bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
P2X2, P2X2–2 and P2X5 receptor subunit expression and function in rat thoracolumbar sympathetic neurons
Authors: Schädlich H. , Wirkner K. , Franke H. , Bauer S. , Grosche J. , Burnstock G. , Reichenbach A. , Illes P. , Allgaier C.
Published: March 2003

The present study investigated the pharmacological properties of excitatory P2X receptors and P2X2 and P2X5 receptor subunit expression in rat‐cultured thoracolumbar sympathetic neurons. In patch‐clamp recordings, ATP (3–1000 µm; applied for 1 s) induced inward currents in a concentration‐dependent manner. Pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS; 30 µm) counteracted the ATP response. In contrast to ATP, α,β‐meATP (30 µm; for 1 s) was virtually ineffective. Prolonged application of ATP (100 µm; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X2−2 splice variant‐mediated responses. Using single‐cell RT‐PCR, P2X2, P2X2−2 and P2X5 mRNA expression was detectable in individual tyrosine hydroxylase‐positive neurons; coexpression of both P2X2 isoforms was not observed. Laser scanning microscopy revealed both P2X2 and P2X5 immunoreactivity in virtually every TH‐positive neuron. P2X2 immunoreactivity was largely distributed over the cell body, whereas P2X5 immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X2, P2X2−2 and P2X5 receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X2/P2X2−2 compared with P2X5 suggest that the excitatory P2X responses are mediated by P2X2 and P2X2−2 receptors. Apparently there exist two types of P2X2 receptor‐bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X2−2 splice variant.
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

Group 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

Midbrain 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

In 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

Human 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

This 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.
Characterization of a cysteine proteinase inhibitor induced during neuronal cell differentiation
Authors: Hong Jia , Yoshida Keiko , Rosner Marsha Rich
Published: March 2003

A rat homolog of human cystatin E/M was identified by differential display of transcripts induced during neuronal cell differentiation. A member of the family 2 cystatins, rat cystatin E/M is secreted, glycosylated and developmentally regulated. Rat cystatin E/M is expressed in brain, and is induced during differentiation of a conditionally immortalized E17 rat hippocampal cell line (H19‐7) by bFGF or activated Raf via MEK‐dependent and ‐independent signaling pathways. Rat cystatin E/M protein is increased post‐transcriptionally in PC12 cells, and the protein is secreted into the medium of primary embryonal hippocampal cultures. Analysis of the Ki of recombinant His‐tagged rat cystatin E/M toward cathepsins B and H revealed that rat cystatin E/M has an inhibitor profile distinct from that of other members of the cystatin family. Motif swapping between rat cystatin E/M and human cystatin C, a well‐characterized cystatin, identified some residues that can contribute to the specificity of inhibition. Taken together, these results describe a member of the cystatin family that has a distinct inhibitor profile and may play a role in neuronal development.
Detergent‐insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane
Authors: Taylor Christopher M. , Coetzee Timothy , Pfeiffer Steven E.
Published: March 2003

Glycosphingolipids and cholesterol form lateral assemblies, or lipid ‘rafts’, within biological membranes. Lipid rafts are routinely studied biochemically as low‐density, detergent‐insoluble complexes (in non‐ionic detergents at 4°C; DIGs, detergent‐insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re‐evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/cholesterol‐rich myelin membrane, using four detergents representing different chemical classes (TX‐100, CHAPS, Brij 96 and TX‐102), under four conditions: detergent extraction of myelin either at (i) 4°C or (ii) 37°C, or at 4°C after pre‐extraction with (iii) saponin or (iv) methyl‐β‐cyclodextrin (MβCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre‐extracted with saponin were denser than DIGs pre‐extracted with MβCD. Furthermore, pre‐extraction with MβCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre‐extraction with saponin did not, suggesting that saponin is less effective as a cholesterol‐perturbing agent than is MβCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent‐specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent.
PI 3‐kinase regulation of dopamine uptake
Authors: Carvelli Lucia , Morón José A. , Kahlig Kristopher M. , Ferrer Jasmine V. , Sen Namita , Lechleiter James D. , Leeb‐Lundberg L. M. Fredrik , Merrill Gerald , Lafer Eileen M. , Ballou Lisa M. , Shippenberg Toni S. , Javitch Jonathan A. , Lin Richard Z. , Galli Aurelio
Published: March 2003

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3‐kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3‐kinase, increased [3H]DA uptake and blocked the amphetamine‐induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3‐kinase. The LY294002‐induced reduction in [3H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin‐dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.
D3 receptor ligands modulate extracellular dopamine clearance in the nucleus accumbens
Authors: Zapata A. , Shippenberg T. S.
Published: March 2003

An involvement of the D3 dopamine receptor in the regulation of extracellular dopamine has been suggested. However, the mechanisms mediating this effect are unclear. We have used the technique of no net flux microdialysis under transient conditions to examine the influence of the D3‐preferring agonist (+)‐PD128907 upon extracellular dopamine levels in the nucleus accumbens of the mouse. (+)‐PD 128907 (0.1 mg/kg intraperitoneally) significantly decreased extracellular dopamine. This decrease was associated with a marked increase in the extraction fraction, which suggests an increase in dopamine clearance. The ability of D3‐preferring compounds to modulate dopamine uptake was investigated in vitro using rotating disk electrode voltammetry. (+)‐PD 128907 (10 nm) significantly increased the initial clearance rate of 3 μm dopamine in rat nucleus accumbens tissue suspensions. Kinetic analysis revealed no change in the apparent Km of uptake but it showed a 33% increase in Vmax. In contrast, the D3 antagonist GR 103691 (10 nm) significantly decreased dopamine uptake. Consistent with the low levels of D3 receptors in the dorsal striatum, neither compound affected uptake in tissue suspensions from this brain region. These data indicate that D3 receptor activation increases dopamine uptake in the nucleus accumbens and suggest that this receptor subtype can regulate extracellular dopamine by modulating the DA transporter activity.
Heterogeneity of normal prion protein in two‐dimensional immunoblot: presence of various glycosylated and truncated forms
Authors: Pan Tao , Li Ruliang , Wong Boon‐Seng , Liu Tong ,   Pierluigi Gambetti and Man‐Sun Sy
Published: March 2003

The common use of one‐dimensional (1‐D) immunoblot with a single monoclonal antibody (Mab) engenders the notion that the normal or cellular prion protein (PrPC) comprises few and simple forms. In this study we used two‐dimensional (2‐D) immunoblot with a panel Mabs to various regions of the prion protein to demonstrate the complexity of the PrPC present in human brain. We distinguished over 50 immunoblot spots, each representing a distinct PrPC species based on combinations of different molecular weights and isoelectric points (pIs). The PrPC heterogeneity is due to the presence of a full‐length and two major truncated forms as well as to the diversity of the glycans linked to most of these forms. The two major truncated forms result from distinct cleavage sites located at the N‐terminus. In addition, enzymatic removal of sialic acid and lectin binding studies indicate that the glycans linked to the full‐length and truncated PrPC forms differ in their structure and ratios of the glycoforms. The truncation of PrPC and the heterogeneity of the linked glycans may play a role in regulating PrPC function. Furthermore, the presence of relatively large quantities of different PrPC species may provide additional mechanisms by which the diversity of prion strains could be generated.
Alternative splicing isoform of tau protein kinase I/glycogen synthase kinase 3β
Authors: Mukai Fumiko , Ishiguro Koichi , Sano Yumiko , Fujita Shinobu C.
Published: March 2003

Glycogen synthase kinase 3 (GSK3) plays important roles in Wnt and insulin signaling, cell fate determination, and Alzheimer‐like tau phosphorylation. We discovered an isoform of tau protein kinase I (TPKI)/GSK3β with a 13 amino acid insert in the catalytic domain owing to alternative splicing. The alternative transcripts were found in the brains of the mouse, rat and human, with highly conserved sequences. The variant protein, named TPKI2/GSK3β2, was abundant in the brain. Immunohistochemistry indicated differential distribution of the conventional and the new TPKI/GSK3β isoforms within young neurons. TPKI2/GSK3β2 showed decreased kinase activities towards two phosphorylation sites on tau compared with the conventional isoform. Immunohistochemistry indicated that TPKI2/GSK3β2 occurs predominantly in the neuronal soma, while TPKI1/GSK3β1 is found both in the soma and processes. These results indicate that the new splice isoform has a different function. Because the amino acid insert occurs in the domain implicated in interaction with a protein phosphatase in a homologous kinase cdk‐2, the alternative splicing can regulate multiprotein complex formation and function involving TPKI/GSK3β.
Poly(ADP‐ribose) polymerase inhibition prevents both apoptotic‐like delayed neuronal death and necrosis after H2O2 injury
Authors: Cole Kasie K. , Perez‐Polo J. Regino
Published: March 2003

Toxic reactive oxygen species (ROS) such as hydrogen peroxide, nitric oxide, superoxide, and the hydroxyl radical are generated in a variety of neuropathological conditions and cause significant DNA damage. We determined the effects of 3‐aminobenzamide (AB), an inhibitor of the DNA repair enzyme poly(ADP‐ribose) polymerase (PARP), on cell death in differentiated PC12 cells, a model of sympathetic neurons, after H2O2 injury. Exposure to 0.5 mm H2O2 resulted in a significant decrease in intracellular NAD(H), NADP(H), and ATP levels. This injury resulted in the death of 90% of the cells with significant necrosis early (2 h) after injury and increased apoptosis (12–24 h after injury), as measured by PS exposure and the presence of cytoplasmic oligonucleosomal fragments. Treatment with 2.5 mm AB restored pyridine nucleotide and ATP levels and ameliorated cell death (65% versus 90%) by decreasing the extent of both necrosis and apoptosis. Interestingly, we observed that H2O2‐induced injury caused a delayed cell death exhibiting features of apoptosis but in which caspase‐3 like activity was absent. Moreover, pretreatment with AB restored caspase‐3‐like activity. Our results suggest that apoptosis and necrosis are both triggered by PARP overactivation, and that maintenance of cellular energy levels after injury by inhibiting PARP shifts cell death from necrosis to apoptosis.
Effect of ethanol on protein kinase Cζ and p70S6 kinase activation by carbachol: a possible mechanism for ethanol‐induced inhibition of glial cell proliferation
Authors: Guizzetti Marina , Costa Lucio G.
Published: March 2003

The signal transduction pathways that mediate the mitogenic response of muscarinic acetylcholine receptors in astroglial cells have not been fully elucidated. In this study we investigated the activation of p70S6 kinase (p70S6K) by carbachol in 1321 N1 astroctyoma cells. Carbachol induced a dose‐ and time‐dependent activation of p70S6K, as evidenced by increased phosphorylation at Thr‐389, Thr‐421 and Ser‐424, by increased p70S6K activity, and by a shift in its molecular weight. Activation of p70S6K was mediated by M3 muscarinic acetylcholine receptors (mAChRs) and was inhibited by two phosphatidylinositol‐3‐kinase (PI3‐K) inhibitors, by a pseudosubstrate to protein kinase C (PKC) ζ, and by the p70S6K inhibitor rapamycin. Carbachol‐induced DNA synthesis was strongly inhibited by rapamycin, suggesting that p70S6K activation plays an important role in carbachol‐induced cell proliferation. Ethanol (25–100 mm) has been shown to inhibit carbachol‐induced proliferation of astroglial cells. In the same range of concentrations, ethanol also inhibits carbachol‐induced activation of PKCζ and of p70S6K. On the other hand, inhibition of PI3‐kinase was only observed at higher ethanol concentrations. These results indicate that activation of the PKCζ→ p70S6K pathway by M3 mAChRs may play a role in the increased DNA synthesis and may represent a target for ethanol‐induced inhibition of astroglial cell proliferation.
Disruptive effects of glucocorticoids on glutathione peroxidase biochemistry in hippocampal cultures
Authors: Patel Ravi , McIntosh Laura , McLaughlin John , Brooke Sheila , Nimon Vitaliy , Sapolsky Robert
Published: March 2003

Glucocorticoids (GCs), the adrenal steroids secreted during stress, compromise the ability of hippocampal neurons to survive various necrotic insults. We have previously observed that GCs enhance the hippocampal neurotoxicity of reactive oxygen species and, as a potential contributor to this, decrease the activity of the antioxidant enzyme, glutathione peroxidase (GSPx). In this report, we have studied the possible mechanisms underlying this GC effect upon GSPx in primary hippocampal cultures and have observed several results. (i) Corticosterone (the GC of rats) decreased glutathione levels; this was predominately a result of a decrease in levels of reduced glutathione (GSH), the form of glutathione which facilitates GSPx activity. (ii) Corticosterone also decreased levels of NADPH; this may help explain the effect on GSH as NADPH is required for regeneration of GSH from oxidized glutathione. (iii) However, the corticosterone effect on total glutathione levels could not just be caused by the NADPH effect, as there were also reduced levels of oxidized glutathione. (iv) Corticosterone caused a small but significant decrease in GSPx activity over a range of glucose concentrations; this occurred under circumstances of an excess of glutathione as a substrate, suggesting a direct effect of corticosterone on GSPx activity. (v) This corticosterone effect was likely to have functional implications, in that enhancement of GSPx activity (to the same magnitude as activity was inhibited by corticosterone) by GSPx overexpression protected against an excitotoxin. Thus, GCs have various effects, both energetic and non‐energetic in nature, upon steps in GSPx biochemistry that, collectively, may impair hippocampal antioxidant capacity.
Lithium inhibits aluminum‐induced apoptosis in rabbit hippocampus, by preventing cytochrome c translocation, Bcl‐2 decrease, Bax elevation and caspase‐3 activation
Authors: Ghribi Othman , Herman Mary M. , Spaulding Natalie K. , Savory John
Published: March 2003

A variety of studies on neuronal death models suggest that lithium has neuroprotective properties. In the present investigation, we have examined the effect of chronic lithium treatment on hippocampus, as monitored by changes at the subcellular level of apoptosis‐regulatory proteins which have been induced by the neurotoxin, aluminum maltolate. Intracisternal administration of aluminum into rabbit brain induces cytochrome c release, decreases levels of the anti‐apoptotic proteins Bcl‐2 and Bcl‐XL, increases levels of the pro‐apoptotic Bax, activates caspase‐3, and causes DNA fragmentation as measured by the TUNEL assay. Pretreatment for 14 days with 7 mm of lithium carbonate in drinking water prevents aluminum‐induced translocation of cytochrome c, and up‐regulates Bcl‐2 and Bcl‐XL, down‐regulates Bax, abolishes caspase‐3 activity and reduces DNA damage. The regulatory effect of lithium on the apoptosis‐controlling proteins occurs in both the mitochondria and endoplasmic reticulum. We propose that the neuroprotective effect of lithium involves the modulation of apoptosis‐regulatory proteins present in the subcellular organelles of rabbit brain.
Isolation of various forms of sterol β‐d‐glucoside from the seed of Cycas circinalis: neurotoxicity and implications for ALS‐parkinsonism dementia complex
Authors: Khabazian I. , Bains J. S. , Williams D. E. , Cheung J. , Wilson J. M. B. , Pasqualotto B. A. , Pelech S. L. , Andersen R. J. , Wang Y.‐T. , Liu L. , Nagai A. , Kim S. U. , Craig U‐K. , Shaw C. A.
Published: March 2003

The factors responsible for ALS‐parkinsonism dementia complex (ALS‐PDC), the unique neurological disorder of Guam, remain unresolved, but identification of causal factors could lead to clues for related neurodegenerative disorders elsewhere. Earlier studies focused on the consumption and toxicity of the seed of Cycas circinalis, a traditional staple of the indigenous diet, but found no convincing evidence for toxin‐linked neurodegeneration. We have reassessed the issue in a series of in vitro bioassays designed to isolate non‐water soluble compounds from washed cycad flour and have identified three sterol β‐d‐glucosides as potential neurotoxins. These compounds give depolarizing field potentials in cortical slices, induce alterations in the activity of specific protein kinases, and cause release of glutamate. They are also highly toxic, leading to release of lactate dehydrogenase (LDH). Theaglycone form, however, is non‐toxic. NMDA receptor antagonists block the actions of the sterol glucosides, but do not compete for binding to the NMDA receptor. The most probable mechanism leading to cell death may involve glutamate neuro/excitotoxicity. Mice fed cycad seed flour containing the isolated sterol glucosides show behavioral and neuropathological outcomes, including increased TdT‐mediated biotin–dUTP nick‐end labelling (TUNEL) positivity in various CNS regions. Astrocytes in culture showed increased caspase‐3 labeling after exposure to sterol glucosides. The present results support the hypothesis that cycad consumption may be an important factor in the etiology of ALS‐PDC and further suggest that some sterol glucosides may be involved in other neurodegenerative disorders.
Oligodendroglial cells in culture effectively dispose of exogenous hydrogen peroxide: comparison with cultured neurones, astroglial and microglial cells
Authors: Hirrlinger Johannes , Resch Alexandra , Gutterer Jan Mirko , Dringen Ralf
Published: March 2003

To investigate the antioxidative capacities of oligodendrocytes, rat brain cultures enriched for oligodendroglial cells were prepared and characterized. These cultures contained predominantly oligodendroglial cells as determined by immunocytochemical staining for the markers galactocerebroside and myelin basic protein. If oligodendroglial cultures were exposed to exogenous hydrogen peroxide (100 µm), the peroxide disappeared from the incubation medium following first order kinetics with a half‐time of approximately 18 min. Normalization of the disposal rate to the protein content of the cultures by calculation of the specific hydrogen peroxide detoxification rate constant revealed that the cells in oligodendroglial cultures have a 60% to 120% higher specific capacity to dispose of hydrogen peroxide than cultures enriched for astroglial cells, microglial cells or neurones. Oligodendroglial cultures contained specific activities of 133.5 ± 30.4 nmol × min−1 × mg protein−1 and 27.5 ± 5.4 nmol × min−1 ×  mg protein−1 of glutathione peroxidase and glutathione reductase, respectively. The specific rate constant of catalase in these cultures was 1.61 ± 0.54 min−1 × mg protein−1. Comparison with data obtained by identical methods for cultures of astroglial cells, microglial cells and neurones revealed that all three of the enzymes which are involved in hydrogen peroxide disposal were present in oligodendroglial cultures in the highest specific activities. These results demonstrate that oligodendroglial cells in culture have a prominent machinery for the disposal of hydrogen peroxide, which is likely to support the protection of these cells in brain against peroxides when produced by these or by surrounding brain cells.
Nitric oxide regulates cGMP‐dependent cAMP‐responsive element binding protein phosphorylation and Bcl‐2 expression in cerebellar neurons: implication for a survival role of nitric oxide
Authors: Ciani Elisabetta , Guidi Sandra , Bartesaghi Renata , Contestabile Antonio
Published: March 2003

Nitric oxide (NO) is a small, diffusible, highly reactive molecule with a dichotomous regulatory role in the brain: an intra‐ and intercellular messenger under physiological conditions and a neurodegenerative agent under pathological conditions. We have recently demonstrated that long‐lasting exposure to an neuronal nitric oxide synthase (nNOS) inhibitor down‐regulated serine/threonine kinase (Akt) survival pathway and caused apoptosis in cerebellar granule cell cultures. The present study further substantiates the role of NO in neuronal survival by demonstrating that blocking its production down‐regulates the activity of cAMP‐responsive element binding protein (CREB), a transcription factor involved in cell survival and synaptic plasticity. Pharmacological dissection of the pathway linking NO to CREB shows that cGMP and its kinase are intermediate effectors. We also identify Bcl‐2 as one of the anti‐apoptotic genes down‐regulated by NO shortage and decreased CREB phosphorylation. These results not only confirm the role of CREB in neuronal survival but also provide circumstantial evidence for a novel link among NO, CREB activation and survival.
Macrophage colony stimulating factor prevents NMDA‐induced neuronal death in hippocampal organotypic cultures
Authors: Vincent Valerie A. M. , Robinson Christopher C. , Simsek Dilek , Murphy Greer M.
Published: March 2003

Macrophage colony stimulating factor (M‐CSF) and its receptor are up‐regulated in the brain in Alzheimer's disease (AD), in transgenic mouse models for AD, and experimental models for traumatic and ischemic brain injury. M‐CSF induces activation and proliferation of microglial cells and expression of proinflammatory cytokines. We examined the role of M‐CSF in excitotoxic neuronal cell death in organotypic hippocampal cultures. NMDA treatment induced neuronal apoptosis and caspase‐3 activation in organotypic hippocampal cultures, whereas treatment with M‐CSF protected hippocampal neurons from NMDA‐induced apoptosis. Caspase‐3 activation was inhibited by M‐CSF treatment to the same degree as with the caspase inhibitor Z‐VAD‐FMK. These results suggest that M‐CSF has neuroprotective properties through inhibition of caspase‐3 that could promote neuronal survival after excitotoxic insult. The role of M‐CSF in neurological disease should be reevaluated as a microglial activator with potentially neuroprotective effects.
Platelet activating factor‐induced apoptosis is inhibited by ectopic expression of the platelet activating factor G‐protein coupled receptor
Authors: Brewer Cynthia , Bonin Fanny , Bullock Paula , Nault Marie‐Christine , Morin Jennifer , Imbeault Sophie , Shen T. Y. , Franks D. J. , Bennett Steffany A. L.
Published: March 2003

The pro‐inflammatory lipid mediator platelet activating factor (PAF: 1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine) accumulates in ischemia, epilepsy, and human immunodeficiency virus‐1‐associated dementia and is implicated in neuronal loss. The present study was undertaken to establish a role for its G‐protein coupled receptor in regulating neurotoxicity. PC12 cells do not express PAF receptor mRNA as demonstrated by northern analysis and RT‐PCR. In the absence of the G‐protein coupled receptor, PAF (0.1−1 µm) triggered chromatin condensation, DNA strand breaks, oligonucleosomal fragmentation, and nuclear disintegration characteristic of apoptosis. Lyso‐PAF (0.001–1 µm), the immediate metabolite of PAF, did not elicit apoptotic death. Concentrations of PAF or lyso‐PAF that exceeded critical micelle concentration had physicochemical effects on plasma membrane resulting in necrosis. Apoptosis but not necrosis was inhibited by the PAF antagonist BN52021 (1–100 µm) but not CV3988 (0.2–20 µm). Ectopic PAF receptor expression protected PC12 transfectants from ligand‐induced apoptosis. PAF receptor‐mediated protection was inhibited by CV3988 (1 µm). These data provide empirical evidence that: (i) PAF can initiate apoptosis independently of its G‐protein coupled receptor; (ii) PAF signaling initiated by its G‐protein coupled receptor is cytoprotective to PC12 cells; (iii) the pro‐ and anti‐apoptotic effects of PAF on PC12 cells can be pharmacologically distinguished using two different PAF antagonists.
Role of blood–brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain
Authors: Ohtsuki Sumio , Asaba Hiroshi , Takanaga Hitomi , Deguchi Tsuneo , Hosoya Ken‐ichi , Otagiri Masaki , Terasaki Tetsuya
Published: March 2003

Renal impairment is associated with CNS dysfunctions and the accumulation of uremic toxins, such as indoxyl sulfate, in blood. To evaluate the relevance of indoxyl sulfate to CNS dysfunctions, we investigated the brain‐to‐blood transport of indoxyl sulfate at the blood–brain barrier (BBB) using the Brain Efflux Index method. [3H]Indoxyl sulfate undergoes efflux transport with an efflux transport rate of 1.08 × 10−2/min, and the process is saturable with a Km of 298 µm. This process is inhibited by para‐aminohippuric acid, probenecid, benzylpenicillin, cimetidine and uremic toxinins, such as hippuric acidand 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid. RT–PCR revealed that an OAT3 mRNA is expressed in conditionally immortalized rat brain capillary endothelial cell lines and rat brain capillary fraction. Xenopus oocytes expressing OAT3 were found to exhibit [3H]indoxyl sulfate uptake, which was significantly inhibited by neurotransmitter metabolites, such as homovanillic acid and 3‐methoxy‐4‐hydroxymandelic acid, and by acyclovir, cefazolin, baclofen, 6‐mercaptopurine, benzoic acid, and ketoprofen. These results suggest that OAT3 mediates the brain‐to‐blood transport of indoxyl sulfate, and is also involved in the efflux transport of neurotransmitter metabolites and drugs. Therefore, inhibition of the brain‐to‐blood transport involving OAT3 would occur in uremia and lead to the accumulation of neurotransmitter metabolites and drugs in the brain.
Brief, repeated exposure to substrates down‐regulates dopamine transporter function in Xenopus oocytes in vitro and rat dorsal striatum in vivo
Authors: Gulley Joshua M. , Doolen Suzanne , Zahniser Nancy R.
Published: March 2003

In heterologous expression systems, dopamine transporter (DAT) cell‐surface localization is reduced after relatively prolonged exposure to d‐amphetamine (AMPH) or dopamine (DA), suggesting a role for substrate‐mediated regulation of transporter function. Here, we investigated whether brief, repeated periods of substrate exposure modulated transporter function, first, in an in vitro model system and, second, in intact rat brain. In human DAT‐expressing Xenopus laevis oocytes, repeated exposure to low micromolar concentrations of DA, AMPH or tyramine markedly reduced transport‐mediated currents. This functional down‐regulation was attenuated by inclusion of a protein kinase C (PKC) inhibitor and probably reflects DAT redistribution, as cell‐surface [3H]WIN 35 428 binding was significantly lower following DA exposure. High‐speed chronoamperometry was used to measure clearance of exogenously applied DA in dorsal striatum (STR) and nucleus accumbens (NAc) of anesthetized rats. In STR, frequent (every 2 min) applications of DA altered DA clearance parameters in a manner consistent with profound down‐regulation of DAT function. Similar changes were not observed in NAc or after repeated vehicle (ascorbic acid) application. Together, our results suggest that brief, repeated periods of substrate exposure lead to rapid down‐regulation of DAT activity and that this type of regulation can occur in vivo in STR, but not NAc.
Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachadonic acid release in primary murine astrocytes
Authors: Xu Jianfeng , Weng Yu‐I. , Simoni Agnes , Krugh Brent W. , Liao Zhongji , Weisman Gary A. , Sun Grace Y.
Published: March 2003

Although Group IV cytosolic phospholipase A2 (cPLA2) in astrocytes has been implicated in a number of neurodegenerative diseases, mechanisms leading to its activation and release of arachidonic acid (AA) have not been clearly elucidated. In primary murine astrocytes, phorbol myristate acetate (PMA) and ATP stimulated phosphorylation of ERK1/2 and cPLA2 as well as evoked AA release. However, complete inhibition of phospho‐ERK by U0126, an inhibitor of mitogen‐activated protein kinase kinase (MEK), did not completely inhibit PMA‐stimulated cPLA2 and AA release. Epidermal growth factor (EGF) also stimulated phosphorylation of ERK1/2 and cPLA2[largely through a protein kinase C (PKC)‐independent pathway], but EGF did not evoke AA release. These results suggest that phosphorylation of cPLA2 due to phospho‐ERK is not sufficient to evoke AA release. However, complete inhibition of ATP‐induced cPLA2 phosphorylation and AA release was observed when astrocytes were treated with GF109203x, a general PKC inhibitor, together with U0126, indicating the important role for both PKC and ERK in mediating the ATP‐induced AA response. There is evidence that PMA and ATP stimulated AA release through different PKC isoforms in astrocytes. In agreement with the sensitivity of PMA‐induced responses to PKC down‐regulation, prolonged treatment with PMA resulted in down‐regulation of PKCα and ε in these cells. Furthermore, PMA but not ATP stimulated rapid translocation of PKCα from cytosol to membranes. Together, our results provided evidence for an important role of PKC in mediating cPLA2 phosphorylation and AA release in astrocytes through both ERK1/2‐dependent and ERK1/2‐independent pathways.
Selective reduction of a PDZ protein, SAP‐97, in the prefrontal cortex of patients with chronic schizophrenia
Authors: Toyooka Kazuhiko , Iritani Shuji , Makifuchi Takao , Shirakawa Osamu , Kitamura Noboru , Maeda Kiyoshi , Nakamura Ryosuke , Niizato Kazuhiro , Watanabe Masahiko , Kakita Akiyoshi , Takahashi Hitoshi , Someya Toshiyuki , Nawa Hiroyuki
Published: March 2003

Many postsynaptic density proteins carrying postsynaptic density‐95/discs large/zone occludens‐1 (PDZ) domain(s) interact with glutamate receptors to control receptor dynamics and synaptic plasticity. Here we examined the expression of PDZ proteins, synapse‐associated protein (SAP) 97, postsynaptic density (PSD)‐95, chapsyn‐110, GRIP1 and SAP102, in post‐mortem brains of schizophrenic patients and control subjects, and evaluated their contribution to schizophrenic pathology. Among these PDZ proteins, SAP97 exhibited the most marked change: SAP97 protein levels were decreased to less than half that of the control levels specifically in the prefrontal cortex of schizophrenic patients. In parallel, its binding partner, GluR1, similarly decreased in the same brain region. The correlation between SAP97 and GluR1 levels in control subjects was, however, altered in schizophrenic patients. SAP102 levels were also significantly reduced in the hippocampus of schizophrenic patients, but this reduction was correlated with sample storage time and post‐mortem interval. There were no changes in the levels of the other PDZ proteins in any of the regions examined. In addition, neuroleptic treatment failed to mimic the SAP97 change. These findings suggest that a phenotypic loss of SAP97 is associated with the postsynaptic impairment in prefrontal excitatory circuits of schizophrenic patients.
Assembly of tau in transgenic animals expressing P301L tau: alteration of phosphorylation and solubility
Authors: Sahara Naruhiko , Lewis Jada , DeTure Michael , McGowan Eileen , Dickson Dennis W. , Hutton Mike , Yen Shu‐Hui
Published: March 2003

Transgenic mice (JNPL3), which develop neurofibrillary degeneration and express four‐repeat human tau with P301L missense mutation, were characterized biochemically to determine whether the development of aggregated tau from soluble tau involves an intermediate stage. Homogenates from mice of different ages were separated into buffer‐soluble (S1), sarkosyl‐ and salt‐extractable (S2) and sarkosyl‐insoluble pellet (P3) fractions, and analyzed for human tau distribution, phosphorylation and filament formation. S1 and S2 fractions contained 50–60‐kDa tau whereas the S2 fraction also had 64‐kDa tau. The level of tau in the P3 fraction increased in an age‐dependent manner and correlated positively with the soluble tau concentration. The P3 fraction from 2.5–6.5‐month‐old mice contained 64‐ and 50–60‐kDa tau, whereas that from 8.5‐month and older transgenic animals contained mostly 64‐kDa and higher molecular weight tau. The S2 and P3 fractions contained comparable amounts of 64‐kDa tau. The 64‐kDa tau was predominantly human, and phosphorylated at multiple sites: Thr181, Ser202/Thr205, Thr212, Thr231, Ser262, Ser396/Ser404, Ser409 and Ser422. Most of these sites were phosphorylated to a lesser extent in S2 than in P3 fractions. Tau polymers were detected in P3 fractions from 3‐month and older female JNPL3 mice, but not in non‐transgenic controls. The results suggest that tau in S2 represents an intermediate from which insoluble tau is derived, and that phosphorylation may play a role in filament formation and/or stabilization.
Participation of protein kinase C α isoform and extracellular signal‐regulated kinase in neurite outgrowth of GT1 hypothalamic neurons
Authors: Choe Youngshik , Lee Byung Ju , Kim Kyungjin
Published: March 2003

In the present study, we investigated the selective role of protein kinase C (PKC) isoforms on neurite outgrowth of the GT1 hypothalamic neurons using several PKC isoform‐selective inhibitors and transfection‐based expression of enhanced green fluorescence protein (EGFP)‐fused PKC isoforms. 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA) induced neurite outgrowth and growth cone formation, effects that were blocked by GF 109203X (a PKC inhibitor), safingolTM(a PKCα‐selective inhibitor), but not by rottlerinTM (a PKCδ‐selective inhibitor), indicating that PKCα may be selectively involved in neurite outgrowth and cytoskeletal changes of filamentous actin and β‐tubulin. To define the differential localization of PKC isoforms, EGFP‐tagged PKCα, PKCγ, and PKCδ were transfected into GT1 neuronal cells. TPA treatment induced relocalization of PKCα‐EGFP to growth cones and cell–cell adhesion sites, PKCγ‐EGFP to the nucleus, and PKCδ‐EGFP to the membrane ruffle, respectively. An EGFP chimera of the catalytic domain of PKCα (PKCα‐Cat‐EGFP), the expression of which was inducible by doxycycline, was employed to directly ascertain the effect of PKCα enzymatic activity on neurite outgrowth of GT1 cells. Transient transfection of PKCα‐Cat‐EGFP alone increased the neurite‐outgrowth and doxycycline treatment further augmented the number of neurite‐containing cells. We also examined the involvement of the extracellular signal‐regulated kinase (ERK) MAP kinase in TPA‐induced neurite outgrowth. TPA treatment increased phosphorylated ERK MAP kinase, but not p38 MAP kinase. Specific inhibition of PKCα with safingol blocked the phosphorylation of ERK induced by TPA. More importantly, both neurite outgrowth and phosphorylation of ERK by TPA were blocked by PD 098059, a specific inhibitor of MEK (MAP kinase/ERK kinase‐1), but not by SB203580, a specific inhibitor of p38 MAP kinase. These results demonstrate that PKCα isoform‐specific activation is involved in neurite outgrowth of GT1 hypothalamic neuronal cells via ERK, but not the p38 MAP kinase signal pathway.