Involvement of serotonin 2C receptor RNA editing in accumbal neuropeptide Y expression and behavioural despair.

Serotonin 2C receptors (5-HT2 C Rs) are widely expressed in the central nervous system, and are associated with various neurological disorders. 5-HT2 C R mRNA undergoes adenosine-to-inosine RNA editing at five sites within its coding sequence, resulting in expression of 24 different isoforms. Several edited isoforms show reduced activity, suggesting that RNA editing modulates serotonergic systems in the brain with causative relevance to neuropsychiatric disorders. Transgenic mice solely expressing the non-edited 5-HT2 C R INI-isoform (INI) or the fully edited VGV-isoform exhibit various phenotypes including metabolic abnormalities, aggressive behaviour, anxiety-like behaviour, and depression-like behaviour. Here, we examined the behavioural phenotype and molecular changes of INI mice on a C57BL/6J background. INI mice showed an enhanced behavioural despair in the forced swimming test, elevated sensitivity to the tricyclic antidepressant desipramine, and significantly decreased serotonin in the nucleus accumbens (NAc), amygdala, and striatum. They also showed reduced expression of neuropeptide Y (NPY) mRNA in the NAc. In addition, by stereotactic injection of adeno-associated virus encoding NPY into the NAc, we demonstrated that accumbal NPY overexpression relieved behavioural despair. Our results suggest that accumbal NPY expression may be regulated by 5-HT2 C R RNA editing, and its impairment may be linked to mood disorders.

Lysosomal enzyme cathepsin B enhances the aggregate forming activity of exogenous α-synuclein fibrils.

The formation of intracellular aggregates containing α-synuclein (α-Syn) is one of the key steps in the progression of Parkinson's disease and dementia with Lewy bodies. Recently, it was reported that pathological α-Syn fibrils can undergo cell-to-cell transmission and form Lewy body-like aggregates. However, little is known about how they form α-Syn aggregates from fibril seeds. Here, we developed an assay to study the process of aggregate formation using fluorescent protein-tagged α-Syn-expressing cells and examined the aggregate forming activity of exogenous α-Syn fibrils. α-Syn fibril-induced formation of intracellular aggregates was suppressed by a cathepsin B specific inhibitor, but not by a cathepsin D inhibitor. α-Syn fibrils pretreated with cathepsin B in vitro enhanced seeding activity in cells. Knockdown of cathepsin B also reduced fibril-induced aggregate formation. Moreover, using LAMP-1 immunocytochemistry and live-cell imaging, we observed that these aggregates initially occurred in the lysosome. They then rapidly grew larger and moved outside the boundary of the lysosome within one day. These results suggest that the lysosomal protease cathepsin B is involved in triggering intracellular aggregate formation by α-Syn fibrils.

Efferent and Afferent Connections of Neuropeptide Y Neurons in the Nucleus Accumbens of Mice.

Neuropeptide Y (NPY) is a neural peptide distributed widely in the brain and has various functions in each region. We previously reported that NPY neurons in the nucleus accumbens (NAc) are involved in the regulation of anxiety behavior. Anterograde and retrograde tracing studies suggest that neurons in the NAc project to several areas, such as the lateral hypothalamus (LH) and ventral pallidum (VP), and receive afferent projections from the cortex, thalamus, and amygdala. However, the neural connections between accumbal NPY neurons and other brain areas in mice remain unclear. In this study, we sought to clarify these anatomical connections of NPY neurons in the NAc by investigating their neural outputs and inputs. To selectively map NPY neuronal efferents from the NAc, we injected Cre-dependent adeno-associated viruses (AAVs) into the NAc of NPY-Cre mice. This revealed that NAc NPY neurons exclusively projected to the LH. We confirmed this by injecting cholera toxin b subunit (CTb), a retrograde tracer, into the LH and found that approximately 7-10% of NPY neurons in the NAc were double-labeled for mCherry and CTb. Moreover, retrograde tracing using recombinant rabies virus (rRABV) also identified NAc NPY projections to the LH. Finally, we investigated monosynaptic input to the NPY neurons in the NAc using rRABV. We found that NPY neurons in the NAc received direct synaptic connections from the midline thalamic nuclei and posterior basomedial amygdala. These findings provide new insight into the neural networks of accumbal NPY neurons and should assist in elucidating their functional roles.

α-Synuclein Promotes Maturation of Immature Juxtaglomerular Neurons in the Mouse Olfactory Bulb.

α-Synuclein (αSyn), the major constituent of Lewy bodies and Lewy neurites, is generally expressed in presynapses and is involved in synaptic function. However, we previously demonstrated that some neurons, including those in the olfactory bulb, show high αSyn expression levels in the cell body under normal conditions. αSyn is also known to be important for adult neurogenesis. Thus, in present study, we examined the role of αSyn in juxtaglomerular neurons (JGNs) with high αSyn expression in the mouse olfactory bulb. Most αSyn-enriched JGNs expressed sex-determining region Y-box 2 (Sox2), which functions to maintain neural immature identity. Interestingly, in αSyn homozygous (-/-) knockout (KO) mice, Sox2-positive JGNs were significantly increased compared with heterozygous (+/-) KO mice. Following global brain ischemia using wild-type mice, there was also a significant decrease in Sox2-positive JGNs, and in the co-expression ratio of Sox2 in αSyn-enriched JGNs. By contrast, the co-expression ratio of neuronal nuclei (NeuN, mature neuronal marker) was significantly increased in αSyn-enriched JGNs. However, this ischemia-induced decrease of Sox2-positive JGNs was not observed in αSyn homozygous KO mice. Overall, these data suggest that αSyn functions to promote the maturation of immature JGNs in the mouse olfactory bulb.

Neuropeptide Y neurons in the nucleus accumbens modulate anxiety-like behavior.

Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that is widely expressed in the central nervous system, including the cerebral cortex, nucleus accumbens (NAc) and hypothalamus. We previously analyzed the behavior of transgenic mice exclusively expressing an unedited RNA isoform of the 5-HT2C receptor. These mice showed decreased NPY gene expression in the NAc and exhibited behavioral despair, suggesting that NAc NPY neurons may be involved in mood disorder; however, their role in this behavior remained unknown. Therefore, in the present study, we investigated the functional role of NAc NPY neurons in anxiety-like behavior by examining the impact of specific ablation or activation of NAc NPY neurons using NPY-Cre mice and Cre-dependent adeno-associated virus. Diphtheria toxin-mediated ablation of NAc NPY neurons significantly increased anxiety-like behavior in the open field and elevated plus maze tests, compared with before toxin treatment. Moreover, chemogenetic activation of NAc NPY neurons reduced anxiety-like behavior in both behavioral tests compared with control mice. These results suggest that NPY neurons in the NAc are involved in the modulation of anxiety in mice.

Expression of α-synuclein is regulated in a neuronal cell type-dependent manner.

α-Synuclein, the major component of Lewy bodies (LBs) and Lewy neurites (LNs), is expressed in presynapses under physiologically normal conditions and is involved in synaptic function. Abnormal intracellular aggregates of misfolded α-synuclein such as LBs and LNs are pathological hallmarks of synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). According to previous studies using pathological models overexpressing α-synuclein, high expression of this protein in neurons is a critical risk factor for neurodegeneration. Therefore, it is important to know the endogenous expression levels of α-synuclein in each neuronal cell type. We previously reported differential expression profiles of α-synuclein in vitro and in vivo. In the wild-type mouse brain, particularly in vulnerable regions affected during the progression of idiopathic PD, α-synuclein is highly expressed in neuronal cell bodies of some early PD-affected regions, such as the olfactory bulb, the dorsal motor nucleus of the vagus, and the substantia nigra pars compacta. Synaptic expression of α-synuclein is mostly accompanied by expression of vesicular glutamate transporter-1, an excitatory synapse marker protein. In contrast, α-synuclein expression in inhibitory synapses differs among brain regions. Recently accumulated evidence indicates the close relationship between differential expression profiles of α-synuclein and selective vulnerability of certain neuronal populations. Further studies on the regulation of α-synuclein expression will help to understand the mechanism of LB pathology and provide an innovative therapeutic strategy to prevent PD and DLB onset.

Phosphorylated NF-κB subunit p65 aggregates in granulovacuolar degeneration and neurites in neurodegenerative diseases with tauopathy.

Granulovacuolar degeneration (GVD) was originally reported in Alzheimer's disease (AD) and later found in aging brains. Pathologically, GVD is thought to be associated with the development of tauopathy, but the precise mechanism remains unknown. Previous studies have suggested that GVD contains proteins associated with an inflammatory signal. In this study, we examined phosphorylated p65 (pp65), which is the activated form of a subunit of nuclear factor-kappa B (NF-κB), in the hippocampus of 21 autopsied cases, including AD, amyotrophic lateral sclerosis cases with optineurin mutation (ALS-OPTN), and a variety of other neurodegenerative disorder cases and normal controls. In all cases, GVDs were immunopositive for pp65. The density of pp65-positive GVDs statistically correlated with that of casein kinase 1 delta (CK1δ), which is known as GVD marker. pp65 was also detected in neurites in AD and ALS-OPTN. The number of neurons with pp65-immunoreactive GVD was significantly higher in the AD group than in the non-AD group. Double immunostaining showed the colocalization of CK1δ and pp65. pp65-positive GVD was found in a neuron with AT8-positive neurofibrillary tangles. Moreover, pp65 was also found in neurites that were immunostained with phosphorylated tau, phosphorylated α-synuclein, or TDP-43 (transactivation response element DNA-binding protein 43 kDa). Therefore, the activation of the NF-κB pathway may be related to the pathology of GVD formation and dementia with tauopathy, including AD and ALS-OPTN. We propose that pp65 is useful as a GVD marker, and that the NF-κB pathway could be a therapeutic target not only for AD, but for age-related neurodegenerative diseases in general.

Characterization of nuclear import of the domain-specific androgen receptor in association with the importin alpha/beta and Ran-guanosine 5'-triphosphate systems.

Androgen induces androgen receptor (AR) nuclear import, which allows AR to act as a transcriptional factor and ultimately leads to biological activity. However, the mechanism of AR translocation to the nucleus is still unclear. In the present study, we assessed the nuclear import abilities of each domain of AR and their mechanisms related to Ran and importin alpha/beta using green fluorescent protein real-time imaging. The localization of AR to the nucleus in the absence and presence of ligands was dependent upon a complex interplay of the amino terminal transactivation domain (NTD), the DNA binding domain (DBD), and the ligand binding domain (LBD). NTD and DBD showed ligand-independent nuclear import ability, whereas LBD had ligand-dependent transport. In addition, AR deletion mutant lacking DBD was distributed in the cytoplasm regardless of ligand existence, suggesting that the remaining domains, NTD and LBD, are responsible for AR cytoplasmic localization. Cotransfection with a dominant negative form of Ran dramatically inhibited the nuclear import of all AR domains, and a dominant negative form of importin alpha prevented AR and DBD import. Importin beta-knockdown strongly blocked DBD import. These results indicate that there are two additional nuclear localization signals (NLSs) in the NTD and LBD, and there are distinct pathways used to attain domain-specific AR nuclear import: the NLS of DBD is Ran and importin alpha/beta-dependent, whereas the NLSs of NTD and LBD are Ran dependent but importin alpha/beta-independent. Our data suggest that the nuclear import of AR is regulated by the interplay between each domain of the AR.

Ethanol Preference and Drinking Behavior Are Controlled by RNA Editing in the Nucleus Accumbens.

RNA editing plays critical roles in normal brain function, and alteration of its activity causes various disorders. We previously found that chronic consumption of ethanol was associated with increased levels of RNA editing of serotonin 2C receptor in the nucleus accumbens (NAc). However, it remains unknown whether RNA editing in the NAc modulates alcohol addiction through the brain reward system. To investigate the involvement of NAc RNA editing in alcohol addiction, we generated NAc-specific knockout mice of the double-stranded RNA-specific adenosine deaminase ADAR2 using AAV-GFP/Cre and conducted a battery of behavioral tests including anxiety- and depression-like behaviors. In addition, NAc-specific ADAR2 knockout mice were exposed to ethanol vapor for 20 days, followed by ethanol-drinking and conditioned place preference (CPP) tests. NAc-specific ADAR2 knockout mice showed a significant decrease in locomotor activity in the open field test although they did not develop anxiety- and depression-like behaviors. In addition, the enhancements of ethanol intake and ethanol preference that are usually observed after chronic ethanol vapor exposure were significantly reduced in these mice. These results suggest that ADAR2-mediated RNA editing in the NAc is involved in determination of alcohol preference after chronic alcohol consumption.

Up-regulation of L-type high voltage-gated calcium channel subunits by sustained exposure to 1,4- and 1,5-benzodiazepines in cerebrocortical neurons.

The aim of this study is to examine how sustained exposure to two 1,4-benzodiazepines (BZDs) with different action period, diazepam and brotizolam, and a 1,5-BZD, clobazam, affects L-type high voltage-gated calcium channel (HVCC) functions and its mechanisms using primary cultures of mouse cerebral cortical neurons. The sustained exposure to these three BZDs increased [(45)Ca2+] influx, which was due to the enhanced [(45)Ca2+] entry through L-type HVCCs but not through of Cav2.1 and Cav2.2. Increase in [(3)H]diltiazem binding after the exposure to these three BZDs was due to the increase in the binding sites of [(3)H]diltiazem. Western blot analysis showed increase of Cav1.2 and Cav1.3 in association with the increased expression of alpha2/delta1 subunit. Similar changes in [(3)H]diltiazem binding and L-type HVCC subunit expression were found in the cerebral cortex from mouse with BZD physical dependence. These results indicate that BZDs examined here have the potential to increase L-type HVCC functions mediated via the enhanced expression of not only Cav1.2 and Cav1.3 but also alpha2/delta1 subunit after their sustained exposure, which may participate in the development of physical dependence by these BZDs.

HSF1 stress response pathway regulates autophagy receptor SQSTM1/p62-associated proteostasis.

Proteostasis is important for protecting cells from harmful proteins and is mainly controlled by the HSF1 (heat shock transcription factor 1) stress response pathway. This pathway facilitates protein refolding by molecular chaperones; however, it is unclear whether it functions in autophagy or inclusion formation. The autophagy receptor SQSTM1/p62 is involved in selective autophagic clearance and inclusion formation by harmful proteins, and its phosphorylation at S349, S403, and S407 is required for binding to substrates. Here, we demonstrate that casein kinase 1 phosphorylates the SQSTM1 S349 residue when harmful proteins accumulate. Investigation of upstream factors showed that both SQSTM1 S349 and SQSTM1 S403 residues were phosphorylated in an HSF1 dependent manner. Inhibition of SQSTM1 phosphorylation suppressed inclusion formation by ubiquitinated proteins and prevented colocalization of SQSTM1 with aggregation-prone proteins. Moreover, HSF1 inhibition impaired aggregate-induced autophagosome formation and elimination of protein aggregates. Our findings indicate that HSF1 triggers SQSTM1-mediated proteostasis.

Expression of beta-adrenergic receptor up-regulation is mediated by two different processes.

Mechanisms of up-regulation of beta-adrenergic receptors (beta-ARs) induced by sustained exposure to 10(-8) M nadolol, a non-selective beta-AR antagonist, were examined using mouse cerebrocortical neurons. Nadolol dose- and time-dependently increased [3H]CGP-12177 bindings to the particulate fractions. This increase occurred 6 h and attained its plateau 12 h after the exposure, whereas beta1- and beta2-AR mRNA significantly increased 24 h and attained their plateaus 3 days after the exposure. Scatchard analysis revealed that the increased bindings were due to increase of receptor density. The [3H]CGP-12177 bindings to beta1- and beta2-ARs increased both 12 h and 5 days after the exposure. Although cycloheximide (CHX) decreased the bindings with or without nadolol, the extent of increase of the bindings induced by nadolol was not affected by CHX. Actinomycin D (AD) with nadolol showed no affects on the bindings 12 h after nadolol exposure, while AD treated 6 h after nadolol exposure significantly reduced the bindings 48 h after nadolol exposure. During 24 h after nadolol exposure, the increase in proteins of beta1- and beta2-ARs in the neuronal membrane was due to the increased receptor protein translocation from cytosol to membrane. These results indicate that the up-regulation of beta-ARs induced by nadolol is mediated by, at least, two different processes, one is increase in translocation of receptor proteins from cytosol to membrane with no changes in synthesis of receptor proteins and their mRNA and another is dependent on receptor protein synthesis with increased synthesis of their mRNA.

Up-regulated L-type high voltage-gated calcium channels cause increase in diazepam binding inhibitor induced by sustained morphine exposure in mouse cerebrocortical neurons.

Mechanisms of increase in diazepam binding inhibitor (DBI) mRNA expression in mouse cerebrocortical neurons after sustained morphine exposure were investigated. Increases in DBI and its mRNA expressions induced by sustained morphine (0.3 microM) exposure for 3 days were completely abolished by naloxone and nifedipine, but not by omega-agatoxin VIA and omega-conotoxin GIVA. Increase in [(3)H]diltiazem binding to the particulate fractions from the morphine-treated neurons was due to increased B(max) value with no changes in K(d) value. Western blot analysis on L-type high voltage-gated calcium channel (HVCC) subunits revealed the increased expressions of alpha1C, alpha1D, and alpha2/delta1 subunits and decreased of beta4 subunit expression, whereas expression of N- and P/Q-type HVCC subunits was not changed. These results indicate that morphine-induced increase in DBI mRNA expression is mediated via increased Ca(2+) entry through up-regulated L-type HVCCs.

Brain region-dependent differential expression of alpha-synuclein.

α-Synuclein, the major constituent of Lewy bodies (LBs), is normally expressed in presynapses and is involved in synaptic function. Abnormal intracellular aggregation of α-synuclein is observed as LBs and Lewy neurites in neurodegenerative disorders, such as Parkinson's disease (PD) or dementia with Lewy bodies. Accumulated evidence suggests that abundant intracellular expression of α-synuclein is one of the risk factors for pathological aggregation. Recently, we reported differential expression patterns of α-synuclein between excitatory and inhibitory hippocampal neurons. Here we further investigated the precise expression profile in the adult mouse brain with special reference to vulnerable regions along the progression of idiopathic PD. The results show that α-synuclein was highly expressed in the neuronal cell bodies of some early PD-affected brain regions, such as the olfactory bulb, dorsal motor nucleus of the vagus, and substantia nigra pars compacta. Synaptic expression of α-synuclein was mostly accompanied by expression of vesicular glutamate transporter-1, an excitatory presynaptic marker. In contrast, expression of α-synuclein in the GABAergic inhibitory synapses was different among brain regions. α-Synuclein was clearly expressed in inhibitory synapses in the external plexiform layer of the olfactory bulb, globus pallidus, and substantia nigra pars reticulata, but not in the cerebral cortex, subthalamic nucleus, or thalamus. These results suggest that some neurons in early PD-affected human brain regions express high levels of perikaryal α-synuclein, as happens in the mouse brain. Additionally, synaptic profiles expressing α-synuclein are different in various brain regions.

Development of the 5-HT2CR-Tango System Combined with an EGFP Reporter Gene.

The serotonin 2C receptor (5-HT2CR) is a G-protein-coupled receptor implicated in emotion, feeding, reward, and cognition. 5-HT2CRs are pharmacological targets for mental disorders and metabolic and reward system abnormalities, as alterations in 5-HT2CR expression, RNA editing, and SNPs are involved in these disturbances. To date, 5-HT2CR activity has mainly been measured by quantifying inositol phosphate production and intracellular Ca(2+) release, but these assays are not suitable for in vivo analysis. Here, we developed a 5-HT2CR-Tango assay system, a novel analysis tool of 5-HT2CR activity based on the G-protein-coupled receptor (GPCR)-arrestin interaction. With desensitization of activated 5-HT2CR by arrestin, this system converts the 5-HT2CR-arrestin interaction into EGFP reporter gene signal via the LexA transcriptional activation system. For validation of our system, we measured activity of two 5-HT2CR RNA-editing isoforms (INI and VGV) in HEK293 cells transfected with EGFP reporter gene. The INI isoform displayed both higher basal- and 5-HT-stimulated activities than the VGV isoform. Moreover, an inhibitory effect of 5-HT2CR antagonist SB242084 was also detected by 5-HT2CR-Tango system. This novel tool is useful for in vitro high-throughput targeted 5-HT2CR drug screening and can be applied to future in vivo brain function studies associated with 5-HT2CRs in transgenic animal models.

Increased expression of L-type high voltage-gated calcium channel alpha1 and alpha2/delta subunits in mouse brain after chronic nicotine administration.

We investigated the effect of chronic nicotine administration on high voltage-gated calcium channels (HVCCs) in the mouse cerebral cortex. The treatment significantly increased expression of alpha1C, alpha1D, alpha1F, and alpha2/delta1 subunits with no changes of beta4 subunit of L-type HVCCs. [(3)H]Diltiazem binding to the particulate fractions increased with increased Bmax value. These results indicate that chronic nicotine treatment up-regulates L-type HVCCs, which is due to increased expression of alpha1 and alpha2/delta1 subunits.

Ethanol physical dependence is accompanied by up-regulated expression of L-type high voltage-gated calcium channel alpha1 subunits in mouse brain.

We investigated how functional changes in high voltage-gated calcium channels (HVCCs) occurred in the cerebral cortex of mouse with ethanol physical dependence. The continuous treatment of mice with ethanol vapor for 8 days significantly increased the expressions of alpha1C, alpha1D, and alpha2/delta1 subunits of L-type HVCCs in association with decreased level of beta4 subunit of L-type HVCCs, although alpha1A and alpha1B subunits of P/Q- and N-type HVCCs, respectively, showed no alterations. [(3)H]Diltiazem binding to the particulate fractions increased with increased Bmax value and no changes of Kd value. These results indicate that ethanol physical dependence accompanies the up-regulation of L- type HVCCs, which is due to increased expression of alpha1C, alpha1D, and alpha2/delta1 subunits.

Differential expression of presenilin-alpha and -beta (PSalpha and PSbeta) in Xenopus laevis: embryonic phosphorylation of PSalpha.

Mutations in genes encoding the highly homologous proteins, presenilin-1 and -2 (PS1 and PS2), are linked to the development of early-onset Alzheimer's disease. On the other hand, presenilins are known to play a critical role(s) in cell fate decisions during embryonic development in Caenorhabditis elegans. The messenger RNAs (mRNAs) of amphibian presenilin homologues PSalpha and PSbeta are most abundantly synthesized in the brain and the ovary, but are differentially degraded upon oocyte maturation and at the midblastula transition (MBT), respectively. In this study, we examined the spatiotemporal distribution of PSalpha and PSbeta proteins and their post-translational modification. The results were essentially consistent with the mRNA data and revealed moreover that PSalpha was present exclusively as processed molecules in the early embryos, while PSbeta was present mainly as unprocessed molecules (90%). Furthermore, the C-terminal fragment (CTF) of PSalpha was phosphorylated upon oocyte maturation and dephosphorylated at MBT, while no phosphorylation of the PSbeta CTF was detectable. Human PS1 CTF exogenously injected was also phosphorylated in Xenopus oocytes induced to mature in vitro by progesterone treatment. Two phosphorylation loci were mapped at Thr(320) and Ser(334) in the hydrophilic loop region of PSalpha. Our results suggest that PS1 and PS2 may play different roles under physiological conditions despite their high structural similarity.

A unifying model for functional difference and redundancy of presenilin-1 and -2 in cell apoptosis and differentiation.

Mutations in genes encoding the highly homologous proteins presenilin-1 and -2 (PS1 and PS2) are linked to the early onset of Alzheimer's disease (AD). Here, we report that polyclonal antibodies against Xenopus PSbeta (PS2), but not PSalpha (PS1), suppress the in vitro apoptotic activation of Xenopus egg extracts. To clarify the relationship between structural and functional differences in presenilins, we searched for presenilin homologues in various living sources, and found that presenilins were divided into three distinct groups, named alpha-, beta- and gamma-types, based on the size of the large hydrophilic loop (HL) regions as follows: HLalpha/HLbeta/HLgamma=4:3:6. No such size conservations were found in the N-terminal (NT) hydrophilic regions. Phylogenetic studies revealed that the presenilin genes were duplicated independently in different lineages of phyla/divisions, suggesting that there were functional requirements for and constraints on the generation and conservation of these HL sizes. On the basis of these findings, we propose a model postulating that both PS1 and PS2 can be differentiative or apoptotic when they are proteolytically processed within the HL regions or not, respectively, and PS1 may be more sensitive than PS2 to auto-proteolytic cleavage due to the larger size of the HL region of the former. Furthermore, the model assumes that C-terminal fragments (CTF) stabilized by phosphorylation may inhibit both the activities due to the dominant-negative effect. The model explains not only the functional redundancy but also apparently conflicting observations reported so far for PS1 and PS2.

Continuous exposure to nitric oxide enhances diazepam binding inhibitor mRNA expression in mouse cerebral cortical neurons.

Effects of sustained exposure to nitric oxide (NO) formed by long-term activation of N-methyl-D-aspartate (NMDA) receptors and liberated from a long-lasting NO generator, DETA NONOate, on diazepam binding inhibitor (DBI) and its mRNA expressions were examined using mouse cerebral cortical neurons. Long-term exposure to NMDA increased DBI mRNA expression, and NO synthase inhibitors dose-dependently inhibited this increase. DETA NONOate dose-dependently increased DBI mRNA expression when exposing the neurons to this agent for 3 days and a maximal enhancement of the expression was found at 100 microM of the NO generator. In addition, a significant increase in DBI mRNA expression was observed 1 day after the exposure to 100 microM DETA NONOate, and the maximal expression was observed 2 days after the exposure, whereas transient exposure for less than 3 h to 100 microM DETA NONOate produced no changes in the expression. DETA NONOate (100 microM)-induced increase in DBI mRNA expression was completely abolished by concomitant exposure to hemoglobin. DBI content was also dose-dependently increased by DETA NONOate after the exposure for 3 days. The inhibition of cGMP formation by 1H-[1,2,4] oxadiazolo [4,3-alpha]quinoxalin-1-one (ODQ) showed no affects on the DETA NONOate-induced expression, suggesting that the increased expression of DBI mRNA is mediated via processes independent of cGMP. These results indicate that continuous exposure of the neurons to NO is an essential factor for increasing DBI mRNA expression in the neurons.