treatment with cutamesine in rat B104 neuroblastoma caused a time- and dose-dependent potentiation of the secretion of BDNF without affecting the mRNA level of BDNF. Cutamesine decreased the intracellular level of pro-BDNF and mature BDNF whereas increased the extracellular level of mature BDNF. The pulse-chase experiment indicated that the knockdown of Sig-1Rs decreased the secreted mature BDNF in B104 cells without affecting the synthesis of BDNF. Our findings indicate that, in contrast to clinically used antidepressants that promote the transcriptional upregulation of BDNF, the Sig-1R agonist cutamesine potentiates the post-translational processing of neurotrophins. This unique pharmacological profile may provide a novel therapeutic opportunity for the treatment of neuropsychiatric disorders.

Sigma-1 receptors regulate hippocampal dendritic spine formation via a free radical-sensitive mechanism involving Rac1xGTP pathway.

Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER)-resident proteins known to be involved in learning and memory. Dendritic spines in hippocampal neurons play important roles in neuroplasticity and learning and memory. This study tested the hypothesis that Sig-1Rs might regulate denritic spine formation in hippocampal neurons and examined potential mechanisms therein. In rat hippocampal primary neurons, the knockdown of Sig-1Rs by siRNAs causes a deficit in the formation of dendritic spines that is unrelated to ER Ca(2+) signaling or apoptosis, but correlates with the mitochondrial permeability transition and cytochrome c release, followed by caspase-3 activation, Tiam1 cleavage, and a reduction in Rac1.GTP. Sig-1R-knockdown neurons contain higher levels of free radicals when compared to control neurons. The activation of superoxide dismutase or the application of the hydroxyl-free radical scavenger N-acetyl cysteine (NAC) to the Sig-1R-knockdown neurons rescues dendritic spines and mitochondria from the deficits caused by Sig-1R siRNA. Further, the caspase-3-resistant TIAM1 construct C1199DN, a stable guanine exchange factor able to constitutively activate Rac1 in the form of Rac1.GTP, also reverses the siRNA-induced dendritic spine deficits. In addition, constitutively active Rac1.GTP reverses this deficit. These results implicate Sig-1Rs as endogenous regulators of hippopcampal dendritic spine formation and suggest a free radical-sensitive ER-mitochondrion-Rac1.GTP pathway in the regulation of dendritic spine formation in the hippocampus.

Identification of Scytalone Dehydratase Inhibitors Effective against Melanin Biosynthesis Dehydratase Inhibitor-Resistant Pyricularia oryzae.

Melanin is a secondary metabolite required for the infection of the rice blast fungus Pyricularia oryzae. Melanin biosynthesis enzymes are targets for controlling rice blast disease, and three types of commercial melanin biosynthesis inhibitors (MBIs) including MBI-R, MBI-D, and MBI-P have been developed. However, the occurrence of MBI-D-resistant strains containing scytalone dehydratase (SDH1/RSY1) with V75M mutations has been recently reported. In this study, we aimed to identify inhibitors of SDH1-V75M. We screened the RIKEN Natural Products Depository chemical library using chemical array technology and evaluated the inhibition of SDH1-V75M by candidate compounds. NPD13731 strongly inhibited the activity of wild-type and mutant SDH1. The structure-activity relationship data were used to create a more potent inhibitor 16, which controlled rice blast disease in rice plants infected with MBI-D-resistant P. oryzae. Compound 16, which we named melabiostin, may be used to develop fungicides for controlling rice blast infections.

Antigen retrieval to improve the immunocytochemistry detection of sigma-1 receptors and ER chaperones.

Molecular chaperones localized at the endoplasmic reticulum (ER) lumen constitutively or cellular stress-dependently associate with a variety of proteins to promote their proper folding or to inhibit protein misfolding. ER chaperones preferentially form large complexes with co-chaperones and/or misfolded proteins in a highly crowded cellular environment that often hampers their detection by immunocytochemistry (ICC). This study establishes an antigen retrieval (AR) protocol to improve the ICC detection of ER chaperones in cultured cells using widely available antibodies against synthetic peptides. Among ten different antigen retrieval/fixation conditions, only the AR with Tris-HCl (pH 9.5) containing 6 M urea (80°C for 10 min) significantly improved the ICC detection of the novel ER chaperone sigma-1 receptor (Sig-1R) in Chinese hamster ovary cells. Extended fixation with 4% paraformaldehyde for 1 h effectively preserved the morphology of the ER under the AR condition. This method greatly enhanced the signal-to-noise ratio in Sig-1R ICC, thus allowing for semi-quantitative detection of protein upregulation under ER stress. The AR similarly improved the ICC detection of a series of other major ER chaperones, including BiP/GRP78, GRP94, calnexin, calreticulin, ERp57, protein disulfide isomerase, and cyclophilin B. The improved ICC methodology using the urea AR at 80°C may improve ICC of ER molecules as well as visualization of ER structure and substructures.

Cholesterol at the endoplasmic reticulum: roles of the sigma-1 receptor chaperone and implications thereof in human diseases.

Despite substantial data elucidating the roles of cholesterol in lipid rafts at the plasma membrane, the roles of cholesterol and related lipids in lipid raft microdomains at the level of subcellular membrane, such as the endoplasmic reticulum (ER) membrane, remain less understood. Growing evidence, however, begins to unveil the importance of cholesterol and lipids on the lipid raft at the ER membrane. A few ER proteins including the sigma-1 receptor chaperone were identified at lipid raft-like microdomains of the ER membrane. The sigma-1 receptor, which is highly expressed at a subdomain of ER membrane directly apposing mitochondria and known as the mitochondria-associated ER membrane or MAM, has been shown to associate with steroids as well as cholesterol. The sigma-1 receptor has been implicated in ER lipid metabolisms/transports, lipid raft reconstitution at the plasma membrane, trophic factor signalling, cellular differentiation, and cellular protection against beta-amyloid-induced neurotoxicity. Recent studies on sigma-1 receptor chaperones and other ER proteins clearly suggest that cholesterol, in concert with those ER proteins, may regulate several important functions of the ER including folding, degradation, compartmentalization, and segregation of ER proteins, and the biosynthesis of sphingolipids.

Detergent-resistant microdomains determine the localization of sigma-1 receptors to the endoplasmic reticulum-mitochondria junction.

Sigma-1 receptors (Sig-1Rs) that bind diverse synthetic and endogenous compounds have been implicated in the pathophysiology of several human diseases such as drug addiction, depression, neurodegenerative disorders, pain-related disorders, and cancer. Sig-1Rs were identified recently as novel ligand-operated molecular chaperones. Although Sig-1Rs are predominantly expressed at endoplasmic reticulum (ER) subdomains apposing mitochondria [i.e., the mitochondria-associated ER membrane (MAM)], they dynamically change the cellular distribution, thus regulating both MAM-specific and plasma membrane proteins. However, what determines the location of Sig-1R at the MAM and how the receptor translocation is initiated is unknown. Here we report that the detergent-resistant membranes (DRMs) play an important role in anchoring Sig-1Rs to the MAM. The MAM, which is highly capable of accumulating ceramides, is enriched with both cholesterol and simple sphingolipids, thus forming Triton X-114-resistant DRMs. Sig-1Rs associate with MAM-derived DRMs but not with those from microsomes. A lipid overlay assay found that solubilized Sig-1Rs preferentially associate with simple sphingolipids such as ceramides. Disrupting DRMs by lowering cholesterol or inhibiting de novo synthesis of ceramides at the ER largely decreases Sig-1R at DRMs and causes translocation of Sig-1R from the MAM to ER cisternae. These findings suggest that the MAM, bearing cholesterol and ceramide-enriched microdomains at the ER, may use the microdomains to anchor Sig-1Rs to the location; thus, it serves to stage Sig-1R at ER-mitochondria junctions.