bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Egr‐1 in cAMP‐dependent protein kinase regulation of the phenylethanolamine N‐methyltransferase gene
Authors: Tai T. C. , Morita Kyoji , Wong Dona L.
Published: March 2003

The molecular mechanism by which cAMP activates the rat phenylethanolamine N‐methyltransferase (PNMT) gene was examined by transient transfection of the wild‐type rat PNMT promoter‐luciferase reporter gene construct pGL3RP893 into PC12 cells. Forskolin treatment (10 μm) of the transfected cells for 3–6 h maximally induced luciferase threefold. Induction by forskolin was mimicked by the cAMP analog, 8‐Br‐cAMP, and prevented in PC12 cells pretreated with the protein kinase A (PKA) inhibitor H‐89 or co‐transfected with an expression construct for PKI, a polypeptide inhibitor of PKA. Furthermore, forskolin did not activate the PNMT promoter when the 893 bp PNMT promoter‐reporter gene construct was transfected into the PKA‐deficient cell line, A126. Detailed examination of the forskolin responsiveness of PNMT constructs harboring ≥ 60 bp and < 893 bp of PNMT promoter demonstrated that the cAMP‐responsive element(s) lay between < 392 bp and ≥60 bp. Within this region of the promoter lies a functional binding element for Egr‐1, a transcriptional activator of the PNMT gene. Forskolin treatment of PC12 cells also rapidly increased nuclear levels of Egr‐1 and the catalytic subunit of PKA (PKA‐C), with the rise in PKA‐C preceding that of Egr‐1. Mutation of the −165 bp Egr‐1 site markedly decreased forskolin activation of the PNMT promoter. These findings demonstrate that the rat PNMT gene promoter can be activated via the cAMP–PKA signal transduction pathway, mediated by the immediate early gene transcription factor, Egr‐1.
Steroidogenic factor‐1 expression in marmoset and rat hippocampus: co‐localization with StAR and aromatase
Authors: Wehrenberg U. , Prange‐Kiel J. , Rune G. M.
Published: March 2003

Steroidogenic factor‐1 (SF‐1), an orphan nuclear receptor, was studied with respect to the expression of steroidogenic enzymes in the hippocampus of rat and marmoset, since SF‐1 is a regulator of steroid biosynthesis in the gonads. We used the steroidogenic acute regulatory protein (StAR) as a marker of the first step in the cascade of oestrogen synthesis and aromatase as a marker of the last. StAR transports cholesterol to the inner mitochondrial membrane where it is converted by the cytochrome P‐450 enzyme complex. This is the rate‐limiting step in steroid biosynthesis. Aromatase metabolizes testosterone to oestrogen. Using an anti‐SF‐1 antibody we show that SF‐1 is highly expressed in neuronal cells of the pyramidal layer (CA1–CA3) and in the dentate gyrus of rat and marmoset hippocampi. Binding of the antibody was seen in more than 60% of all cells in the pyramidal layer and in the fascia dentata. In situ hybridization studies revealed the same expression pattern for StAR and aromatase. StAR and aromatase‐positive cells were strictly correlated with SF‐1 as shown by computer‐assisted confocal microscopy in double labelling experiments (immunohistochemistry and in situ hybridization). This coexpression may imply SF‐1 as a possible regulator of steroidogenesis in the hippocampus. However, a few interneurones express solely SF‐1 and aromatase but are negative for StAR. Since the expression of StAR represents the first step in steroidogenesis its expression is suggestive for a de novo synthesis of steroids. A small population of interneurones must import precursors for oestrogen synthesis from other sources. Responsive cells, as evidenced by the presence of oestrogen receptor transcripts, were also found in the pyramidal layer and dentate gyrus. In conclusion, (1) SF‐1 could play a regulatory role in steroidogenesis in the hippocampus of marmoset and rat and (2) with respect to the capacity of steroidogenesis two populations of hippocampal neurones coexist.
Identity of nuclear high‐mobility‐group protein, HMG‐1, and sulfoglucuronyl carbohydrate‐binding protein, SBP‐1, in brain
Authors: Chou Denise K. H. , Evans James E. , Jungalwala Firoze B.
Published: March 2003

High‐mobility‐group (HMG) proteins are a family of non‐histone chromosomal proteins which bind to DNA. They have been implicated in multiple aspects of gene regulation and cellular differentiation. Sulfoglucuronyl carbohydrate binding protein, SBP‐1, which is also localized in the neuronal nuclei, was shown to be required for neurite outgrowth and neuronal migration during development of the nervous system. In order to establish relationship between SBP‐1 and HMG family proteins, two HMG proteins were isolated and purified from developing rat cerebellum by heparin–sepharose and sulfatide‐octyl–sepharose affinity column chromatography and their biochemical and biological properties were compared with those of SBP‐1. Characterization by high performance liquid chromatography–mass spectrometry (HPLC–MS), partial peptide sequencing and western blot analysis showed the isolated HMG proteins to be HMG‐1 and HMG‐2. Isoelectric focusing, HPLC–MS and peptide sequencing data also suggested that HMG‐1 and SBP‐1 were identical. Similar to SBP‐1, both HMG proteins bound specifically to sulfated glycolipids, sulfoglucuronylglycolipids (SGGLs), sulfatide and seminolipid in HPTLC‐immuno‐overlay and solid‐phase binding assays. The HMG proteins promoted neurite outgrowth in dissociated cerebellar cells, which was inhibited by SGGLs, anti‐Leu7 hybridoma (HNK‐1) and anti‐SBP‐1 peptide antibodies, similar to SBP‐1. The proteins also promoted neurite outgrowth in explant cultures of cerebellum. The results showed that the cerebellar HMG‐1 and ‐2 proteins have similar biochemical and biological properties and HMG‐1 is most likely identical to SBP‐1.
Distinct molecular mechanisms lead to diminished myelin basic protein and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase in qkv dysmyelination
Authors: Zhang Youyi , Feng Yue
Published: March 2003

The genetic lesion of quakingviable (qkv) causes diminished expression of the QKI RNA‐binding protein in myelin producing cells. Consequently, several structural myelin proteins are severely reduced. Among these affected proteins, the reduction of the myelin basic protein (MBP) results from post‐transcriptional abnormalities of the MBP mRNA, presumably due to the lack of interactions with QKI. However, whether this is the common mechanism for reduced expression of other myelin proteins in qkv dysmyelination remains unclear. Here we report that distinct molecular mechanisms underlie the reduction of MBP and the 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) in qkv dysmyelination. MBP transcripts bind QKI strongly and are markedly reduced in the qkv/qkv oligodendrocytes in which QKI is almost completely lost. In contrast, CNP transcripts bind QKI weakly and are only slightly affected by the lack of QKI. None the less, CNP proteins are severely reduced in the qkv/qkv brain. Since CNP transcripts are predominantly associated with translating polyribosomes, diminished CNP expression in qkv dysmyelination is unlikely to be due to translational failures, but more likely results from accelerated protein degradation.
Direct determination of the N‐acetyl‐l‐aspartate synthesis rate in the human brain by 13C MRS and [1‐13C]glucose infusion
Authors: Moreno Angel , Ross Brian D. , Blüml Stefan
Published: March 2003

A non‐invasive 13C magnetic resonance spectroscopy (MRS) technique is described for the determination of the N‐acetyl‐l‐aspartate (NAA) synthesis rate, VNAA, in the human brain in vivo. In controls, the mean VNAA was 9.2 ± 3.9 nmol/min/g. In Canavan disease, where [NAA] is increased (p < 0.001) and [aspartate] is deceased (p < 0.001), VNAA was significantly reduced to 3.6 ± 0.1 nmol/min/g (p < 0.001). These rates are in close agreement with the activity of the biosynthetic enzyme measured in vitro in animals, and with the rate of urinary excretion of NAA in human subjects with Canavan disease. The present result is consistent with the regulation of NAA synthesis by the activity of a single enzyme, l‐aspartate‐N‐acetyltransferase, in vivo, and with its control in Canavan disease by limited substrate supply and/or product inhibition. The 13C MRS technique provides the means for further determination of abnormal rates of neuronal NAA synthesis among neurological disorders in which low cerebral [NAA] has been identified.
Anandamide degradation and N‐acylethanolamines level in wild‐type and CB1 cannabinoid receptor knockout mice of different ages
Authors: Maccarrone Mauro , Attinà Marina , Bari Monica , Cartoni Antonella , Ledent Catherine , Finazzi‐Agrò Alessandro
Published: March 2003

CD1 mice lacking the CB1 receptors (knockout, KO) were compared with wild‐type littermates for their ability to degrade N‐arachidonoylethanolamine (anandamide, AEA) through a membrane transporter (AMT) and a fatty acid amide hydrolase (FAAH). The regional distribution and age‐dependence of AMT and FAAH activity were investigated. Anandamide membrane transporter and FAAH increased with age in knockout mice, whereas they showed minor changes in wild‐type animals. Remarkably, they were higher in all brain areas of 6‐month‐old knockout versus wild‐type mice, and even higher in 12‐month‐old animals. The molecular mass (≈ 67 kDa) and isoelectric point (≈ 7.6) of mouse brain FAAH were determined and the FAAH protein content was shown to parallel the enzyme activity. The kinetic constants of AMT and FAAH in the cortex of wild‐type and knockout mice at different ages suggested that different amounts of the same proteins were expressed. The cortex and hippocampus of wild‐type and knockout mice contained the following N‐acylethanolamines: AEA (8% of total), 2‐arachidonoylglycerol (5%), N‐oleoylethanolamine (20%), N‐palmitoylethanolamine (53%) and N‐stearoylethanolamine (14%). These compounds were twice as abundant in the hippocampus as in the cortex. Minor differences were observed in AEA or 2‐arachidonoylglycerol content in knockout versus wild‐type mice, whereas the other compounds were lower in the hippocampus of knockout versus wild‐type animals.
Androgen‐induced up‐regulation of tubulin isoforms in neuroblastoma cells
Authors: Butler Rachel , Leigh P. Nigel , Gallo Jean‐Marc
Published: March 2003

Androgens regulate the physiology of motor neurones both during development and in adult life. In particular, androgens increase the rate of axonal regeneration after axotomy, an effect correlated with the up‐regulation of tubulin. In order to determine whether this was the result of a direct hormone action on neurones, we examined the effect of testosterone on microtubular proteins in human neuroblastoma SH‐SY5Y cells. Treatment of proliferating SH‐SY5Y cells with testosterone resulted in an up‐regulation of α‐ and β‐tubulin. By contrast, no change in tubulin was observed either in cells differentiated into a neuronal phenotype by retinoic acid or in adrenal SW13 cells. We also show that an up‐regulation of the ubiquitous βII‐tubulin and of the neurone‐specific βIII‐tubulin isoforms contributes to the overall increase in tubulin in response to androgen treatment. The increase in tubulin levels following testosterone treatment was abolished by co‐incubation with antiandrogens, indicating that this effect is mediated through a classical mechanism of steroid action. The two microtubule‐associated proteins, tau and MAP2b, remained unchanged following testosterone exposure. Thus, these results demonstrate that tubulin is a direct neuronal target of androgen regulation and suggest that dysregulation of tubulin expression may contribute to the pathogenesis of some motor neuronopathies.
Exploitation of the HIV‐1 coat glycoprotein, gp120, in neurodegenerative studies in vivo
Authors: Tiziana Corasaniti M. , Maccarrone Mauro , Nistico Robert , Malorni Walter , Rotiroti Domenicantonio , Bagetta Giacinto
Published: March 2003

Neuronal loss has often been described at post‐mortem in the brain neocortex of patients suffering from AIDS. Neuroinvasive strains of HIV infect macrophages, microglial cells and multinucleated giant cells, but not neurones. Processing of the virus by cells of the myelomonocytic lineage yields viral products that, in conjunction with potentially neurotoxic molecules generated by the host, might initiate a complex network of events which lead neurones to death. In particular, the HIV‐1 coat glycoprotein, gp120, has been proposed as a likely aetiologic agent of the described neuronal loss because it causes death of neurones in culture. More recently, it has been shown that brain neocortical cell death is caused in rat by intracerebroventricular injection of a recombinant gp120 coat protein, and that this occurs via apoptosis. The latter observation broadens our knowledge in the pathophysiology of the reported neuronal cell loss and opens a new lane of experimental research for the development of novel therapeutic strategies to limit damage to the brain of patients suffering from HIV‐associated dementia.
Neuroprotection of striatal neurons against kainate excitotoxicity by neurotrophins and GDNF family members
Authors: Gratacòs Elena , Pérez‐Navarro Esther , Tolosa Eduard , Arenas Ernest , Alberch Jordi
Published: March 2003

Neurotrophic factors are regarded as potential therapeutic tools in neurodegenerative disorders. Here, we analysed the protective effects of brain‐derived neurotrophic factor, neurotrophin‐3, glial cell line‐derived neurotrophic factor and neurturin against the excitotoxic damage induced by kainate in striatal neurons in vitro and in vivo. Our results show that the decrease in the number of cultured striatal calbindin‐positive neurons induced by kainate was prevented by treatment with any of these factors. To characterize their protective effects in vivo, cell lines overexpressing brain‐derived neurotrophic factor, neurotrophin‐3, glial cell line‐derived neurotrophic factor or neurturin were grafted into the striatum. We found that the numbers of striatal projection neurons (calbindin‐positive) and striatal interneurons (parvalbumin‐ or choline acetyltransferase‐positive) were differentially decreased after kainate lesion. These neurotrophic factors prevented the loss of striatal projection neurons and interneurons with differing efficiency: brain‐derived neurotrophic factor was the most efficient, whereas neurturin was the least. Our findings show that brain‐derived neurotrophic factor, neurotrophin‐3, glial cell line‐derived neurotrophic factor and neurturin have specific neuroprotective profiles in striatal neurons and indicate that they are specific modulators of the survival of distinct subsets of striatal neurons in pathophysiological conditions.
Noradrenaline provides long‐term protection to dopaminergic neurons by reducing oxidative stress
Authors: Troadec Jean‐Denis , Marien Marc , Darios Frédéric , Hartmann Andreas , Ruberg Merle , Colpaert Francis , Michel Patrick P.
Published: March 2003

To better understand the neurotrophic function of the neurotransmitter noradrenaline, we have developed a model of mesencephalic cultures in which we find low concentrations (0.3–10 µm) of noradrenaline to be remarkably effective in promoting long‐term survival and function of dopaminergic neurons. This protective action reproduced the effect of caspase inhibition. It was atypical in that it occurred independently of adrenoceptor activation and was mimicked by some antioxidants, redox metal chelators and the hydroxyl radical detoxifying enzyme catalase. Interestingly, intracellular reactive oxygen species (ROS) were drastically reduced by treatment with noradrenaline, indicating that the neurotransmitter itself acted as an antioxidant. Prevention of oxidative stress was, however, independent of the glutathione antioxidant defense system. Chemical analogues of noradrenaline bearing two free hydroxyl groups in the ortho position of the aromatic ring (o‐catechols), as well as o‐catechol itself, mimicked the survival promoting effects of the neurotransmitter, suggesting that this diphenolic structure was critical for both neuroprotection and reduction of ROS production. Paradoxically, the autoxidation of noradrenaline and the ensuing production of quinone metabolites may be required for both effects, as the neurotransmitter was spontaneously and rapidly degraded over time in the culture medium. These results support the concept that central noradrenergic mechanisms have a neuroprotective role, perhaps in part by reducing oxidative stress.
Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD‐95
Authors: Rong Yongqi , Lu Xiaoying , Bernard Anne , Khrestchatisky Michel , Baudry Michel
Published: March 2003

Both tyrosine phosphorylation and calpain‐mediated truncation of ionotropic glutamate receptors are important mechanisms for synaptic plasticity. Previous work from our laboratory has shown that calpain activation results in truncation of the C‐terminal domains of several glutamate receptor subunits. To test whether and how tyrosine phosphorylation of glutamate ionotropic receptor subunits modulates calpain susceptibility, synaptic membranes were phosphorylated by Fyn or Src, two members of the Src family tyrosine kinases. Tyrosine phosphorylation of synaptic membranes by Src significantly reduced calpain‐mediated truncation of both NR2A and NR2B subunits of NMDA receptors, but not of GluR1 subunits of AMPA receptors. In contrast, phosphorylation with Fyn significantly protected calpain‐mediated truncation of GluR1 subunits of AMPA receptors, but enhanced calpain‐mediated truncation of NR2A subunits of NMDA receptors. Similar results were observed with NR2A and NR2B C‐terminal domain fusion proteins phosphorylated by Fyn or Src before incubation with calpain and calcium. In addition, phosphorylation of NR2A and NR2B C‐terminal fusion proteins by Fyn or Src enhanced their binding to spectrin and PSD‐95. Thus, tyrosine phosphorylation impairs or facilitates calpain‐mediated truncation of glutamate receptor subunits, depending on which tyrosine kinase is activated. Such mechanisms could serve to regulate receptor integrity and location, in addition to modulating channel properties.
Inhibition of the rat blood–brain barrier choline transporter by manganese chloride
Authors: Lockman Paul R. , Roder Karen E. , Allen David D.
Published: March 2003

Choline transport has been characterized by multiple mechanisms including the blood–brain barrier (BBB), and high‐ and low‐affinity systems. Each mechanism has unique locations and characteristics yet retain some similarities. Previous studies have demonstrated cationic competition by monovalent cations at the BBB and cation divalent manganese in the high‐affinity system. To evaluate the effects of divalent manganese inhibition as well as other cationic metals at the BBB choline transporter, brain choline uptake was evaluated in the presence of certain metals of interest in Fischer‐344 rats using the in situ brain perfusion technique. Brain choline uptake was inhibited in the presence of Cd2+ (73 ± 2%) and Mn2+ (44 ± 6%), whereas no inhibition was observed with Cu2+ and Al3+. Furthermore, it was found that manganese caused a reduction in brain choline uptake and significant regional choline uptake inhibition in the frontal and parietal cortex, the hippocampus and the caudate putamen (45 ± 3%, 68 ± 18%, 58 ± 9% and 46 ± 15%, respectively). These results suggest that choline uptake into the CNS can be inhibited by divalent cationic metals and monovalent cations. In addition, the choline transporter may be a means by which manganese enters the brain.
The cytoplasmic tail of the D1A receptor subtype: identification of specific domains controlling dopamine cellular responsiveness
Authors: Chaar Ziad Y. , Jackson Adele , Tiberi Mario
Published: March 2003

In this study the rat D1A receptor (wild‐type, WT) and truncation mutants thereof, are utilized to delineate specific cytoplasmic tail (CT) domains responsible for regulating ligand binding and receptor‐mediated adenylyl cyclase activation. In human embryonic kidney (HEK) cells, all truncation mutants of the D1A receptor (Δ425, Δ379, Δ351) display cell surface localization and express at high but different receptor numbers. Binding studies suggest that residues located between Cys351 and Asp425 may serve to restrain the agonist binding conformation of the D1A receptor. This contention is supported by the observation that the constitutive activation of Δ351 is significantly increased in comparison with WT, Δ425 and Δ379. Furthermore, we demonstrate that the extent of dopamine‐mediated maximal activation of adenylyl cyclase is significantly augmented in cells expressing Δ351 when compared with WT or mutants harboring shorter truncations. These results suggest that in addition to restraining receptor conformation, determinants located downstream of Cys351 may act as negative regulators of the G protein coupling efficiency and adenylyl cyclase activation. Interestingly, all truncated receptors used in the present study display a decrease in dopamine potency when compared with WT. We show that inhibition of protein kinase A (PKA) activity leads also to a reduction in dopamine potency in cells expressing WT but not Δ351 receptors. These results hint at a potential previously unanticipated role for PKA in facilitating D1A receptor coupling efficiency in HEK cells. Overall, the present study has uncovered specific CT domains involved in regulating discrete aspects of the D1A receptor signaling.
Role of phosphatidylinositide 3‐kinase in brain‐derived neurotrophic factor‐induced DARPP‐32 expression in medium size spiny neurons in vitro
Authors: Stroppolo Antonella , Guinea Barbara , Tian Chai , Sommer John , Ehrlich Michelle E.
Published: March 2003

Brain‐derived neurotrophic factor (BDNF) regulates several properties of striatal dopaminoceptive medium‐sized spiny neurons (MSNs) in vivo and in vitro, including expression levels of DARPP‐32 (dopamine and cyclic adenosine 3′,5′‐monophosphate‐regulated phosphoprotein, 32 kDa). DARPP‐32 is expressed in 96% of the MSNs, and is a key modulator of dopamine actions. We investigated the intracellular signal transduction pathways activated by BDNF in MSNs and via which BDNF induces DARPP‐32 expression. We found that phosphorylation of the cyclic AMP response element binding protein (CREB) is only transiently increased following stimulation of MSNs by BDNF, whereas increased phosphorylation of the extracellular signal regulated kinases 1 and 2 (Erk1/2) and Akt is sustained for longer than 4 h. Treatment of cultures with inhibitors of mitogen‐activated protein kinase kinase (MEK) or phosphatidylinositide 3‐kinase (PI3K) showed that the majority of the BDNF‐induced increase in DARPP‐32 requires the PI3K pathway. We also found that inhibition of PI3K reduces BDNF‐induced Erk phosphorylation, indicating that cross‐talk between these pathways may play a prominent role in MSNs.
Chronic nutritional deprivation of n‐3 α‐linolenic acid does not affect n‐6 arachidonic acid recycling within brain phospholipids of awake rats
Authors: Contreras Miguel A. , Chang Michael C. J. , Rosenberger Thad A. , Greiner Rebecca Sheaff , Myers Carol S. , Salem Norman , Rapoport Stanley I.
Published: March 2003

Using an in vivo fatty acid model and operational equations, we reported that esterified and unesterified concentrations of docosahexaenoic acid (DHA, 22 : 6 n‐3) were markedly reduced in brains of third‐generation (F3) rats nutritionally deprived of α‐linolenic acid (18 : 3 n‐3), and that DHA turnover within phospholipids was reduced as well. The concentration of docosapentaenoic acid (DPA, 22 : 5 n‐6), an arachidonic acid (AA, 20 : 4 n‐6) elongation/desaturation product, was barely detectable in control rats but was elevated in the deprived rats. In the present study, we used the same in vivo model, involving the intravenous infusion of radiolabeled AA to demonstrate that concentrations of unesterified and esterified AA, and turnover of AA within phospholipids, were not altered in brains of awake F3‐generation n‐3‐deficient rats, compared with control concentrations. Brain DPA‐CoA could be measured in the deprived but not control rats, and AA‐CoA was elevated in the deprived animals. These results indicated that AA and DHA are recycled within brain phospholipids independently of each other, suggesting that recycling is regulated independently by AA‐ and DHA‐selective enzymes, respectively. Competition among n‐3 and n‐6 fatty acids within brain probably does not occur at the level of recycling, but at levels of elongation and desaturation (hence greater production of DPA during n‐3 deprivation), or conversion to bioactive eicosanoids and other metabolites.
Nε‐(γ‐l‐Glutamyl)‐l‐lysine (GGEL) is increased in cerebrospinal fluid of patients with Huntington's disease
Authors: Jeitner Thomas M. , Bogdanov Mikhail B. , Matson Wayne R. , Daikhin Yevgeny , Yudkoff Marc , Folk John E. , Steinman Lawrence , Browne Susan E. , Beal M. Flint , Blass John P. , Cooper Arthur J. L.
Published: March 2003

Pathological‐length polyglutamine (Qn) expansions, such as those that occur in the huntingtin protein (htt) in Huntington's disease (HD), are excellent substrates for tissue transglutaminasein vitro, and transglutaminase activity is increased in post‐mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patientsin vivois scarce. We now report that levels ofNε‐(γ‐l‐glutamyl)‐l‐lysine (GGEL)– a ‘marker’ isodipeptide produced by the transglutaminase reaction – are elevated in the CSF of HD patients (708 ± 41 pmol/mL, SEM,n = 36) vs. control CSF (228 ± 36,n = 27);p < 0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brainin vivo.
Increased phosphorylation of the NR1 subunit of the NMDA receptor following cerebral ischemia
Authors: Cheung Herman H. , Teves Lucy , Wallace M. Christopher , Gurd James W.
Published: March 2003

The effects of transient cerebral ischemia on phosphorylation of the NR1 subunit of the NMDA receptor by protein kinase C (PKC) and protein kinase A (PKA) were investigated. Adult rats received 15 min of cerebral ischemia followed by various times of recovery. Phosphorylation was examined by immunoblotting hippocampal homogenates with antibodies that recognized NR1 phosphorylated on the PKC phosphorylation sites Ser890 and Ser896, the PKA phosphorylation site Ser897, or dually phosphorylated on Ser896 and Ser897. The phosphorylation of all sites examined increased following ischemia. The increase in phosphorylation by PKC was greater than by PKA. The ischemia‐induced increase in phosphorylation was predominantly associated with the population of NR1 that was insoluble in 1% deoxycholate. Enhanced phosphorylation of NR1 by PKC and PKA may contribute to alterations in NMDA receptor function in the postischemic brain.
Quantitative dual‐probe microdialysis: evaluation of [3H]mannitol diffusion in agar and rat striatum
Authors: Höistad Malin , Chen Kevin C. , Nicholson Charles , Fuxe Kjell , Kehr Jan
Published: March 2003

Dual‐probe microdialysis was used to study interstitial diffusion in the rat brain. A radiolabelled tracer, [3H]mannitol, was continuously infused at different concentrations via a probe acutely implanted into the striatum of an anaesthetized male rat or into a dilute agar gel. Samples were collected by a second probe placed 1 mm away from the first, and the recovered [3H]mannitol was measured by liquid scintillation counting. In the striatum, the delivery of [3H]mannitol was counteracted by its removal from the extracellular space by passive uptake into cells and clearance into the microcirculation, causing the diffusion profile to approach quasi steady‐state levels within 2 h. Diffusion data from brain and agar were analysed using a mathematical model. The apparent (effective) diffusion coefficient for [3H]mannitol wasD* = 2.9 × 10−6cm2/s, the effective volume fraction α* = 0.30 and the clearance rate constant κ = 2.3 × 10−5/s. A tortuosity, λ = 1.81, and penetration distance Γ = 4.2 mm, were calculated. We conclude that, using dual‐probe microdialysis, parameters reflecting geometric and dynamic tissue properties may be obtained using appropriate mathematical analysis. Quantitative dual‐probe microdialysis will be valuable in characterizing interstitial diffusion and the clearance processes underpinning volume transmission in the brain.
Contribution of Ca2+ calmodulin‐dependent protein kinase II and mitogen‐activated protein kinase kinase to neural activity‐induced neurite outgrowth and survival of cerebellar granule cells
Authors: Borodinsky Laura N. , Coso Omar A. , Fiszman Mónica L.
Published: March 2003

In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long‐term survival of cerebellar granule cells. The effect of voltage‐gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin‐dependent protein kinase II and mitogen‐activated protein kinase kinase (MEK1), to the effects of high [K+]e under serum‐free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24–48 h under low‐density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin‐dependent protein kinase II. This also produced an increase in long‐term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin‐dependent protein kinase II and of the extracellular signal‐regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin‐dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.
Involvement of glycogen synthase kinase‐3β and tau phosphorylation in neuronal Golgi disassembly
Authors: Elyaman Wassim , Yardin Catherine , Hugon Jacques
Published: March 2003

The dissociation of the neuronal Golgi complex is a classical feature observed in neurodegenerative disorders including Alzheimer's disease. The goal of this study is to determine if the phosphorylation of tau protein is involved in neuronal Golgi disassembly. Primary cortical cultures were exposed to two Golgi toxins, brefeldin A (BFA) or nordihydroguaiaretic acid (NDGA). Immunocytochemical studies using the anti58 k antibody revealed that Golgi disassembly started in exposed neurons a few minutes after treatment. BFA and NDGA induced a rapid and transient increase in tau phosphorylation in a site‐specific manner on immunoblots. In addition, the increase in tau phosphorylation directly correlated with a transient dissociation of tau from the cytoskeleton and a decrease of the acetylated tubulin. Furthermore, the activity of glycogen synthase kinase‐3β (GSK‐3β) increased transiently, as demonstrated by the kinase activity assay and by immunoblottings of serine‐9 and tyrosine‐216 phosphorylated of GSK‐3β. A decrease of the Akt phosphorylated form was also shown. The increase in tau phosphorylation was inhibited by the GSK‐3β inhibitor, lithium. Finally, morphometric studies showed that lithium partially blocked the Golgi disassembly caused by BFA or NDGA. Together these findings indicate that GSK‐3β activity and tau phosphorylation state are involved in the maintenance of the neuronal Golgi organization.
A novel seven transmembrane receptor induced during the early steps of astrocyte differentiation identified by differential expression
Authors: De Smet Charles , Nishimori Hiroyuki , Furnari Frank B. , Bögler Oliver , Huang H.‐J. Su , Cavenee Webster K.
Published: March 2003

The rat glial progenitor cell line CG‐4 can be induced to differentiate into either oligodendrocytes or type‐2 astrocytes. In order to identify genes whose expression varies coincident with such phenotypic differentiation, we employed representational difference analysis (RDA) of mRNA. Here, we report 38 cDNAs induced in type‐2 astrocytes, oligodendrocytes, or both differentiated states. Among these were known transcription factors, membrane receptors, extracellular matrix proteins, secreted signaling modulators, chromatin regulators and myelin sheath components. In addition several novel genes were identified; among these was a gene induced during the very early stages of astrocyte differentiation that we have named Ieda (induced early in differentiating astrocytes). Several Ieda transcripts were detected by RT‐PCR, and appeared to be produced by alternative splicing and promoter usage. The protein deduced from the longest Ieda mRNA exhibited sequence features characteristic of G‐protein coupled receptors, including seven putative transmembrane domains, while the shorter Ieda transcripts encoded proteins that lacked several transmembrane segments. In the adult rat, Ieda transcripts were found exclusively in brain and testis. In the developing rat brain, Ieda expression was first detected at embryonic day 16, that is two days before the first appearance of mature astrocytes. Thus, this approach has yielded a potential source of markers for differentiation states of these two cellular types as well as genes predicted to be functionally involved in the differentiation process itself.
ATP‐driven, Na+‐independent inward Cl– pumping in neuroblastoma cells
Authors: Bettendorff Lucien , Lakaye Bernard , Margineanu Ilca , Grisar Thierry , Wins Pierre
Published: March 2003

In immature neurones, the steady‐state intracellular Cl− concentration [Cl−]i is generally higher than expected for passive distribution, and this is believed to be due to Na+–K+−2Cl– co‐transport. Here, we show that N2a neuroblastoma cells, incubated in HEPES‐buffered NaCl medium maintain a [Cl−]i around 60 mm, two‐ to threefold higher than expected for passive distribution at a membrane potential of − 49 mV. When the cells were transferred to a Cl−‐free medium, [Cl−]i decreased quickly (t1/2 < 5 min), suggesting a high Cl− permeability. When the intracellular ATP concentration was reduced to less than 1 mm by metabolic inhibitors, the initial rate of 36Cl− uptake was strongly inhibited (60–65%) while steady‐state [Cl−]i decreased to 24 mm, close to the value predicted from the Nernst equilibrium. Moreover, after reduction of [ATP]i and [Cl−]i by rotenone, the subsequent addition of glucose led to a reaccumulation of Cl−, in parallel with ATP recovery. Internal bicarbonate did not affect Cl− pumping, suggesting that Cl−/HCO3– exchange does not significantly contribute to active transport. Likewise, Na+–K+−2Cl– co‐transport also appeared to play a minor role: although mRNA for the NKCC1 form of the co‐transporter was detected in N2a cells, neither the initial rate of 36Cl− uptake nor steady‐state [Cl−]i were appreciably decreased by 10 µm bumetanide or replacement of external Na+ by choline. These results suggest that a highly active ATP‐dependent mechanism, distinct from Na+–K+−2Cl– co‐transport, is responsible for most of the inward Cl− pumping in N2a cells.
Modification of tyrosine hydroxylase activity by chloral derived β‐carbolines in vitro
Authors: Riederer Franz , Luborzewski Alexander , God Ralf , Bringmann Gerhard , Scholz Joachim , Feineis Doris , Moser Andreas
Published: March 2003

β‐Carbolines have been suggested to be involved in the pathogenesis of Parkinson's disease as a result of their structural similarity to the neurotoxin N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). The chloral‐derived β‐carboline derivative 1‐trichloromethyl‐1,2,3,4‐tetrahydro‐β‐carboline (TaClo) causes cell loss in neuronal and glial cell cultures and induces a slowly developing neurodegenerative process in rats. In our experiments, effects of TaClo and its derivatives 2‐methyl‐TaClo (2‐Me‐TaClo), and 1‐dichloromethylene‐1,2,3,4‐tetrahydro‐β‐carboline (1‐CCl2‐THβC) on tyrosine hydroxylase (TH) activity were investigated in TH assays using homogenate preparations of the rat nucleus accumbens and recombinant human TH (hTH1). TH activity was determined in vitro by measuring l‐DOPA production with HPLC‐ECD. Using homogenate preparations, TaClo, 2‐Me‐TaClo, and 1‐CCl2‐THβC inhibited TH in concentrations of 0.1 mm, while 1‐CCl2‐THβC in low concentrations enhanced TH activity. When TH was activated by PACAP‐27, TaClo, 2‐Me‐TaClo, or 1‐CCl2‐THβC also inhibited activated enzyme activity in high concentrations. However, in the case of 2‐Me‐TaClo and 1‐CCl2‐THβC a biphasic effect was observed with a marked increase of TH activity in the nanomolar range. In our experiments using recombinant hTH1, TaClo, 2‐Me‐TaClo, or 1‐CCl2‐THβC did not modify enzyme activity. After activation of hTH1 by PKA all the tetrahydro‐β‐carbolines investigated in this study decreased l‐DOPA formation. We suggest that these β‐carbolines modulate dopamine synthesis by interacting with a protein kinase TH‐activating system.
The neuropeptide FF analogue, 1DMe, reduces in vivo dynorphin release from the rat spinal cord
Authors: Ballet Sébastien , Braz Joao , Mauborgne Annie , Bourgoin Sylvie , Zajac Jean‐Marie , Hamon Michel , Cesselin François
Published: March 2003

Intrathecal infusion of the neuropeptide FF analogue, [D‐Tyr1, (NMe)Phe3]neuropeptide FF (1DMe; 0.1 µm−0.1 mm) in anaesthetized rats produced a concentration‐dependent decrease in the spinal outflow of dynorphin A (1–8)‐like material, which persisted for at least 90 min after treatment with 10 µm−0.1 mm of the compound. Co‐administration of d‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP; 1 µm) to block spinal µ‐opioid receptors did not modify this effect, whereas naltrindole (10 µm) totally prevented it and nor‐binaltorphimine (10 µm) reduced the post‐effect. These data suggest that 1DMe triggers the release of endogenous opioids that stimulate mainly δ‐opioid receptors, and secondarily κ‐opioid receptors, thereby exerting a negative influence on dynorphin A (1–8)‐like material outflow. Because dynorphin has pronociceptive properties, such a decrease in spinal dynorphin A (1–8)‐like material release might underlie the long‐lasting antinociceptive effects of intrathecally administered neuropeptide FF and analogues.
The role of muscarinic acetylcholine receptor‐mediated activation of extracellular signal‐regulated kinase 1/2 in pilocarpine‐induced seizures
Authors: Berkeley Jennifer L. , Decker Michael J. , Levey Allan I.
Published: March 2003

Pilocarpine‐induced seizures are mediated by the M1 subtype of muscarinic acetylcholine receptor (mAChR), but little is known about the signaling mechanisms linking the receptor to seizures. The extracellular signal‐regulated kinase (ERK) signaling cascade is activated by M1 mAChR and is elevated during status epilepticus. Yet, the role of ERK activation prior to seizure has not been evaluated. Here, we examine the role of pilocarpine‐induced ERK activation in the induction of seizures in mice by pharmacological and behavioral approaches. We show that pilocarpine induces ERK activation prior to the induction of seizures by both western blot and immunocytochemistry with an antibody to phosphorylated ERK. In addition, we show that the ERK pathway inhibitor SL327 effectively blocks the pilocarpine‐induced ERK activation. However, SL327 pretreatment has no effect on the initiation of seizures. In fact, animals treated with SL327 had higher seizure‐related mortality than vehicle‐treated animals, suggesting activated ERK may serve a protective role during seizures. In addition, ERK inhibition had no effect on the development of the long‐term sequelae of status epilepticus (SE), including mossy fiber sprouting, neuronal death and spontaneous recurrent seizures.
Mayhem of the multiple mechanisms: modelling neurodegeneration in prion disease
Authors: Brown David R.
Published: March 2003

This review examines recent attempts to advance the understanding of the mechanism by which neurones die in prion disease. Prion diseases or transmissible spongiform encephalopathies are characterized by the conversion of a normal glycoprotein, the prion protein, to a protease‐resistant form that is suggested to be both the infectious agent and the cause of the rapid neurodegeneration in the disease. Death of the patient results from this widespread neuronal loss. Thus understanding the mechanism by which the abnormal form of the prion protein causes neuronal death might lead to treatments that would prevent the life‐threatening nature of these diseases.
Identification of three cAMP‐dependent protein kinase (PKA) phosphorylation sites within the major intracellular domain of neuronal nicotinic receptor α4 subunits
Authors: Guo Xiang , Wecker Lynn
Published: March 2003

This study determined whether all protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites on the α4 subunit of rat α4β2 neuronal nicotinic receptors could be localized to the M3/M4 cytoplasmic domain of the protein, and investigated specific amino acid substrates for the kinases through two‐dimensional phosphopeptide mapping and site‐directed mutagenesis. Experiments were conducted using α4β2 receptors expressed in Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of α4 (α4333−594). When oocytes expressing α4β2 receptors were incubated with [32P]orthophosphate in order to label endogenous ATP stores, phosphorylation of α4 subunits was evident. Incubation of either immunoprecipitated receptors or the fusion protein with [32P]ATP and either PKA or PKC followed by trypsinization of the samples demonstrated that the kinases phosphorylated α4 subunits on multiple phosphopeptides, and that the phosphorylated full‐length α4 protein and fusion protein produced identical phosphopeptide maps. Site‐directed mutagenesis of Ser365, Ser472 and Ser491 to alanines in the fusion protein eliminated phosphopeptides phosphorylated by PKA, but not by PKC. Other mutations investigated, Ser470, Ser493, Ser517 and Ser590, did not alter the phosphopeptide maps. Results indicate that Ser365, Ser472 and Ser491 on neuronal nicotinic receptor α4 subunits are phosphorylated by PKA and are likely to represent post‐translational regulatory sites on the receptor.
The role of NADPH oxidase, neuronal nitric oxide synthase and poly(ADP ribose) polymerase in oxidative neuronal death induced in cortical cultures by brain‐derived neurotrophic factor and neurotrophin‐4/5
Authors: Hwang Jung‐Jin , Choi So‐Young , Koh Jae‐Young
Published: March 2003

Certain neurotrophins promote or induce oxidative neuronal death in cortical cultures. However, the effector mechanisms mediating this phenomenon have not been delineated. In this study, we investigated the possibility that NADPH oxidase and nitric oxide synthase (NOS) function as such effectors. Western blot analysis showed that treatment with brain‐derived neurotrophic factor (BDNF) and neurotrophin (NT)‐4/5 increased the levels of NADPH oxidase subunits. Moreover, neurotrophin treatment resulted in membrane translocation of p67phox, a characteristic feature of NADPH oxidase activation. Administration of the specific NADPH oxidase inhibitor, 4‐(2‐aminoethyl)benzenesulfonylfluoride (AEBSF), attenuated increases in oxygen free radicals thereby suggesting that NADPH oxidase contributes to the oxidative stress induced by neurotrophins. Furthermore, neuronal death induced by BDNF or NT‐4/5 was significantly attenuated by AEBSF. Treatment with BDNF has previously been shown to induce neuronal NOS (nNOS). Our data indicated that inhibitors of nNOS attenuated neuronal death induced by BDNF or NT‐4/5, consistent with an active role of nNOS in the mediation of neurotrophin neurotoxicity. As in other models of oxidative cell death, BDNF‐induced neuronal death was accompanied by poly(ADP ribose) polymerase (PARP) activation. AEBSF or N‐nitro‐l‐arginine (NNA) reduced BDNF‐mediated PARP activation. PARP and poly(ADP ribose) glycohydrolase (PARG) are actively involved in mediating neurotrophin neurotoxicity since inhibitors of PARP and PARG significantly reduced levels of cell death. These results suggest that NADPH oxidase and nNOS contribute to increased oxidative stress, subsequent activation of PARP/PARG, and neuronal death induced by prolonged neurotrophin exposure.
Arachidonic acid and docosahexaenoic acid suppress thrombin‐evoked Ca2+ response in rat astrocytes by endogenous arachidonic acid liberation
Authors: Sergeeva Marina , Strokin Mikhail , Wang Hong , Ubl Joachim J. , Reiser Georg
Published: March 2003

Arachidonic (AA) and docosahexaenoic acid (DHA) are the major polyunsaturated fatty acids (PUFAs) in the brain. However, their influence on intracellular Ca2+ signalling is still widely unknown. In astrocytes, the amplitude of thrombin‐ induced Ca2+ response was time‐dependently diminished by AA and DHA, or by the AA tetraynoic analogue ETYA, but not by eicosapentaenoic acid (EPA). Thrombin‐elicited Ca2+ response was reduced (20–30%) by 1‐min exposure to AA or DHA. Additionally, 1‐min application of AA or DHA together with thrombin in Ca2+‐free medium blocked Ca2+ influx, which followed after readdition of extracellular Ca2+. EPA and ETYA, however, were ineffective. Long‐term treatment of astrocytes with AA and DHA, but not EPA reduced the amplitude of the thrombin‐induced Ca2+ response by up to 80%. AA and DHA caused a comparable decrease in intracellular Ca2+ store content. Only DHA and AA, but not EPA or ETYA, caused liberation of endogenous AA by cytosolic phospholipase A2 (cPLA2). Therefore, we reasoned that the suppression of Ca2+ response to thrombin by AA and DHA could be due to release of endogenous AA. Possible participation of AA metabolites, however, was excluded by the finding that specific inhibitors of the different oxidative metabolic pathways of AA were not able to abrogate the inhibitory AA effect. In addition, thrombin evoked AA release via activation of cPLA2. From our data we propose a novel model of positive/negative‐feed‐back in which agonist‐induced release of AA from membrane phospholipids promotes further AA release and then suppresses agonist‐induced Ca2+ responses.
Mixed lineage kinase 3 mediates gp120IIIB‐induced neurotoxicity
Authors: Bodner Amos , Maroney Anna C. , Finn James P. , Ghadge Ghanashyam , Roos Raymond , Miller Richard J.
Published: March 2003

Overexpression of gp120, the major coat protein of the HIV‐1 virus, in central glial cells, or treatment of neurons with gp120 in culture, produces apoptotic neuronal death. Here we demonstrate that CEP‐1347 (KT7515), an inhibitor of mixed lineage kinase 3 (MLK3), an upstream activator of JNK, inhibits gp120IIIB‐induced apoptosis of hippocampal neurons. Furthermore, expression of wild type MLK3 in hippocampal pyramidal neurons enhanced gp120IIIB‐induced neurotoxicity, whereas expression of a dominant negative MLK3 protected neurons from the toxic effects of the glycoprotein. These results indicate a role for MLK3 signaling in gp120IIIB‐induced neuronal death, and suggest potential clinical utility of CEP‐1347 in inhibiting the progression of AIDS dementia.
NMDA‐induced acetylcholine release in mouse striatum: role of NO synthase isoforms
Authors: Buchholzer Marie‐Luise , Klein Jochen
Published: March 2003

Striatal cholinergic interneurons are stimulated by glutamatergic inputs from thalamus and cortex via NMDA receptors. The present microdialysis study was designed to characterize the role of nitric oxide (NO) in this process and to identify the NO synthase (NOS) isoform responsible for this effect. For this purpose, we studied the effects of NMDA and 3‐morpholino sydnonimine (SIN‐1) perfusions on the release of acetylcholine (ACh) in mouse striatum. In wild‐type C57/Bl6 mice, perfusion of NMDA (100 µm) induced a two‐fold stimulation of ACh release. This effect was attenuated in mice lacking endothelial NOS but was completely absent in mice lacking neuronal NOS. Local perfusion of SIN‐1 (300 µm), an NO donor, increased ACh release by more than two‐fold in all three mouse lines. We conclude that NO synthesized by neuronal NOS provides a nitrergic link in the glutamatergic stimulation of striatal cholinergic interneurons.