bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective Effects of Riluzole on Dopamine Neurons
Authors: Storch Alexander , Burkhardt Katrin , Ludolph Albert C. , Schwarz Johannes
Published: March 2003

Abstract: Riluzole is neuroprotective in patients with amyotrophiclateral sclerosis and may also protect dopamine (DA) neurons in Parkinson'sdisease. We examined the neuroprotective potential of riluzole on DA neuronsusing primary rat mesencephalic cultures and human dopaminergic neuroblastomaSH‐SY5Y cells. Riluzole (up to 10 μM) alone affected neither thesurvival of DA neurons in primary cultures nor the growth of SH‐SY5Y cellsafter up to 72 h. Riluzole (1‐10 μM) dose‐dependently reduced DAcell loss caused by exposure to MPP+ in both types of cultures.These protective effects were accompanied by a dose‐dependent decrease ofintracellular ATP depletion caused by MPP+ (30‐300 μM)in SH‐SY5Y cells without affecting intracellular net NADH content, suggestinga reduction of cellular ATP consumption rather than normalization ofmitochondrial ATP production. Riluzole (1‐10 μM) also attenuatedoxidative injury in both cell types induced by exposure to L‐DOPA and6‐hydroxydopamine, respectively. Consistent with its antioxidative effects,riluzole reduced lipid peroxidation induced by Fe3+ and L‐DOPA inprimary mesencephalic cultures. Riluzole (10 μM) did not alterhigh‐affinity uptake of either DA or MPP+. However, in the samecell systems, riluzole induced neuronal and glial cell death withconcentrations higher than those needed for maximal protective effects(≥100 μM). These data demonstrate that riluzole has protectiveeffects on DA neurons in vitro against neuronal injuries induced by (a)impairment of cellular energy metabolism and/or (b) oxidative stress. Theseresults provide further impetus to explore the neuroprotective potential ofriluzole in Parkinson's disease.
Effect of α‐Phenyl‐N‐tert‐Butylnitrone on Brain Cell Membrane Function and Energy Metabolism in Experimental Escherichia coli Meningitis in the Newborn Piglet
Authors: Park Won Soon , Chang Yun Sil , Lee Munhyang
Published: March 2003

Abstract: We evaluated the efficacy of α‐phenyl‐N‐tert‐butylnitrone as an adjunctive therapy in experimental bacterial meningitis in the newborn piglet. Meningitis was induced by intracisternal injection of 108 colony‐forming units of Escherichia coli in 100 μl of saline. α‐Phenyl‐N‐tert‐butylnitrone 100 mg/kg was given as a bolus intravenous injection 30 min before induction of meningitis. Although it completely abolished the elevated CSF tumor necrosis factor‐α level observed in the meningitis group, α‐phenyl‐N‐tert‐butylnitrone did not down‐modulate parameters of inflammatory responses such as increased intracranial pressure, hypoglycorrhachia, elevated CSF lactate level, and CSF leukocytosis observed in this group. However, α‐phenyl‐N‐tert‐butylnitrone treatment mitigated alterations in brain cell membrane structure and function during meningitis, evidenced by amelioration of increased brain cell membrane lipid peroxidation products (conjugated dienes) and decreased Na+,K+‐ATPase activity. Reduced mean arterial blood pressure, cerebral perfusion pressure, brain glucose concentration, and cerebral energy stores and marginally increased brain lactate level observed in the meningitis group were also ameliorated. These results suggest that although it failed to attenuate the inflammatory responses, α‐phenyl‐N‐tert‐butylnitrone was effective in ameliorating brain injury in neonatal bacterial meningitis.
An Investigation of Noradrenaline Uptake and Release by the CATH.a Cell Line
Authors: Bundey Richard A. , Jones Philip G. , Kendall David A.
Published: March 2003

Abstract: The cell bodies of ascending noradrenergic neurons in the brain are located predominantly in the locus coeruleus. An in vitro model of locus coeruleus neurons could prove to be a useful tool in the investigation of noradrenergic neural networks and their associated pathophysiologies. The CATH.a cell line demonstrates some of the properties expected of locus coeruleus neurons, and the present study investigated the neurotransmitter uptake and release properties of the CATH.a cells. It was surprising that the CATH.a cells failed to accumulate [3H]noradrenaline ([3H]NA), suggesting the lack of a functional NA transporter. RT‐PCR supported this finding by demonstrating the absence of NA transporter mRNA. Treatment of CATH.a cells with various differentiating agents failed to increase the [3H]NA uptake. Endogenous NA release was studied using HPLC detection, which revealed a lack of depolarisation‐induced increases in endogenous NA release. A human NA transporter‐transfected CATH.a cell line was generated (termed RUNT), and a study of the [3H]NA uptake revealed that the RUNT cells displayed significant uptake that could be blocked by cocaine (10 μM). Furthermore, the uptake capacity could be dramatically increased by differentiation of the cells with dibutyryl cyclic AMP (1 mM) for 24 h. Using dibutyryl cyclic AMP‐differentiated RUNT cells, high K+ concentrations (50 mM) significantly increased [3H]NA release above basal levels.
Frontal Lobe Dysfunction in Progressive Supranuclear Palsy
Authors: Albers David S. , Augood Sarah J. , Park Larry C. H. , Browne Susan E. , Martin Debroah M. , Adamson Jennifer , Hutton Michael , Standaert David G. , Vonsattel Jean Paul G. , Gibson Gary E. , Beal M. Flint
Published: March 2003

Abstract: Recent data from our laboratory have shown a regionally specific increase in lipid peroxidation in postmortem progressive supranuclear palsy (PSP) brain. To extend this finding, we measured activities of mitochondrial enzymes as well as tissue malondialdehyde (MDA) levels in postmortem superior frontal cortex (Brodmann’s area 9; SFC) from 14 pathologically confirmed cases of PSP and 13 age‐matched control brains. Significant decreases (‐39%) in α‐ketoglutarate dehydrogenase complex/glutamate dehydrogenase ratio and significant increases (+36%) in tissue MDA levels were observed in the SFC in PSP; no differences in complex I or complex IV activities were detected. Together, these results suggest that mitochondrial dysfunction and lipid peroxidation may underlie the frontal metabolic and functional deficits observed in PSP.
Site‐Directed Mutations in the Third Intracellular Loop of the Serotonin 5‐HT1A Receptor Alter G Protein Coupling from Gi to Gs in a Ligand‐Dependent Manner
Authors: Malmberg À , Strange Philip G.
Published: March 2003

Abstract: The effect of mutations (V344E and T343A/V344E) in the third intracellular loop of the serotonin 5‐HT1A receptor expressed transiently in human embryonic kidney 293 cells have been examined in terms of receptor/G protein interaction and signaling. Serotonin, (R)‐8‐hydroxy‐2‐dipropylaminotetralin [(R)‐8‐OH‐DPAT], and buspirone inhibited cyclic AMP production in cells expressing native and mutant 5‐HT1A receptors. Serotonin, however, produced inverse bell‐shaped cyclic AMP concentration‐response curves at native and mutant 5‐HT1A receptors, indicating coupling not only to Gi/Go, but also to Gs. (R)‐8‐OH‐DPAT, however, induced stimulation of cyclic AMP production only after inactivation of Gi/Go proteins by pertussis toxin and only at the mutant receptors. The partial agonist buspirone was unable to induce coupling to Gs at any of the receptors, even after pertussis toxin treatment. The basal activities of native and mutant 5‐HT1A receptors in suppressing cyclic AMP levels were not found to be significantly different. The receptor binding characteristics of the native and mutant receptors were investigated using the novel 5‐HT1A receptor antagonist [3H]NAD‐299. For other receptors, analogous mutations have produced constitutive activation. This does not occur for the 5‐HT1A receptor, and for this receptor the mutations seem to alter receptor/G protein coupling, allowing ligand‐dependent coupling of receptor to Gs in addition to Gi/Go proteins.
Functional gamma‐secretase inhibitors reduce beta‐amyloid peptide levels in brain
Authors: Dovey H. F. , John V. , Anderson J. P. , Chen L. Z. , De Saint Andrieu P. , Fang L. Y. , Freedman S. B. , Folmer B. , Goldbach E. , Holsztynska E. J. , Hu K. L. , Johnson‐Wood K. L. , Kennedy S. L. , Kholodenko D. , Knops J. E. , Latimer L. H. , Lee M. , Liao Z. , Lieberburg I. M. , Motter R. N. , Mutter L. C. , Nietz J. , Quinn K. P. , Sacchi K. L. , Seubert P. A. , Shopp G. M. , Thorsett E. D. , Tung J. S. , Wu J. , Yang S. , Yin C. T. , Schenk D. B. , May P. C. , Altstiel L. D. , Bender M. H. , Boggs L. N. , Britton T. C. , Clemens J. C. , Czilli D. L. , Dieckman‐McGinty D. K. , Droste J. J. , Fuson K. S. , Gitter B. D. , Hyslop P. A. , Johnstone E. M. , Li W‐Y. , Little S. P. , Mabry T. E. , Miller F. D. , Ni B. , Nissen J. S. , Porter W. J. , Potts B. D. , Reel J. K. , Stephenson D. , Su Y. , Shipley L. A. , Whitesitt C. A. , Yin T. , Audia J. E.
Published: March 2003

Converging lines of evidence implicate the beta‐amyloid peptide (Aβ) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce Aβ production by functionally inhibiting γ‐secretase, the activity responsible for the carboxy‐terminal cleavage required for Aβ production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon Aβ production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester, to mice transgenic for human APPV717F reduces brain levels of Aβ in a dose‐dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain Aβin vivo. Development of such novel functional γ‐secretase inhibitors will enable a clinical examination of the Aβ hypothesis that Aβ peptide drives the neuropathology observed in Alzheimer's disease.
Parameters related to lipid metabolism as markers of myelination in mouse brain
Authors: Muse Evan D. , Jurevics Helga , Toews Arrel D. , Matsushima G. K. , Morell Pierre
Published: March 2003

Myelination, during both normal development and with respect to disorders of myelination, is commonly studied by morphological and/or biochemical techniques that assay as their end‐points the extent of myelination. The rate of myelination is potentially a more useful parameter, but it is difficult and time‐consuming to establish, requiring a complete developmental study with labor‐intensive methodology. We report herein development of methodology to assay the absolute rate of myelination at any desired time during development. This involves intraperitoneal injection of 3H2O to label body water pools, followed by determination of label in the myelin‐specific lipid, cerebroside. The absolute amount of cerebroside synthesized can then be calculated from the specific radioactivity of body water and knowledge of the number of hydrogens from water incorporated into cerebroside. During development, the rate of cerebroside synthesis correlated well with the rate of accumulation of the myelin‐specific components, myelin basic protein and cerebroside. For purposes of control, we also tested other putative, albeit less quantitative, indices of the rate of myelination. Levels of mRNA for ceramide galactosyltransferase (rate‐limiting enzyme in cerebroside synthesis) and for myelin basic protein did not closely correlate with myelination at all times. Cholesterol synthesis closely matched the rate of cholesterol accumulation but did not track well with myelination. Synthesis of fatty acids did not correlate well with accumulation of either fatty acids (phospholipids) or myelin markers. We conclude that measurement of cerebroside synthesis rates provides a good measure of the rate of myelination. This approach may be useful as an additional parameter for examining the effects of environmental or genetic alterations on the rate of myelination.
Site of injury‐directed induction of heme oxygenase‐1 and ‐2 in experimental spinal cord injury: differential functions in neuronal defense mechanisms?
Authors: Panahian Nariman , Maines Mahin D.
Published: March 2003

The heme oxygenase (HO) isozymes catalyze oxidation of the heme molecule to biliverdin and carbon monoxide (CO) with the release of chelated iron. Presently, we have defined, for the first time, propensity for site of injury‐directed induction of isozymes – the stress‐inducible isozyme, HO‐1, responds distal (below) and the glucocorticoid (GC)‐inducible HO‐2 responds proximal (above) to the site of injury. We have also shown that reactive iron (Fe3+) and cGMP staining spatially resemble that of HO‐1; which, in turn, colocalizes in motor neurons with transcription factors: Fas‐associated protein containing death domain (FADD), tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) and p53. Spinal cord injury (SCI) was inflicted by clip compression for 30 min, and analyses were carried out after 4 h or 16 h. When compared with spinal cord segments proximal to the site of injury, northern blot analysis showed remarkably higher levels of HO‐1 mRNA distal (below) to the site of injury at both time points. In contrast, HO‐2 mRNA levels were elevated proximal (above) to the site of injury and more prominently at 16 h post SCI. Immunohistochemical analyses were carried out using 2 × 5 mm segments above and below the compression site. When compared with segments above the site of injury, the intensity of HO‐1 immunostaining and the number of HO‐1 positive neurons in the ventral horn motor neurons were prominently increased in segments below the injury. Western blot analysis confirmed the observations. HO‐2 protein was mapped to the ventral horn motor neurons, oligodendrocytes, the Clarke's nucleus neurons and the ependymal cells. When compared with segments below the site of injury, neuronal HO‐2 staining intensity was increased above the site of injury, and most notably at 16 h. These observations were also confirmed by western blotting and HO activity measurements. Tissue Fe3+ and cGMP staining were increased and prominently mapped below the site of injury, where cGMP colocalized with HO‐1 in the nucleus of the motor neurons. Also, a site of injury‐directed pattern of induction of FADD, TRAIL, and p53 immunoreactivity, and a widespread colocalization of the oncogenes with HO‐1 protein, were found within motor neurons below the level of injury.

We forward the hypothesis that HO‐1 and HO‐2 have different roles in the defense mechanisms of the injured nervous system. We hypothesize that HO‐1 protects against further damage by contributing to controlled cell death through their intrinsic suicide program, while HO‐2 is involved in suppression of inflammatory response by NO derived radicals.
Modulation of electrically evoked acetylcholine release in cultured rat septal neurones
Authors: Ehret Andreas , Haaf Andreas , Jeltsch Hélène , Heimrich Bernd , Feuerstein Thomas J. , Jackisch Rolf
Published: March 2003

The electrically evoked release of acetylcholine and its modulation via auto‐ and heteroreceptors were studied in primary cell cultures prepared from embryonic rat septum (ED 17). Cultures were grown for 1, 2 or 3 weeks on circular, poly D‐lysine‐coated glass coverslips. They developed a dense network of non‐neuronal and neuronal cells, only some of which were immunopositive for choline acetyltransferase. To measure acetylcholine release, the cells on the coverslips were pre‐incubated with [3H]choline (0.1 µmol/L), superfused with modified Krebs–Henseleit buffer at 25°C and electrically stimulated twice for 2 min (S1, S2; 3 Hz, 0.5 ms, 90–100 mA). The electrically evoked overflow of [3H] from the cells consisted of ≈ 80% of authentic [3H]Ach, was largely Ca2+‐dependent and tetrodotoxin sensitive, and hence represents an action potential‐evoked, exocytotic release of acetylcholine. Using pairs of selective agonists and antagonists added before S2, muscarinic autoreceptors, as well as inhibitory adenosine A1‐ and opioid µ‐receptors, could be detected, whereas δ‐opioid receptors were not found. Evoked [3H] overflow from cultures grown for 1 week, although Ca2+ dependent and tetrodotoxin sensitive, was insensitive to the muscarinic agonist oxotremorine, whereas the effect of oxotremorine on cells grown for 3 weeks was even more pronounced than that in 2‐week‐old cultures. In conclusion, similar to observations on rat septal tissue in vivo, acetylcholine release from septal cholinergic neurones grown in vitro is inhibited via muscarinic, adenosine A1 and µ‐opioid receptors. This in vitro model may prove useful in the exploration of regulatory mechanisms underlying the expression of release modulating receptors on septal cholinergic neurones.
Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor
Authors: Grosshans David R. , Browning Michael D.
Published: March 2003

The N‐methyl‐d‐aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non‐receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini‐slices with 500 nm phorbol 12‐myristate 13‐acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti‐NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 ± 11% above control) and NR2B (41 ± 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.
The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory
Authors: Sweatt J. David
Published: March 2003

The mitogen‐activated protein kinase (MAP kinase, MAPK) cascade, as the name implies, was originally discovered as a critical regulator of cell division and differentiation. As further details of this signaling cascade were worked out, it became clear that the MAPK cascade is in fact a prototype for a family of signaling cascades that share the motif of three serially linked kinases regulating each other by sequential phosphorylation. Thus, a revised nomenclature arose that uses the term MAPK to refer to the entire superfamily of signaling cascades (comprising the erks, the JNKs and the p38 stress activated protein kinases), and specifies the prototype MAPK as the extracellular signal‐regulated kinase (erk). The two erk MAPK isoforms, p44 MAPK and p42 MAPK, are referred to as erk1 and erk2, respectively.The erks are abundantly expressed in neurons in the mature central nervous system, raising the question of why the prototype molecular regulators of cell division and differentiation are present in these non‐dividing, terminally differentiated neurons. This review will describe the beginnings of an answer to this question. Interestingly, the general model has begun to emerge that the erk signaling system has been co‐opted in mature neurons to function in synaptic plasticity and memory. Moreover, recent insights have led to the intriguing prospect that these molecules serve as biochemical signal integrators and molecular coincidence detectors for coordinating responses to extracellular signals in neurons. In this review I will first outline the essential components of this signal transduction cascade, and briefly describe recent results implicating the erks in mammalian synaptic plasticity and learning. I will then proceed to outline recent results implicating the erks as molecular signal integrators and, potentially, coincidence detectors. Finally, I will speculate on what the critical downstream effectors of the erks are in neurons, and how they might provide a readout of the integrated signal.
Group II metabotropic glutamate receptor activation protects striatal dopaminergic nerve terminals against MPP+‐induced neurotoxicity along with brain‐derived neurotrophic factor induction
Authors: Matarredona Esperanza R. , Santiago Marti , Venero Jose L. , Cano Josefina , Machado Alberto
Published: March 2003

We have studied the in vivo effect of the selective agonist for group II metabotropic glutamate receptors (2S, 2′R, 3′R)‐2‐(2′3′‐dicarboxycyclopropyl)glycine (DCG‐IV) against MPP+‐induced toxicity on rat striatal dopaminergic nerve terminals by using both microdialysis and immunohistochemical techniques. Perfusion of 1 mm DCG‐IV during 1 h protected dopaminergic nerve terminals against the degeneration induced by a 15‐minute perfusion of 1 mm MPP+. In addition, the microglial cell population was markedly activated 24 h after DCG‐IV perfusion. The astroglial cell population was only markedly activated around the microdialysis probe. This protective effect seems to be dependent on protein synthesis since 1 mm cycloheximide, an inhibitor of protein synthesis, abolished the neuroprotective effect of 1 mm DCG‐IV against MPP+ toxicity. Perfusion of DCG‐IV induced an upregulation of striatal brain‐derived neurotrophic factor (BDNF) mRNA expressing cells which were confined precisely around the microdialysis probe. Taken together, our results suggest that the induction and release of brain‐derived neurotrophic factor (BDNF) by activated glial cells induced by DCG‐IV perfusion may account for its protective action against MPP+‐induced dopaminergic terminal degeneration.
Protection from MPTP‐induced neurotoxicity in differentiating mouse N2a neuroblastoma cells
Authors: De Girolamo Luigi A. , Hargreaves Alan J. , Billett E. Ellen
Published: March 2003

We have shown previously that subcytotoxic concentrations of MPTP (1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF‐H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 µm) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP‐induced cell death (cytotoxicity) and MPTP‐induced NF‐H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 µm) reduced the cytotoxic effect of MPTP, but blocked di‐butyryl cyclic AMP (dbcAMP)‐induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine osidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP‐induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF‐H hyper‐phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP‐induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.
In vivo13C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle flux in rat brain during [2‐13C]glucose infusion
Authors: Sibson Nicola R. , Mason Graeme F. , Shen Jun , Cline Gary W. , Herskovits A. Zara , Wall Joyce E. M. , Behar Kevin L. , Rothman Douglas L. , Shulman Robert G.
Published: March 2003

The aims of this study were twofold: (i) to determine quantitatively the contribution of glutamate/glutamine cycling to total astrocyte/neuron substrate trafficking for the replenishment of neurotransmitter glutamate; and (ii) to determine the relative contributions of anaplerotic flux and glutamate/glutamine cycling to total glutamine synthesis. In this work in vivo and in vitro13C NMR spectroscopy were used, with a [2‐13C]glucose or [5‐13C]glucose infusion, to determine the rates of glutamate/glutamine cycling, de novo glutamine synthesis via anaplerosis, and the neuronal and astrocytic tricarboxylic acid cycles in the rat cerebral cortex. The rate of glutamate/glutamine cycling measured in this study is compared with that determined from re‐analysis of 13C NMR data acquired during a [1‐13C]glucose infusion. The excellent agreement between these rates supports the hypothesis that glutamate/glutamine cycling is a major metabolic flux (∼0.20 µmol/min/g) in the cerebral cortex of anesthetized rats and the predominant pathway of astrocyte/neuron trafficking of neurotransmitter glutamate precursors. Under normoammonemic conditions anaplerosis was found to comprise 19–26% of the total glutamine synthesis, whilst this fraction increased significantly during hyperammonemia (∼32%). These findings indicate that anaplerotic glutamine synthesis is coupled to nitrogen removal from the brain (ammonia detoxification) under hyperammonemic conditions.
Stimulatory role of dopamine on fibroblast growth factor‐2 expression in rat striatum
Authors: Roceri Mila , Molteni Raffaella , Fumagalli Fabio , Racagni Giorgio , Gennarelli Massimo , Corsini Giovanni U. , Maggio Roberto , Riva Marco A.
Published: March 2003

We have previously shown that systemic injection of (–)nicotine produces a selective up‐regulation of fibroblast growth factor (FGF)‐2 mRNA levels in rat striatum. Because (–)nicotine can increase striatal release of dopamine and glutamate, in the present study we have investigated the contribution of these neurotransmitters in the modulation of FGF‐2 expression. We found that coinjection of dopaminergic D1 (SCH23390) or D2 (haloperidol) receptor antagonists prevents nicotine‐induced elevation of FGF‐2 expression. However, injection of the NMDA receptor antagonist MK‐801 produced a significant increment of FGF‐2 mRNA and protein levels in rat striatum similar to the effect produced by (–)nicotine alone. Interestingly this effect of MK‐801 could also be prevented by D1 or D2 receptor antagonists, suggesting that an elevation of dopamine levels may be required for the regulation of the trophic molecule. Accordingly we found that the non‐selective dopaminergic agonist apomorphine can similarly increase striatal FGF‐2 mRNA levels. Despite the observation that both D1 and D2 receptors appear to contribute to the modulation of FGF‐2 expression, only a direct activation of D2 receptors, through quinpirole administration, was able to mimic the effect of apomorphine. On the basis of FGF‐2 neurotrophic activity, these results suggest that direct or indirect activation of dopaminergic system can be neuroprotective and might reduce cell vulnerability in degenerative disorders.
β‐Amyloid efflux mediated by p‐glycoprotein
Authors: Lam Fred C. , Liu Ronghua , Lu Peihua , Shapiro Adam B. , Renoir Jack‐Michel , Sharom Frances J. , Reiner Peter B.
Published: March 2003

A large body of evidence suggests that an increase in the brain β‐amyloid (Aβ) burden contributes to the etiology of Alzheimer's disease (AD). Much is now known about the intracellular processes regulating the production of Aβ, however, less is known regarding its secretion from cells. We now report that p‐glycoprotein (p‐gp), an ATP‐binding cassette (ABC) transporter, is an Aβ efflux pump. Pharmacological blockade of p‐gp rapidly decrease extracellular levels of Aβ secretion.In vitrobinding studies showed that addition of synthetic human Aβ1–40 and Aβ1–42 peptides to hamster mdr1‐enriched vesicles labeled with the fluorophore MIANS resulted in saturable quenching, suggesting that both peptides interact directly with the transporter. Finally, we were able to directly measure transport of Aβ peptides across the plasma membranes of p‐gp enriched vesicles, and showed that this phenomenon was both ATP‐ and p‐gp‐dependent. Taken together, our study suggests a novel mechanism of Aβ detachment from cellular membranes, and represents an obvious route towards identification of such a mechanism in the brain.
Constitutive α‐secretase cleavage of the β‐amyloid precursor protein in the furin‐deficient LoVo cell line: involvement of the pro‐hormone convertase 7 and the disintegrin metalloprotease ADAM10
Authors: Lopez‐Perez Elvira , Zhang Yue , Frank Stuart J , Creemers John , Seidah Nabil , Checler Frédéric
Published: March 2003

The β‐amyloid precursor protein (βAPP) undergoes a physiological cleavage triggered by one or several proteolytic activities referred to as α‐secretases, leading to the secretion of sAPPα. Several lines of evidence indicate that the α‐secretase cleavage is a highly regulated process. Thus, besides constitutive production of sAPPα, several studies have reported on protein kinase C‐regulated sAPPα secretion. Studies aimed at identifying α‐secretase(s) candidates suggest the involvement of enzymes belonging to the pro‐hormone convertases and disintegrin families. The delineation of respective contributions of proteolytic activities in constitutive and regulated sAPPα secretion is rendered difficult by the fact that the overall regulated response always includes the basal constitutive counterpart that cannot be selectively abolished. Here we report on the fact that the furin‐deficient LoVo cells are devoid of regulated PKC‐dependent sAPPα secretion and therefore represent an interesting model to study exclusively the constitutive sAPPα secretion. We show here, by a pharmacological approach using selective inhibitors, that pro‐hormone convertases and proteases of the ADAM (disintegrin metalloproteases) family participate in the production/secretion of sAPPαs in LoVo cells. Transfection analysis allowed us to further establish that the pro‐hormone convertase 7 and ADAM10 but not ADAM17 (TACE, tumour necrosis factor α‐converting enzyme) likely contribute to constitutive sAPPα secretion by LoVo cells.
Rapid transferrin efflux from brain to blood across the blood–brain barrier
Authors: Zhang Yun , Pardridge William M.
Published: March 2003

The brain efflux index method is used to examine the extent to which transferrin effluxes from brain to blood across the blood–brain barrier (BBB) following intracerebral injection. Whereas high‐molecular‐weight dextran is nearly 100% retained in brain for up to 90 min after intracerebral injection in the Par2 region of the parietal cortex of brain, there is rapid efflux of transferrin from brain to blood across the BBB. The efflux of apotransferrin is 3.5‐fold faster than the efflux of holo‐transferrin. The brain to blood efflux of apotransferrin is completely saturable by unlabeled transferrin, but is not inhibited by other plasma proteins. These studies provide evidence for reverse transcytosis of transferrin from brain to blood across the BBB. As circulating transferrin is known to undergo transcytosis across the BBB in the blood‐to‐brain direction, these studies support the model of bidirectional transcytosis of transferrin through the BBB in vivo.
Identification of mouse NMDA receptor subunit NR2A C‐terminal tyrosine sites phosphorylated by coexpression with v‐Src
Authors: Yang Ming , Leonard John P.
Published: March 2003

The protein tyrosine kinase Src is known to regulate NMDA receptors in native neurons. While NR2A, NR2B and NR2D are known to be phosphorylated on tyrosine residues, the exact sites have remained unidentified. Immunoprecipitation of NMDA receptor subunits followed by western blotting was used to analyze the state of tyrosine phosphorylation of recombinant NMDA receptor subunits expressed in HEK293 cells. Using antiphosphotyrosine antibody PY20, we find that on expression in HEK cells, v‐Src and Fyn cause detectable tyrosine phosphorylation only of NR2A. Because a stronger signal was produced by the constitutively active v‐Src, the general region of v‐Src phosphorylation was delimited by expression of a series of truncation mutants of NR2A. Site‐directed mutagenesis on candidate sites within the likely region allowed identification of three sites, Y1292, Y1325, and Y1387 that account for a significant fraction of the total PY20 signal. Two of these sites, Y1292 and Y1387, were suggested to control current modulation by Src in previous studies of HEK cells expressing NR1/NR2A. One of these sites, Y1325, has not yet been evaluated for effects on receptor current. A unique tyrosine site, Y1267, was shown not to be a site of detectable phosphorylation, in accordance with its Src‐independent regulation of receptor currents.
Cell‐specific caspase expression by different neuronal phenotypes in transient retinal ischemia
Authors: Singh Manjeet , Savitz Sean I. , Hoque Romy , Gupta Gaurav , Roth Steven , Rosenbaum Pearl S. , Rosenbaum Daniel M.
Published: March 2003

Emerging evidence supports an important role for caspases in neuronal death following ischemia‐reperfusion injury. This study assessed whether cell specific caspases participate in neuronal degeneration and whether caspase inhibition provides neuroprotection following transient retinal ischemia. We utilized a model of transient global retinal ischemia. The spatial and temporal pattern of the active forms of caspase 1, 2 and 3 expression was determined in retinal neurons following ischemic injury. Double‐labeling with cell‐specific markers identified which cells were expressing different caspases. In separate experiments, animals received various caspase inhibitors before the induction of ischemia. Sixty minutes of ischemia resulted in a delayed, selective neuronal death of the inner retinal layers at 7 days. Expression of caspase 1 was not detected at any time point. Maximal expression of caspase 2 was found at 24 h primarily in the inner nuclear and ganglion cell layers of the retina and localized to ganglion and amacrine neurons. Caspase 3 also peaked at 24 h in both the inner nuclear and outer nuclear layers and was predominantly expressed in photoreceptor cells and to a lesser extent in amacrine neurons. The pan caspase inhibitor, Boc‐aspartyl fmk, or an antisense oligonucleotide inhibitor of caspase 2 led to significant histopathologic and functional improvement (electroretinogram) at 7 days. No protection was found with the caspase 1 selective inhibitor, Y‐vad fmk. These observations suggest that ischemia‐reperfusion injury activates different caspases depending on the neuronal phenotype in the retina and caspase inhibition leads to both histologic preservation and functional improvement. Caspases 2 and 3 may act in parallel in amacrine neurons following ischemia‐reperfusion. These results in the retina may shed light on differential caspase specificity in global cerebral ischemia.
Control of serotonergic neurons in rat brain by dopaminergic receptors outside the dorsal raphe nucleus
Authors: Martín‐Ruiz Raúl , Ugedo Luisa , Honrubia María A. , Mengod Guadalupe , Artigas Francesc
Published: March 2003

We studied the control of dorsal raphe (DR) serotonergic neurons by dopaminergic transmission in rat brain using microdialysis and single unit extracellular recordings. Apomorphine (0.5–3.0 mg/kg s.c.) and quinpirole (0.5 mg/kg s.c.) increased serotonin (5‐HT) output in the DR and (only apomorphine) in striatum. These effects were antagonized by 0.3 mg/kg s.c. SCH 23390 (in DR and striatum) and 1 mg/kg s.c. raclopride (in DR). 5‐HT1A receptor blockade potentiated the 5‐HT increase produced by apomorphine in the DR. Apomorphine (50–400 µg/kg i.v.) increased the firing rate of most 5‐HT neurons, an effect prevented by SCH 23390 and raclopride. Quinpirole (40–160 µg/kg i.v.) also enhanced the firing rate of 5‐HT neurons. When applied in the DR, neither drug increased the 5‐HT output in the DR or striatum. Likewise, micropressure injection of quinpirole (0.2–8 pmol) failed to increase the firing rate of 5‐HT neurons. In situ hybridization showed that the dopamine (DA) D2 receptor transcript was almost absent in the DR and abundant in the substantia nigra (SN) and the periaqueductal grey matter (PAG). Using dual probe microdialysis, the application of tetrodotoxin or apomorphine in SN significantly increased the DR 5‐HT output. Thus, the discrepancy between local and systemic effects of dopaminergic agonists and the absence of DA D2 receptor transcript in 5‐HT neurons suggest that DA D2 receptors outside the DR control serotonergic activity.
Enhanced neuronal protection from oxidative stress by coculture with glutamic acid decarboxylase‐expressing astrocytes
Authors: Lamigeon C. , Bellier J. P. , Sacchettoni S. , Rujano M. , Jacquemont B.
Published: March 2003

Astrocytes expressing glutamic acid decarboxylase GAD67 directed by the glial fibrillary acidic protein promoter were shown to provide enhanced protection of PC12 cells from H2O2 treatment and serum deprivation in the presence of glutamate. In addition, they protected non‐differentiated, but not differentiated, embryonic rat cortical neurons from glutamate toxicity. Glutamic acid decarboxylase (GAD)‐expressing astrocytes showed increased glutathione synthesis and release compared to control astrocytes. These changes were due to GAD transgene expression, as transient expression of a GAD antisense plasmid resulted in partial suppression of the increase in glutathione release. In addition to the previously demonstrated increases in NADH and ATP levels and lactate release, GAD‐expressing astrocytes show increased antioxidant activity, explaining their ability to protect neurons from various injuries.
Sustained extracellular signal‐regulated kinase activation by 6‐hydroxydopamine: implications for Parkinson's disease
Authors: Kulich Scott M. , Chu Charleen T.
Published: March 2003

Although the toxin 6‐hydroxydopamine (6‐OHDA) is utilized extensively in animal models of Parkinson's disease, the underlying mechanism of its toxic effects on dopaminergic neurons is not completely understood. We examined the effects of 6‐OHDA on the CNS‐derived tyrosine hydroxylase expressing B65 cell line, with particular attention to the regulation of the extracellular signal‐regulated protein kinases (ERK). 6‐OHDA elicited a dose‐dependent cytotoxicity in B65 cells. Toxic doses of 6‐OHDA also elicited a biphasic pattern of ERK phosphorylation with a prominent sustained phase, a pattern that differed from that observed with hydrogen peroxide (H2O2) treatment. 6‐OHDA‐elicited ERK phosphorylation was blocked by PD98059, an inhibitor of the upstream mitogen activated protein kinase kinase (MEK) that phosphorylates and activates ERK. PD98059 also conferred protection against 6‐OHDA cytotoxicity, but did not affect H2O2 toxicity in B65 cells. These results suggest that ERK activation plays a direct mechanistic role in 6‐OHDA toxicity, rather than representing a protective compensatory response, and raise the possibility that abnormal patterns of ERK activation may contribute to dopaminergic neuronal cell death.
Transfer of glutamine between astrocytes and neurons
Authors: Bröer Stefan , Brookes Neville
Published: March 2003

The export of glutamine from astrocytes, and the uptake of glutamine by neurons, are integral steps in the glutamate‐glutamine cycle, a major pathway for the replenishment of neuronal glutamate. We review here the functional and molecular identification of the transporters that mediate this transfer. The emerging picture of glutamine transfer in adult brain is of a dominant pathway mediated by system N transport (SN1) in astrocytes and system A transport (SAT/ATA) in neurons. The participating glutamine transporters are functionally and structurally related, sharing the following properties: (a) unlike many neutral amino acid transporters which have proven to be obligate exchangers, these glutamine transporters mediate net substrate transfer energized by coupling to ionic gradients; (b) they are sensitive to small pH changes in the physiological range; (c) they are susceptible to adaptive and humoral regulation; (d) they are related structurally to the AAAP (amino acid and auxin permeases) family of transporters. A key difference between SN1 and the SAT/ATA transporters is the ready reversibility of glutamine fluxes via SN1 under physiological conditions, which allows SN1 both to sustain a glutamine concentration gradient in astrocytes and to mediate the net outward flux of glutamine. It is likely that the ASCT2 transporter, an obligate exchanger of neutral amino acids, displaces the SN1 transporter as the main carrier of glutamine export in proliferating astrocytes.
Inverse agonists and neutral antagonists at µ opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence
Authors: Wang Danxin , Raehal Kirsten M. , Bilsky Edward J. , Sadée Wolfgang
Published: March 2003

The µ opioid receptor, MOR, displays spontaneous agonist‐independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPγS binding to cell membranes and cAMP levels in intact cells. β‐CNA, C‐CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6α‐ and 6β‐naltrexol and ‐naloxol, and 6β‐naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine‐induced dependence, 6β‐naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone‐induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine‐dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.
Glutamate receptor subunit expression in primary neuronal and secondary glial cultures
Authors: Janssens N. , Lesage A. S. J.
Published: March 2003

We report on the expression of ionotropic glutamate receptor subunits in primary neuronal cultures from rat cortex, hippocampus and cerebellum and of metabotropic glutamate (mGlu) receptor subtypes in these neuronal cultures as well as in cortical astroglial cultures. We found that the NMDA receptor (NR) subunits NR1, NR2A and NR2B were expressed in all three cultures. Each of the three cultures showed also expression of the four AMPA receptor subunits. Although RT‐PCR detected mRNA of all kainate (KA) subunits in the three cultures, western blot showed only expression of Glu6 and KA2 receptor subunits. The expression analysis of mGlu receptors indicated the presence of all mGlu receptor subtype mRNAs in the three neuronal cultures, except for mGlu2 receptor mRNA, which was not detected in the cortical and cerebellar culture. mGlu1a/α, ‐2/3 and ‐5 receptor proteins were present in all three cultures, whereas mGlu4a and mGlu8a receptor proteins were not detected. Astroglial cultures were grown in either serum‐containing or chemically defined medium. Only mGlu5 receptor protein was found in astroglial cultures grown in serum‐containing medium. When astrocytes were cultured in chemically defined medium, mGlu3, ‐5 and ‐8 receptor mRNAs were detected, but at the protein level, still only mGlu5 receptor was found.
Brain ischemia and reperfusion activates the eukaryotic initiation factor 2α kinase, PERK
Authors: Kumar Rita , Azam Salman , Sullivan Jonathan M. , Owen Cheri , Cavener Douglas R. , Zhang Peichuan , Ron David , Harding Heather P. , Chen Jane‐Jane , Han Anping , White Blaine C. , Krause Gary S. , DeGracia Donald J.
Published: March 2003

Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons, which persists in vulnerable neurons, that is caused by the inhibition of translation initiation as a result of the phosphorylation of the α‐subunit of eukaryotic initiation factor 2 (eIF2α). To identify kinases responsible for eIF2α phosphorylation [eIF2α(P)] during brain reperfusion, we induced ischemia by bilateral carotid artery occlusion followed by post‐ischemic assessment of brain eIF2α(P) in mice with homozygous functional knockouts in the genes encoding the heme‐regulated eIF2α kinase (HRI), or the amino acid‐regulated eIF2α kinase (GCN2). A 10‐fold increase in eIF2α(P) was observed in reperfused wild‐type mice and in the HRI–/– or GCN2–/– mice. However, in all reperfused groups, the RNA‐dependent protein kinase (PKR)‐like endoplasmic reticulum eIF2α kinase (PERK) exhibited an isoform mobility shift on SDS–PAGE, consistent with the activation of the kinase. These data indicate that neither HRI nor GCN2 are required for the large increase in post‐ischemic brain eIF2α(P), and in conjunction with our previous report that eIF2α(P) is produced in the brain of reperfused PKR–/– mice, provides evidence that PERK is the kinase responsible for eIF2α phosphorylation in the early post‐ischemic brain.
HIV‐1 Tat through phosphorylation of NMDA receptors potentiates glutamate excitotoxicity
Authors: Haughey N. J. , Nath A. , Mattson M. P. , Slevin J. T. , Geiger J. D.
Published: March 2003

Toxic effects of HIV‐1 proteins contribute to altered function and decreased survival of select populations of neurons in HIV‐1‐infected brain. One such HIV‐1 protein, Tat, can activate calcium release from IP3‐sensitive intracellular pools, induce calcium influx in neural cells, and, as a result, can increase neuronal cell death. Here, we provide evidence that Tat potentiates excitatory amino acid (glutamate and NMDA) triggered calcium flux, as well as glutamate‐ and staurosporine‐mediated neurotoxicity. Calcium flux in cultured rat hippocampal neurons triggered by the transient application of glutamate or NMDA was facilitated by pre‐exposure to Tat. Facilitation of glutamate‐triggered calcium flux by Tat was prevented by inhibitors of ADP‐ribosylation of Gi/Go proteins (pertussis toxin), protein kinase C (H7 and bisindolymide), and IP3‐mediated calcium release (xestospongin C), but was not prevented by an activator of Gs (cholera toxin) or an inhibitor of protein kinase A (H89). Facilitation of NMDA‐triggered calcium flux by Tat was reversed by inhibitors of tyrosine kinase (genestein and herbimycin A) and by an inhibitor of NMDA receptor function (zinc). Tat increased 32P incorporation into NMDA receptor subunits NR2A and NR2B and this effect was blocked by genestein. Subtoxic concentrations of Tat combined with subtoxic concentrations of glutamate or staurosporine increased neuronal cell death significantly. Together, these findings suggest that NMDA receptors play an important role in Tat neurotoxicity and the mechanisms identified may provide additional therapeutic targets for the treatment of HIV‐1 associated dementia.