bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased expression of p62 in expanded polyglutamine‐expressing cells and its association with polyglutamine inclusions
Authors: Nagaoka Utako , Kim Ken , Jana Nihar Ranjan , Doi Hiroshi , Maruyama Mieko , Mitsui Kenichi , Oyama Fumitaka , Nukina Nobuyuki
Published: March 2006

Huntington's disease is a progressive neurodegenerative disorder that is associated with a CAG repeat expansion in the gene encoding huntingtin. We found that a 60‐kDa protein was increased in Neuro2a cells expressing the N‐terminal portion of huntingtin with expanded polyglutamine. We purified this protein, and, using mass spectrometry, identified it as p62, an ubiquitin‐associated domain‐containing protein. A specific p62 antibody stained the ubiquitylated polyQ inclusions in expanded polyglutamine‐expressing cells, as well as in the brain of the huntingtin exon 1 transgenic mice. Furthermore, the level of p62 protein and mRNA was increased in expanded polyglutamine‐expressing cells. We also found that p62 formed aggresome‐like inclusions when p62 was increased in normal Neuro2a cells by a proteasome inhibitor. Knock‐down of p62 does not affect the formation of aggresomes or polyglutamine inclusions, suggesting that p62 is recruited to the aggresome or inclusions secondary to their formation. These results suggest that p62 may play important roles as a responsive protein to a polyglutamine‐induced stress rather than as a cross‐linker between ubiquitylated proteins.
Multiple promoters drive tissue‐specific expression of the human M2muscarinic acetylcholine receptor gene
Authors: Krejci Alena , Bruce Alexander W. , Dolezal Vladimir , Tucek Stanislav , Buckley Noel J.
Published: March 2006

Despite the wealth of information on the functional and pharmacological properties of the M2muscarinic receptor, we know relatively little of structure and regulation of the M2receptor gene. Here, we describe the organisation of the human M2gene and its promoters. Four exons are present in the 5′ untranslated region of the human M2mRNA distributed over 146 kb on chromosome 7 which produce eight different splice variants in the IMR‐32 neuroblastoma cell line. The unexpectedly large size of this gene indicates that transcription initiates much further upstream of the coding region than earlier studies had indicated. We present evidence that there are three distinct human M2promoters. Analysis of endogenous transcripts revealed that promoter 2 is preferentially used in neuroblastoma cells, whereas promoter 1 in cardiac cells. All promoters are highly conserved across human, mouse, rat and pig. They contain multiple start sites and none possess a TATA‐box. In addition, we describe another M2promoter that is specific for rat. We show that GATA‐4 transcription factor binds to two sites within the regulatory regions of the M2gene using reporter gene assays, electromobility shift assays and mutational analysis.
Plasma membrane oestrogen receptor mediates neuroprotection against β‐amyloid toxicity through activation of Raf‐1/MEK/ERK cascade in septal‐derived cholinergic SN56 cells
Authors: Guerra Borja , Díaz Mario , Alonso Rafael , Marin Raquel
Published: March 2006

Rapid oestrogen neuroprotection against β‐amyloid peptide (Aβ)‐induced toxicity, a main feature of Alzheimer's disease, may be partially initiated at the plasma membrane. However, the mechanism by which this oestrogen effect occurs is unknown. In a septal murine cell line (SN56), we observed that short exposures to either 17β‐oestradiol (E2) or membrane impermeant E2 bound to horseradish peroxidase (E‐HRP) induced a biphasic stimulation of extracellular‐signal regulated protein kinase (ERK1/2) phosphorylation, with peak inductions detected around 4–8 min in the early phase and a second maximum around 8 h after treatment. ERK1/2 phosphorylation was abolished by ERK1/2 kinase (MEK) inhibitors PD98059 and U0126. Interestingly, PD98059 was also shown to block rapid E2‐related prevention of death in cells exposed to Aβ fragment 1–40 (Aβ1−40) for 24 h. In contrast, no neuroprotective effects were obtained when MEK inhibitor was used to selectively abolish the late phosphorylation phase. Furthermore, both ERK1/2 activation and E2‐associated protection were blocked by an inhibitor of Raf‐1 kinase. Raf‐1 may be involved in these effects because oestrogen caused the rapid serine 338 (Ser338) phosphorylation of this protein. In addition, the oestrogen receptor (ER) antagonist ICI 182 780 was also observed to block ERK1/2 phosphorylation. We propose a novel mechanism in SN56 cells by which rapid effects of oestrogen leading to neuroprotection are signalled through Raf‐1/MEK/ERK1/2 pathway, possibly by activation of a membrane‐related ER.
Effects of morphine on gene expression in the rat amygdala
Authors: Rodriguez Parkitna J. M. , Bilecki W. , Mierzejewski P. , Stefanski R. , Ligeza A. , Bargiela A. , Ziolkowska B. , Kostowski W. , Przewlocki R.
Published: March 2006

Influence of morphine self‐administration on gene expression in the rat amygdala was studied using rat genome DNA arrays U34A from Affymetrix. Animals were trained to self‐administer morphine, each having two ‘yoked’ control animals, receiving passive injections of either morphine or saline. After 40 sessions of self‐administration, amygdalae were removed, total RNA was isolated and used to prepare probes for Genechip® arrays. The treatment was found to significantly change abundance of 29 transcripts. Analysis by means of reverse transcription real‐time PCR showed significant changes in abundance of five transcripts: γ protein kinase C (PKC), upstream binding factor 2 (UBF2), lysozyme, noggin and heat shock protein 70 (hsp70). After 30 days of forced abstinence from morphine self‐administration, abundance of hsp70 and lysozyme returned to basal levels. Changes in abundance of UBF2 persisted, and abundance of three additional genes, namely nuclear factor I/A, γ1 subunit of GABAA receptor and the neuronal calcium sensor 1, changed. Additionally, acute as well as chronic intraperitoneal morphine administration changed the abundance of PKC γ, γ1 subunit of GABAA and hsp70 genes.
The arachidonic acid 5‐lipoxygenase inhibitor nordihydroguaiaretic acid inhibits tumor necrosis factor α activation of microglia and extends survival of G93A‐SOD1 transgenic mice
Authors: West Melinda , Mhatre Molina , Ceballos Alex , Floyd Robert A. , Grammas Paula , Gabbita S. Prasad , Hamdheydari Ladan , Mai Tammy , Mou Shenyun , Pye Quentin N. , Stewart Charles , West Stuart , Williamson Kelly S. , Zemlan Frank , Hensley Kenneth
Published: March 2006

Familial forms of amyotrophic lateral sclerosis (ALS) can be caused by mutations in copper, zinc‐superoxide dismutase (SOD1). Mice expressing SOD1 mutants demonstrate a robust neuroinflammatory reaction characterized, in part, by up‐regulation of tumor necrosis factor alpha (TNFα) and its primary receptor TNF‐RI. In an effort to identify small molecule inhibitors of neuroinflammation useful in treatment of ALS, a microglial culture system was established to identify TNFα antagonists. Walker EOC‐20 microglia cells were stimulated with recombinant TNFα, with or without inhibitors, and the cell response was indexed by NO2– output. Three hundred and fifty‐five rationally selected compounds were included in this bioassay. The arachidonic acid 5‐lipoxygenase (5LOX) and tyrosine kinase inhibitor nordihydroguaiaretic acid (NDGA), a natural dicatechol, was one of the most potent non‐cytotoxic antagonists tested (IC50 8 ± 3 μm). Investigation of the G93A‐SOD1 mouse model for ALS revealed increased message and protein levels of 5LOX at 120 days of age. Oral NDGA (2500 p.p.m.) significantly extended lifespan and slowed motor dysfunction in this mouse, when administration was begun relatively late in life (90 days). NDGA extended median total lifespan of G93A‐SOD1 mice by 10%, and life expectancy following start of treatment was extended by 32%. Disease‐associated gliosis and cleaved microtubule‐associated tau protein, an indicator of axon damage, were likewise reduced by NDGA. Thus, TNFα antagonists and especially 5LOX inhibitors might offer new opportunities for treatment of ALS.
Static, transient and permanent organization of GABAA receptor expression in calbindin‐positive interneurons in response to amygdala kindled seizures
Authors: Meguro R. , Lu J. , Gavrilovici C. , Poulter M. O.
Published: March 2006

We tested the hypothesis that experimentally produced epilepsy (by kindling) may induce changes in GABAA receptor expression in some but not all interneuron populations. Using laser capture microdissection and quantitative polymerase chain reaction (QPCR) analysis, GABAA receptor α subunit expression in calbindin‐ (CBir) and parvalbumin‐ (Parvir) immunoreactive interneurons was compared between normal brains and brains in which amygdala kindled seizure responses were permanently established. Two weeks after the last seizure response, Cbir neurons in the hilus and/or perirhinal cortex up‐regulated the expression of α2, α3 and α5 subunit mRNAs up to 900%. In contrast, no changes were found in Parvir neurons. In Cbir neurons contralateral to the amygdala kindling site α1 subunit mRNA expression was increased. In both Cbir and Parvir neurons, the coordinated subunit expression patterns ipsilateral (fully kindled) and contralateral (partially kindled) to the kindling site suggested that permanent and transient co‐expressional relationships occur respectively. In the perirhinal cortex α2 protein was up‐regulated in the processes but not in the cell somas of calbindin‐positive neurons, whereas α3 subunit protein expression was up‐regulated on the cell bodies of Cbir neurons in the hilus. These data indicate that different interneuron populations may selectively reorganize their GABAA subunit expression in response to seizures.
Glutamate‐dependent transcriptional regulation of GLAST: role of PKC
Authors: López‐Bayghen Esther , Ortega Arturo
Published: March 2006

The Na+‐dependent glutamate/aspartate transporter GLAST plays a major role in the removal of glutamate from the synaptic cleft. Short‐term, as well as long‐term changes in transporter activity are triggered by glutamate. An important locus of regulation is the density of transporter molecules present at the plasma membrane. A substrate‐dependent change in the translocation rate of the transporter molecules accounts for the short‐term effect, whereas the long‐term modulation apparently involves transcriptional regulation. Using cultured chick cerebellar Bergmann glial cells, we report here that glutamate receptors activation mediate a substantial reduction in the transcriptional activity of the chglast promoter through the Ca2+/diacylglicerol‐dependent protein kinase (PKC) signaling cascade. Overexpression of constitutive active PKC isoforms of mimic the glutamate effect. Accordingly, increased levels of c‐Jun or c‐Fos, but not Jun‐B, Jun‐D or Fos‐B, lower the chglast promoter activity. Serial deletions and electrophorectic mobility shift assays were used to define a specific region within the 5′ proximal region of the chglast promoter, associated with transcriptional repression. A putative glutamate response element could be defined in the proximal promoter stretch more likely between nts – 40 and − 78. These results demonstrate that GLAST is under glutamate‐dependent transcriptional control through PKC, and support the notion of a pivotal role of this neurotransmitter in the regulation of its own removal from the synaptic cleft, thereby modulating, mainly in the long term, glutamatergic transmission.
Voltammetric study of the control of striatal dopamine release by glutamate
Authors: Borland Laura M. , Michael Adrian C.
Published: March 2006

The central dopamine systems are involved in several aspects of normal brain function and are implicated in a number of human disorders. Hence, it is important to understand the mechanisms that control dopamine release in the brain. The striatum of the rat receives both dopaminergic and glutamatergic projections that synaptically target striatal neurons but not each other. Nevertheless, these afferents do form frequent appositional contacts, which has engendered interest in the question of whether they communicate with each other despite the absence of a direct synaptic connection. In this study, we used voltammetry in conjunction with carbon fiber microelectrodes in anesthetized rats to further examine the effect of the ionotropic glutamate antagonist, kynurenate, on extracellular dopamine levels in the striatum. Intrastriatal infusions of kynurenate decreased extracellular dopamine levels, suggesting that glutamate acts locally within the striatum via ionotropic receptors to regulate the basal extracellular dopamine concentration. Infusion of tetrodotoxin into the medial forebrain bundle or the striatum did not alter the voltammetric response to the intrastriatal kynurenate infusions, suggesting that glutamate receptors control a non‐vesicular release process that contributes to the basal extracellular dopamine level. However, systemic administration of the dopamine uptake inhibitor, nomifensine (20 mg/kg i.p.), markedly decreased the amplitude of the response to kynurenate infusions, suggesting that the dopamine transporter mediates non‐vesicular dopamine release. Collectively, these findings are consistent with the idea that endogenous glutamate acts locally within the striatum via ionotropic receptors to control a tonic, impulse‐independent, transporter‐mediated mode of dopamine release. Although numerous prior in vitro studies had suggested that such a process might exist, it has not previously been clearly demonstrated in an in vivo experiment.
Insulin exerts neuroprotection by counteracting the decrease in cell‐surface GABAA receptors following oxygen–glucose deprivation in cultured cortical neurons
Authors: Mielke John G. , Wang Yu Tian
Published: March 2006

A loss of balance between excitatory and inhibitory signaling leads to excitoxicity, and contributes to ischemic cell death. Reduced synaptic inhibition as a result of dysfunction of the ionotropic GABAA receptor has been suggested as one of the major causes for this imbalance, although the underlying mechanisms remain poorly understood. In the present study, we investigated whether oxygen–glucose deprivation (OGD), an ischemia‐like challenge, alters cell‐surface expression of GABAA receptors in cultured hippocampal neurons, and thereby leads to excitotoxic cell death. Using cell culture ELISA as a cell surface receptor assay, we found that OGD produced a marked decrease in cell surface GABAA receptors, without altering the total amount of receptors. Furthermore, the reduction could be prevented by inhibition of receptor endocytosis with hypertonic sucrose treatment. Notably, insulin significantly limited OGD‐induced changes in cell‐surface GABAA receptors. In parallel, insulin protected cultured neurons against both glutamate toxicity and OGD, as assayed by mitochondrial reduction of Alamar Blue. Importantly, insulin‐mediated neuroprotection was eliminated when bicuculline, a GABAA receptor antagonist, was co‐applied with insulin during OGD. Together, our results strongly suggest that ischemia‐like insults decrease cell surface GABAA receptors in neurons via accelerated internalization, and that insulin provides neuroprotection by counteracting this reduction.
Evidence on extracellular dopamine level in rat striatum: implications for the validity of quantitative microdialysis
Authors: Chen Kevin C.
Published: March 2006

Microdialysis zero‐net‐flux (ZNF) method is commonly used to monitor drug‐induced changes in neurotransmitter baseline and release/uptake processes. Recent studies in this field suggest that microdialysis ZNF method seriously underestimates the resting concentration of extracellular dopamine in the rat neostriatum because probe implantation preferentially damages nearby dopamine release sites and that dopamine uptake inhibition increases the relative recovery of dopamine by microdialysis. This study assessed the validity of these claims by examining current data on extracellular dopamine levels at rest and after drug application obtained by voltammetry, a technique thought to induce less tissue disruption than microdialysis. To obtain the extracellular baseline value for dopamine from the evoked overflow data, we modified the existing dopamine kinetic model to suit both the resting and stimulated circumstances. It was found that dopamine uptake inhibition did in fact decrease the microdialysis relative recovery of dopamine, implying that the average basal extracellular dopamine level is within the range of 7–20 nmin rat striatum. This study concludes that the microdialysis ZNF method indeed underestimates the extracellular dopamine concentration, although not by as much as had been thought. Chronic microdialysis damages both neurotransmitter release and uptake, but it does so in a somewhat relative and proportional way for both processes. Thus the validity of the microdialysis ZNF method is not seriously undermined.
Increased mitochondrial respiration maintains the mitochondrial membrane potential and promotes survival of cerebellar neurons in an endogenous model of glutamate receptor activation
Authors: García Octavio , Almeida Angeles , Massieu Lourdes , Bolaños Juan P.
Published: March 2006

It is thought that the combination of extracellular glutamate accumulation and mitochondrial damage is involved in neuronal death associated with brain ischemia and hypoglycemia, and some neurodegenerative diseases such as Huntington's disease. However, the mechanism whereby those two factors interact together to trigger neurodegeneration in this and other neurodegenerative disorders is still elusive. Here, we have addressed this issue using a model of mild and sustained accumulation of extracellular glutamate in cerebellar cultured neurons, which are mostly glutamatergic and commonly used to study glutamate neurotoxicity. The resulting stimulation of glutamate receptors triggered a ∼ 50% persistent increase in mitochondrial respiration that was associated with free radicals formation, and which was found to be necessary to prevent the collapse of the mitochondial membrane potential (Δψm) and apoptotic cell death. In fact, hampering the glutamate‐mediated increase in mitochondrial respiration with an inhibitor of the mitochondrial respiratory chain stopped neurons from producing free radicals, but led them to undergo rapid and profound Δψm collapse and apoptotic cell death. Thus, we suggest that the formation of reactive oxygen species by glutamate receptor activation is the unavoidable consequence of an increase in the mitochondrial respiration aimed to prevent Δψm collapse and neurodegeneration. These results may be relevant to understand the pathophysiology of those neurodegenerative diseases associated with both mitochondrial respiratory chain and glutamate transporter defects.
Biochemical fingerprints of prion diseases: scrapie prion protein in human prion diseases that share prion genotype and type
Authors: Pan Tao , Li Ruliang , Kang Shin‐Chung , Pastore Manuela , Wong Boon‐Seng , Ironside James , Gambetti Pierluigi , Sy Man‐Sun
Published: March 2006

The phenotype of human prion diseases is influenced by the prion protein (PrP) genotype as determined by the methionine (M)/valine (V) polymorphism at codon 129, the scrapie PrP (PrPSc) type and the etiology. To gain further insight into the mechanisms of phenotype determination, we compared two‐dimensional immunoblot profiles of detergent insoluble and proteinase K‐resistant PrP species in a type of sporadic Creutzfeldt‐Jakob disease (sCJDMM2), variant CJD (vCJD) and sporadic fatal insomnia (sFI). Full‐length and truncated PrP forms present in the insoluble fractions were also separately analyzed. These three diseases were selected because they have the same M/M PrP genotype at codon 129 and the same type 2 PrPSc, but different etiologies, also sCJDMM2 and sFI are sporadic, whereas vCJD is acquired by infection. We observed minor differences in the PrP detergent‐insoluble fractions between sCJDMM2 and vCJD, although both differ in the corresponding fractions from sFI. We detected more substantial heterogeneity between sCJDMM2 and vCJD in the two‐dimensional blots of the proteinase K‐resistant PrP fraction suggesting that different PrP species are selected for conversion to proteinase K‐resistant PrP in sCJDMM2 and vCJD. These differences are mostly, but not exclusively, due to variations in the type of the N‐linked glycans. We also show that the over‐representation of the highly glycosylated forms distinctive of the proteinase K‐resistant PrPSc of vCJD in one‐dimensional blots is due to differences in both the amount and the natures of the glycans. Overall, these findings underline the complexity of phenotypic determination in human prion diseases.
Molecular cloning, localization and circadian expression of chicken melanopsin (Opn4): differential regulation of expression in pineal and retinal cell types
Authors: Chaurasia S. S. , Rollag M. D. , Jiang G. , Hayes W. P. , Haque R. , Natesan A. , Zatz M. , Tosini G. , Liu C. , Korf H. W. , Iuvone P. M. , Provencio I.
Published: March 2006

The avian retina and pineal gland contain autonomous circadian oscillators and photo‐entrainment pathways, but the photopigment(s) that mediate entrainment have not been definitively identified. Melanopsin (Opn4) is a novel opsin involved in entrainment of circadian rhythms in mammals. Here, we report the cDNA cloning of chicken melanopsin and show its expression in retina, brain and pineal gland. Like the melanopsins identified in amphibians and mammals, chicken melanopsin is more similar to the invertebrate retinaldehyde‐based photopigments than the retinaldehyde‐based photopigments typically found in vertebrates. In retina, melanopsin mRNA is expressed in cells of all retinal layers. In pineal gland, expression was strong throughout the parenchyma of the gland. In brain, expression was observed in a few discrete nuclei, including the lateral septal area and medial preoptic nucleus. The retina and pineal gland showed distinct diurnal expression patterns. In pineal gland, melanopsin mRNA levels were highest at night at Zeitgeber time (ZT) 16. In contrast, transcript levels in the whole retina reached their highest levels in the early morning (ZT 0–4). Further analysis of melanopsin mRNA expression in retinal layers isolated by laser capture microdissection revealed different patterns in different layers. There was diurnal expression in all retinal layers except the ganglion cell layer, where heavy expression was localized to a small number of cells. Expression of melanopsin mRNA peaked during the daytime in the retinal pigment epithelium and inner nuclear layer but, like in the pineal, at night in the photoreceptors. Localization and regulation of melanopsin mRNA in the retina and pineal gland is consistent with the hypothesis that this novel photopigment plays a role in photic regulation of circadian function in these tissues.
Matrix metalloproteinase‐7 disrupts dendritic spines in hippocampal neurons through NMDA receptor activation
Authors: Bilousova Tina V. , Rusakov Dmitri A. , Ethell Douglas W. , Ethell Iryna M.
Published: March 2006

Dendritic spines are protrusions from the dendritic shaft that host most excitatory synapses in the brain. Although they first emerge during neuronal maturation, dendritic spines remain plastic through adulthood, and recent advances in the molecular mechanisms governing spine morphology have shown them to be exquisitely sensitive to changes in the micro‐environment. Among the many factors affecting spine morphology are components and regulators of the extracellular matrix (ECM). Modification of the ECM is critical to the repair of injuries throughout the body, including the CNS. Matrix metalloproteinase (MMP)‐7/matrilysin is a key regulator of the ECM during pathogen infection, after nerve crush and in encephalitogenic disorders. We have investigated the effects of MMP‐7 on dendritic spines in hippocampal neuron cultures and found that it induces the transformation of mature, short mushroom‐shaped spines into long, thin filopodia reminiscent of immature spines. These changes were accompanied by a dramatic redistribution of F‐actin from spine heads into thick, rope‐like structures in the dendritic shaft. Strikingly, MMP‐7 effects on dendritic spines were similar to those of NMDA treatment, and both could be blocked by channel‐specific antagonists. These findings are the first direct evidence that MMPs can influence the morphology of mature dendritic spines, and hence synaptic stability.
Mnk is a negative regulator of cap‐dependent translation in Aplysia neurons
Authors: Ross Gabriel , Dyer John R. , Castellucci Vincent F. , Sossin Wayne S.
Published: March 2006

To investigate the mechanisms underlying regulation of eukaryotic initiation factor 4E (eIF4E) phosphorylation in Aplysia neurons, we have cloned the Aplysia homolog of the vertebrate eIF4E kinases, Mnk1 and ‐2. Aplysia Mnk shares many conserved regions with vertebrate Mnk, including putative eukaryotic initiation factor 4G binding regions, activation loop phosphorylation sites, and a carboxy‐terminal anchoring site for MAP kinases. As expected, purified Aplysia Mnk phosphorylated Aplysia eIF4E at a conserved carboxy‐terminal serine and over‐expression of Aplysia Mnk in sensory neurons led to increased phosphorylation of endogenous eIF4E. Over‐expression of Aplysia Mnk led to strong decreases in cap‐dependent translation, while generally sparing internal ribosomal entry site (IRES)‐dependent translation. However, decreases in cap‐dependent translation seen after expression of Aplysia Mnk could only be partly explained by increases in eIF4E phosphorylation. In Aplysia sensory neurons, phosphorylation of eIF4E is reduced during intermediate memory formation. However, we found that this physiological regulation of eIF4E phosphorylation was independent of changes in Aplysia Mnk phosphorylation. We propose that changes in eIF4E phosphorylation in Aplysia neurons are a consequence of changes in cap‐dependent translation that are independent of regulation of Aplysia Mnk.
Visualization of APP dimerization and APP‐Notch2 heterodimerization in living cells using bimolecular fluorescence complementation
Authors: Chen Ci‐Di , Oh Sun‐Young , Hinman Jason D. , Abraham Carmela R.
Published: March 2006

We previously demonstrated that the amyloid precursor protein (APP) interacts with Notch receptors. Here, we confirmed the APP/Notch1 endogenous interaction in embryonic day 17 rat brain tissue, suggesting the interaction was not as a result of over‐expression artifacts. To investigate potential homodimeric and heterodimeric interactions of APP and Notch2 (N2), we have visualized the subcellular localization of the APP/N2 complexes formed in living cells using bimolecular fluorescence complementation (BiFC) analysis. BiFC was accomplished by fusing the N‐terminal fragment or the C‐terminal fragment of yellow fluorescent protein (YFP) to APP, N2, and a C‐terminally truncated form of N2. When expressed in COS‐7 cells, these tagged proteins alone did not produce a fluorescent signal. The tagged APP homodimer produced a weak fluorescent signal, while neither full‐length N2, nor a truncated N2 alone, produced a visible signal, suggesting that N2 receptors do not form homodimers. The strongest fluorescent signal was obtained with co‐expression of the C‐terminal fragment of YFP fused to APP and the N‐terminal fragment of YFP fused to the truncated form of N2. This heterodimer localized to plasma membrane, endoplasmic reticulum (ER), Golgi and other compartments. The results were confirmed and quantified by flow cytometry. The BiFC method of specifically visualizing APP/Notch interactions can be applied to study APP and Notch signaling during development, aging and neurodegeneration.
Modulation of bradykinin signaling by EP24.15 and EP24.16 in cultured trigeminal ganglia
Authors: Jeske Nathaniel A. , Berg Kelly A. , Cousins Joanne C. , Ferro Emer S. , Clarke William P. , Glucksman Marc J. , Roberts James L.
Published: March 2006

Metalloendopeptidases expressed in neural tissue are characterized in terms of their neuropeptide substrates. One such neuropeptide, bradykinin (BK), is an important inflammatory mediator that activates the type‐2 BK receptor (B2R) on the terminal endings of specialized pain‐sensing neurons known as nociceptors. Among several metalloendopeptidases that metabolize and inactivate BK, EP24.15 and EP24.16 are known to associate with the plasma membrane in several immortalized cell lines. Potentially, the colocalization of EP24.15/16 and B2R at plasma membrane microdomains known as lipid rafts in a physiologically relevant nociceptive system would allow for discrete, peptidase regulation of BK signaling. Western blot analysis of crude subcellular fractions and lipid raft preparations of cultured rat trigeminal ganglia demonstrate similar expression profiles between EP24.15/16 and B2R on a subcellular level. Furthermore, the treatment of primary cultures of trigeminal ganglia with inhibitors of EP24.15/16 led to the potentiation of several bradykinin‐induced events that occur downstream of B2R activation. EP24.15/16 inhibition byN‐[1(R,S)‐carboxy‐3‐phenylpropyl]‐Ala‐AlalTyr‐p‐aminobenzoate (cFP) resulted in a 1000‐fold increase in B2R sensitivity to BK as measured by inositol phosphate accumulation. In addition, cFP treatment resulted in a 31.1 ± 5.0% potentiation of the ability of BK to inhibit protein kinase B (Akt) activity. Taken together, these data demonstrate that EP24.15/16 modulate intracellular, peptidergic signaling cascades through B2R in a physiologically relevant nociceptive system.
Ion interaction at the pore of Lc‐type Ca2+ channel is sufficient to mediate depolarization‐induced exocytosis
Authors: Lerner Immanuel , Trus Michael , Cohen Roy , Yizhar Ofer , Nussinovitch Itzhak , Atlas Daphne
Published: March 2006

The coupling of voltage‐gated Ca2+ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization‐evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca2+ with La3+ or other lanthanide ions supported exocytosis in divalent ion‐free solution. Cd2+, nifedipine, or verapamil inhibited depolarization‐evoked secretion in La3+, indicating specific binding of La3+ at the pore of L‐type VGCC, probably at the poly‐glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La3+ > Ce3+ > Pr3+, consistent with a size‐selective binding interface of trivalent cations at the channel pore. La3+ inward currents were not detected and the highly sensitive La3+/fura‐2 imaging assay (∼1 pm) detected no La3+ entry, cytosolic La3+ build‐up, or alterations in cytosolic Ca2. These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build‐up (intracellular Ca2+ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca2+ ion build‐up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca2+ sensor protein in the process of neuroendocrine secretion.
The dopamine transporter proteome
Authors: Torres Gonzalo E.
Published: March 2006

Dopamine (DA) uptake through the neuronal plasma membrane DA transporter (DAT) is essential for the maintenance of normal DA homeostasis in the brain. The DAT‐mediated re‐uptake system limits not only the intensity but also the duration of DA actions at presynaptic and postsynaptic receptors. This protein is the primary target for cocaine and amphetamine, both highly addictive and major substances of abuse worldwide. DAT is also the molecular target for therapeutic agents used in the treatment of mental disorders, such as attention deficit hyperactivity disorder and depression. Given the role played by the DAT in regulation of DA neurotransmission and its contribution to the abuse potential of psychostimulants, it becomes not only important but also necessary to understand the functional regulation of this protein. To investigate the cellular and molecular mechanisms associated with DAT function and regulation, our laboratory and others have embarked on a systematic search for DAT protein–protein interactions. Recently, a growing number of proteins have been shown to interact with DAT. These novel interactions might be important in the assembly, targeting, trafficking and/or regulation of transporter function. In this review, I summarize the main findings obtained from the characterization of DAT‐interacting proteins and discuss the functional implications of these novel interactions. Based on these new data, I propose to use the term DAT proteome to explain how interacting proteins regulate DAT function. These novel interactions might help define new mechanisms associated with the function of the transporter.
Spermidine synthase is prominently expressed in the striatal patch compartment and in putative interneurones of the matrix compartment
Authors: Krauss M. , Langnaese K. , Richter K. , Brunk I. , Wieske M. , Ahnert‐Hilger G. , Veh R. W. , Laube G.
Published: March 2006

The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate‐limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase‐like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the µ opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase‐like immunoreactivity was seen in patch compartment‐apposed putative interneurones. These spermidine synthase‐positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.
The JAK3 inhibitor WHI‐P154 prevents PDGF‐evoked process outgrowth in human neural precursor cells
Authors: Richards G. R. , Smith A. J. , Cuddon P. , Ma Q. P. , Leveridge M. , Kerby J. , Roderick H. L. , Bootman M. D. , Simpson P. B.
Published: March 2006

The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits offers a novel mechanism for treating a variety of CNS disorders. The aim of this study was to investigate pathways controlling neurite outgrowth in human neural precursor cells, in particular in response to platelet‐derived growth factor (PDGF). PDGF‐AA, ‐AB and ‐BB were found to initiate calcium signalling and produce robust increases in neurite outgrowth. PDGF‐induced outgrowth of Tuj1‐positive precursors was abolished by the addition of EGTA, suggesting that calcium entry is a critical part of the signalling pathway. Wortmannin and PD098059 failed to inhibit PDGF‐induced outgrowth. Clostridium Toxin B increased the amount of PDGF‐induced neurite branching but had no effect on basal levels. In contrast, WHI‐P154, an inhibitor of Janus protein tyrosine kinase (JAK3), Hck and Syk, prevented PDGF‐induced neurite outgrowth. PDGF activates multiple signalling pathways with considerable potential for cross‐talk. This study has highlighted the complexity of the pathways leading to neurite outgrowth in human neural precursors, and provided initial evidence to suggest that calcium entry is critical in producing the morphological changes observed.
Modulated splicing‐associated gene expression in P19 cells expressing distinct acetylcholinesterase splice variants
Authors: Ben‐Ari Shani , Toiber Debra , Sas Aldema S. , Soreq Hermona , Ben‐Shaul Yoram
Published: March 2006

Alternative splicing configurations and acetylcholinesterase (AChE) gene expression are both modified in neurons under stress. However, it is unclear if these phenomena are functionally interrelated. Using a home‐made spotted microarray focused on splicing‐associated transcripts, we tested the effects of excess 3′ splice variants of human AChE on splicing‐related gene expression in semi‐differentiated neuronal P19 cells. Of the tested transcripts, 17.3% and 20.2% showed modified expression levels (log2 of the ratio < − 0.3 or > 0.3) in transfected P19 cells overexpressing the stress‐inducible AChE‐R variant or the synaptic AChE‐S protein, respectively. Multiple transcripts encoding serine‐arginine rich (SR) and SR‐related splicing regulators were suppressed in cells expressing either of these variants, whereas the gene groups including splicing‐related helicases and transcripts involved in apoptosis displayed variant‐specific changes. Our findings are compatible with the assumption that both neuronal overexpression and alternative splicing of pre‐AChE mRNA may be causally involved in initiating global changes in neuronal alternative splicing, causing subsequent modifications in the expression patterns of numerous target genes.
Critical role of ASK1 in the 6‐hydroxydopamine‐induced apoptosis in human neuroblastoma SH‐SY5Y cells
Authors: Ouyang Mingxing , Shen Xun
Published: March 2006

6‐Hydroxydopamine (6‐OHDA)‐induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal‐regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH‐SY5Y cells exposed to 6‐OHDA. Over‐expressing kinase‐dead mutant ASK1(K709M) or knock‐down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1‐knock‐down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6‐OHDA‐induced cell apoptosis could be effectively prevented by over‐expressing the dominant‐negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6‐OHDA‐generated reactive oxygen species (ROS) by pre‐incubation of cells with N‐acetyl‐l‐cysteine effectively inhibited the 6‐OHDA‐induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6‐OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.
Big dynorphin as a putative endogenous ligand for the κ‐opioid receptor
Authors: Merg Florence , Filliol Dominique , Usynin Ivan , Bazov Igor , Bark Niklas , Hurd Yasmin L. , Yakovleva Tatjana , Kieffer Brigitte L. , Bakalkin Georgy
Published: March 2006

The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphin A (Dyn A), and dynorphin B (Dyn B) are endogenous opioid peptides that activate the κ‐opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32‐amino acid prodynorphin‐derived peptide consisting of Dyn A and Dyn B, with human KOR, μ‐ (hMOR) and δ‐ (hDOR) opioid receptors and opioid receptor‐like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR‐mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dyn B being approximately 1 : 3. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR‐mediated activation of G proteins.
Neuronal cell death caused by inhibition of intracellular cholesterol trafficking is caspase dependent and associated with activation of the mitochondrial apoptosis pathway
Authors: Huang Zhili , Hou Qingsong , Cheung Nam Sang , Li Qiu‐Tian
Published: March 2006

An elevated level of cholesterol in mitochondrial membranes of Niemann‐Pick disease type C1 (NPC1) mouse brains and neural cells has been found to cause mitochondrial dysfunction. In this study, we demonstrate that inhibition of intracellular cholesterol trafficking in primary neurons by class 2 amphiphiles, which mimics the major biochemical and cellular feature of NPC1, led to not only impaired mitochondrial function but also activation of the mitochondrial apoptosis pathway. In activation of this pathway both cytochrome c and Smac/Diablo were released but apoptosis‐inducing factor (AIF) was not involved. Treatment of the neurons with taurine, a caspase 9‐specific inhibitor, could prevent the amphiphile‐induced apoptotic cell death, suggesting that formation of apoptosome, followed by caspase 9 and caspase 3 activation, might play a critical role in the neuronal death pathway. Taken together, the mitochondria‐dependent death cascade induced by blocking intracellular cholesterol trafficking was caspase dependent. The findings provide clues for both understanding the molecular basis of neurodegeneration in NPC1 disease and developing therapeutic strategies for treatment of this disorder.
Mass spectrometric characterization and physiological actions of GAHKNYLRFamide, a novel FMRFamide‐like peptide from crabs of the genus Cancer
Authors: Cruz‐Bermúdez Nelson D. , Fu Qiang , Kutz‐Naber Kimberly K. , Christie Andrew E. , Li Lingjun , Marder Eve
Published: March 2006

The stomatogastric ganglion (STG) and the cardiac ganglion (CG) of decapod crustaceans are modulated by neuroactive substances released locally and by circulating hormones released from neuroendocrine structures including the pericardial organs (POs). Using nanoscale liquid chromatography electrospray ionization quadrupole‐time‐of‐flight tandem mass spectrometry and direct tissue matrix‐assisted laser desorption/ionization Fourier transform mass spectrometry we have identified and sequenced a novel neuropeptide, GAHKNYLRFamide (previously misassigned as KHKNYLRFamide in a study that did not employ peptide derivatization), from the POs and/or the stomatogastric nervous system (STNS) of the crabs, Cancer borealis, Cancer productus and Cancer magister. In C. borealis, exogenous application of GAHKNYLRFamide increased the burst frequency and number of spikes per burst of the isolated CG and re‐initiated bursting activity in non‐bursting ganglia, effects also elicited by the FMRFamide‐like peptides (FLPs) SDRNFLRFamide and TNRNFLRFamide. In the intact STNS (which contains the STG), exogenous application of GAHKNYLRFamide increased the frequency of the pyloric rhythm and activated the gastric mill rhythm, effects also similar to those elicited by SDRNFLRFamide and TNRNFLRFamide. FLP‐like immunoreactivity in the POs and the STNS was abolished by pre‐adsorption with the synthetic GAHKNYLRFamide. Different members of the FLP family exhibited differential degradation in the presence of extracellular peptidases. Taken collectively, the amino acid sequence of GAHKNYLRFamide, the blocking of FLP‐like immunostaining, and its physiological effects on the CG and STNS suggest that this peptide is a novel member of the FLP superfamily.
Up‐regulation of tyrosine hydroxylase gene transcription by tetradecanoylphorbol acetate is mediated by the transcription factors Ets‐like protein‐1 (Elk‐1) and Egr‐1
Authors: Stefano Luisa , Al Sarraj Jude , Rössler Oliver G. , Vinson Charles , Thiel Gerald
Published: March 2006

Tyrosine hydroxylase is the rate‐limiting enzyme in the biosynthesis of catecholamines. Expression of the tyrosine hydroxylase gene is regulated at the transcriptional level by extracellular signalling molecules, including epidermal growth factor (EGF), nerve growth factor (NGF) and glucocorticoids. We have analysed the stimulation of tyrosine hydroxylase gene transcription by the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in noradrenergic locus coeruleus‐like CATH.a cells and observed a striking enhancement of the transcriptional activation potential of the ternary complex factor Ets‐like protein‐1 (Elk‐1), a key transcriptional regulator of serum response element‐driven gene transcription. Likewise, TPA strongly up‐regulated the biosynthesis of the transcription factor Egr‐1 via distal serum response elements within the Egr‐1 5′‐flanking region. Subsequently, enhancement of the transcriptional activation potential of Egr‐1 was observed. Overexpression of Egr‐1 was sufficient to activate transcription of a tyrosine hydroxylase promoter/reporter gene, corroborating the view that the tyrosine hydroxylase gene is a target gene of Egr‐1. Expression of dominant‐negative mutants of Elk‐1 or Egr‐1 impaired TPA‐induced stimulation of a tyrosine hydroxylase promoter/reporter gene transcription. In contrast, dominant‐negative mutants of the transcription factors activating transcription factor (ATF)‐2, ATF4, cAMP response element‐binding protein, c‐Jun and CCAAT/enhancer binding protein (C/EBP) did not change TPA‐induced tyrosine hydroxylase promoter activity, indicating that these proteins are not part of the TPA‐mediated signalling cascade directed towards the tyrosine hydroxylase gene.