bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of Endothelin B Receptor Modulates the Inflammatory Activation of Rat Astrocytes
Authors: Morga Eleonora , Faber Chantal , Heuschling Paul
Published: March 2003

Abstract: Inside the brain tissue, endothelins play numerous important biological roles. One of the targets, astrocytes, predominantly display endothelin receptor subtype B (ETB). On cultured primary rat astroglial cells, we analyzed the effect of IRL 1620, a selective ETB receptor agonist, on the production of nitric oxide (NO) and the synthesis of interleukin (IL)‐6 and tumor necrosis factor (TNF)‐α. We performed these experiments in the presence or absence of interferon‐γ (IFN‐γ) and/or lipopolysaccharide (LPS). IRL 1620 decreases NO production under basal conditions and after IFN‐γ stimulation. However, during LPS‐induced NO production, IRL 1620 enhances this release. The basal IL‐6 secretion and especially the LPS‐induced synthesis are enhanced by the IRL 1620 stimulation. The LPS‐dependent TNF‐α production is increased by the ETB stimulation. The IRL 1620‐induced decrease of basal NO production is not dependent on Ca2+ entry or on phospholipase C (PLC) activation, as shown by the use of LaCl3 and U73122, respectively. In the presence of LPS, the IRL 1620 potentiation of NO production is inhibited by LaCl3 and U73122. The IRL 1620‐induced increase of IL‐6 is dependent on PLC activation. These results suggest that endothelins can have dual effects depending on the costimulatory factors present. Endothelins thus have important immunomodulatory functions in the brain.
Kainic Acid‐Induced Apoptosis in Rat Striatum Is Associated with Nuclear Factor‐κB Activation
Authors: Nakai Masami , Qin Zheng‐Hong , Chen Jiang‐Fan , Wang Yumei , Chase Thomas N.
Published: March 2003

Abstract: The present study evaluated whether nuclear factor‐κB (NF‐κB) activation contributes to the apoptotic‐like death of striatal neurons induced by kainic acid (KA) receptor stimulation. Intrastriatally infused KA (1.25‐5.0 nmol) produced substantial neuronal loss as indicated by an 8‐73% decrease in 67‐kDa glutamic acid decarboxylase (p < 0.05). KA (1.25‐5.0 nmol) elicited internucleosomal DNA fragmentation that was inhibited by the AMPA/KA receptor antagonist NBQX (1,2,3,4‐tetrahydro‐6‐nitro‐2,3‐dibenzo[f]quinoxaline‐7‐sulfonamide) but not by the NMDA receptor antagonist MK‐801. A decrease in IκB‐α protein levels, which was accompanied by an increase in NF‐κB binding activity, was found from 6 to 72 h after KA (2.5 nmol) infusion. NF‐κB was composed mainly of p65 and c‐Rel as revealed by supershift assay. In addition, c‐Myc and p53 increased from five‐ to sevenfold from 24 to 72 h after KA (2.5 nmol) administration. Immunohistochemistry revealed high levels of c‐Myc and p53 immunoreactivity, mainly in medium‐sized striatal neurons. Pretreatment with the cell‐permeable recombinant peptide NF‐κB SN50 (5‐20 μg) blocked NF‐κB nuclear translocation, but had no effect on AP‐1 binding. NF‐κB SN50 also inhibited the KA‐induced up‐regulation of c‐Myc and p53, as well as internucleosomal DNA fragmentation. The apoptotic‐like destruction of rat striatal neurons induced by KA receptor stimulation thus appears to involve biochemical mechanisms similar to those mediating the excitotoxic response to NMDA receptor stimulation. The present results provide additional support for the view that NF‐κB activation contributes to c‐Myc and p53 induction and subsequent apoptosis in an excitotoxic model of Huntington’s disease.
Neurotrophin‐3 mediates the autocrine survival of the catecholaminergic CAD CNS neuronal cell line
Authors: Horton Cheryl D. , Qi Yanping , Chikaraishi Dona , Wang James K. T.
Published: March 2003

The mechanisms for neuronal survival in the CNS are not well understood, but are likely to be complex due to possible autocrine and redundant neurotrophic support. Most studies have focused on the nerve growth factor (NGF)/TrkA pathway in peripheral neurons, and little is known regarding the other neurotrophins, particularly neurotrophin‐3 (NT3)/TrkC. Progress has also been hampered by the paucity of homogenous and accessible CNS neuronal experimental models. We now report that the novel catecholaminergic CNS cell line, CAD, is capable of autocrine survival mediated by NT3. The CAD cell is of CNS neuronal origin and can survive and morphologically differentiate in the absence of exogenously provided trophic factors. However, neutralizing reagents against NT3 (the neutralizing TrkC‐IgG fusion protein and anti‐NT3 antibodies), but not those that block the other neurotrophins, inhibited survival of differentiating CAD cells. Moreover, Trk phosphorylation was detected in CAD cells and its inhibition by K252a was correlated with K252a‐induced apoptosis. Finally, endogenous NT3 was detectable in CAD cell extracts by a specific ELISA assay. Thus, CAD cells possess an autocrine survival capability mediated by NT3, and may provide a valuable model system for studying the signaling pathways that mediate the actions of this little understood neurotrophin.
Clozapine increases apolipoprotein D expression in rodent brain: towards a mechanism for neuroleptic pharmacotherapy
Authors: Thomas Elizabeth A. , Danielson Patria E. , Nelson P. Austin , Pribyl Thomas M. , Hilbush Brian S. , Hasel Karl W. , Sutcliffe J. Gregor
Published: March 2003

In contrast to typical neuroleptic drugs, which have high affinities for dopamine D2 receptors, clozapine binds to multiple neurotransmitter receptors. The mechanisms responsible for its superior clinical efficacy over typical neuroleptics remain unknown. Using an automated genomics approach, total gene expression analysis (TOGA), we found an approximately threefold increase in the accumulation of the mRNA encoding apolipoprotein D (apoD) in mouse striatum in response to chronic treatment with clozapine. While in control animals, apoD is expressed predominantly in astrocytes, in situ hybridization and immunohistochemical studies indicated a substantial increase in apoD expression in neurons of the striatum, globus pallidus and thalamus after 2 weeks of clozapine treatment. Clozapine‐induced increases in apoD expression were also observed in some white matter regions. These results suggest that apoD is a mediator in the mechanisms of clozapine and thus that deficiencies in aspects of lipid metabolism may be responsible for psychoses.
Progressive depletion of complexin II in a transgenic mouse model of Huntington's disease
Authors: Morton A. J. , Edwardson J. M.
Published: March 2003

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, emotional and cognitive dysfunction. There is no treatment or cure for this disease, and after the onset of symptoms, usually in the fourth decade of life, there is an inexorable decline to death. In many patients there is a complex deterioration of function before the onset of neuronal loss and, at least in mouse models, abnormalities in neurotransmission represent early events in the development of the disease. Here we describe the specific and progressive loss of complexin II from the brains of mice carrying the HD mutation (R6/2 line), and the later appearance of this protein in a subpopulation of neuronal intranuclear inclusions. Although the precise role of complexin II is still unclear, it is known to bind to the SNARE complex, and is therefore likely to be involved in the control of exocytosis. Our results suggest that changes in neurotransmitter release might contribute to the neuronal dysfunction seen in these mice.
Dopamine D4receptor‐mediated regulation of rod opsin mRNA expression in tiger salamander
Authors: Alfinito Peter D. , Townes‐Anderson Ellen
Published: March 2003

Light stimulates dopamine release in the retina and has been shown to rapidly up‐regulate rod opsin mRNA. In the present study, we tested the effect of dopamine on rod opsin mRNA expression and examined the hypothesis that dopamine can mediate a light‐evoked increase in opsin gene expression. Northern blots showed that a 30‐min light‐exposure increased rod opsin mRNA expression 27%.In situhybridization on isolated rods showed that 500 nmdopamine and 1 µmquinpirole (dopamine D2/D3/D4agonist) increased opsin mRNA 45% and 26%, respectively. The effect of quinpirole was selectively blocked by the D4antagonist, L750,667 (20 µm). In very low density cultures, quinpirole increased opsin expression 46%, suggesting a direct effect on rod photoreceptors. Consistent with a dopamine D4receptor mechanism, 1 µmH‐89 (protein kinase A inhibitor) increased opsin mRNA 39%. Finally, intravitreal injection of quinpirole increased opsin mRNA 21% whereas injection of L750,667 (10 µm) blocked the light‐evoked increase in opsin expression. These data show that rod opsin mRNA is up‐regulated by dopamine binding a D4‐like receptor on rods, possibly through inhibition of protein kinase A, and that endogenous dopamine can mediate the light‐evoked increase in opsin mRNA expression.
5‐HT2A and D2 receptor blockade increases cortical DA release via 5‐HT1A receptor activation: a possible mechanism of atypical antipsychotic‐induced cortical dopamine release
Authors: Ichikawa Junji , Ishii Hideo , Bonaccorso Stefania , Fowler Wiley L. , O'Laughlin Ian A. , Meltzer Herbert Y.
Published: March 2003

Atypical antipsychotic drugs (APDs), all of which are relatively more potent as serotonin (5‐HT)2A than dopamine D2 antagonists, may improve negative symptoms and cognitive dysfunction in schizophrenia, in part, via increasing cortical dopamine release. 5‐HT1A agonism has been also suggested to contribute to the ability to increase cortical dopamine release. The present study tested the hypothesis that clozapine, olanzapine, risperidone, and perhaps other atypical APDs, increase dopamine release in rat medial prefrontal cortex (mPFC) via 5‐HT1A receptor activation, as a result of the blockade of 5‐HT2A and D2 receptors. M100907 (0.1 mg/kg), a 5‐HT2A antagonist, significantly increased the ability of both S(–)‐sulpiride (10 mg/kg), a D2 antagonist devoid of 5‐HT1A affinity, and R(+)‐8‐OH‐DPAT (0.05 mg/kg), a 5‐HT1A agonist, to increase mPFC dopamine release. These effects of M100907 were abolished by WAY100635 (0.05 mg/kg), a 5‐HT1A antagonist, which by itself has no effect on mPFC dopamine release. WAY100635 (0.2 mg/kg) also reversed the ability of clozapine (20 mg/kg), olanzapine (1 mg/kg), risperidone (1 mg/kg), and the R(+)‐8‐OH‐DPAT (0.2 mg/kg) to increase mPFC dopamine release. Clozapine is a direct acting 5‐HT1A partial agonist, whereas olanzapine and risperidone are not. These results suggest that the atypical APDs via 5‐HT2A and D2 receptor blockade, regardless of intrinsic 5‐HT1A affinity, may promote the ability of 5‐HT1A receptor stimulation to increase mPFC DA release, and provide additional evidence that coadministration of 5‐HT2A antagonists and typical APDs, which are D2 antagonists, may facilitate 5‐HT1A agonist activity.
Integrin signaling via the PI3‐kinase–Akt pathway increases neuronal resistance to glutamate‐induced apoptosis
Authors: Gary Devin S. , Mattson Mark P.
Published: March 2003

Integrins are integral membrane proteins that mediate adhesive interactions of cells with the extracellular matrix and with other cells. Integrin engagement results in activation of intracellular signaling cascades that effect several different cellular responses including motility, proliferation and survival. Although integrins are known to provide cell survival signaling in various types of non‐neuronal cells, the possibility that integrins modulate neuron survival has not been explored. We now report data demonstrating a neuroprotective function of integrins in embryonic hippocampal neurons. Neurons grown on laminin, an integrin ligand, exhibit increased resistance to glutamate‐induced apoptosis compared with neurons grown on polylysine. Neurons expressed integrin β1 and treatment of cultures with an antibody against integrin β1 abolished the protective effect of laminin. Neurons maintained on laminin exhibited a sustained activation of the Akt signaling pathway demonstrated in immunoblot analyses using an antibody that selectively recognizes phosphorylated Akt. The neuroprotective effect of integrin engagement by laminin was mimicked by an IKLLI‐containing integrin‐binding peptide and was abolished by treatment of neurons with the PI3 kinase inhibitor wortmanin. Levels of the anti‐apoptotic protein Bcl‐2 were increased in neurons grown on laminin and decreased by wortmanin, suggesting a mechanism for the neuroprotective effect of integrin‐mediated signaling. The ability of integrin‐mediated signaling to prevent glutamate‐induced apoptosis suggests a mechanism whereby neuron–substrate interactions can promote neuron survival under conditions of glutamate receptor overactivation.
Acute and chronic effects of citalopram on postsynaptic 5‐hydroxytryptamine1A receptor‐mediated feedback: a microdialysis study in the amygdala
Authors: Bosker F. J. , Cremers T. I. F. H. , Jongsma M. E. , Westerink B. H. C. , Wikström H. V. , Den Boer J. A.
Published: March 2003

Microdialysis was used to assess the involvement of postsynaptic 5‐hydroxytryptamine1A (5‐HT1A) receptors in the regulation of extracellular 5‐HT in the amygdala. Local infusion of the 5‐HT1A receptor agonist flesinoxan (0.3, 1, 3 µm) for 30 min into the amygdala maximally decreased 5‐HT to 50% of basal level. Systemic administration of citalopram (10 µmol/kg) increased 5‐HT to 175% of basal level. Local infusion of 1 µm of the 5‐HT1A receptor antagonist WAY 100.635 into the amygdala augmented the effect of citalopram to more than 500% of basal 5‐HT level. 5‐HT1A receptor responsiveness after chronic citalopram treatment was determined in two ways. First, by local infusion of 1 µm flesinoxan for 30 min into the amygdala, which showed a significant 63% reduction in response (area under the concentration–time curve; AUC) for the citalopram group compared to the saline group. Second, by systemic administration of citalopram (10 µmol/kg), which increased 5‐HT to 350% of basal level. The effect was larger than in untreated animals, but more important, local infusion of 1 µm WAY 100.635 into the amygdala now failed to augment the effect of citalopram. Both the flesinoxan and WAY 100.635 data suggest an involvement of postsynaptic 5‐HT1A receptor‐mediated feedback in the amygdala, which diminishes following chronic citalopram treatment.
Plasminogen expression in the neonatal and adult mouse brain
Authors: Basham Mark E. , Seeds Nicholas W.
Published: March 2003

Tissue‐type plasminogen activator (tPA) has been implicated in a variety of types of neural plasticity, including cell migration, occlusion‐induced visual system plasticity, and learning. In the periphery, plasminogen serves as tPA's primary substrate; however, studies attempting to identify plasminogen in the central nervous system have produced mixed results. We have performed a comprehensive, multitechnique study examining plasminogen expression in the neonatal and adult mouse brain. Reverse transcription polymerase chain reaction (RT‐PCR) and in situ hybridization reveal plasminogen mRNA in the cortex, hippocampus and cerebellum of both neonatal and adult C57BL/6 mice. Immunocytochemistry reveals plasminogen protein expression in these same brain regions. Notably, plasminogen expression in the cerebellum occurs in the granule cell and the Purkinje cell layers. tPA activity in these same regions is involved in granule cell migration during development and motor learning in adulthood. Therefore, these findings demonstrate that plasminogen is present in the central nervous system and localized to areas where it could serve as a substrate for plasticity‐related increases in tPA activity.
D1–D2 dopamine receptor interaction within the nucleus accumbens mediates long‐loop negative feedback to the ventral tegmental area (VTA)
Authors: Rahman Shafiqur , McBride William J.
Published: March 2003

The objective of the present study was to examine the effects of perfusion of dopamine (DA) D1‐ and D2‐like receptor agonists in the nucleus accumbens (ACB) on the long‐loop negative feedback regulation of mesolimbic somatodendritic DA release in the ventral tegmental area (VTA) of Wistar rats employing ipsilateral dual probe in vivo microdialysis. Perfusion of the ACB for 60 min with the D1‐like receptor agonist SKF 38393 (SKF, 1–100 µm) dose‐dependently reduced the extracellular levels of DA in the ACB, whereas the extracellular levels of DA in the VTA were not changed. Similarly, application of the D2‐like receptor agonist quinpirole (Quin, 1–100 µm) through the microdialysis probe in the ACB reduced the extracellular levels of DA in the ACB in a concentration‐dependent manner, whereas extracellular levels of DA in the VTA were not altered. Co‐application of SKF (100 µm) and Quin (100 µm) produced concomitant reductions in the extracellular levels of DA in the ACB and VTA. The reduction in extracellular levels of DA in the ACB and VTA produced by co‐infusion of SKF and Quin was reversed in the presence of either 100 µm SCH 23390 (D1‐like antagonist) or 100 µm sulpiride (D2‐like antagonist). Overall, the results suggest that (a) activation of dopamine D1‐ or D2‐like receptors can independently regulate local terminal DA release in the ACB, whereas stimulation of both subtypes is required for activation of the negative feedback pathway to the VTA.
Expression of Rho‐family GTPases (Rac, cdc42, RhoA) and their association with p‐21 activated kinase in adult rat peripheral nerve
Authors: Terashima Tomoya , Yasuda Hitoshi , Terada Masahiko , Kogawa Shuro , Maeda Kengo , Haneda Masakazu , Kashiwagi Atsunori , Kikkawa Ryuichi
Published: March 2003

To clarify the presence of the Rho family of small GTPases p21‐activated kinase (pak) signaling pathway in the PNS, we have examined their expression, the association between the small GTPases and pak and the pak kinase activity in the PNS using immunoblot analysis, immunohistochemistry, co‐immunoprecipitation study, and in vitro kinase assay. Immunoblot analysis showed the expression of Rac, cdc42, RhoA and pak in the dorsal root ganglion (DRG) and sciatic nerve. The localization of these proteins in the DRG neurons and axons and Schwann cells of the sciatic nerve was confirmed by immunohistochemistry. Co‐immunoprecipitation studies indicated the in vivo associations of pak with Rac and cdc42, but not with RhoA, in both the DRG and sciatic nerve. The autophosphorylation of pak and phosphorylation of histone H4 by pak were also found in the DRG and sciatic nerve as well as in the CNS. These results suggest that the Rac/cdc42‐pak signaling pathway exists and functions in the PNS and may mediate some intracellular signals.
Bax κ, a novel Bax splice variant from ischemic rat brain lacking an ART domain, promotes neuronal cell death
Authors: Jin Kun Lin , Graham Steven H. , Mao Xiao Ou , He Xiangjun , Nagayama Tetsuya , Simon Roger P. , Greenberg David A.
Published: March 2003

Bax is a pro‐apoptotic Bcl‐2 family protein that regulates programmed cell death through homodimerization and through heterodimerization with Bcl‐2. Bax α is encoded by six exons and undergoes alternative splicing. Bax κ, a splice variant of Bax with conserved BH1, BH2 and BH3 binding domains and a C‐terminal transmembrane domain (TM), but with an extra 446‐bp insert between exons 1 and 2 leading to loss of an N‐terminal ART domain, was identified from an ischemic rat brain cDNA library. Expression of Bax κ mRNA and protein was up‐regulated in hippocampus after cerebral ischemic injury. The increased Bax κ mRNA was distributed mainly in selectively vulnerable hippocampal CA1 neurons that are destined to die after global ischemia. Overexpression of Bax κ protein in HN33 mouse hippocampal neuronal cells induced cell death, which was partially abrogated by co‐overexpression of Bcl‐2. Moreover, co‐overexpression of Bax κ and Bax α increased HN33 cell death. The results suggest that the Bax κ may have a role in ischemic neuronal death.
Gene expression analysis in N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine mice model of Parkinson's disease using cDNA microarray: effect of R‐apomorphine
Authors: Grünblatt Edna , Mandel Silvia , Maor Gila , Youdim Moussa B. H.
Published: March 2003

To establish the possible roles of oxidative stress, inflammatory processes and other unknown mechanisms in neurodegeneration, we investigated brain gene alterations in N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mice model of Parkinson's disease using Atlas mouse cDNA expression array membrane. The expression of 51 different genes involved in oxidative stress, inflammation, glutamate and neurotrophic factors pathways as well as in still undefined processes, such as cell cycle regulators and signal transduction molecules, was differentially affected by the treatment. The present study indicates the involvement of an additional cascade of events that might act in parallel to oxidative stress and inflammation to converge eventually into a common pathway leading to neurodegeneration. The attenuation of these gene changes by R‐apomorphine, an iron chelator‐radical scavenger drug, supports our previous findings in vivo where R‐apomorphine was neuroprotective.
Changes in mACh, NMDA and GABAA receptor binding after lateral fluid‐percussion injury: in vitro autoradiography of rat brain frozen sections
Authors: Sihver Sven , Marklund Niklas , Hillered Lars , Långström Bengt , Watanabe Yasuyoshi , Bergström Mats
Published: March 2003

Adult rats were subjected to a moderate lateral fluid percussion injury (FPI), followed by survival periods of 2 and 12 h. Regional NMDA subtype glutamate, muscarinic acetylcholine and GABAA receptor binding in various brain regions was analysed by quantitative in vitro autoradiography and short‐lived positron emission tomography tracers [11C]cyano‐dizocilpine, 4‐N‐[11C]methylpiperidylbenzilate (4‐N‐[11C]MPB), and [11C]flumazenil, respectively. The binding potential (BP, Bmax/KD) was calculated. The data with [11C]cyano‐dizocilpine showed a significant decrease in BP bilaterally for the frontoparietal cortex and hippocampus at both time points, in comparison with that of the sham‐operated controls. At 12 h the decrease was significantly more prominent for the ipsilateral cortex and hippocampus than for the contralateral side. The BP of 4‐N‐[11C]MPB was significantly decreased after 2 h for the trauma‐side hippocampus, and after 12 h it had decreased for the trauma‐site cortex and the bilateral hippocampus. The [11C]flumazenil exhibited a significant decrease in BP for the trauma‐site cortex and the underlying hippocampus by 2 h after the traumatic brain injury. After 12 h a significantly decreased BP was observed only for the trauma‐site cortex. The finding of a decreased BP demonstrates the involvement of these receptor systems in the development of cellular dysfunction, which is widespread and not limited to the site of lateral FPI.
Zn2+‐induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells
Authors: Seo Su Ryeon , Chong Seon Ah , Lee Syng‐Ill , Sung Jee Young , Ahn Young Soo , Chung Kwang Chul , Seo Jeong Taeg
Published: March 2003

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia‐ and seizure‐induced neuronal death. However, the intracellular signaling pathways involved in Zn2+‐induced cell death are largely unknown. In the present study, we investigated the roles of mitogen‐activated protein kinases (MAPKs), such as c‐Jun N‐terminal kinase (JNK), p38 MAPK and extracellular signal‐regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+‐induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 µm), a Zn2+ ionophore, and Zn2+ (10 µm) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+‐induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol‐3 kinase had little effect on Zn2+‐induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+‐induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.
Preconditioning‐induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor‐κB
Authors: Ravati Alexander , Ahlemeyer Barbara , Becker Andreas , Klumpp Susanne , Krieglstein Josef
Published: March 2003

Preconditioning by a sublethal stimulus induces tolerance to a subsequent, otherwise lethal insult and it has been suggested that reactive oxygen species (ROS) are involved in this phenomenon. In the present study, we determined whether preconditioning activates the transcription factor nuclear factor‐κB (NF‐κB) and how this activation contributes to preconditioning‐induced inhibition of neuronal apoptosis. Preconditioning was performed by incubating mixed cultures of neurons and astrocytes from neonatal rat hippocampus with xanthine/xanthine oxidase or FeSO4 for 15 min followed by 24 h of recovery which protected the neurons against subsequent staurosporine‐induced (200 nm, 24 h) apoptosis. The cellular ROS content increased during preconditioning, but returned to basal levels after removal of xanthine/xanthine oxidase or FeSO4. We detected a transient activation of NF‐κB 4 h after preconditioning as shown by immunocytochemistry, by a decrease in the protein level of IκBα as well as by electrophoretic mobility shift assay. Preconditioning‐mediated neuroprotection was abolished by antioxidants, inhibitors of NF‐κB activation and cycloheximide suggesting the involvement of ROS, an activation of NF‐κB and de novo protein synthesis in preconditioning‐mediated rescue pathways. Furthermore, preconditioning increased the protein level of Mn‐superoxide dismutase which could be blocked by antioxidants, cycloheximide and κB decoy DNA. Our data suggest that inhibition of staurosporine‐induced neuronal apoptosis by preconditioning with xanthine/xanthine oxidase or FeSO4 involves an activation of NF‐κB and an increase in the protein level of Mn‐superoxide dismutase.
Neurotrophins prevent HIV Tat‐induced neuronal apoptosis via a nuclear factor‐κB (NF‐κB)‐dependent mechanism
Authors: Ramirez Servio H. , Sanchez Joseph F. , Dimitri Christopher A. , Gelbard Harris A. , Dewhurst Stephen , Maggirwar Sanjay B.
Published: March 2003

HIV‐1 associated dementia is thought to be caused by neuronal damage and death in response to the production of soluble neurotoxic factors by virally infected mononuclear phagocytes. These neurotoxins include HIV‐1 Tat. The ability of neurotrophins to promote cell survival prompted us to examine whether neurotrophins might also be capable of opposing the pro‐apoptotic effects of Tat. Here, we show that Tat‐induced neuronal apoptosis in primary cultures of rat cerebellar granule cells and in neuronally differentiated human SK‐N‐MC cells is profoundly inhibited by brain‐derived neurotrophic factor, nerve growth factor and activity‐dependent neurotrophic factor nonamer peptide. These neurotrophins activated the transcription factor NF‐κB, and inhibition of NF‐κB activation using a super‐repressor IκB‐α mutant was found to block the survival‐promoting activity of the neurotrophins. Reporter gene assays and immunoblot experiments revealed that the neurotrophins also up‐regulated the expression of Bcl‐2, at both the transcriptional and protein levels. Overexpression of the super‐repressor IκB‐α mutant prevented this induction of Bcl‐2 expression. Moreover, overexpression of either Bcl‐2, alone, or the RelA subunit of NF‐κB, alone, protected neurons from Tat‐induced apoptosis. These findings suggest that the activation of NF‐κB by neurotrophic factors may promote survival of neurons exposed to Tat, via regulation of anti‐apoptotic genes including Bcl‐2.
Effect of the Alzheimer amyloid fragment Aβ(25–35) on Akt/PKB kinase and survival of PC12 cells
Authors: Martín Daniel , Salinas Marta , López‐Valdaliso Raquel , Serrano Elena , Recuero María , Cuadrado Antonio
Published: March 2003

The phosphatidylinositol 3 kinase (PI3K)‐Akt/PKB pathway protects neurons from apoptosis caused by diverse stress stimuli. However, its protective role against the amyloid beta peptide (Aβ), a major constituent of Alzheimer's disease plaques, has not been studied. We investigated the effect of the Aβ‐derived Aβ(25–35) peptide on apoptosis and on the Akt survival pathway in PC12 cells. Cells submitted to micromolar concentrations of Aβ(25–35) exhibited increased production of reactive oxygen species (ROS) and morphological alterations consistent with apoptosis. Akt1 was activated shortly after incubation with Aβ(25–35) and Aβ(1–40) with a kinetics different to that of nerve‐derived growth factor. Akt1 activation was blocked by the PI3K inhibitor wortmannin. We tested the hypothesis that Akt1 might modify the vulnerability of neural cells to apoptosis induced by Aβ(25–35). Overexpression of an active version of Akt1 attenuated the apoptotic effect of Aβ(25–35) as determined by flow cytometry. Moreover, PC12 cells overexpressing a membrane‐targeted N‐myristylated fusion protein of enhanced green fluorescence protein (EGFP) and mouse Akt1 exhibited lower levels of ROS than control EGFP‐transfected cells. The present findings demonstrate that Akt1 is activated in response to Aβ(25–35) in a PI3K‐dependent manner and that active Akt1 protects PC12 cells against the pro‐apoptotic action of this peptide.
Partial resistance to malonate‐induced striatal cell death in transgenic mouse models of Huntington's disease is dependent on age and CAG repeat length
Authors: Hansson Oskar , Castilho Roger F. , Korhonen Laura , Lindholm Dan , Bates Gillian P. , Brundin Patrik
Published: March 2003

Transgenic Huntington's disease (HD) mice, expressing exon 1 of the HD gene with an expanded CAG repeat, are totally resistant to striatal lesion induced by excessive NMDA receptor activation. We now show that striatal lesions induced by the mitochondrial toxin malonate are reduced by 70–80% in transgenic HD mice compared with wild‐type littermate controls. This occurred in 6‐ and 12‐week‐old HD mice with 150 CAG repeats (line R6/2) and in 18‐week‐old, but not 6‐week‐old, HD mice with 115 CAG repeats (line R6/1). Therefore, we show for the first time that the resistance to neurotoxin in transgenic HD mice is dependent on both the CAG repeat length and the age of the mice. Importantly, most HD patients develop symptoms in adulthood and exhibit an inverse relationship between CAG repeat length and age of onset. Transgenic mice expressing a normal CAG repeat (18 CAG) were not resistant to malonate. Although endogenous glutamate release has been implicated in malonate‐induced cell death, glutamate release from striatal synaptosomes was not decreased in HD mice. Malonate‐induced striatal cell death was reduced by 50–60% in wild‐type mice when they were treated with either the NMDA receptor antagonist MK‐801 or the caspase inhibitor zVAD‐fmk. These two compounds did not reduce lesion size in transgenic R6/1 mice. This might suggest that NMDA receptor‐ and caspase‐mediated cell death pathways are inhibited and that the limited malonate‐induced cell death still occurring in HD mice is independent of these pathways. There were no changes in striatal levels of the two anti cell death proteins Bcl‐XL and X‐linked inhibitor of apoptosis protein (XIAP), before or after the lesion in transgenic HD mice. We propose that mutant huntingtin causes a sublethal grade of metabolic stress which is CAG repeat length‐dependent and results in up‐regulation over time of cellular defense mechanisms against impaired energy metabolism and excitotoxicity.
Stimulation of prefrontal cortex at physiologically relevant frequencies inhibits dopamine release in the nucleus accumbens
Authors: Jackson Mark E. , Frost Adam S. , Moghaddam Bita
Published: March 2003

The prefrontal cortex (PFC) is thought to provide an excitatory influence on the output of mesoaccumbens dopamine neurons. The evidence for this influence primarily arises from findings in the rat that chemical or high‐intensity and high‐frequency (60–200 Hz) electrical stimulations of PFC increase burst activity of midbrain dopamine neurons, and augment terminal release of dopamine in the nucleus accumbens. However, PFC neurons in animals that are engaged in PFC‐dependent cognitive tasks increase their firing frequency from a baseline of 1–3 Hz to 7–10 Hz, suggesting that the commonly used high‐frequency stimulation parameters of the PFC may not be relevant to the behavioral states that are associated with PFC activation. We investigated the influence of PFC activation at lower physiologically relevant frequencies on the release of dopamine in the nucleus accumbens. Using rapid (5‐min) microdialysis measures of extracellular dopamine in the nucleus accumbens, we found that although PFC stimulation at 60 Hz produces the expected increases in accumbal dopamine release, the same amplitude of PFC stimulation at 10 Hz significantly decreased these levels. These results indicate that activation of PFC, at frequencies that are associated with increased cognitive demand on this region, inhibits the mesoaccumbens dopamine system.
Dopamine mediates striatal malonate toxicity via dopamine transporter‐dependent generation of reactive oxygen species and D2 but not D1 receptor activation
Authors: Xia Xu Gang , Schmidt Nicole , Teismann Peter , Ferger Boris , Schulz Jörg B.
Published: March 2003

Intrastriatal injection of the reversible succinate dehydrogenase inhibitor malonate results in both chemically induced hypoxia and striatal lesions that are similar to those seen in Huntington's disease and cerebral ischaemia. The mechanisms leading to neuronal death involve secondary excitotoxicity, the release of dopamine from nigrostriatal fibres and the generation of reactive oxygen species (ROS) including nitric oxide (NO) and hydroxyl radicals. Here, we further investigated the contribution and mechanism of dopamine on malonate‐induced striatal lesions. Prior lesions of the nigrostriatal pathway with 6‐OHDA or the depletion of striatal dopamine stores by pretreatment with reserpine, an inhibitor or the vesicular monoamine transporter type‐2 (VMAT2), in combination with α‐methyl‐p‐tyrosine resulted in a significant reduction of malonate‐induced striatal lesion volumes. This was paralleled by block or reduction of the malonate‐induced generation of ROS, as measured by the conversions of salicylate to 2,3‐dihydroxybenzoic acid (2,3‐DHBA) using microdialysis. Systemic or intrastriatal application of l‐DOPA or dopamine, respectively, reconstituted malonate toxicity and the generation of ROS in 6‐OHDA‐lesioned rats. Block of the dopamine transporter by GBR12909 did not result in a reduction of malonate‐induced dopamine release, but significantly reduced the generation of hydroxyl radicals. The D2 receptor agonist lisuride and the mixed D1 and D2 receptor agonist apomorphine, but not the D1 receptor agonist SKF38393, partially restored malonate toxicity in 6‐OHDA‐lesioned rats without increasing the generation of ROS. In line with these results sulpiride, an inhibitor of D2 receptors, reduced the malonate‐induced lesion volume, whereas SCH23390, an inhbitor of D1 receptors, was ineffective. Our data suggest that malonate‐induced dopamine toxicity to energetically impaired neurons is mediated by two independent pathways: (i) dopamine transporter uptake‐dependent, dopamine receptor‐independent generation of ROS, and (ii) excessive stimulation of D2 receptors.
Increased infarct size and exacerbated apoptosis in the glutathione peroxidase‐1 (Gpx‐1) knockout mouse brain in response to ischemia/reperfusion injury
Authors: Crack Peter J. , Taylor Juliet M. , Flentjar Nicole J. , De Haan Judy , Hertzog Paul , Iannello Rocco C. , Kola Ismail
Published: March 2003

Glutathione peroxidase is an antioxidant enzyme that is involved in the control of cellular oxidative state. Recently, unregulated oxidative state has been implicated as detrimental to neural cell viability and involved in both acute and chronic neurodegeneration. In this study we have addressed the importance of a functional glutathione peroxidase in a mouse ischemia/reperfusion model. Two hours of focal cerebral ischemia followed by 24 h of reperfusion was induced via the intraluminal suture method. Infarct volume was increased three‐fold in the glutathione peroxidase‐1 (Gpx‐1) –/– mouse compared with the wild‐type mouse; this was mirrored by an increase in the level of apoptosis found at 24 h in the Gpx‐1 –/– mouse compared with the wild‐type mouse. Neuronal deficit scores correlated to the histologic data. We also found that activated caspase‐3 expression is present at an earlier time point in the Gpx‐1 –/– mice when compared with the wild‐type mice, which suggests an enhanced susceptibility to apoptosis in the Gpx‐1 –/– mouse. This is the first known report of such a dramatic increase, both temporally and in level of apoptosis in a mouse stroke model. Our results suggest that Gpx‐1 plays an important regulatory role in the protection of neural cells in response to the extreme oxidative stress that is released during ischemia/reperfusion injury.
Circulating cell wall components derived from gram‐negative, not gram‐positive, bacteria cause a profound induction of the gene‐encoding Toll‐like receptor 2 in the CNS
Authors: Laflamme Nathalie , Soucy Geneviève , Rivest Serge
Published: March 2003

The recent characterization of human homologs of Toll may be the missing link for the transduction events leading to nuclear factor‐κB (NF‐κB) activity and proinflammatory gene transcription during innate immune response. Mammalian cells may express as many as 10 distinct Toll‐like receptors (TLRs), although TLR2 is a key receptor for recognizing cell wall components of Gram‐positive bacteria. The present study investigated the effects of circulating bacterial cell wall components on the expression of the gene‐encoding TLR2 across the mouse brain. Surprisingly, while Gram‐negative components caused a robust increase in TLR2 transcription within the cerebral tissue, peptidoglycan (PGN) and lipoteichoic acid (LTA), either alone or combined, failed to modulate the receptor transcript. Indeed, the mRNA levels for TLR2 in the choroid plexus and few other regions of the brain remained similar between vehicle‐, LTA‐, PGN‐, and LTA/PGN‐administered mice at all the times evaluated (i.e. 30 min to 24 h post‐intraperitoneal injection). This contrasts with the profound de novo expression of TLR2 following a single systemic injection of the lipopolysaccharide (LPS). The signal was first detected in regions devoid of blood–brain barrier and few blood vessels and microcapillaries. A second wave of TLR2 expression was also detected from these structures to their surrounding parenchymal cells that stained for a microglial marker iba1. The rapid induction of IκBα (index of NF‐κB activity) and up‐regulation of the adaptor protein MyD88 suggest that LPS‐induced TLR2 transcription may be dependent on the NF‐κB pathway. These data provide the evidence that TLR2 is not only present in the brain, but its encoding gene is regulated by cell wall components derived from Gram‐negative, not Gram‐positive, bacteria. The robust wave of TLR2‐expressing microglial cells may have a determinant impact on the innate immune response that occurs in the brain during systemic infection by Gram‐negative, not Gram‐positive, bacteria.
Endoplasmic reticulum dysfunction – a common denominator for cell injury in acute and degenerative diseases of the brain?
Authors: Paschen Wulf , Frandsen Aase
Published: March 2003

Various physiological, biochemical and molecular biological disturbances have been put forward as mediators of neuronal cell injury in acute and chronic pathological states of the brain such as ischemia, epileptic seizures and Alzheimer's or Parkinson's disease. These include over‐activation of glutamate receptors, a rise in cytoplasmic calcium activity and mitochondrial dysfunction. The possible involvement of the endoplasmic reticulum (ER) dysfunction in this process has been largely neglected until recently, although the ER plays a central role in important cell functions. Not only is the ER involved in the control of cellular calcium homeostasis, it is also the subcellular compartment in which the folding and processing of membrane and secretory proteins takes place. The fact that blocking of these processes is sufficient to cause cell damage indicates that they are crucial for normal cell functioning. This review presents evidence that ER function is disturbed in many acute and chronic diseases of the brain. The complex processes taken place in this subcellular compartment are however, affected in different ways in various disorders; whereas the ER‐associated degradation of misfolded proteins is affected in Parkinson's disease, it is the unfolded protein response which is down‐regulated in Alzheimer's disease and the ER calcium homeostasis that is disturbed in ischemia. Studying the consequences of the observed deteriorations of ER function and identifying the mechanisms causing ER dysfunction in these pathological states of the brain will help to elucidate whether neurodegeneration is indeed caused by these disturbances, and will help to fascilitate the search for drugs capable of blocking the pathological process directly at an early stage.
Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ
Authors: Bull Natalie D. , Barnett Nigel L.
Published: March 2003

Neuronal and glial high‐affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non‐metabolisable glutamate transporter substrate, d‐aspartate. In the rat retina, pan‐isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Müller cells. This effect was mimicked by rottlerin but not by Gö6976, suggesting the involvement of the PKCδ isoform, but not PKCα, β or γ. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCδ selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform‐specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.
Metabotropic glutamate receptor 5 and calcium signaling in retinal amacrine cells
Authors: Sosa Romina , Hoffpauir Brian , Rankin Michele L. , Bruch Richard C. , Gleason Evanna L.
Published: March 2003

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)‐dependent Ca2+ elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both Gq and Gs. Selective activation of mGluR5 stimulated Ca2+ elevations that varied in waveform from cell to cell. Experiments using high external K+ revealed that the mGluR5‐dependent Ca2+ elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca2+‐free external solution demonstrated that the variability in the time course of mGluR5‐dependent Ca2+ elevations is largely due to the influx of extracellular Ca2+. The sensitivity of the initial phase of the Ca2+ elevation to thapsigargin indicates that this phase of the response is due to the release of Ca2+ from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5‐mediated Ca2+ elevations are dependent upon the activation of phospholipase C. We rule out a role for L‐type Ca2+ channels and cAMP‐gated channels as pathways for Ca2+ entry, but provide evidence of transient receptor potential (TRP) channel‐like immunoreactivity, suggesting that Ca2+ influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C‐dependent Ca2+ release and Ca2+ influx, possibly through TRP channels.
Alteration of amino acid metabolism in neuronal aggregate cultures exposed to hypoglycaemic conditions
Authors: Honegger Paul , Braissant Olivier , Henry Hugues , Boulat Olivier , Bachmann Claude , Zurich Marie‐Gabrielle , Pardo Beatriz
Published: March 2003

The neuronal effects of glucose deficiency on amino acid metabolism was studied on three‐dimensional cultures of rat telencephalon neurones. Transient (6 h) exposure of differentiated cultures to low glucose (0.25 mm instead of 25 mm) caused irreversible damage, as judged by the marked decrease in the activities of two neurone‐specific enzymes and lactate dehydrogenase, 1 week after the hypoglycemic insult. Quantification of amino acids and ammonia in the culture media supernatants indicated increased amino acid utilization and ammonia production during glucose‐deficiency. Measurement of intracellular amino acids showed decreased levels of alanine, glutamine, glutamate and GABA, while aspartate was increased. Added lactate (11 mm) during glucose deficiency largely prevented the changes in amino acid metabolism and ammonia production, and attenuated irreversible damage. Higher media levels of glutamine (4 mm instead of 0.25 mm) during glucose deprivation prevented the decrease of intracellular glutamate and GABA, while it further increased intracellular aspartate, ammonia production and neuronal damage. Both lactate and glutamine were readily oxidized in these neuronal cultures. The present results suggest that in neurones, glucose deficiency enhances amino acid deamination at the expense of transamination reactions. This results in increased ammonia production and neuronal damage.
Alpha7 nicotinic receptor activation inhibits ethanol‐induced mitochondrial dysfunction, cytochrome c release and neurotoxicity in primary rat hippocampal neuronal cultures
Authors: Li Yangxin , Meyer Edwin M. , Walker Don W. , Millard William J. , He Yun‐Ju , King Michael A.
Published: March 2003

Primary hippocampal neuronal cultures exhibited a concentration‐ and time‐dependent loss of cells when exposed to ethanol (EtOH). EtOH‐induced neurotoxicity was attenuated by 2,4‐dimethoxybenzilidene anabaseine (DMXB) which selectively activates alpha7 nicotinic receptors in a concentration‐dependent manner. We further investigated the mechanisms of the protective effect of DMXB on EtOH‐ induced neurotoxicity. We found that EtOH decreased the mitochondrial membrane potential and released cytochrome c from mitochondria at neurotoxic concentrations. DMXB (3 μm) attenuated both of these actions in a manner that was in turn blocked with the nicotinic antagonist methyllyconitine (MLA) 100 nm. Neither DMXB nor MLA alone affected these parameters. These results suggest that the neuroprotection conferred by alpha7 nicotinic receptor activation may be mediated, at least in part, through preventing the decrease in the mitochondrial membrane potential and the increase in the release of cytochrome c caused by EtOH.