bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuregulin‐1 proteins in rat brain and transfected cells are localized to lipid rafts
Authors: Frenzel Kristen E. , Falls Douglas L.
Published: March 2003

Neuregulin‐1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin‐1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N‐terminal fragment containing the bioactive EGF‐like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140‐kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X‐100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low‐density fractions enriched in PSD‐95, a known lipid raft protein. In transfected cells expressing the neuregulin I‐β1a or the III‐β1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X‐100, and neuregulin proproteins and C‐terminal fragments were detected in lipid raft fractions. In contrast, the III‐β1a N‐terminal fragment was detected only in the detergent‐soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.
Induction of brain‐derived neurotrophic factor by convulsant drugs in the rat brain: involvement of region‐specific voltage‐dependent calcium channels
Authors: Katoh‐Semba Ritsuko , Takeuchi Ikuo K. , Inaguma Yutaka , Ichisaka Satoshi , Hata Yoshio , Tsumoto Tadaharu , Iwai Miwako , Mikoshiba Katsuhiko , Kato Kanefusa
Published: March 2003

A high level of hippocampal brain‐derived neurotrophic factor (BDNF) in normally aged as compared with young rats suggests that it is important to maintain a considerable level of hippocampal BDNF during aging in order to keep normal hippocampal functions. To elucidate possible mechanisms of endogenous BDNF increase, changes in levels of BDNF were studied in the rat brain following systemic administration of various convulsant agents; excitotoxic glutamate agonists, NMDA, kainic acid and (+/–)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA); GABA receptor antagonists, picrotoxin, pentylenetetrazole (PTZ) and lindane (γ‐hexachlorocyclohexane); and l‐type voltage‐dependent calcium channel agonist, BAY‐K 8644. Kainic acid and AMPA, but not NMDA, caused remarkable increases in BDNF protein in the rat hippocampus and entorhinal cortex. Picrotoxin, PTZ and lindane stimulated BDNF production in the entorhinal cortex and also in the hippocampus of rats showing very severe convulsions. On the other hand, BAY‐K 8644 treatment increased BDNF levels in the neocortex and entorhinal cortex. Maximal levels of BDNF protein were observed at 12–24 h, 8–16 h and 6 h following administration of kainic acid, PTZ and BAY‐K 8644, respectively. Kainic acid stimulated BDNF synthesis in presynaptic hippocampal granule neurons, but not in postsynaptic neurons with its receptors, while PTZ and BAY‐K 8644 produced the same effects in postsynaptic neurons in the entorhinal cortex (in granule neurons in the hippocampus) and in the whole cortex, respectively. Nifedipine inhibited almost completely BAY‐K 8644, but not PTZ, effects. ω‐Conotoxin GVIA and DCG‐IV partially blocked kainic acid‐induced enhancement of BDNF, indicating involvement of l‐type and N‐type voltage‐dependent calcium channels, respectively. In addition, BDNF levels in the hippocampus of mice deficient in d‐myo‐inositol‐1,4,5‐triphosphate receptor gene were scarcely different from those in the same region of controls, suggesting little involvement of intracellular calcium increase through this receptor. BAY‐K 8644, but not kainic acid or PTZ, stimulated the phosphorylation of cyclic AMP responsive element binding protein. Our results indicate convulsant‐dependent stimulation of BDNF production and involvement of region‐specific voltage‐dependent calcium channels.
Microglial secreted cathepsin B induces neuronal apoptosis
Authors: Kingham P. J. , Pocock J. M.
Published: March 2003

Activated microglia release a number of substances that can influence neuronal signalling and survival. Here we report that microglia stimulated with the peptide chromogranin A (CGA), secreted the cysteine protease, cathepsin B. Conditioned medium from CGA exposed microglia was neurotoxic to the HT22 hippocampal cell line and to primary cultures of cerebellar granule neurones. In both neuronal cell types, the neurotoxicity could be significantly attenuated with z‐FA‐fmk or by depletion of microglial conditioned medium with cathepsin B antibody. Conditioned medium from activated microglia or cathepsin B alone induced neuronal apoptosis and caspase 3 activation. Our data indicate that CGA‐activated microglia can trigger neuronal apoptosis and that this may be mediated through the secretion of cathepsin B. Since cathepsins may also play a role in the amyloidogenic processing of amyloid precursor protein, these results may have significance for tissue damage and neuronal loss in the neuropathology of Alzheimer's disease.
Evidence for non‐oxidative dopamine cytotoxicity: potent activation of NF‐κ B and lack of protection by anti‐oxidants
Authors: Weingarten Paul , Bermak Jason , Zhou Qun‐Yong
Published: March 2003

A stable aromatic acid decarboxylase expressing the Chinese hamster ovary cell line was developed to study the cytotoxic properties of intracellular and extracellular dopamine. The relative impermeability of cells to dopamine, but not to l‐DOPA, allows the differentiation of extracellular and intracellular dopamine cytotoxicity. In contrast to extracellular dopamine, intracellular dopamine toxicity was resistant to antioxidant protection, and did not require melanin formation for its toxicity. Furthermore, we demonstrated a rapid and potent activation of the stress‐inducible transcription factor NF‐κB by intracellular dopamine, which was also largely insensitive to antioxidant inhibition. A distinctly slower and less potent NF‐κB activation by extracellular dopamine was blocked by antioxidants and acetylsalicylic acid. Our results indicate the existence of a non‐oxidative mechanism of dopamine cytotoxicity. Mitigating intracellular dopamine toxicity could be a novel strategy of slowing the progressive degeneration of dopaminergic neurons in Parkinson's disease.
ARPP‐16/ARPP‐19: a highly conserved family of cAMP‐regulated phosphoproteins
Authors: Dulubova Irina , Horiuchi Atsuko , Snyder Gretchen L. , Girault Jean‐Antoine , Czernik Andrew J. , Shao Li , Ramabhadran Ram , Greengard Paul , Nairn Angus C.
Published: March 2003

ARPP‐16 and ARPP‐19 are closely related cAMP‐regulated phosphoproteins that were initially discovered in mammalian brain as in vitro substrates for protein kinase A (PKA). ARPP‐16 is enriched in dopamine‐responsive medium spiny neurons in the striatum, while ARPP‐19 is ubiquitously expressed. ARPP‐19 is highly homologous to α‐endosulfine and database searches allowed the identification of novel related proteins in D. melanogaster, C. elegans, S. mansoni and yeast genomes. Using isoform‐specific antibodies, we now show that ARPP‐19 is composed of at least two differentially expressed isoforms (termed ARPP‐19 and ARPP‐19e/endosulfine). All ARPP‐16/19 family members contain a conserved consensus site for phosphorylation by PKA (RKPSLVA in mammalian ARPP‐16 and ARPP‐19), and this site was shown to be efficiently phosphorylated in vitro by PKA. An antibody that specifically recognized the phosphorylated form of ARPP‐16/19/19e was used to examine the phosphorylation of ARPP‐16/19 family members in intact cells. In striatal slices, the phosphorylation of ARPP‐16 was increased in response to activation of D1‐type dopamine receptors, and decreased in response to activation of D2‐type dopamine receptors. In non‐neuronal cells, ARPP‐19 was highly phosphorylated in response to activation of PKA. These results establish that ARPP‐16/19 proteins constitute a family of PKA‐dependent intracellular messengers that function in all cells. The high levels of ARPP‐16 in striatal neurons and its bi‐directional regulation by dopamine suggest a specific role in dopamine‐dependent signal transduction. The conservation of this protein family through evolution suggests that it subserves an important cellular function that is regulated by PKA.
Effects of glutathione depletion by 2‐cyclohexen‐1‐one on excitatory amino acids‐induced enhancement of activator protein‐1 DNA binding in murine hippocampus
Authors: Ogita Kiyokazu , Kitayama Tomoya , Okuda Hiroaki , Yoneda Yukio
Published: March 2003

We have investigated the role of glutathione in mechanisms associated with excitatory amino acid signaling to the nuclear transcription factor activator protein‐1 (AP1) in the brain using mice depleted of endogenous glutathione by prior treatment with 2‐cyclohexen‐1‐one (CHX). In the hippocampus of animals treated with CHX 2 h before, a significant increase was seen in enhancement of AP1 DNA binding when determined 2 h after the injection of kainic acid (KA) at low doses. The sensitization to KA was not seen in animals injected with CHX 24 h before, in coincidence with the recovery of glutathione contents to the normal levels. By contrast, CHX did not significantly affect the potentiation by NMDA of AP1 binding under any experimental conditions. Prior treatment with CHX resulted in facilitation of behavioral changes induced by KA without affecting those induced by NMDA. These results suggest that endogenous glutathione may be at least in part involved in molecular mechanisms underlying transcriptional control by KA, but not by NMDA, signals of cellular functions.
DNase I disinhibition is predominantly associated with actin hyperpolymerization after traumatic brain injury
Authors: Bareyre Florence M. , Raghupathi Ramesh , Saatman Kathryn E. , McIntosh Tracy K.
Published: March 2003

To elucidate a role for the cytoskeletal protein actin in post‐traumatic apoptotic cell death, the ability of actin‐containing tissue extracts to inhibit exogenous DNase I was evaluated. In addition, cortical, hippocampal and thalamic extracts were examined for caspase‐mediated actin cleavage and changes in actin polymerization state. Rats were anesthetized, subjected to lateral fluid percussion brain injury of moderate severity, and euthanized at 1 h, 6 h, 24 h, 1 week or 3 weeks post‐injury (n = 3 per time‐point). Tissue extracts from all brain regions of sham (uninjured) animals inhibited exogenous DNase I activity to a significant extent. However, inhibition of DNase I was significantly reduced at 1 h and 6 h in the injured hippocampus, and at 1 h, 6 h and 3 weeks in the thalamus. DNase I in cortical extracts of all injured animals was inhibited to a similar extent as that in uninjured animals. Actin fragments consistent with caspase‐mediated proteolysis were observed in immunoblots of the injured hippocampus and thalamus at 1 h and 24 h, respectively, and were present up to 3 weeks post‐injury. Transient actin hyperpolymerization was observed at 1 and 6 h post‐injury in the thalamus and hippocampus, while actin depolymerization was observed at 1 and 3 weeks in the cortex and thalamus. Collectively our data suggest that DNase I disinhibition following brain trauma is associated predominantly with actin hyperpolymerization but also with actin depolymerization and concomitant caspase‐mediated actin proteolysis.
3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes
Authors: Isobe Ichiro , Yanagisawa Katsuhiko , Michikawa Makoto
Published: March 2003

3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti‐Rab5 antibody, dissociation of early endosomal auto‐antigen (EEA1) from the membrane fraction, and phosphorylation of Akt probably through a phosphatidylinositol‐3‐OH kinase [PI(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or PI(3)K is activated.
Roles of fibroblast growth factor 2 during innervation of the avian inner ear
Authors: Carnicero Estela , Garrido Juan José , Alonso Maria Teresa , Schimmang Thomas
Published: March 2003

The importance of individual members of the fibroblast growth factor gene family during innervation of the vertebrate inner ear is not clearly defined. Here we address the role of fibroblast growth factor 2 (FGF‐2 or basic FGF) during development of the chicken inner ear. We found that FGF‐2 stimulated survival of isolated cochlear and vestibular neurons during distinct phases of inner ear innervation. The potential neurotrophic role of FGF‐2 was confirmed by its expression in the corresponding sensory epithelia and the detection of one of its high‐affinity receptors in inner ear neurons. Finally, we have analysed the potential of the amplicon system based on defective herpes simplex virus type 1 (HSV‐1) vectors to express FGF‐2 in cochlear neurons. Overexpression of FGF‐2 in cochlear neurons resulted in neuronal differentiation demonstrating the presence of biologically active growth factor. This study underlines the potential of FGF‐2 to control innervation and development of sensory epithelia in the avian inner ear. Furthermore, amplicon vectors may provide a useful tool to analyse gene function in isolated neurons of the vertebrate inner ear.
Extracellular ATP stimulates an inhibitory pathway towards growth factor‐induced cRaf‐1 and MEKK activation in astrocyte cultures
Authors: Lenz Guido , Gonçalves Daniela , Luo Zhijun , Avruch Joseph , Rodnight Richard , Neary Joseph T.
Published: March 2003

ATP, acting via P2Y, G protein‐coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances fibroblast growth factor‐2 (FGF‐2)‐induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF‐2 cotreatment on the main components of the extracellular‐signal regulated protein kinase (ERK) cascade, cRaf‐1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf‐1 by FGF‐2 in primary cultures of rat cortical astrocytes. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf‐1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf‐1 by other growth factors, epidermal growth factor and platelet‐derived growth factor, as well as other MEK1 activators stimulated by FGF‐2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF‐2‐induced cRaf‐1 activation, thereby indicating specificity in the ATP‐induced inhibitory cross‐talk. These findings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor‐induced activation of cRaf‐1 and MEKKs. Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y‐GPCRs.
Proteasome inhibition in oxidative stress neurotoxicity: implications for heat shock proteins
Authors: Ding Qunxing , Keller Jeffrey N.
Published: March 2003

Recent studies have demonstrated that inhibition of the proteasome, an enzyme responsible for the majority of intracellular proteolysis, may contribute to the toxicity associated with oxidative stress. In the present study we demonstrate that exposure to oxidative injury (paraquat, H2O2, FeSO4) induces a rapid increase in reactive oxygen species (ROS), loss of mitochondrial membrane potential, inhibition of proteasome activity, and induction of cell death in neural SH‐SY5Y cells. Application of proteasome inhibitors (MG115, epoxomycin) mimicked the effects of oxidative stressors on mitochondrial membrane potential and cell viability, and increased vulnerability to oxidative injury. Neural SH‐SY5Y cells stably transfected with human HDJ‐1, a member of the heat shock protein family, were more resistant to the cytotoxicity associated with oxidative stressors. Cells expressing increased levels of HDJ‐1 displayed similar degrees of ROS formation following oxidative stressors, but demonstrated a greater preservation of mitochondrial function and proteasomal activity following oxidative injury. Cells transfected with HDJ‐1 were also more resistant to the toxicity associated with proteasome inhibitor application. These data support a possible role for proteasome inhibition in the toxicity of oxidative stress, and suggest heat shock proteins may confer resistance to oxidative stress, by preserving proteasome function and attenuating the toxicity of proteasome inhibition.
6‐Hydroxykynurenic acid and kynurenic acid differently antagonise AMPA and NMDA receptors in hippocampal neurones
Authors: Weber Marco , Dietrich Dirk , Gräsel Ines , Reuter Gerhard , Seifert Gerald , Steinhäuser Christian
Published: March 2003

6‐Hydroxykynurenic acid (6‐HKA), a derivative of kynurenic acid (KYNA) extracted from Ginkgo biloba leaves, was tested for its putative glutamate receptor (GluR) antagonism in comparison to the scaffold substance. The patch‐clamp method together with fast‐application techniques were used to estimate inhibition by 6‐HKA and KYNA of agonist binding at NMDA and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors (NMDARs and AMPARs) of CA1 pyramidal neurones. 6‐Hydroxykynurenic acid proved to be a low‐affinity antagonist. When comparing with KYNA, 6‐HKA was less potent at NMDARs (IC50 = 136 versus 59 µm), but showed a higher affinity to AMPARs (KB = 22 versus 172 µm). The replacement of 6‐HKA and KYNA by glutamate was investigated on outside‐out patches. Both antagonists competitively inhibited AMPAR responses and displayed fast unbinding kinetics, but the derivative was significantly slower displaced than KYNA (τ = 1.63 versus 1.22 ms). Our findings demonstrate that 6‐hydroxylation considerably changes the pharmacological profile of KYNA. Among the 6‐derivatives of KYNA, 6‐HKA shows the highest affinity to AMPARs. Despite its relatively low lipophily, these properties might be of clinical relevance under conditions that compromise the integrity of the blood–brain barrier. Furthermore, 6‐HKA should be a useful tool to analyse glutamate‐mediated synaptic responses.
Identification of a basolateral membrane targeting signal within the cytoplasmic domain of myelin/oligodendrocyte glycoprotein
Authors: Kroepfl John F. , Gardinier Minnetta V.
Published: March 2003

Oligodendrocytes possess two distinct membrane compartments – uncompacted plasma membrane (cell body, processes) and compact myelin. Specific targeting mechanisms must exist to establish and maintain these membrane domains. Polarized epithelial cells have the best characterized system for targeting components to apical and basolateral compartments. Since oligodendrocytes arise from neuroepithelial cells, we investigated whether they might utilize targeting paradigms similar to polarized epithelial cells. Myelin/oligodendrocyte glycoprotein (MOG) is a transmembrane Ig‐like molecule restricted to uncompacted oligodendroglial plasma membrane. We stably expressed MOG in Madin–Darby canine kidney (MDCK) Type II epithelial cells, which have been extensively used in protein‐targeting studies. Data from surface biotinylation assays and confocal microscopy revealed that MOG sorts exclusively to the basolateral membrane of MDCK cells. Expression vectors containing progressive truncations of MOG from the cytoplasmic C‐terminus were expressed in MDCK cells to localize basolateral sorting signals. A loss of only four C‐terminal residues results in some MOG expression at the apical surface. More strikingly, removal of the C‐terminal membrane associated hydrophobic domain from MOG results in complete loss of basolateral sorting and specific targeting to the apical membrane. These data suggest that myelinating oligodendrocytes may utilize a sorting mechanism similar to that of polarized epithelia.
Genomic structure, developmental distribution and functional properties of the chicken P2X5 receptor
Authors: Ruppelt Anja , Ma Weiyuan , Borchardt Kerstin , Silberberg Shai D. , Soto Florentina
Published: March 2003

We report here the cloning of a chicken cDNA (402 aa) showing high sequence similarity to the previously cloned rat and human P2X5 receptors (67 and 69%, respectively). The chicken P2X5 subunit is encoded by a gene composed of 12 translated exons, which shows conserved genomic structure with mammalian P2X genes. In HEK‐293 cells heterologously expressing chicken P2X5 receptors, ATP activates a current that desensitizes in a way that is dependent on the presence of extracellular divalent cations. ATP and 2‐methylthio ATP are equipotent agonists (EC50 ∼ 2 µm) and suramin and pyridoxal 5‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid are potent antagonists. Additionally, reversal potential measurements indicate that chicken P2X5 is permeable not only to cations but also to chloride (PCs+/PCl‐ ∼ 1.9), as has been described for native P2X receptor mediated responses in embryonic chicken skeletal muscle. mRNA distribution of chicken P2X5 was determined by in situ hybridization analysis in both whole embryos and on tissue slices of heart and skeletal muscle. Our results suggest that chicken P2X5 receptors are expressed in developing muscle and might play a role in early muscle differentiation.
Endogenous neurotensin down‐regulates dopamine efflux in the nucleus accumbens as revealed by SR‐142948A, a selective neurotensin receptor antagonist
Authors: Brun P. , Leonetti M. , Sotty F. , Steinberg R. , Soubrié P. , Renaud B. , Suaud‐Chagny M. F.
Published: March 2003

SR‐142948A belongs to the second generation of potent, selective, non‐peptide antagonists of neurotensin receptors. It was used to investigate the role of endogenous neurotensin in the regulation of dopamine efflux in the nucleus accumbens and striatum of anaesthetized and pargyline‐treated rats. All the data were obtained using in vivo electrochemistry. Electrically evoked (20 Hz, 10 s) dopamine efflux was monitored by differential pulse amperometry, whereas variations in basal (tonic) dopamine efflux were monitored by differential normal pulse voltammetry. Like the first‐generation compound SR‐48692, SR‐142948A did not affect the tonic and evoked dopamine efflux, but dose‐dependently enhanced haloperidol (50 µg/kg, i.p.) induced facilitation of the electrically evoked dopamine release in the nucleus accumbens. In contrast to SR‐48692, SR‐142948A dose‐dependently potentiated haloperidol (50 µg/kg, i.p.) induced increase in the basal dopamine level in the nucleus accumbens. This potentiating effect did not appear in the striatum. When dopaminergic and/or neurotensinergic transmissions were modified by a higher dose of haloperidol (0.5 mg/kg, i.p.), apomorphine, amphetamine or nomifensine, SR‐142948A pre‐treatment affected only the effect of apomorphine on the basal dopamine level in the nucleus accumbens. These results strengthen the hypothesis that endogenous neurotensin could exert a negative control on mesolimbic dopamine efflux.
Pharmacological and functional characterization of muscarinic receptor subtypes in developing oligodendrocytes
Authors: Ragheb Fadi , Molina‐Holgado Eduardo , Cui Qiao‐Ling , Khorchid Amani , Liu Hsueh‐Ning , Larocca Jorge N. , Almazan Guillermina
Published: March 2003

This study focused on the molecular and pharmacological characterization of muscarinic acetylcholine receptors expressed by progenitors and differentiated oligodendrocytes. We also analyzed the role of muscarinic receptors in regulating downstream signal transduction pathways and the functional significance of receptor expression in oligodendrocytes. RT‐PCR analysis revealed the expression of transcripts for M3, and to a lesser extent M4, followed by M1, M2 and M5 receptor subtypes in both progenitors and differentiated oligodendrocytes. Competition binding experiments using [3H]N‐methylscopolamine and several antagonists, as well as inhibition of carbachol‐mediated phosphoinositide hydrolysis, showed that M3 is the main subtype expressed in these cells. In progenitors the activation of p42/44‐mitogen‐activated protein kinase (MAPK) and cAMP‐response element binding protein (CREB) as well as c‐fos mRNA expression were blocked by the M3 relatively selective antagonist, 4‐DAMP, and its irreversible analogue, 4‐DAMP‐mustard. Carbachol increased proliferation of progenitors, an effect prevented by atropine and 4‐DAMP, as well as by the MAPK kinase inhibitor PD98059. These results indicate that carbachol modulates oligodendrocyte progenitor proliferation through M3 receptors, involving activation of a MAPK signaling pathway. Receptor density and phosphoinositide hydrolysis are down‐regulated during oligodendrocyte differentiation. Functional consequences of these events are a reduction in carbachol‐stimulated p42/44MAPK and CREB phosphorylation, as well as induction of c‐fos.
Sensitivity to MPTP is not increased in Parkinson's disease‐associated mutant α‐synuclein transgenic mice
Authors: Rathke‐Hartlieb Silvia , Kahle Philipp J. , Neumann Manuela , Ozmen Laurence , Haid Sibylle , Okochi Masayasu , Haass Christian , Schulz Jörg B.
Published: March 2003

Environmental and genetic factors that contribute to the pathogenesis of Parkinson's disease are discussed. Mutations in the α‐synuclein (αSYN ) gene are associated with rare cases of autosomal‐dominant Parkinson's disease. We have analysed the dopaminergic system in transgenic mouse lines that expressed mutant [A30P]αSYN under the control of a neurone‐specific Thy‐1 or a tyrosine hydroxylase (TH) promoter. The latter mice showed somal and neuritic accumulation of transgenic [A30P]αSYN in TH‐positive neurones in the substantia nigra. However, there was no difference in the number of TH‐positive neurones in the substantia nigra and the concentrations of catecholamines in the striatum between these transgenic mice and non‐transgenic littermates. To investigate whether forced expression of [A30P]αSYN increased the sensitivity to putative environmental factors we subjected transgenic mice to a chronic 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) regimen. The MPTP‐induced decrease in the number of TH‐positive neurones in the substantia nigra and the concentrations of catecholamines in the striatum did not differ in any of the [A30P]αSYN transgenic mouse lines compared with wild‐type controls. These results suggest that mutations and forced expression of αSYN are not likely to increase the susceptibility to environmental toxins in vivo.
JNK3 contributes to c‐Jun activation and apoptosis but not oxidative stress in nerve growth factor‐deprived sympathetic neurons
Authors: Bruckner Shane R. , Tammariello Steven P. , Kuan Chia‐Yi , Flavell Richard A. , Rakic Pasko , Estus Steven
Published: March 2003

The stress activated protein kinase pathway culminates in c‐Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP‐1347, a compound known to inhibit this overall pathway but not JNKsper se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3‐deficient mice and quantified nerve growth factor (NGF) deprivation‐induced neuronal death, oxidative stress, c‐Jun phosphorylation, andc‐juninduction. Here, we report that oxidative stress in neurons from JNK3‐deficient mice is normal after NGF deprivation. In contrast, NGF‐deprivation‐induced increases in the levels of phosphorylated c‐Jun,c‐jun, and apoptosis are each inhibited in JNK3‐deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c‐Jun and apoptosis in a classic model of cell‐autonomous programmed neuron death.
Agonist‐induced internalization of the metabotropic glutamate receptor 1a is arrestin‐ and dynamin‐dependent
Authors: Mundell Stuart J. , Matharu Anne‐Lise , Pula Giordano , Roberts Peter J. , Kelly Eamonn
Published: March 2003

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N‐terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA‐mGluR1a, the epitope‐tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 µm; 30 min) the HA‐mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist‐induced internalization of the HA‐mGluR1a. To determine whether agonist‐induced mGluR1a internalization is an arrestin‐ and dynamin‐dependent process, cells were cotransfected with HA‐mGluR1a and either of these dynamin‐K44A or arrestin‐2 (319–418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate‐induced (10 µm; 30 min) HA‐mGluR1a internalization. In addition, wild‐type arrestin‐2−green fluorescent protein (arrestin‐2−GFP) or arrestin‐3−GFP underwent agonist‐induced translocation from cytosol to membrane in HEK293 cells coexpressing HA‐mGluR1a. Taken together our observations demonstrate that agonist‐induced internalization of mGluR1a is an arrestin‐ and dynamin‐dependent process.
The binding of zinc and copper ions to nerve growth factor is differentially affected by pH: implications for cerebral acidosis
Authors: Ross Gregory M. , Shamovsky Igor L. , Woo Sang B. , Post Joan I. , Vrkljan Philip N. , Lawrance Gail , Solc Mark , Dostaler Suzanne M. , Neet Kenneth E. , Riopelle Richard J.
Published: March 2003

It has recently been shown that transition metal cations Zn2+ and Cu2+ bind to histidine residues of nerve growth factor (NGF) and other neurotrophins (a family of proteins important for neuronal survival) leading to their inactivation. Experimental data and theoretical considerations indicate that transition metal cations may destabilize the ionic form of histidine residues within proteins, thereby decreasing their pKa values. Because the release of transition metal cations and acidification of the local environment represent important events associated with brain injury, the ability of Zn2+ and Cu2+ to bind to neurotrophins in acidic conditions may alter neuronal death following stroke or as a result of traumatic injury. To test the hypothesis that metal ion binding to neurotrophins is influenced by pH, the effects of Zn2+ and Cu2+ on NGF conformation, receptor binding and NGF tyrosine kinase (trkA) receptor signal transduction were examined under conditions mimicking cerebral acidosis (pH range 5.5–7.4). The inhibitory effect of Zn2+ on biological activities of NGF is lost under acidic conditions. Conversely, the binding of Cu2+ to NGF is relatively independent of pH changes within the studied range. These data demonstrate that Cu2+ has greater binding affinity to NGF than Zn2+ at reduced pH, consistent with the higher affinity of Cu2+ for histidine residues. These findings suggest that cerebral acidosis associated with stroke or traumatic brain injury could neutralize the Zn2+‐mediated inactivation of NGF, whereas corresponding pH changes would have little or no influence on the inhibitory effects of Cu2+. The importance of His84 of NGF for transition metal cation binding is demonstrated, confirming the involvement of this residue in metal ion coordination.
Selective down‐regulation of the astrocyte glutamate transporters GLT‐1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy
Authors: Hazell Alan S. , Rao K. V. Rama , Danbolt Niels C. , Pow David V. , Butterworth Roger F.
Published: March 2003

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT‐1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT‐1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non‐vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT‐3 were unaffected in the medial thalamus. In addition, astrocyte‐specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT‐1 or GLAST protein was not observed on day 12 of treatment, indicating that down‐regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT‐1 content was positively correlated with levels of the neurofilament protein α‐internexin, suggesting that early neuronal drop‐out may contribute to the down‐regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT‐1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency‐induced cell death.
Overexpression of dopamine D2 receptors reduces alcohol self‐administration
Authors: Thanos Panayotis K. , Volkow Nora D. , Freimuth Paul , Umegaki Hiroyuki , Ikari Hiroyuki , Roth George , Ingram Donald K. , Hitzemann Robert
Published: March 2003

The mechanism(s) underlying predisposition to alcohol abuse are poorly understood but may involve brain dopamine system(s). Here we used an adenoviral vector to deliver the dopamine D2 receptor (DRD2) gene into the nucleus accumbens of rats, previously trained to self‐administer alcohol, and to assess if DRD2 levels regulated alcohol preference and intake. We show that increases in DRD2 (52%) were associated with marked reductions in alcohol preference (43%), and alcohol intake (64%) of ethanol preferring rats, which recovered as the DRD2, returned to baseline levels. In addition, this DRD2 overexpression similarly produced significant reductions in ethanol non‐preferring rats, in both alcohol preference (16%) and alcohol intake (75%). This is the first evidence that overexpression of DRD2 reduces alcohol intake and suggests that high levels of DRD2 may be protective against alcohol abuse.
Trafficking of green fluorescent protein tagged‐vesicular acetylcholine transporter to varicosities in a cholinergic cell line
Authors: Santos M. S. , Barbosa J. , Veloso G. S. , Ribeiro F. , Kushmerick C. , Gomez M. V. , Ferguson S. S. G. , Prado V. F. , Prado M. A. M.
Published: March 2003

Synaptic vesicle proteins are suggested to travel from thetrans‐Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)‐tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP–VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP–VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4‐64 and GFP–Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP–VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis‐defective GFP–VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin‐mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.
Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina
Authors: Ientile Riccardo , Macaione Vincenzo , Teletta Maria , Pedale Santa , Torre Valerio , Macaione Salvatore
Published: March 2003

Excitotoxic studies using isolated chick embryo retina indicated that such an in vitro model provides a valid tool to characterize the effect of different agonists for subtypes of glutamate ionotropic receptors. In retinas maintained for 24 h in a Krebs medium, after a brief exposure (30 min) to glutamate agonists, we compared the effects produced by NMDA and non‐NMDA‐agonists, such as kainic acid (KA) or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA). Delayed retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium after exposure to the previously named agonists. Although at high concentrations, both KA and AMPA produced more relevant release than NMDA, 7–8% of total retinal LDH was released after exposure to a 50 µm concentration of non‐NMDA agonists. These values were similar to those obtained after 100 µm NMDA. In this regard, retinal tissue appeared to be less sensitive to excitotoxicity based on the activation of NMDA receptor subtype. All three agents produced histopathological lesions typical for excitotoxic damage. A delayed form of excitotoxicity observed in retina segments was predominated by necrotic features. However, the activation of apoptotic machinery early during the incubation period subsequent to brief exposure to NMDA (100 µm) was also present. The activation of caspase enzymes was studied by a fluorometric protease activity assay as well as by western blot analysis. Caspase‐3‐like activity reached the highest value within 3 h of incubation after exposure to excitotoxin, then the level of enzyme activity declined to lower values. As confirmed by a time‐related appearance of TUNEL‐positive nuclei, apoptotic features appeared to be specific for retina response to NMDA. In contrast, the exposure to a 50 µm concentration of KA or AMPA induced necrotic cell damage which was evident through the incubation, leading to a delayed mechanism of excitotoxicity. These observations provide evidence that in the retinal model, with regard to agonist concentrations and subtype of glutamate receptors, the cascade of events leading to excitotoxicity may result in either apoptotic or necrotic neuronal cell damage.
Axotomy and nerve growth factor regulate levels of neuronal nicotinic acetylcholine receptor α3 subunit protein in the rat superior cervical ganglion
Authors: Yeh Jenny J. , Ferreira Manuel , Ebert Steven , Yasuda Robert P. , Kellar Kenneth J. , Wolfe Barry B.
Published: March 2003

Neuronal nicotinic acetylcholine receptors (nAChRs) play a significant role in sympathetic transmission in the superior cervical ganglia (SCG), with most of the signal carried by a nAChR containing an α3 subunit. Work has shown that transection of the postganglionic nerves (axotomy) of the SCG results in a decrease in mRNA transcripts for α3, α5, α7 and β4 and in protein expression of α7 and β4. To evaluate effects of axotomy on α3 protein in the SCG, quantitative immunoblotting was used to demonstrate a dramatic decrease (> 80%) in the levels of this subunit 4 days after axotomy. Similarly, immunocytochemistry showed a marked decline in the number and the intensity of stained neurons for the α3 subunit as well as tyrosine hydroxylase. Ganglia explanted into culture for 4 days also showed a substantial decrease in α3 subunit protein. This decrease was partially prevented by the addition of nerve growth factor (NGF) to the culture medium at the time of explantation. Additionally, this decrease was reversed by the addition of NGF to the culture medium following 4 days in culture in the absence of NGF. These findings suggest that the loss of α3 subunit contributes to the reported decrease in ganglionic synaptic transmission that follows axotomy, and that NGF plays an important role in regulating the expression of α3‐containing nAChRs in the SCG.
Brain‐derived neurotrophic factor stimulates β‐amyloid gene promoter activity by a Ras‐dependent/AP‐1‐independent mechanism in SH‐SY5Y neuroblastoma cells
Authors: Ruiz‐León Yolanda , Pascual Angel
Published: March 2003

The β‐amyloid peptide, the major component of Alzheimer‐associated plaques, derives from a larger β‐amyloid precursor protein (APP), that is expressed in both neural and non‐neural cells. Overexpression of APP actively contributes to the development of senile plaques and is considered a risk factor for the disease. APP expression is regulated by a variety of cellular mediators, among them ligands of tyrosine kinase receptors. In this study, we present evidence that brain‐derived neurotrophic factor (BDNF) modulates, in a dose‐ and time‐dependent fashion, APP promoter activity in SH‐SY5Y neuroblastoma cells transiently expressing the receptor TrkB. The APP promoter contains two potential AP‐1 sites, and we examined whether or not protein kinase C (PKC) and the AP‐1 sites of the promoter mediate the BDNF‐induced stimulation of APP. Stimulation of APP promoter activity by BDNF was not affected by the PKC inhibitor bisindolylmaleimide, or by dominant negative mutants of the AP‐1 components Fos and Jun, which, however, blocked the response to phorbol esters. These results suggest that activation of the APP promoter by BDNF is largely independent of PKC and AP‐1. In contrast, activated Ras increased APP promoter activity in SH‐SY5Y cells, and a dominant negative mutant of Ras abolished BDNF‐mediated promoter stimulation. Taken together, our results suggest a mechanism that involves activation of the Ras/MAP kinase signaling pathway, and phosphorylation of as yet unidentified effectors which in turn can activate response elements within the APP promoter.
IL‐10 and IL‐4 regulate type‐I and type‐II IL‐1 receptors expression on IL‐1β‐activated mouse primary astrocytes
Authors: Pousset F. , Cremona S. , Dantzer R. , Kelley K. W. , Parnet P.
Published: March 2003

When activated by its ligand, the interleukin receptor type I (IL‐1RI) transduces signals in cooperation with the IL‐1 receptor accessory protein (IL‐1RacP). In contrast, IL‐1RII functions as a decoy receptor without participating in IL‐1 signalling. Brain astrocytes are cellular targets of IL‐1 and play a pivotal role in brain responses to inflammation. The regulation of IL‐1 receptors on astrocytes by anti‐inflammatory cytokines such as IL‐4 and IL‐10 has not been studied, despite its importance for understanding the way these cells respond to IL‐1. Using RT‐PCR, we first showed that the expression of IL‐1RI and IL‐1RII, but not IL‐1RacP, mRNAs are up‐regulated by IL‐1β in a time‐dependent manner. Using a radioligand binding technique, we then showed that astrocytes display an equivalent number of IL‐1RI and IL‐1RII. IL‐1β decreases the number of IL‐1RI binding sites, whereas it increases those of IL‐1RII. IL‐4 and IL‐10 both up‐regulate IL‐1RII IL‐1β‐induced, but only IL‐4 does so for IL‐1RI. At the protein level, IL‐4 and IL‐10 dramatically reverse the ability of IL‐1β to inhibit expression of IL‐1RI but neither affects the ability of IL‐1β to enhance the number of IL‐1RII. Collectively, these results establish the existence of receptor cross‐talk between pro‐ and anti‐inflammatory cytokines on a critical type of cell that regulates inflammatory events in the brain.
Activation of MKK6, an upstream activator of p38, in Alzheimer's disease
Authors: Zhu Xiongwei , Rottkamp Catherine A. , Hartzler Anthony , Sun Zheng , Takeda Atsushi , Boux Heather , Shimohama Shun , Perry George , Smith Mark A.
Published: March 2003

Mitogen‐activated protein kinase (MAPK) p38 has been implicated in the pathogenesis of Alzheimer's disease, but the upstream cascade leading to p38 activation has not been elucidated in the disease. In the present study, we focused on mitogen‐activated protein kinase kinase 6 (MKK6), one of the upstream activators of p38 MAPK. We found that MKK6 was not only increased but also specifically associated with granular structures in the susceptible neurons in the hippocampus and cortex of Alzheimer's disease patients, but was only weakly diffuse in the cytoplasm in neurons in control cases. Immunoblot analysis demonstrated a significant increase of MKK6 level in Alzheimer's disease compared with age‐matched controls. In this regard, in hippocampal and cortical regions of individuals with Alzheimer's disease, the activated phospho‐MKK6 was localized exclusively in association with pathological alterations including neurofibrillary tangles, senile plaques, neuropil threads and granular structures, overlapping with activated p38 MAPK suggesting both a functional and mechanic link. By immunoblot analysis, phospho‐MKK6 is also significantly increased in AD compared with control cases. Together, these findings lend further credence to the notion that the p38 MAPK pathway is dysregulated in Alzheimer's disease and also indicates an active role for this pathway in disease pathogenesis.
The transcription factors CREB and c‐Fos play key roles in NCAM‐mediated neuritogenesis in PC12‐E2 cells
Authors: Jessen Ulla , Novitskaya Vera , Pedersen Nina , Serup Palle , Berezin Vladimir , Bock Elisabeth
Published: March 2003

The neural cell adhesion molecule (NCAM) stimulates axonal outgrowth by activation of the Ras‐mitogen activated protein kinase (MAPK) pathway and by generation of arachidonic acid. We investigated whether the transcription factors, cyclic‐AMP response‐element binding protein (CREB) and c‐Fos play roles in this process by estimating NCAM‐dependent neurite outgrowth from PC12‐E2 cells grown in co‐culture with NCAM‐negative or NCAM‐positive fibroblasts. PC12‐E2 cells were transiently transfected with expression plasmids encoding wild‐type or dominant negative forms of CREB and c‐Fos or an activated form of the MAPK kinase, MEK2. Alternatively, PC12‐E2 cells were treated with arachidonic acid, the cAMP analogue dBcAMP, or protein kinase A (PKA) inhibitors. The negative forms of CREB and c‐Fos inhibited neurite outgrowth mediated by NCAM, arachidonic acid, dBcAMP, or MEK2. Neither CREB nor c‐Fos could compensate for the inactivation of the other, indicating that both factors are important in NCAM‐mediated neuritogenesis. Treatment of primary hippocampal neurons with a synthetic NCAM peptide ligand known to stimulate neurite outgrowth induced phosphorylation of CREB and expression of c‐fos. We thus present evidence that NCAM‐mediated neurite outgrowth involves a series of signal transduction pathways, including the cAMP/PKA pathway, targeting c‐Fos and CREB.