bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
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.
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.
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.
Serotonin‐1B receptor‐mediated inhibition of [3H]GABA release from rat ventral tegmental area slices
Authors: Yan Qing‐Shan , Yan Shu‐E 
Published: March 2003

In order to assess a role of 5‐HT1B receptors for regulation of GABA transmission in the ventral tegmental area (VTA), VTA slices from the rat were incubated with [3H]GABA and β‐alanine, and superfused in the presence of nipecotic acid and aminooxyacetic acid. [3H]GABA release was induced by exposures to the medium containing 30 mm potassium for 2 min. The results showed that high potassium‐evoked [3H]GABA release was sensitive to calcium withdrawal or blockade of sodium channels by tetrodotoxin, suggesting that tritium overflow induced by high potassium derived largely from neuronal stores. Administration of CP 93129 (0.15 and 0.45 µm), a 5‐HT1B receptor agonist, or RU 24969 (0.15 and 0.45 µm), a 5‐HT1B/1A receptor agonist, but not 8‐OH‐DPAT (0.45 µm), a 5‐HT1A receptor agonist, inhibited high potassium‐evoked [3H]GABA release in a concentration‐related manner. The RU 24969‐induced inhibition of [3H]GABA release was antagonized by either SB 216641, a 5‐H1B receptor antagonist, or cyanopindolol, a 5‐HT1B/1A receptor antagonist, but not by WAY 100635, a 5‐HT1A receptor antagonist. Pre‐treatment with SB 216641 also antagonized CP 93129‐induced inhibition of [3H]GABA release. The results support the hypothesis that 5‐HT1B receptors within the VTA can function as heteroreceptors to inhibit GABA release.
Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis
Authors: Yagami Tatsurou , Ueda Keiichi , Asakura Kenji , Hayasaki‐Kajiwara Yoko , Nakazato Hitoshi , Sakaeda Toshiyuki , Hata Satoshi , Kuroda Takayuki , Takasu Nobuo , Hori Yozo 
Published: March 2003

AbstractGroup IB secretory phospholipase A2 (sPLA2‐IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high‐affinity binding sites of sPLA2‐IB have been documented. However, it remains obscure whether sPLA2‐IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2‐IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2‐IB induced neuronal cell death in a concentration‐dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2‐IB‐induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2‐IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Δ12‐PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase‐2 (COX‐2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2‐IB‐induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2‐IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Δ12‐PGJ2, might mediate sPLA2‐IB‐induced apoptosis.
Synaptic regulation of somatodendritic dopamine release by glutamate and GABA differs between substantia nigra and ventral tegmental area
Authors: Chen Billy T. , Rice Margaret E. 
Published: March 2003

AbstractMidbrain dopamine (DA) cells of the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) exhibit somatodendritic release of DA. To address how somatodendritic release is regulated by synaptic glutamatergic and GABAergic input, we examined the effect of ionotropic‐ receptor antagonists on locally evoked extracellular DA concentration ([DA]o) in guinea pig midbrain slices. Evoked [DA]owas monitored with carbon‐fiber microelectrodes and fast‐scan cyclic voltammetry. In SNc, evoked [DA]owas 160% of control in the presence of the AMPA‐receptor antagonist, GYKI‐52466, or the NMDA‐receptor antagonist, AP5. Similar increases were seen with the GABAA‐receptor antagonist, picrotoxin, or the GABAB‐receptor antagonist, saclofen. The increase seen with GYKI‐52466 was prevented when both picrotoxin and saclofen were present, consistent with normal, AMPA‐receptor mediated activation of GABAergic inhibition. The increase with AP5 persisted, however, implicating NMDA‐receptor mediated activation of another inhibitory circuit in SNc. In the VTA, by contrast, evoked [DA]owas unaffected by GYKI‐52466 and fell slightly with AP5. Neither picrotoxin nor saclofen alone or in combination had a significant effect on evoked [DA]o. When GABA receptors were blocked in the VTA, evoked [DA]owas decreased by 20% with either GYKI‐52466 or AP5. These data suggest that in SNc, glutamatergic input acts predominantly on GABAergic or other inhibitory circuits to inhibit somatodendritic DA release, whereas in VTA, the timing or strength of synaptic input will govern whether the net effect on DA release is excitatory or inhibitory.
Functional and molecular characterization of metabotropic glutamate receptors expressed in rat striatal cholinergic interneurones
Authors: Bell Matt I. , Richardson Peter J. , Lee Kevin 
Published: March 2003

AbstractIn the present study we have used single‐cell RT‐PCR in conjunction with electrophysiology to examine the expression and functional properties of metabotropic glutamate receptors (mGluRs) expressed within biochemically identified cholinergic interneurones in the rat striatum. Using single‐cell RT‐PCR, it was possible to demonstrate the presence of mGluR1, mGluR2, mGluR3, mGluR5 and mGluR7 mRNAs within single cholinergic interneurones. Bath application of the non‐selective mGluR agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) or the group‐I mGluR agonist 3,5‐dihydroxyphenylglycine (DHPG) depolarized all cholinergic neurones tested by activation of an inward current at − 60 mV. The effects of DHPG were partially inhibited by the mGluR5 selective antagonist 6‐methyl‐2‐(pherazo)‐3‐pyridinol and by the non‐selective group‐I antagonist α‐methyl‐4‐carboxyphenylglycine but were not mimicked by the group‐II and group‐III selective mGluR agonists 2‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV) and l‐2‐amino‐4‐phosphonobutanoate (l‐AP4), respectively. Intrastriatal stimulation evoked an excitatory postsynaptic current within cholinergic neurones that was reversibly inhibited by bath application of the group‐II and group‐III selective mGluR agonists DCG‐IV and l‐AP4, respectively, via presynaptic actions.In summary, we have identified the mGluRs expressed by striatal cholinergic interneurones and demonstrated that their activation produces modulatory effects via both pre‐ and postsynaptic mechanisms.
Highly conserved and disease‐specific patterns of carboxyterminally truncated Aβ peptides 1–37/38/39 in addition to 1–40/42 in Alzheimer's disease and in patients with chronic neuroinflammation
Authors: Wiltfang J. , Esselmann H. , Bibl M. , Smirnov A. , Otto M. , Paul S. , Schmidt B. , Klafki H.‐W. , Maler M. , Dyrks T. , Bienert M. , Beyermann M. , Rüther E. , Kornhuber J. 
Published: March 2003

AbstractHuman lumbar CSF patterns of Aβ peptides were analysed by urea‐based β‐amyloid sodium dodecyl sulphate polyacrylamide gel electrophoresis with western immunoblot (Aβ‐SDS–PAGE/immunoblot). A highly conserved pattern of carboxyterminally truncated Aβ1–37/38/39 was found in addition to Aβ1–40 and Aβ1–42. Remarkably, Aβ1–38 was present at a higher concentration than Aβ1–42, being the second prominent Aβ peptide species in CSF. Patients with Alzheimer's disease (AD, n = 12) and patients with chronic inflammatory CNS disease (CID, n = 10) were differentiated by unique CSF Aβ peptide patterns from patients with other neuropsychiatric diseases (OND, n = 37). This became evident only when we investigated the amount of Aβ peptides relative to their total Aβ peptide concentration (Aβ1–x%, fractional Aβ peptide pattern), which may reflect disease‐specific γ‐secretase activities. Remarkably, patients with AD and CID shared elevated Aβ1–38% values, whereas otherwise the patterns were distinct, allowing separation of AD from CID or OND patients without overlap. The presence of one or two ApoE ε4 alleles resulted in an overall reduction of CSF Aβ peptides, which was pronounced for Aβ1–42. The severity of dementia was significantly correlated to the fractional Aβ peptide pattern but not to the absolute Aβ peptide concentrations.
Regulation of cAMP‐specific phosphodiesterases type 4B and 4D (PDE4) splice variants by cAMP signaling in primary cortical neurons
Authors: D'Sa Carrol , Tolbert Lara M. , Conti Marco , Duman Ronald S. 
Published: March 2003

AbstractThis study examined the regulation of all known phosphodiesterase (PDE) type PDE4A, PDE4B and PDE4D splice variants in cortical neurons by cAMP signaling. Treatment with dibutyryl‐cAMP (db‐cAMP) caused the induction of two of the known splice variants, PDE4B2 and PDE4D1/PDE4D2. Although the splice variants PDE4A1, PDE4A5/PDE4A10, PDE4B3, PDE4B1, PDE4D3 and PDE4D4 were present in cortical neurons, their mRNA was not regulated at the transcriptional level by db‐cAMP. To assess the increase in PDE4B2 and PDE4D1/D2 mRNA expression, the promoters containing these genes were characterized. Transcription from both promoters was stimulated by db‐cAMP. Because chronic antidepressant treatment increases PDE4B, and not PDE4D, mRNA expression, we focused on the regulation of the PDE4B2 promoter by cAMP and CREB. Dominant negative mutants of CREB suppressed PDE4B2 promoter activity and a constitutively active form of CREB robustly stimulated it. These data demonstrate that in cortical neurons, a short PDE4B2 intronic promoter is regulated by CREB, confers cAMP responsitivity and directs PDE4B2 mRNA and protein expression.