bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a Dopamine‐Releasing Action of 6R‐l‐erythro‐Tetrahydrobiopterin: Comparison with a 6S‐Form
Authors: Koshimura Kunio , Takagi Yasutaka , Miwa Soichi , Kido Tsuneo , Watanabe Yasuyoshi , Murakami Yoshio , Kato Yuzuru , Masaki Tomoh
Published: July 2010

Abstract: 6R‐l‐erythro‐Tetrahydrobiopterin (6R‐BH4) is a cofactor for aromatic l‐amino acid hydroxylases and nitric oxide synthase. Recently, we have reported that independently of its cofactor activities, 6R‐BH4 acts from the outside of neurons in the brain to enhance the release of monoamine neurotransmitters such as dopamine. To characterize the pharmacological properties of the action, we examined the effects of 6S‐BH4, a diastereoisomer of 6R‐BH4, on dopamine release in the rat striatum by using brain microdialysis and compared its effects with those of 6R‐BH4. Perfusion of 6S‐BH4 or 6R‐BH4 through the dialysis probe increased extracellular dopamine levels (an index of in vivo dopamine release) concentration dependently; the maximal increase by 6S‐BH4, was one‐sixth of that by 6R‐BH4. 6S‐BH4 increased extracellular DOPA levels in the presence of NSD 1015, an inhibitor of aromatic l‐amino acid decarboxylase (an index of in vivo tyrosine hydroxylase activity), to an extent similar to the increase induced by 6R‐BH4. The increase in the DOPA levels induced by either of the pteridines was abolished after pretreatment of rats with α‐methyl‐p‐tyrosine (an inhibitor of tyrosine hydroxylase). Under the same conditions, the 6S‐BH4‐induced dopamine release was abolished, but most of the 6R‐BH4‐induced increase persisted. Coadministration of 6S‐BH4 with 6R‐BH4 inhibited the increase in dopamine release induced by 6R‐BH4 alone. These results show that 6R‐BH4 stimulates dopamine release by acting at the specific recognition site on the neuronal membrane, and that 6S‐BH4 acts as an antagonist of 6R‐BH4 at this site, although it has cofactor activities.
Defective Herpes Simplex Virus Vectors Expressing the Rat Brain Glucose Transporter Protect Cultured Neurons from Necrotic Insults
Authors: Ho Dora Y. , Saydam Tippi C. , Fink Sheri L. , Lawrence Matthew S. , Sapolsky Robert M.
Published: July 2010

Abstract: Because neurons are postmitotic, they are irreplaceable once they succumb to necrotic insults such as hypoglycemia, ischemia, and seizure. A paucity of energy can exacerbate the toxicities of these insults; thus, a plausible route to protect neurons from necrotic injury would be to enhance their glucose uptake capability. We have demonstrated previously that defective herpes simplex virus (HSV) vectors overexpressing the rat brain glucose transporter (GT) gene (gt) can enhance glucose uptake in adult rat hippocampus and in hippocampal cultures. Furthermore, we have observed that such vectors can maintain neuronal metabolism during hypoglycemia and reduce kainic acid‐induced seizure damage. In this study, we have developed bicistronic vectors that coexpressed gt and Escherichia coli lacZ as a reporter gene, which allows us to identify directly neurons that are infected with the vectors. Overexpression of GT from these vectors protected cultured hippocampal, spinal cord, and septal neurons against various necrotic insults, including hypoglycemia, glutamate, and 3‐nitropropionic acid. Our observations demonstrate the feasibility of using HSV vectors to protect neurons from necrotic insults. Although this study has concentrated on the delivery of gt, other genes with therapeutic or protective capability might also be used.
Different GABAB‐Mediated Effects on Protein Kinase C Activity and Immunoreactivity in Neonatal and Adult Rat Hippocampal Slices
Authors: Tremblay E. , Ben‐Ari Y. , Roisin M. P.
Published: July 2010

Abstract: The effects of GABA on protein kinase C (PKC) were investigated in rat hippocampal slices at various postnatal ages [postnatal day (P) 1‐P60]. At P4, GABA (300 µM) induced a rapid (in 1–2 min) 40–50% increase of PKC activity in the membrane fraction and a decrease in the cytosol. These effects were mediated by GABAB receptors because (a) they were neither blocked by 10 µM bicuculline nor reproduced by 10 µM isoguvacine and (b) they were mimicked by the GABAB agonist baclofen (3–30 µM), an effect fully antagonized by the GABAB antagonist 2‐hydroxysaclofen (10 µM). A baclofen‐induced increased PKC activity in the membrane fraction was only present during the early postnatal period (P1–P14); it was associated with a translocation from the cytosol to the membrane of the immunoreactivity of some PKC isoforms (α‐, β‐, and ε‐PKCs). In contrast, after P21, PKC activity and α‐, β‐, ε‐, and γ‐PKC immunoreactivities were decreased by baclofen in the membrane fraction and increased in the cytosol. These results suggest that the stimulation of GABAB receptors differentially modulates PKC activity via distinct second messenger pathways in developing and mature hippocampi.
Phosphorylation of τ In Situ: Inhibition of Calcium‐Dependent Proteolysis
Authors: Litersky Joel M. , Johnson Gail V. W.
Published: July 2010

Abstract: In this study, the in situ phosphorylation and subsequent calcium‐activated proteolysis of τ protein were examined in human neuroblastoma (LA‐N‐5) cells, which were differentiated into a neuronal phenotype. The phosphorylation of τ was increased by treating the cells with forskolin and rolipram, which elevate cyclic AMP levels, by treating with the phosphatase inhibitor okadaic acid, or by treating with a combination of both treatments. Phosphorylated τ migrated slightly slower on sodium dodecyl sulfate‐polyacrylamide gels than τ from untreated cells. Immunostaining with the phosphate‐sensitive monoclonal antibody Tau‐1 was also decreased in cells treated with okadaic acid, indicating an increase in the phosphorylation of specific Ser‐Pro motifs within the molecule. Calcium‐dependent, in situ proteolysis of τ protein was induced by treating the cells with the calcium ionophore A23187. τ protein was proteolyzed to a significantly lesser extent in cells treated with forskolin and rolipram, okadaic acid, or both than in cells in which phosphorylation was not increased. Partially purified τ protein from cells treated with a combination of forskolin, rolipram, and okadaic acid was also more resistant to proteolysis by calpain in vitro compared with τ isolated from control cells. These data suggest a possible role for phosphorylation in the regulation of τ metabolism and in pathological conditions in which the balance between protein kinases and phosphatases is disrupted.
Mössbauer Spectroscopic Studies of Purified Human Neuromelanin Isolated from the Substantia Nigra
Authors: Gerlach M. , Trautwein A. X. , Zecca L. , Youdim M. B. H. , Riederer P.
Published: July 2010

Abstract: 57Fe Mössbauer spectroscopy at different temperatures has been used to characterize the nature of purified human neuromelanin isolated from the substantia nigra. The quantitative determination of iron(III) by estimation of the overall area of the Mössbauer spectrum at room temperature reveals an iron content of 2.8 ± 1.4%. No subspectra corresponding to divalent iron could be observed in these spectra. The derived Mössbauer parameters lead to the conclusion that the iron sites in the human neuromelanin are similar to those of human hemosiderin (or ferritin). However, owing to the water insolubility of the purified neuromelanin, it must be concluded that the neuromelanin hemosiderin (or ferritin) is bound in a protein matrix that makes it insoluble and difficult to stain histochemically. This protein attachment to neuromelanin is important in that it is what makes it different from synthetic dopamine melanin.
A Role for Amplified Protein Kinase C Activity in the Pathogenesis of Amyotrophic Lateral Sclerosis
Authors: Lanius R. A. , Paddon H. B. , Mezei M. , Wagey R. , Krieger C. , Pelech S. L. , Shaw C. A.
Published: July 2010

Abstract: Amyotrophic lateral sclerosis (ALS) is a human neurodegenerative disorder of unknown origin that is characterized by progressive degeneration of corticospinal tracts and anterior horn cells in the brainstem and spinal cord. Previous studies have indicated that motoneuron degeneration associated with ALS may be triggered by mechanisms leading to increased intracellular Ca2+. In the present report, Ca2+‐activated phospholipid‐dependent protein kinase C (PKC) was evaluated in cervical spinal cords from ALS patients and control subjects. In patients who died with ALS, PKC histone H1 phosphotransferase activity was significantly increased by 330% in cytosolic‐ and 118% in particulate‐derived extracts compared with controls. This increase in PKC phosphotransferase activity appeared to be partially due to an increase in the amount of PKC protein present in ALS spinal cord tissue. PKC histone H1 phosphotransferase activities of cytosolic‐ and particulate‐derived extracts from motor and visual cortex of ALS patients and controls were not statistically different, nor were there differences in PKC histone H1 phosphotransferase activity in platelets and leukocytes. The specific nature of PKC alterations in affected regions of the CNS supports a role for PKC in the events leading to motoneuron death in sporadic ALS.
Coregulation of Two Embedded Gene Products, Choline Acetyltransferase and the Vesicular Acetylcholine Transporter
Authors: Berrard Sylvie , Varoqui Hélène , Cervini Riccardo , Israël Maurice , Mallet Jacques , Diebler Marie‐Françoise
Published: July 2010

Abstract: The gene encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the gene encoding choline acetyltransferase (ChAT) and is in the same transcriptional orientation. These two genes, whose products are required for the expression of the cholinergic phenotype, could therefore be coregulated. We thus tested the effects on VAChT gene expression of the cholinergic differentiation factor/leukemia inhibitory factor and retinoic acid, both of which induce ChAT activity and increase ChAT mRNA levels in cultured sympathetic neurons. These factors increased both the number of binding sites for vesamicol, a specific ligand of VAChT, and VAChT immunoreactivity. This increase in the number of VAChT molecules resulted from an increase in the amount of VAChT mRNA, as assessed by reverse transcription‐PCR and which paralleled that of ChAT mRNAs. These data suggest a functional role for ChAT and VAChT gene organization and are consistent with the existence of a coregulatory mechanism for the embedded ChAT and VAChT genes.
Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) Stimulates Adenylyl Cyclase and Phospholipase C Activity in Rat Cerebellar Neuroblasts
Authors: Basille Magali , Gonzalez Bruno J. , Desrues Laurence , Demas Myriam , Fournier Alain , Vaudry Hubert
Published: July 2010

Abstract: The presence of receptors for the novel neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27‐(PACAP27) and 38‐(PACAP38) amino‐acid forms of PACAP, induced a dose‐dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED50 = 0.12 ± 0.01 and 0.23 ± 0.07 nM, respectively), whereas vasoactive intestinal polypeptide (VIP) was ∼100 times less potent. PACAP27 and PACAP38 also induced a dose‐dependent stimulation of polyphosphoinositide breakdown (ED50 = 19.1 ± 6.3 and 13.4 ± 6.0 nM, respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U‐73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin‐sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP‐induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.
Expression of Inducible Nitric Oxide Synthase in Cerebral Endothelial Cells Is Regulated by Cytokine‐Activated Astrocytes
Authors: Borgerding Rose A. , Murphy Sean
Published: July 2010

Abstract: A nitric oxide (NO) synthase (NOS) can be induced in both astrocytes and cerebral endothelial cells with a combination of interleukin‐1β/interferon‐γ or lipopolysaccharide/interferon‐γ, respectively. Exogenous NO, either from the chemical donor spermine NONOate or from activated astrocytes, affected the expression of inducible NOS in cerebral endothelial cells. In cerebral endothelial cells pretreated with spermine NONOate the induction of NOS was reduced, as revealed by mRNA expression and nitrite accumulation. Cytokine‐treated astrocytes generating NO and placed in close proximity to endothelial cells decreased the expression of NOS induced by cytokines in endothelial cells. In addition, it was apparent that cytokine‐activated astrocytes released a factor(s) that initiated transcriptional induction of NOS in cerebral endothelium. This suggests that astrocytes activated by cytokines in vivo could influence expression of inducible NOS in cells of the adjacent microvasculature.
Evidence for a Glutamatergic Pathway from the Guinea Pig Auditory Cortex to the Inferior Colliculus
Authors: Feliciano M. , Potashner S. J.
Published: July 2010

Abstract: We attempt to provide evidence that the projection from the guinea pig auditory cortex (AC) to the inferior colliculus (IC) may contain glutamatergic or GABAergic fibers. Seven days after unilateral AC aspiration, histological studies indicated almost complete AC destruction and preterminal degeneration of fibers and terminal fields in the dorsal cortex (DCIC), external cortex (ECIC), and central nucleus (CNIC) of the IC ipsilateral to the ablated AC. Contralaterally, degeneration appeared in the DCIC. AC ablation depressed the electrically evoked Ca2+‐dependent release of d‐[3H]aspartate (d‐[3H]Asp) in the ipsilateral DCIC, ECIC, and CNIC, and d‐[3H]Asp uptake in the CNIC. Together with other evidence that the corticocollicular pathway is excitatory, these findings suggest that this projection may contain glufamatergic and/or aspartatergic (Glu/Asp‐ergic) fibers. Glutamic acid decarboxylase immunoreactivity was not apparent in presumed pyramidal cells of layer V of the AC retrogradely labeled with biotinylated dextran injected into the ipsilateral IC. Thus, corticocollicular neurons probably do not synthesize GABA and may not be GABAergic. However, AC ablation depressed [14C]GABA release from the ipsilateral DCIC and ECIC, and [14C]GABA uptake in the DCIC. These findings are consistent with the atrophy or down‐regulation of some subcortical neurons that mediate GABAergic transmission in the IC.
Selective Inhibition of Cholesterol Biosynthesis in Brain Cells by Squalestatin 1
Authors: Crick Dean C. , Suders Julie , Kluthe Catherine M. , Andres Douglas A. , Waechter Charles J.
Published: July 2010

Abstract: The effect of squalestatin 1 (SQ) on squalene synthase and other enzymes utilizing farnesyl pyrophosphate (F‐P‐P) as substrate was evaluated by in vitro enzymological and in vivo metabolic labeling experiments to determine if the drug selectively inhibited cholesterol biosynthesis in brain cells. Direct in vitro enzyme studies with membrane fractions from primary cultures of embryonic rat brain (IC50 = 37 nM), pig brain (IC50 = 21 nM), and C6 glioma cells (IC50 = 35 nM) demonstrated that SQ potently inhibited squalene synthase activity but had no effect on the long‐chain cis‐isoprenyltransferase catalyzing the conversion of F‐P‐P to polyprenyl pyrophosphate (Poly‐P‐P), the precursor of dolichyl phosphate (Dol‐P). SQ also had no effect on F‐P‐P synthase; the conversion of [3H]F‐P‐P to geranylgeranyl pyrophosphate (GG‐P‐P) catalyzed by partially purified GG‐P‐P synthase from bovine brain; the enzymatic farnesylation of recombinant H‐p21ras by rat brain farnesyltransferase; or the enzymatic geranylgeranylation of recombinant Rab1A, catalyzed by rat brain geranylgeranyltransferase. Consistent with SQ selectively blocking the synthesis of squalene, when C6 glial cells were metabolically labeled with [3H]mevalonolactone, the drug inhibited the incorporation of the labeled precursor into squalene and cholesterol (IC50 = 3–5 µM) but either had no effect or slightly stimulated the labeling of Dol‐P, ubiquinone (CoQ), and isoprenylated proteins. These results indicate that SQ blocks cholesterol biosynthesis in brain cells by selectively inhibiting squalene synthase. Thus, SQ provides a useful tool for evaluating the obligatory requirement for de novo cholesterol biosynthesis in neurobiological processes without interfering with other critical reactions involving F‐P‐P.
Opposing Actions of Thrombin and Protease Nexin‐1 on Amyloid β‐Peptide Toxicity and on Accumulation of Peroxides and Calcium in Hippocampal Neurons
Authors: Smith‐Swintosky Virginia L. , Zimmer Stephen , Fenton John W. , Mattson Mark P.
Published: July 2010

Abstract: Amyloid β‐peptide (Aβ) is the principal component of neuritic plaques in the brain in Alzheimer's disease (AD). Recent studies revealed that Aβ can be neurotoxic by a mechanism involving free radical production and loss of cellular ion homeostasis, thus implicating Aβ as a key factor in the pathogenesis of AD. However, other proteins are present in plaques in AD, including the protease thrombin and protease nexin‐1 (PN1), a thrombin inhibitor. We therefore tested the hypothesis that thrombin and PN1 modify neuronal vulnerability to Aβ toxicity. In dissociated rat hippocampal cell cultures the toxicity of Aβ was significantly enhanced by coincubation with thrombin, whereas PN1 protected neurons against Aβ toxicity. Aβ induced an increase in levels of intracellular peroxides and calcium. Thrombin enhanced, and PN1 attenuated, the accumulation of peroxides and calcium induced by Aβ. Taken together, these data demonstrate that thrombin and PN1 have opposing effects on neuronal vulnerability to Aβ and suggest that thrombin and PN1 play roles in the pathogenesis of neuronal injury in AD.
Learning‐Induced Expression of Meningeal Ependymin mRNA and Demonstration of Ependymin in Neurons and Glial Cells
Authors: Rother Stefan , Schmidt Rupert , Brysch Wolfgang , Schlingensiepen Karl‐Hermann
Published: July 2010

Abstract: The turnover of a CNS‐specific cell adhesion glycoprotein, ependymin, has earlier been found to increase during periods of neuronal plasticity. Here, ependymin mRNA expression was analyzed by semiquantitative in situ hybridization in goldfish. Learning of an active avoidance response resulted in a significant increase in ependymin mRNA expression 20 min to 4 h after acquisition of the task. In contrast, yoked control animals that were exposed to the same numbers of conditioned and unconditioned stimuli in a random, unpaired manner exhibited a strong down‐regulation of ependymin mRNA. Hybridization signals were also increased by injection of anti‐ependymin antiserum into brain ventricles. Ependymin mRNA was exclusively localized to reticular‐shaped fibroblasts of the inner endomeningeal cell layer. Immunoelectron microscopic investigation, however, revealed ependymin also in distinct neuronal and glial cell populations in which no ependymin mRNA had been detected. Uptake of meningeal protein factors into glial and neuronal cells may therefore be of functional importance for plastic adaptations of the CNS.
Inhibition of Memory Consolidation After Active Avoidance Conditioning by Antisense Intervention with Ependymin Gene Expression
Authors: Schmidt Rupert , Brysch Wolfgang , Rother Stefan , Schlingensiepen Karl‐Hermann
Published: July 2010

Abstract: A rapid increase in ependymin mRNA expression demonstrated by semiquantitative in situ hybridization after avoidance conditioning on goldfish suggested a molecular demand for newly synthesized ependymin translation product. To inhibit de novo synthesis of ependymin molecules without interference with preexisting ones, 18 mer anti‐ependymin mRNA‐phosphorothioate oligodeoxynucleotides (S‐ODNs) were injected into the perimeningeal brain fluid before active avoidance training. S‐ODN‐injected animals learned the avoidance response; however, they were amnesic in the test. When injected into overtrained animals, S‐ODNs did not interfere with retrieval or performance of the avoidance response. Fish treated with randomized S‐ODN sequences served as further controls. Incorporation of S‐ODNs was analyzed by injection of fluorescein isothiocyanate (FITC)‐conjugated oligodeoxynucleotide probes. Microscopic observation revealed strong FITC‐S‐ODN fluorescence in reticular‐shaped fibroblasts, the only known site of ependymin synthesis. Results demonstrate that selective inhibition of ependymin gene expression in vivo can specifically prevent memory formation. We conclude that in particular the newly synthesized ependymin molecules are involved in memory consolidation, possibly because they have not yet undergone irreversible molecular changes, which have been reported of this glycoprotein in a low‐calcium microenvironment.
Three Unique 5′ Untranslated Regions Are Spliced to Common Coding Exons of High‐ and Low‐Molecular‐Weight Microtubule‐Associated Protein‐2
Authors: Kalcheva Nellie , Shafit‐Zagardo Bridget
Published: July 2010

Abstract: Three unique 5′ untranslated regions (UTRs) have been characterized for human microtubule‐associated protein‐2 (MAP‐2) transcripts. All three UTRs shared a common 171‐bp sequence adjacent to the MAP‐2 coding region and then diverged upstream. The size of the unique upstream sequence was 281, 146, or 104 bp. PCR of genomic DNA demonstrated that the 5′ UTRs span multiple exons. The unique region of the UTRs recognizes a 9.5‐ and a 6‐kb MAP‐2 transcript in poly(A)+ mRNA isolated from human MSN cells, and PCR analysis demonstrated that each unique UTR is contained in multiple high‐ and low‐molecular‐weight MAP‐2 transcripts. Reverse transcription‐PCR (RT‐PCR) performed on MSN mRNA isolated from polysomes demonstrated that all three of the UTRs contained within multiple MAP‐2 transcripts were associated with polysomes and hence translated. RT‐PCR from human fetal spinal cord and adult brain mRNA demonstrated that all of the UTRs are expressed at these developmental time points.
Dynorphins Modulate DNA Synthesis in Fetal Brain Cell Aggregates
Authors: Gorodinsky A. , Barg J. , Belcheva M. M. , Levy R. , McHale R. J. , Vogel Z. , Coscia C. J.
Published: July 2010

Abstract: Previously, opioid peptide analogues, β‐endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7‐day fetal rat brain cell aggregates via κ‐and μ‐opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 µM, all dynorphins tested and β‐endorphin inhibited [3H]thymidine incorporation into DNA by 20–38% in 7‐day rat brain cell aggregates. The putative ε‐antagonist β‐endorphin (1–27) did not prevent the effect of β‐endorphin, suggesting that the ε‐receptor is not involved in opioid inhibition of DNA synthesis. The κ‐selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a κ‐opioid receptor. In dose‐dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete κ‐receptor subtype. The IC50 value of 0.1 nM estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the κ‐receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21‐day aggregates were treated with dynorphins, a 33–86% enhancement of thymidine incorporation was observed. Because both 7‐ and 21‐day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express κ‐receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.
Differential Induction of Immediate Early Gene Proteins in Cultured Neurons by β‐Amyloid (Aβ): Association of c‐Jun with Aβ‐Induced Apoptosis
Authors: Anderson Aileen J. , Pike Christian J. , Cotman Carl W.
Published: July 2010

Abstract: β‐Amyloid (Aβ) is a 39–42 amino acid that is the primary component of plaques in Alzheimer's disease (AD). Previous studies from our laboratory and others have shown that Aβ induces neurodegeneration via apoptosis in vitro, suggesting that Aβ may also initiate an apoptotic pathway of cell death in AD. Apoptosis has been suggested to proceed by a gene‐directed program in several systems. Accordingly, we have investigated whether Aβ‐mediated apoptosis is associated with the induction of genes that may regulate or play a role in cell death in vitro. Immediate early genes (IEGs) respond to cellular stimuli and participate in cellular signaling pathways. The protein products of some IEGs, e.g., c‐jun, are capable of forming dimers and acting as transcriptional regulatory proteins, and have been implicated in apoptosis in both nonneuronal and neuronal cells. In this study, we report a selective and abnormally sustained induction of c‐Jun in cultured hippocampal neurons treated with Aβ. In addition, we describe the lack of induction of c‐Jun in neurons that are relatively resistant to Aβ‐mediated toxicity, and a correspondence between immunoreactivity for c‐Jun and changes in nuclear morphology that are indicative of apoptosis. These data demonstrate that c‐Jun is induced in cultured neurons that undergo Aβ‐mediated apoptosis and suggest that c‐Jun may participate in a cell death program in these neurons.
Staurosporine Induces Astrocytic Phenotypes and Differential Expression of Specific PKC Isoforms in C6 Glial Cells
Authors: Kronfeld Ilana , Zsukerman Alexander , Kazimirsky Gila , Brodie Chaya
Published: July 2010

Abstract: In this study we examined the effects of staurosporine, a potent inhibitor of protein kinase C (PKC), on the differentiation of C6 glial cells and on the expression and cellular distribution of specific PKC isoforms. Staurosporine reduced cell proliferation and induced distinctive changes in the morphological appearance of the cells to that characteristic of cells exhibiting astrocytic phenotypes. The differentiative effect of staurosporine was further indicated by the increased expression of two proteins related to astrocytic phenotypes, glial fibrillary acidic protein (GFAP) and glutamine synthetase. Thus, staurosporine induced a dose‐dependent increase both in GFAP immunoreactivity and in the activity and protein levels of glutamine synthetase. Staurosporine also induced a decrease in the expression of PKC‐β2 and an increase in that of PKC‐γ. In addition, it induced translocation of PKC‐ε from the membrane to the cytosol, whereas no differences were observed in the distribution of the other PKC isoforms. The results of our study indicate that staurosporine induced astrocytic phenotypes in glial cells and that changes in the expression and cellular distribution of these PKC isoforms may be related to astrocytic differentiation.
Adenosine Receptors Modulate [Ca2+]i in Hippocampal Astrocytes In Situ
Authors: Porter James T. , McCarthy Ken D.
Published: July 2010

Abstract: Cultured astroglia express both adenosine and ATP purinergic receptors that are coupled to increases in intracellular calcium concentration ([Ca2+]i). Currently, there is little evidence that such purinergic receptors exist on astrocytes in vivo. To address this issue, calcium‐sensitive fluorescent dyes were used in conjunction with confocal microscopy and immunocytochemistry to examine the responsiveness of astrocytes in acutely isolated hippocampal slices to purinergic neuroligands. Both ATP and adenosine induced dynamic increases in astrocytic [Ca2+]i that were blocked by the adenosine receptor antagonist 8‐(p‐sulfophenyl)theophylline. The responses to adenosine were not blocked by tetrodotoxin, 8‐cyclopentyltheophylline, 8‐(3‐chlorostyryl)caffeine, dipyridamole, or removal of extracellular calcium. The P2Y‐selective agonist 2‐methylthioadenosine triphosphate was unable to induce increases in astrocytic [Ca2+]i, whereas the P2 agonist adenosine 5′‐O‐(2‐thiodiphosphate) induced astrocytic responses in a low percentage of astrocytes. These results indicate that the majority of hippocampal astrocytes in situ contain P1 purinergic receptors coupled to increases in [Ca2+]i, whereas a small minority appear to contain P2 purinergic receptors. Furthermore, individual hippocampal astrocytes responded to adenosine, glutamate, and depolarization with increases in [Ca2+]i. The existence of both purinergic and glutamatergic receptors on individual astrocytes in situ suggests that astrocytes in vivo are able to integrate information derived from glutamate and adenosine receptor stimulation.
Components of the Plasminogen Activator System in Astrocytes Are Modulated by Tumor Necrosis Factor‐α and Interleukin‐1β Through Similar Signal Transduction Pathways
Authors: Faber‐Elman Anat , Miskin Ruth , Schwartz Michal
Published: July 2010

Abstract: Migration of astrocytes is thought to play a role in nerve regeneration and to be mediated, at least in part, by inflammation‐associated cytokines. Plasminogen activators are secreted proteases that function in fibrinolysis and participate in cellular migration and invasion and, in some cases, are modulated by cytokines. Here, we show that two cytokines, tumor necrosis factor‐α and interleukin‐1β, can modulate plasminogen activation in astrocytes, each causing 90% reduction of total plasminogen activator activity. Direct and reverse zymography indicated that this reduction resulted from two simultaneous events, a pronounced decrease in tissue‐type plasminogen activator activity and an induction of plasminogen activator inhibitor‐1. Northern hybridization analysis indicated a 30‐fold increase of the steady‐state level of plasminogen activator inhibitor‐1 mRNA following treatment with each of the two cytokines. Both of the cytokine‐induced effects could be blocked by cycloheximide or actinomycin D. When signal transduction pathways were blocked, the results indicated the involvement of reduction in cyclic AMP levels, protein kinase activity, and arachidonic metabolites of the lipoxygenase pathway. The results thus show that the two cytokines reduce the ability of astrocytes to conduct fibrinolysis and extracellular proteolysis, and suggest that the effect of these cytokines on members of the plasminogen activation system is through a common signal transduction pathway.
Peroxynitrite‐Induced Cytotoxicity in PC12 Cells: Evidence for an Apoptotic Mechanism Differentially Modulated by Neurotrophic Factors
Authors: Estévez Alvaro G. , Radi Rafael , Barbeito Luis , Shin Jordan T. , Thompson John A. , Beckman Joseph S.
Published: July 2010

Abstract: Peroxynitrite is a powerful oxidant formed by the near‐diffusion‐limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 mM) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC50 = 850 µM) initially (3–4 h) caused minimal damage to low‐density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite‐treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite‐treated cells demonstrated DNA fragmentation (by terminal‐deoxynucleotide transferase‐mediated dUTP‐digoxigenin nick end‐labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite‐induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite‐initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite‐induced apoptosis in vitro.
Rat Frontal Cortex β1‐Adrenoceptors Are Activated by the β3‐Adrenoceptor Agonists SR 58611A and SR 58878A but Not by BRL 37344 or ICI 215,001
Authors: Nisoli Enzo , Tonello Cristina , Benarese Marina , Carruba Michele O.
Published: July 2010

Abstract: SR 58611A, a selective agonist of gut and brown adipose tissue β3‐adrenoceptors (β3ARs), has been reported to have antidepressant‐like activity in rodents by indicating brain β3ARs as the sites of this property. SR 58611A and its acid metabolite SR 58878A, as opposed to BRL 37344, ICI 215,001, and CGP 12177, increased cyclic AMP levels in rat frontal cortex. ICI 215,001, differently from BRL 37344, at concentrations in the millimolar range antagonized norepinephrine‐ or (−)‐isoproterenol‐stimulated adenylyl cyclase partially. The increase of cyclic AMP levels induced by SR 58878A was blocked selectively by β1AR antagonist CGP 20712A but not by β2AR antagonist ICI 118,551. In addition, PCR analysis did not reveal β3AR mRNA, and no specific β3AR binding sites were detected by [3H]CGP 12177 in rat frontal cortex. When down‐regulation of the β1AR ligand binding and mRNA levels had been induced in frontal cortex by chronic administration of imipramine, SR 58878A as well as norepinephrine and (−)‐isoproterenol increased the cyclic AMP production less markedly. Our findings indicate that β3ARs are absent in the adult rat frontal cortex, and that various β3AR agonists differently affect the frontal cortex β1ARs, indicating that SR 58611A may exert its putative antidepressant effect acting on the frontal cortex β1ARs.
Dopamine Receptor Stimulation Decreases Cytosolic γ Protein Kinase C Immunoreactivity in Rat Hippocampal Slices: Evidence for Increased Ca2+‐Dependent Proteolysis
Authors: Yurko‐Mauro Karin A. , Friedman Eitan
Published: July 2010

Abstract: The effect of dopamine (DA) receptor stimulation on the distribution of γ protein kinase C (γPKC) in hippocampal slices was assessed. Nanomolar concentrations of DA decreased cytosolic γPKC (56%) without altering membrane γPKC levels, resulting in decreased total γPKC immunoreactivity. The maximal decrease in cytosolic γPKC occurred at 20 min of incubation and was significantly blocked by the D1 DA antagonist SCH 23390 (10−6M) but not by the D2 antagonist sulpiride (10−5M). The D1 agonists SKF 38393 and A 77636 mimicked the effect of DA with similar responses produced at 10 µM and 1 nM, respectively. The D2 agonist quinpirole had no effect on γPKC immunoreactivity, thus indicating that this dopaminergic response is mediated through a D1‐like receptor. DA had no effect on α, δ, or ζPKC isozyme immunoreactivity in the same hippocampal preparations. The DA‐induced decrease in cytosolic γPKC immunoreactivity was blocked by the Ca2+‐dependent protease inhibitor N‐acetyl‐Leu‐Leu‐norleucinal (100 µM) and by the inorganic Ca2+ channel blocker Co2+. The data suggest that DA stimulates a D1‐like DA receptor, which increases the influx of Ca2+ and activates the Ca2+‐dependent proteolysis of γPKC.
Sphingolipid Metabolic Disorders in Trembler Mouse Peripheral Nerves In Vivo Result from an Abnormal Substrate Supply
Authors: Heape Anthony M. , Bessoule Jean‐Jacques , Boiron‐Sargueil Françoise , Garbay Bertrand , Cassagne Claude
Published: July 2010

Abstract: Sphingolipid metabolic pathways in the peripheral nerves of dysmyelinating Trembler mice were studied in vivo, using intraneurally injected [3H]palmitate as the exogenous substrate. The kinetic analysis of the experimental data obtained for the mutant revealed that, as in normal nerves, two metabolically and kinetically independent pathways are implicated in the biosynthesis of the major peripheral nerve sphingolipids: the ceramide pathway and another pathway in which there is no detectable labeled intermediate (“direct amidification”). The results also show that, in the Trembler mouse sciatic nerves: (a) The severely deficient sphingolipid biosynthesis results from the constitution of a qualitatively and quantitatively abnormal fatty acid substrate pool destined for metabolism via the ceramide pathway, which ensures the totality of the galactocerebroside labeling and two‐thirds of that of sphingomyelin. The ceramide intermediates of this pathway are labeled only on their fatty acyl moiety, which contains only 16‐carbon atom chains. (b) “Direct amidification” events implicated in sphingolipid labeling are decreased compared with normal and account for the remaining sphingomyelin formation.
Extracellular GABA in the Ventrolateral Thalamus of Rats Exhibiting Spontaneous Absence Epilepsy: A Microdialysis Study
Authors: Richards D. A. , Lemos T. , Whitton P. S. , Bowery N. G.
Published: July 2010

Abstract: There is compelling evidence that excessive GABA‐mediated inhibition may underlie the abnormal electrical activity, initiated in the thalamus, associated with epileptic absence seizures. In particular, the GABAB receptor subtype seems to play a critical role, because its antagonists are potent inhibitors of absence seizures, whereas its agonists exacerbate seizure activity. Using a validated rat model of absence epilepsy, we have previously found no evidence of abnormal GABAB receptor density or affinity in thalamic tissue. In the present study, we have used in vivo microdialysis to monitor changes in levels of extracellular GABA and other amino acids in this brain region. We have shown that basal extracellular levels of GABA and, to a lesser extent, taurine are increased when compared with values in nonepileptic controls. However, modifying GABAergic transmission with the GABAB agonist (−)‐baclofen (2 mg/kg i.p.), the GABAB antagonist CGP‐35348 (200 mg/kg i.p.), or the GABA uptake inhibitor tiagabine (100 µM) did not produce any further alteration in extracellular GABA levels, despite the ability of these compounds to increase (baclofen and tiagabine) or decrease (CGP‐35348) seizure activity. These findings suggest that the increased basal GABA levels observed in this animal model are not simply a consequence of seizure activity but may contribute to the initiation of absence seizures.
Effect of Reperfusion Following Cerebral Ischaemia on the Activity of the Mitochondrial Respiratory Chain in the Gerbil Brain
Authors: Almeida Angeles , Allen Kathryn L. , Bates Timothy E. , Clark John B.
Published: July 2010

Abstract: The effect of reperfusion following 30 min of cerebral ischaemia on brain mitochondrial respiratory chain activity has been studied in the gerbil. The state 3 respiration rates with both FAD‐ and NAD‐linked substrates were reduced after ischaemia. After 5 min of reperfusion, state 3 respiration with FAD‐linked substrates was restored, but levels of NAD‐linked substrates did not return to control values until 30 min of reperfusion. By 120 min of reperfusion state 3 respiration decreased relative to control values with all substrates studied. Measurement of the individual respiratory chain complexes showed that complex I, complex II–III, and complex V activities were reduced after ischaemia. By 5 min of reperfusion complex II–III activity was restored, but the activities of complexes I and V did not return to control values until 30 min of reperfusion. In contrast, complex IV activity was unaffected by ischaemia or 5 and 30 min of reperfusion but was significantly reduced after 120 min of reperfusion, possibly owing to free radical production and lipid peroxidation.
Glutamate Release and Free Radical Production Following Brain Injury: Effects of Posttraumatic Hypothermia
Authors: Globus Mordecai Y.‐T. , Alonso Ofelia , Dietrich W. Dalton , Busto Raul , Ginsberg Myron D.
Published: July 2010

Abstract: Posttraumatic hypothermia reduces the extent of neuronal damage in remote cortical and subcortical structures following traumatic brain injury (TBI). We evaluated whether excessive extracellular release of glutamate and generation of hydroxyl radicals are associated with remote traumatic injury, and whether posttraumatic hypothermia modulates these processes. Lateral fluid percussion was used to induce TBI in rats. The salicylate‐trapping method was used in conjunction with microdialysis and HPLC to detect hydroxyl radicals by measurement of the stable adducts 2,3‐ and 2,5‐dihydroxybenzoic acid (DHBA). Extracellular glutamate was measured from the same samples. Following trauma, brain temperature was maintained for 3 h at either 37 or 30°C. Sham‐trauma animals were treated in an identical manner. In the normothermic group, TBI induced significant elevations in 2,3‐DHBA (3.3‐fold, p < 0.01), 2,5‐DHBA (2.5‐fold, p < 0.01), and glutamate (2.8‐fold, p < 0.01) compared with controls. The levels of 2,3‐DHBA and glutamate remained high for approximately 1 h after trauma, whereas levels of 2,5‐DHBA remained high for the entire sampling period (4 h). Linear regression analysis revealed a significant positive correlation between integrated 2,3‐DHBA and glutamate concentrations (p < 0.05). Posttraumatic hypothermia resulted in suppression of both 2,3‐ and 2,5‐DHBA elevations and glutamate release. The present data indicate that TBI is followed by prompt increases in both glutamate release and hydroxyl radical production from cortical regions adjacent to the impact site. The magnitude of glutamate release is correlated with the extent of the hydroxyl radical adduct, raising the possibility that the two responses are associated. Posttraumatic hypothermia blunts both responses, suggesting a mechanism by which hypothermia confers protection following TBI.
Metabolic and Energetic Changes During Apoptosis in Neural Cells
Authors: Mills Jason C. , Nelson David , Erecińska Maria , Pittman Randall N.
Published: July 2010

Abstract: Changes in cellular energetic and metabolic parameters were analyzed at several time points during apoptosis of differentiated PC12 cells following removal of nerve growth factor (NGF). As approximately 60% of the population died during the period of study (24 h), most of the measured metabolic indicators declined over time. However, this decline paralleled the overall decrease in cellular viability, suggesting that, in individual cells, a compromised metabolic state occurred suddenly and very late in the death process. For example, when expressed as a function of viable cells, protein and RNA synthesis did not decrease until 24 h. Glucose utilization in live cells was never significantly reduced relative to control levels; lactate production decreased slightly within 4–8 h after NGF removal, but eventually rebounded to 122% of control levels by 24 h. ATP levels dropped 27% in an early predeath period, but then returned to near control levels (on a per‐live‐cell basis) once the population actively began to die. The ATP/ADP ratio remained at least 84% of control throughout. UTP/UDP and GTP/GDP ratios did not change significantly at any time point.
Fatty Acids from Degenerating Myelin Lipids Are Conserved and Reutilized for Myelin Synthesis During Regeneration in Peripheral Nerve
Authors: Goodrum Jeffry F. , Weaver Janice E. , Goines Nelson D. , Bouldin Thomas W.
Published: July 2010

Abstract: Following nerve crush, cholesterol from degenerating myelin is conserved and reutilized for new myelin synthesis during nerve regeneration. The possibility that other myelin lipids are salvaged and reutilized has not been investigated previously. We examined the fate of myelin phospholipids and their fatty acyl moieties following nerve crush by electron microscopic autoradiography of myelin lipids prelabeled with [3H]oleate or [2‐3H]‐glycerol. Both precursors were incorporated predominantly (>90%) into phospholipids; >85% of the [3H]oleate was incorporated as oleate, with the remainder in longer‐chain fatty acids. Before nerve crush, both labels were restricted to myelin sheaths. Following nerve crush and subsequent regeneration, over half the label from [3H]oleate, but little from [2‐3H]glycerol, remained in nerve. The oleate label was present as fatty acyl moieties in phospholipids and was localized to newly formed myelin sheaths. Among the extracellular soluble lipids within the degenerating nerve, the bulk of the labeled phospholipids floated at the same density as lipoprotein particles. These data indicate that myelin phospholipids are completely hydrolyzed during nerve degeneration, that at least half the resultant free fatty acids are salvaged and reutilized for new myelin synthesis, and that these salvaged fatty acids are transported by a lipoprotein‐mediated mechanism similar to that functioning in cholesterol reutilization.
Transglutaminase Cross‐Linking of the τ Protein
Authors: Miller Michael L. , Johnson Gail V. W.
Published: July 2010

Abstract: Tissue transglutaminase (EC 2.3.2.13) is a calcium‐activated enzyme that cross‐links specific substrate proteins into insoluble, protease‐resistant, high molecular weight complexes. Because the neurofibrillary tangles in Alzheimer disease have similar biochemical characteristics, and because the microtubule‐associated protein τ is the predominant component of these structures, the substrate properties of τ with respect to transglutaminase were investigated. Bovine τ and recombinant human τ isoforms rapidly form high molecular weight, cross‐linked polymers on incubation with transglutaminase. Polyamine incorporation assays indicate that bovine τ is an excellent substrate of transglutaminase, with a Km of 10.4 ± 2.2 µM and a Vmax of 40.9 ± 4.5 nmol/mg of enzyme/min. Individual recombinant human τ isoforms are not equivalent with respect to transglutaminase, as the smallest isoform T3 (352 amino acids) is not as good a substrate as the larger isoforms T4 (383 amino acids) and T4L (441 amino acids). To determine which segments of the τ protein are susceptible to modification by transglutaminase, τ was labeled with [3H]putrescine by transglutaminase and proteolyzed with α‐chymotrypsin, and the breakdown products were analyzed. These experiments demonstrate that the enzyme modifies τ at only one or a few discrete sites, primarily in the carboxyl half of the molecule. Thus, the reaction is specific for only a small number of the many glutamine residues in τ. Furthermore, a τ deletion construct (T264) containing a portion of the microtubule‐binding domains, which is a substrate of transglutaminase, cannot be cross‐linked by the enzyme. This provides evidence that the cross‐linking reaction is specific, and requires that the substrates be appropriately associated for cross‐linking to occur.