bluuebunny/crossref_metadata_2025 huggingface dataset:
The Metabolism of [3‐13C]Lactate in the Rat Brain Is Specific of a Pyruvate Carboxylase‐Deprived Compartment
Authors: Bouzier Anne‐Karine , Thiaudiere Eric , Biran Marc , Rouland Richard , Canioni Paul , Merle Michel
Published: March 2003

Lactate metabolism in the adult rat brain was investigated in relation with the concept of lactate trafficking between astrocytes and neurons. Wistar rats were infused intravenously with a solution containing either [3‐13C]lactate (534 mM) or both glucose (750 mM)

and [3‐13C]lactate (534 mM). The time courses of both the

concentration and 13C enrichment of blood glucose and lactate were

determined. The data indicated the occurrence of [3‐13C]lactate

recycling through liver gluconeogenesis. The yield of glucose labeling was,

however, reduced when using the glucose‐containing infusate. After a 20‐min or

1‐h infusion, perchloric acid extracts of the brain tissue were prepared and

subsequently analyzed by 13C‐ and

1H‐observed/13C‐edited NMR spectroscopy. The

13C labeling of amino acids indicated that

[3‐13C]lactate was metabolized in the brain. Based on the alanine

C3 enrichment, lactate contribution to brain metabolism amounted to 35% under

the most favorable conditions used. By contrast with what happens with

[1‐13C]glucose metabolism, no difference in glutamine C2 and C3 labeling was evidenced, indicating that lactate was metabolized in a compartment deprived of pyruvate carboxylase activity. This result confirms, for the first time from an in vivo study, that lactate is more specifically a neuronal substrate.
Cultured Granule Cells and Astrocytes from Cerebellum Differ in Metabolizing Sphingosine
Authors: Riboni Laura , Viani Paola , Bassi Rosaria , Giussani Paola , Tettamanti Guido
Published: March 2003

Sphingosine metabolism was studied in primary cultures of differentiated cerebellar granule cells and astrocytes. After a 2‐h pulse with [C3‐3H]sphingosine at different doses (0.1‐200 nmol/mg of

cell protein), both cell types efficiently incorporated the long chain base ;

the percentage of cellular [3H]sphingosine over total label

incorporation was extremely low at sphingosine doses of <10 nmol/mg of cell

protein and increased at higher doses. Most of the [3H]sphingosine

taken up underwent metabolic processing by N‐acylation,

1‐phosphorylation, and degradation (assessed as 3H2O

released in the medium). The metabolic processing of exogenous sphingosine was

extremely efficient in both cells, granule cells and astrocytes being able to

metabolize, respectively, an amount of sphingosine up to 80‐ and 300‐fold the

cellular content of this long chain base in 2 h. At the different doses, the

prevailing metabolic route of sphingosine was different. At lower doses and in

a wide dose range, the major metabolic fate of sphingosine was

N‐acylation. With increasing doses, there was first increased sphingosine degradation and then increased levels of sphingosine‐1‐phosphate. The data demonstrate that, in neurons and astrocytes, the metabolic machinery devoted to sphingosine processing is different, astrocytes possessing an overall higher capacity to synthesize the bioactive compounds ceramide and sphingosine‐1‐phosphate.
Enzymatic Degradation Protects Neurons from Glutamate Excitotoxicity
Authors: Matthews Christopher C. , Zielke H. Ronald , Wollack Jan B. , Fishman Paul S.
Published: March 2003

Abstract: Several enzymes with the capacity to degrade glutamate

have been suggested as possible neuroprotectants. We initially evaluated the

kinetic properties of glutamate pyruvate transaminase (GPT; also known as

alanine aminotransferase), glutamine synthetase, and glutamate dehydrogenase

under physiologic conditions to degrade neurotoxic concentrations of

glutamate. Although all three enzymes initially degraded glutamate rapidly,

only GPT was able to reduce toxic (500 μM) levels of glutamate

into the physiologic (<20 μM) range. Primary cultures of fetal

murine cortical neurons were subjected to paradigms of either exogenous or

endogenous glutamate toxicity to evaluate the neuroprotective value of GPT.

Neuronal survival after exposure to added glutamate ranging from 100 to 500

μM was improved significantly in the presence of GPT (≥1 U/ml).

Cultures were also exposed to the glutamate transporter inhibitor

L‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC), which produces neuronal injury by elevating extracellular glutamate. GPT significantly reduced the toxicity of PDC. This reduction was associated with a reduction in the PDC‐dependent rise in the medium concentration of glutamate. These results suggest that enzymatic degradation of glutamate by GPT can be an alternative to glutamate receptor blockade as a strategy to protect neurons from excitotoxic injury.
Two Mechanisms of Synaptic Vesicle Recycling in Rat Brain Nerve Terminals
Authors: Cousin Michael A. , Robinson Phillip J.
Published: March 2003

Abstract: KCl and 4‐aminopyridine (4‐AP) evoke glutamate release
from rat brain cortical nerve terminals by voltage clamping or by
Na+ channel‐generated repetitive action potentials, respectively. Stimulation by 4‐AP but not KCl is largely mediated by protein kinase C (PKC). To determine whether KCl and 4‐AP utilise the same mechanism to release glutamate, we correlated glutamate release with release of the hydrophobic synaptic vesicle (SV) marker FM2‐10. A strong correlation was observed for increasing concentrations of KCl and after application of phorbol 12‐myristate 13‐acetate (PMA) or staurosporine. The parallel increase in exocytosis measured by two approaches suggested it occurred by a PKC‐independent mechanism involving complete fusion of SVs with the plasma membrane. At low concentrations of 4‐AP, alone or with staurosporine, glutamate and FM2‐10 release also correlated. However, higher concentrations of 4‐AP or of 4‐AP plus PMA greatly increased glutamate release but did not further increase FM2‐10 release. This divergence suggests that 4‐AP recruits an additional mechanism of release during strong stimulation that is PKC dependent and is superimposed upon the first mechanism. This second mechanism is characteristic of kiss‐and‐run, which is not detectable by styryl dyes. Our data suggest that glutamate release in nerve terminals occurs via two mechanisms: (1) complete SV fusion, which is PKC independent; and (2) a kiss‐and‐run‐like mechanism, which is PKC dependent. Recruitment of a second release mechanism may be a widespread means to facilitate neurotransmitter release in central neurons.
Degradation of the Type I Inositol 1,4,5‐Trisphosphate Receptor by Caspase‐3 in SH‐SY5Y Neuroblastoma Cells Undergoing Apoptosis
Authors: Haug Lise Sofie , Walaas S. Ivar , Østvold Anne Carine
Published: March 2003

Abstract: The type I inositol 1,4,5‐trisphosphate (IP3)
receptor is selectively down‐regulated in several neurodegenerative diseases,
including Alzheimer's disease, Huntington's chorea, and ischemia, all
conditions in which apoptotic neuronal loss occurs. In the present study, we
used a neuronal cell line, human neuroblastoma SH‐SY5Y cells, to investigate
whether the levels of IP3 receptor are changed during apoptosis in
these cells. Following induction of apoptosis by staurosporine, the
immunoreactivity of the type I IP3 receptor in microsome
preparations from SH‐SY5Y cells was reduced within 2 h, with a further
reduction during subsequent hours. Immunoblot analyses, using antibodies to
poly(ADP‐ribose) polymerase and spectrin breakdown products, revealed
proteolysis of these caspase‐3 substrates within 3 h, confirming that
IP3 receptor cleavage is an early consequence of apoptosis. In
vitro incubation of SH‐SY5Y microsomes or immunopurified IP3
receptor from rat cerebellum with recombinant caspase‐3 led to generation of
immunoreactive breakdown products similar to those observed in intact cells,
suggesting that the type I IP3 receptor is a potential substrate
for caspase‐3. Preincubation of the neuroblastoma cells with the caspase‐3
inhibitor Z‐Asp‐Glu‐Val‐Asp‐fluoromethyl ketone prevented IP3
receptor degradation. These results show that the type I IP3
receptor is a substrate for caspase‐3 in neuronal cells and indicate that
apoptotic down‐regulation of IP3 receptor levels may contribute to the pathology of neurodegenerative conditions.
Oxidative Stress Following Traumatic Brain Injury in Rats
Authors: Tyurin Vladimir A. , Tyurina Yulia Y. , Borisenko Grigory G. , Sokolova Tatiana V. , Ritov Vladimir B. , Quinn Peter J. , Rose Marie , Kochanek Patrick , Graham Steven H. , Kagan Valerian E.
Published: March 2003

Abstract: Oxidative stress may contribute to many pathophysiologic
changes that occur after traumatic brain injury. In the current study,
contemporary methods of detecting oxidative stress were used in a rodent model
of traumatic brain injury. The level of the stable product derived from
peroxidation of arachidonyl residues in phospholipids,
8‐epi‐prostaglandin F2α, was increased at 6 and 24 h
after traumatic brain injury. Furthermore, relative amounts of fluorescent end
products of lipid peroxidation in brain extracts were increased at 6 and 24 h
after trauma compared with sham‐operated controls. The total antioxidant
reserves of brain homogenates and water‐soluble antioxidant reserves as well
as tissue concentrations of ascorbate, GSH, and protein sulfhydryls were
reduced after traumatic brain injury. A selective inhibitor of
cyclooxygenase‐2, SC 58125, prevented depletion of ascorbate and thiols, the
two major water‐soluble antioxidants in traumatized brain. Electron
paramagnetic resonance (EPR) spectroscopy of rat cortex homogenates failed to
detect any radical adducts with a spin trap, 5,5‐dimethyl‐1‐pyrroline
N‐oxide, but did detect ascorbate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from traumatized brain samples compared with sham‐operated controls. These results along with detailed model experiments in vitro indicate that ascorbate is a major antioxidant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free radicals in brain tissue after traumatic brain injury.
Mitochondrial Carbonic Anhydrase in the Nervous System
Authors: Ghandour M. Said , Parkkila Anna‐Kaisa , Parkkila Seppo , Waheed Abdul , Sly William S.
Published: March 2003

Abstract: Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C‐terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30‐kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell‐specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion‐induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.
A Nonfibrillar Form of the Fusogenic Prion Protein Fragment [118‐135] Induces Apoptotic Cell Death in Rat Cortical Neurons
Authors: Pillot Thierry , Drouet Béatrice , Pinçon‐Raymond Martine , Vandekerckhove Joël , Rosseneu Maryvonne , Chambaz Jean
Published: March 2003

Abstract: Neuronal loss is a salient feature of prion diseases.However, its cause and mechanism, particularly its relationship with theaccumulation and precipitation of the pathogenic, protease‐resistant isoformPrPSc of the cellular prion protein PrPC, are still anenigma. Several studies suggest that neuronal loss could occur through aprocess of programmed cell death, which is consistent with the lack ofinflammation in these conditions. By analogy with the pathological eventsoccurring during the development of Alzheimer's disease, controversies stillexist regarding the relationship between amyloidogenesis, prion aggregation,and neuronal loss. We recently demonstrated that a prion protein fragment(118‐135) displayed membrane‐destabilizing properties and was able to induce,in a nonfibrillar form, the fusion of unilamellar liposomes. To unravel themechanism of prion protein neurotoxicity, we characterize the effects of thehuman Pr[118‐135] peptide on rat cortical neurons. We demonstrate that lowconcentrations of the Pr[118‐135] peptide, in a nonfibrillar form, induce atime‐ and dose‐ dependent apoptotic cell death, including caspase activation,DNA condensation, and fragmentation. This toxicity might involve oxidativestress, because antioxidant molecules, such as probucol and propyl gallate,protect neurons against prion peptide toxicity. By contrast, a nonfusogenicvariant Pr[118‐135, 0°] peptide, which displays the same amino acidcomposition but several amino acid permutations, is not toxic to corticalneurons, which emphasizes the critical role of the fusogenic properties of theprion peptide in its neurotoxicity. Taken together, our results suggest thatthe interaction between the Pr[118‐135] peptide and the plasma membrane ofneurons might represent an early event in a cascade leading toneurodegeneration.
Evidence for a Complex Influence of Nicotinic Acetylcholine Receptors on Hippocampal Serotonin Release
Authors: Kenny Paul J. , File Sandra E. , Neal Michael J.
Published: March 2003

Abstract: The effects of nicotine on 5‐hydroxytryptamine (5‐HT)release from serotonergic nerve endings in rat dorsal hippocampal slices werestudied. Nicotine (50‐500 μM) caused a concentration‐dependentincrease in 5‐HT release. This effect was antagonised by mecamylamine (0.5μM), indicating an action at nicotinic receptors. Nicotine‐evoked5‐HT release was not affected by tetrodotoxin (3 μM), cadmiumchloride (0.1 mM), or the absence of Ca2+ orNa+ in the superfusion medium. Unexpectedly, higher concentrationsof mecamylamine alone (1‐50 μM) increased 5‐HT release. Thissuggested the presence of inhibitory input to 5‐HT neurones and that theseinhibitory neurones possess tonically active nicotinic receptors. The effectof mecamylamine (50 μM) on 5‐HT release was reduced by themuscarinic M1 receptor agonist, McN‐A‐343 (100 μM), butpirenzepine (0.005‐1 μM), which blocks M1 receptors,alone increased 5‐HT release. Hippocampal serotonergic neurones are known topossess both excitatory nicotinic receptors and inhibitory M1receptors. Although there may be several explanations for our results, onepossible explanation is that nicotine stimulates 5‐HT release by activatingnicotinic heteroreceptors on 5‐HT terminals. Mecamylamine (0.5 μM)antagonises this effect, but higher concentrations increase 5‐HT releaseindirectly by blocking the action of endogenous acetylcholine on nicotinicreceptors situated on cholinergic neurones that provide muscarinic inhibitoryinput to 5‐HT neurones.
Transient Expression of NMDA Receptor Subunit NR2B in the Developing Rat Heart
Authors: Seeber Silke , Becker Kristina , Rau Thomas , Eschenhagen Thomas , Becker Cord‐Michael , Herkert Matthias
Published: March 2003

Abstract: NMDA receptors represent a subtype of the ionotropicglutamate receptor family, comprising three classes of subunits (NR1, NR2A‐D,NR3), which exhibit distinct patterns of regional and developmental expressionin the CNS. Recently, some NMDA receptor subunits have also been described inadult extraneuronal tissues and keratinocytes. However, their developmentalexpression patterns are currently unknown. With use of RT‐PCR and western blotanalysis, the expression of NMDA receptor subunit NR2B was investigated in thedeveloping rat heart. NR2B mRNA and protein were detected in heart tissue ofrats from embryonic day 14 until postnatal day 21 but disappeared 10 weeksafter birth. In contrast, no NMDA receptor subunit NR1,α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptor subunitGluR2, or anchoring postsynaptic density protein‐95 could be detected in ratheart at any developmental stage. Confocal microscopy of cultured cardiacmyocytes (CMs) from neonatal rats revealed distinct NR2B staining mainly ofintracellular structures. However, no functional NMDA receptor could bedetected on CMs by whole‐cell recordings. In conclusion, high concentrationsof NR2B protein can be detected in early rat heart development, but itsfunction still remains elusive.
Roles of Nuclear Factor κB in Neuronal Survival and Plasticity
Authors: Mattson Mark P. , Culmsee Carsten , Yu ZaiFang , Camandola Simonetta
Published: March 2003

Abstract: The transcription factor nuclear factor κB (NF‐κB) is moving to the forefront of the fields of apoptosis and neuronal plasticity because of recent findings showing that activation of NF‐κB prevents neuronal apoptosis in various cell culture and in vivo models and because NF‐κB is activated in association with synaptic plasticity. Activation of NF‐κB was first shown to mediate antiapoptotic actions of tumor necrosis factor in cultured neurons and was subsequently shown to prevent death of various nonneuronal cells. NF‐κB is activated by several cytokines and neurotrophic factors and in response to various cell stressors. Oxidative stress and elevation of intracellular calcium levels are particularly important inducers of NF‐κB activation. Activation of NF‐κB can interrupt apoptotic biochemical cascades at relatively early steps, before mitochondrial dysfunction and oxyradical production. Gene targets for NF‐κB that may mediate its anti‐apoptotic actions include the antioxidant enzyme manganese superoxide dismutase, members of the inhibitor of apoptosis family of proteins, and the calcium‐binding protein calbindin D28k. NF‐κB is activated by synaptic activity and may play important roles in the process of learning and memory. The available data identify NF‐κB as an important regulator of evolutionarily conserved biochemical and molecular cascades designed to prevent cell death and promote neuronal plasticity. Because NF‐κB may play roles in a range of neurological disorders that involve neuronal degeneration and/or perturbed synaptic function, pharmacological and genetic manipulations of NF‐κB signaling are being developed that may prove valuable in treating disorders ranging from Alzheimer’s disease to schizophrenia.
Feedback Control of Mesolimbic Somatodendritic Dopamine Release in Rat Brain
Authors: Rahman Shafiqur , McBride William J.
Published: March 2003

Abstract: The objective of this study was to examine the role of dopamine (DA) receptors in the nucleus accumbens (ACB) in controlling feedback regulation of mesolimbic somatodendritic DA release in the ventral tegmental area (VTA) of Wistar rats using ipsilateral dualprobe in vivo microdialysis. Perfusion of the ACB for 60 min with the DA uptake inhibitor GBR‐12909 (10‐1,000 μM) or nomifensine (10‐1,000 μM) dose‐dependently increased the extracellular levels of DA in ACB and concomitantly reduced the extracellular levels of DA in the VTA. Coperfusion of 100 μM nomifensine with either 100 μM SCH‐23390 (SCH), a D1 antagonist, or 100 μM sulpiride (SUL), a D2 receptor antagonist, produced either an additive (for SCH) or a synergistic (for SUL) elevation in the extracellular levels of DA in the ACB, whereas the reduction in the extracellular levels of DA in the VTA produced by nomifensine alone was completely prevented by addition of either antagonist. Application of 100 μM SCH or SUL alone through the microdialysis probe in the ACB increased the extracellular levels of DA in the ACB, whereas the extracellular levels of DA in the VTA remained unchanged. Overall, the results suggest that (a) increasing the synaptic levels of DA in the ACB activates a long‐loop negative feedback pathway to the VTA involving both D1 and D2 postsynaptic receptors and (b) terminal DA release within the ACB is regulated directly by D2 autoreceptors and may be indirectly regulated by D1 receptors, possibly on interneurons and/or through postsynaptic inhibition of the negative feedback loop.
Nerve Growth Factor in Glia and Inflammatory Cells of the Injured Rat Spinal Cord
Authors: Krenz Natalie R. , Weaver Lynne C.
Published: March 2003

Abstract: Nerve growth factor (NGF) is crucial for the development of sympathetic and small‐diameter sensory neurons and for maintenance of their mature phenotype. Its role in generating neuronal pathophysiology is less well understood. After spinal cord injury, central processes of primary afferent fibers sprout into the dorsal horn, contributing to the development of autonomic dysfunctions and pain. NGF may promote these states as it stimulates sprouting of small‐diameter afferent fibers and its concentration in the spinal cord increases after cord injury. The cells responsible for this increase must be identified to develop a strategy to prevent the afferent sprouting. Using immunocytochemistry, we identified cells containing NGF in spinal cord sections from intact rats and from rats 1 and 2 weeks after high thoracic cord transection. In intact rats, this neurotrophin was present in a few ramified microglia and in putative Schwann cells in the dorsal root. Within and close to the lesion of cord‐injured rats, NGF was in many activated, ramified microglia, in a subset of astrocytes, and in small, round cells that were neither glia nor macrophages. NGF‐immunore‐active putative Schwann cells were prevalent throughout the thoracolumbar cord in the dorsal roots and the dorsal root entry zones. Oligodendrocytes were never immunoreactive for this protein. Therapeutic strategies targeting spinal cord cells that produce NGF may prevent primary afferent sprouting and resulting clinical disorders after cord injury.
Caspase‐3 Mediated Neuronal Death After Traumatic Brain Injury in Rats
Authors: Clark Robert S. B. , Kochanek Patrick M. , Watkins Simon C. , Chen Minzhi , Dixon C. Edward , Seidberg Neal A. , Melick John , Loeffert J. Eric , Nathaniel Paula D. , Jin Kun Lin , Graham Steven H.
Published: March 2003

Abstract: During programmed cell death, activation of caspase‐3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase‐activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase‐3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro‐caspase‐3 in cytosolic cellular fractions and an increase in caspase‐3‐like enzyme activity were seen in injured brain versus control. Cleavage of the caspase‐3 substrates DNA‐dependent protein kinase and inhibitor of caspase‐activated deoxyribonuclease and co‐localization of cytosolic caspase‐3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase‐3 inhibitor N‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp‐fluoromethyl ketone (480 ng) after trauma reduced caspase‐3‐like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp‐fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase‐3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.
Decreased Protein Phosphatase 2A Activity in Hippocampal Long‐Term Potentiation
Authors: Fukunaga Kohji , Muller Dominique , Ohmitsu Masao , Bakó Eva , DePaoli‐Roach Anna A. , Miyamoto Eishichi
Published: March 2003

Abstract: Using autophosphorylated Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) as substrate, we now find that long‐term potentian (LTP) induction and maintenance are also associated with a significant decrease in calyculin A‐sensitive protein phosphatase (protein phosphatase 2A) activity, without changes in Mg2+‐dependent protein phosphatase (protein phosphatase 2C) activity. This decrease in protein phosphatase 2A activity was prevented when LTP induction was inhibited by treatment with calmidazolium or D‐2‐amino‐5‐phosphonopentanoic acid. In addition, the application of high‐frequency stimulation to 32P‐labeled hippocampal slices resulted in increases in the phosphorylation of a 55‐kDa protein immunoprecipitated with anti‐phosphatase 2A antibodies. Use of a specific antibody revealed that the 55‐kDa protein is the B′α subunit of protein phosphatase 2A. Following purification of brain protein phosphatase 2A, the B′α subunit was phosphorylated by CaM kinase II, an event that led to the reduction of protein phosphatase 2A activity. These results suggest that the decreased activity in protein phosphatase 2A following LTP induction contributes to the maintenance of constitutively active CaM kinase II and to the long‐lasting increase in phosphorylation of synaptic components implicated in LTP.
Thrombin attenuation is neuroprotective in the injured rat optic nerve
Authors: Friedmann Igor , Yoles Eti , Schwartz Michal
Published: March 2003

The functional loss that often follows injury of the mammalian CNS has been attributed not only to the immediate neural loss, but also to secondary neuronal degeneration caused by toxic biochemical mediators in the environment of the injured nerve. We report here that a high thrombin content, produced as a result of injury‐induced activation of prothrombin, appears to be an important mediator of secondary damage. Measurement of post‐traumatic neuronal survival in vivo revealed that post‐traumatic local application of the thrombin inhibitor N‐α‐(2‐naphthylsulphonylglycyl)‐4‐(d,l)‐amidinophenylalanine piperidide acetate in the rat optic nerve subjected to mild partial crush injury left twice as many retinal ganglion cells with functioning axons as in controls. Thus, by readjusting thrombin activity, thereby possibly obtaining a moderate post‐traumatic increase and thus gaining the benefit of thrombin without its toxic effects, it may be possible to create an environment that is more favourable towards post‐traumatic survival.
Carbohydrate–protein interactions between HNK‐1‐reactive sulfoglucuronyl glycolipids and the proteoglycan lectin domain mediate neuronal cell adhesion and neurite outgrowth
Authors: Miura Ryu , Ethell Iryna M. , Yamaguchi Yu
Published: March 2003

Lecticans, a family of chondroitin sulfate proteoglycans, represent the largest group of proteoglycans expressed in the nervous system. We previously showed that the C‐type lectin domains of lecticans bind two classes of sulfated cell surface glycolipids, sulfatides and HNK‐1‐reactive sulfoglucuronylglycolipids (SGGLs). In this paper, we demonstrate that the interaction between the lectin domain of brevican, a nervous system‐specific lectican, and cell surface SGGLs acts as a novel cell recognition system that promotes neuronal adhesion and neurite outgrowth. The Ig chimera of the brevican lectin domain bind to the surface of SGGL‐expressing rat hippocampal neurons. The substrate of the brevican chimera promotes adhesion and neurite outgrowth of hippocampal neurons. The authentic, full‐length brevican also promotes neuronal cell adhesion and neurite outgrowth. These activities of brevican substrates are neutralized by preincubation of cells with HNK‐1 monoclonal antibodies and by pretreatment of the brevican substrates with purified SGGLs. Brevican and HNK‐1 carbohydrates are coexpressed in specific layers of the developing hippocampus where axons from entorhinal neurons elongate. Our observations suggest that cell surface SGGLs and extracellular lecticans comprise a novel cell‐substrate recognition system operating in the developing nervous system.
Mitochondria deficient in complex I activity are depolarized by hydrogen peroxide in nerve terminals: relevance to Parkinson's disease
Authors: Chinopoulos Christos , Adam‐Vizi Vera
Published: March 2003

Deficiency of complex I in the respiratory chain and oxidative stress induced by hydrogen peroxide occur simultaneously in dopaminergic neurones in Parkinson's disease. Here we demonstrate that the membrane potential of in situ mitochondria (ΔΨm), as measured by the fluorescence change of JC‐l (5,5′,6,6′‐tetrachloro‐1,1,3,3′‐tetraethylbenzimidazolyl‐carbocyanine iodide), collapses when isolated nerve terminals are exposed to hydrogen peroxide (H2O2,100 and 500 µm) in combination with the inhibition of complex I by rotenone (5 nm−1 µm). H2O2 reduced the activity of complex I by 17%, and the effect of H2O2 and rotenone on the enzyme was found to be additive. A decrease in ΔΨm induced by H2O2 was significant when the activity of complex I was reduced to a similar extent as found in Parkinson's disease (26%). The loss of ΔΨm observed in the combined presence of complex I deficiency and H2O2 indicates that when complex I is partially inhibited, mitochondria in nerve terminals become more vulnerable to H2O2‐induced oxidative stress. This mechanism could be crucial in the development of bioenergetic failure in Parkinson's disease.
Rat cerebral cortex corticotropin‐releasing hormone receptors: evidence for receptor coupling to multiple G‐proteins
Authors: Grammatopoulos Dimitris K. , Randeva Harpal S. , Levine Michael A. , Kanellopoulou Korinna A. , Hillhouse Edward W.
Published: March 2003

The wide distribution of corticotrophin‐releasing hormone (CRH) receptors in brain and periphery appear to be important in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli. The type 1 receptors are highly expressed throughout the cerebral cortex, a region involved in cognitive function and modulation of stress responses, where they are coupled to the adenylyl cyclase system. Using techniques that analyse receptor‐mediated guanine‐nucleotide binding protein (G‐proteins) activation, we recently demonstrated that expressed type 1α CRH receptors are capable of activating multiple G‐proteins, which suggests that CRH can regulate multiple signalling pathways. In an effort to characterize the intracellular signals generated by CRH in the rat cerebral cortex we sought to identify G‐proteins activated by CRH in a physiological membrane environment. Rat cerebral cortical membrane suspensions were analysed for the ability of CRH to stimulate incorporation of [α‐32P]‐GTP‐γ‐azidoanilide to various G‐protein α‐chains. Our results show that CRH receptors are coupled to and activate at least five different G‐proteins (Gs, Gi, Gq/11, Go and Gz) with subsequent stimulation of at least two intracellular signalling cascades. In addition, the photoaffinity experiments indicated that the CRH receptors preferentially activate the 45 kDa form of the Gsα‐protein. This data may help elucidate the intracellular signalling pathways mediating the multiple actions of CRH especially under different physiological conditions.
Lipid constituents in oligodendroglial cells alter susceptibility to H2O2‐induced apoptotic cell death via ERK activation
Authors: Brand Annette , Gil Shosh , Seger Rony , Yavin Ephraim
Published: March 2003

The present work examines the effect of membrane lipid composition on activation of extracellular signal‐regulated protein kinases (ERK) and cell death following oxidative stress. When subjected to 50 µm docosahexaenoic acid (DHA, 22 : 6 n‐3), cellular phospholipids of OLN 93 cells, a clonal line of oligodendroglia origin low in DHA, were enriched with this polyunsaturated fatty acid. In the presence of 1 mmN,N‐dimethylethanolamine (dEa) a new phospholipid species analog was formed in lieu of phosphatidylcholine. Exposure of DHA‐enriched cells to 0.5 mm H2O2, caused sustained activation of ERK up to 24 h. At this time massive apoptotic cell death was demonstrated by ladder and TUNEL techniques. H2O2‐induced stress applied to dEa or DHA/dEa co‐supplemented cells showed only a transient ERK activation and no cell death after 24 h. Moreover, while ERK was rapidly translocated into the nucleus in DHA‐enriched cells, dEa supplements completely blocked ERK nuclear translocation. This study suggests that H2O2‐induced apoptotic cell death is associated with prolonged ERK activation and nuclear translocation in DHA‐enriched OLN 93 cells, while both phenomena are prevented by dEa supplements. Thus, the membrane lipid composition ultimately modulates ERK activation and translocation and therefore can promote or prevent apoptotic cell death.
Collapse of extracellular glutamate regulation during epileptogenesis: down‐regulation and functional failure of glutamate transporter function in rats with chronic seizures induced by kainic acid
Authors: Ueda Yuto , Doi Taku , Tokumaru Jun , Yokoyama Hidekatsu , Nakajima Akira , Mitsuyama Yoshio , Ohya‐Nishiguchi Hiroaki , Kamada Hitoshi , Willmore L. James
Published: March 2003

We used northern and western blotting to measure the quantity of glutamate and GABA transporters mRNA and their proteins within the hippocampal tissue of rats with epileptogenesis. Chronic seizures were induced by amygdalar injection of kainic acid 60 days before death. We found that expression of the mRNA and protein of the glial glutamate transporters GLAST and GLT‐1 were down‐regulated in the kainic acid‐administered group. In contrast, EAAC‐1 and GAT‐3 mRNA and their proteins were increased, while GAT‐1 mRNA and protein were not changed. We performed in vivo microdialysis in the freely moving state. During the interictal state, the extracellular glutamate concentration was increased, whereas the GABA level was decreased in the kainic acid group. Following potassium‐induced depolarization, glutamate overflow was higher and the recovery time to the basal release was prolonged in the kainic acid group relative to controls. Our data suggest that epileptogenesis in rats with kainic acid‐induced chronic seizures is associated with the collapse of extracellular glutamate regulation caused by both molecular down‐regulation and functional failure of glutamate transport.
Synaptotagmin I is a molecular target for lead
Authors: Bouton Christopher M. L. S. , Frelin Laurence P. , Forde Cameron E. , Godwin Hilary Arnold , Pevsner Jonathan
Published: March 2003

Lead poisoning can cause a wide range of symptoms with particularly severe clinical effects on the CNS. Lead can increase spontaneous neurotransmitter release but decrease evoked neurotransmitter release. These effects may be caused by an interaction of lead with specific molecular targets involved in neurotransmitter release. We demonstrate here that the normally calcium‐dependent binding characteristics of the synaptic vesicle protein synaptotagmin I are altered by lead. Nanomolar concentrations of lead induce the interaction of synaptotagmin I with phospholipid liposomes. The C2A domain of synaptotagmin I is required for lead‐mediated phospholipid binding. Lead protects both recombinant and endogenous rat brain synaptotagmin I from proteolytic cleavage in a manner similar to calcium. However, lead is unable to promote the interaction of either recombinant or endogenous synaptotagmin I and syntaxin. Finally, nanomolar concentrations of lead are able to directly compete with and inhibit the ability of micromolar concentrations of calcium to induce the interaction of synaptotagmin I and syntaxin. Based on these findings, we conclude that synaptotagmin I may be an important, physiologically relevant target of lead.
Molecular cloning and characterization of hNP22: a gene up‐regulated in human alcoholic brain
Authors: Fan Li , Jaquet Vincent , Dodd Peter R. , Chen Wenbin , Wilce Peter A.
Published: March 2003

An improved differential display technique was used to search for changes in gene expression in the superior frontal cortex of alcoholics. A cDNA fragment was retrieved and cloned. Further sequence of the cDNA was determined from 5′ RACE and screening of a human brain cDNA library. The gene was named hNP22 (human neuronal protein 22). The deduced protein sequence of hNP22 has an estimated molecular mass of 22.4 kDa with a putative calcium‐binding site, and phosphorylation sites for casein kinase II and protein kinase C. The deduced amino acid sequence of hNP22 shares homology (from 67% to 42%) with four other proteins, SM22α, calponin, myophilin and mp20. Sequence homology suggests a potential interaction of hNP22 with cytoskeletal elements. hNP22 mRNA was expressed in various brain regions but in alcoholics, greater mRNA expression occurred in the superior frontal cortex, but not in the primary motor cortex or cerebellum. The results suggest that hNP22 may have a role in alcohol‐related adaptations and may mediate regulatory signal transduction pathways in neurones.
Modulation of the neuronal dopamine transporter activity by the metabotropic glutamate receptor mGluR5 in rat striatal synaptosomes through phosphorylation mediated processes
Authors: Page Guylène , Peeters Magali , Najimi Mustapha , Maloteaux Jean‐Marie , Hermans Emmanuel
Published: March 2003

There is considerable evidence that the activity of the neuronal dopamine transporter (DAT) is dynamically regulated and a putative implication of its phosphorylation in this process has been proposed. However, there is little information available regarding the nature of physiological stimuli that contribute to the endogenous control of the DAT function. Based on the close relationship between glutamatergic and dopaminergic systems in the striatum, we investigated the modulation of the DAT activity by metabotropic glutamate receptors (mGluRs). Short‐term incubations of rat striatal synaptosomes with micromolar concentrations of the group I mGluR selective agonist (S)‐3,5‐dihydroxyphenylglycine were found to significantly decrease the DAT capacity and efficiency. This alteration was completely prevented by a highly selective mGluR5 antagonist, 2‐methyl‐6‐(phenylethynyl)pyridine hydrochloride (MPEP). The effect of (S)‐3,5‐dihydroxyphenylglycine was also inhibited by staurosporine and by selective inhibitors of protein kinase C and calcium calmodulin‐dependent protein kinase II. Co‐application of okadaic acid prolonged the transient effect of the agonist, supporting a critical role for phosphorylation in the modulation of the DAT activity by mGluRs. In conclusion, we propose that striatal mGluR5 contribute to the control of the DAT activity through concomitant activation of both protein kinase C and calcium calmodulin‐dependent protein kinase II.
Nurr1 enhances transcription of the human dopamine transporter gene through a novel mechanism
Authors: Sacchetti Paola , Mitchell Todd R. , Granneman James G. , Bannon Michael J.
Published: March 2003

The importance of the nuclear receptor nurr1 for the appropriate development of mesencephalic dopamine‐synthesizing neurons has been clearly demonstrated through the targeted disruption of the nurr1 gene. The persistence of nurr1 expression in adult tissue suggests a possible role for this transcription factor in the maintenance, as well as development, of the dopaminergic phenotype. To address this issue, we analyzed the effects of nurr1 on the transcriptional expression of the human dopamine transporter gene (hDAT), one of the most specific phenotypic markers for dopaminergic neurons. Nurr1 enhanced the transcriptional activity of hDAT gene constructs transiently transfected into a newly described cell line (SN4741) that expresses a dopaminergic phenotype, whereas other members of the NGFI‐B subfamily of nuclear receptors had lesser or no effects. Nurr1 activation of hDAT was not dependent upon heterodimerization with the retinoid X receptor. Unexpectedly, functional analysis of a series of gene constructs revealed that a region of the hDAT 5′‐flanking sequence devoid of NGFI‐B response element (NBRE)‐like sites mediated nurr1 activation. Additional experiments using a nurr1 mutant construct suggest that nurr1 activates hDAT transcription via a novel NBRE‐independent mechanism.
Coincident signalling between the Gi/Go‐coupled δ‐opioid receptor and the Gq‐coupled m3 muscarinic receptor at the level of intracellular free calcium in SH‐SY5Y cells
Authors: Yeo A. , Samways D. S. K. , Fowler C. E. , Gunn‐Moore F. , Henderson G.
Published: March 2003

In SH‐SY5Y cells, activation of δ‐opioid receptors with [d‐Pen2,5]‐enkephalin (DPDPE; 1 µm) did not alter the intracellular free Ca2+ concentration [Ca2+]i. However, when DPDPE was applied during concomitant Gq‐coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine‐M, it produced an elevation of [Ca2+]i. The DPDPE‐evoked increase in [Ca2+]i was abolished when the carbachol‐sensitive intracellular Ca2+ store was emptied. There was a marked difference between the concentration–response relationship for the elevation of [Ca2+]i by carbachol (EC50 13 µm, Hill slope 1) and the concentration–response relationship for carbachol's permissive action in revealing the δ‐opioid receptor‐mediated elevation of [Ca2+] (EC50 0.7 mm; Hill slope 1.8). Sequestration of free G protein βγ dimers by transient transfection of cells with a βγ binding protein (residues 495–689 of the C terminal tail of G protein‐coupled receptor kinase 2) reduced the ability of δ opioid receptor activation to elevate [Ca2+]i. However, DPDPE did not elevate either basal or oxotremorine‐M‐evoked inositol phosphate production indicating that δ‐opioid receptor activation did not stimulate phospholipase C. Furthermore, δ‐opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the δ‐opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca2+]i in SH‐SY5Y cells by a PLC‐independent mechanism. In SH‐SY5Y cells the coincident signalling between the endogenously expressed δ‐opioid and m3 muscarinic receptors appears to occur in the receptor activation‐Ca2+ release signalling pathway at a step after the activation of phospholipase C.
β‐Amyloid‐induced neuronal apoptosis requires c‐Jun N‐terminal kinase activation
Authors: Troy Carol M. , Rabacchi Sylvia A. , Xu Zhiheng , Maroney Anna C. , Connors Thomas J. , Shelanski Michael L. , Greene Lloyd A.
Published: March 2003

β‐Amyloid (Aβ) has been strongly implicated in the pathophysiology of Alzheimer's disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c‐Jun N‐terminal kinases (JNKs) and of their substrate, c‐Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated Aβ. The activities of JNK family members increased in neuronal PC12 cells within 2 h of Aβ treatment and reached 3–4‐fold elevation by 6 h. To test the role of these changes in death caused by Aβ, we examined the effects of CEP‐1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP‐1347 (100–300 nm) in the culture medium effectively blocked the increases in cellular JNK activity caused by Aβ and, at similar concentrations, protected both PC12 cells and sympathetic neurons from Aβ‐evoked‐death. Effective protection required addition of CEP‐1347 within 2 h of Aβ treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant‐negative c‐Jun construct also conferred protection from Aβ‐evoked death, supporting a model in which JNK activation contributes to death via activation of c‐Jun. Finally, CEP‐1347 blocked Aβ‐stimulated activation of caspase‐2 and ‐3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by Aβ and hence identify it as a potential therapeutic target in AD.
Differential splicing of transcripts encoding the orphanin FQ/nociceptin precursor
Authors: Arjomand Jamshid , Evans Chris J.
Published: March 2003

Orphanin FQ or nociceptin (OFQ/N), the heptadecapeptide agonist for the NOP receptor, is derived by proteolytic processing from a precursor protein, preproOFQ/N. Previous studies have reported alternative splicing between exons 3 and 4 of the mouse OFQ/N transcript, which, upon translation, would yield precursor proteins with different C‐termini. Using RT‐PCR, we identified similar alternative splicing of preproOFQ/N transcripts in humans and rats. In addition, we identified two novel human preproOFQ/N splice variants from which exon 2 has been excised and which also undergo alternative splicing between exons 3 and 4. Exon 2 contains the translational start site for preproOFQ/N and encodes the signal peptide sequence. In vitro translation of cRNAs lacking exon 2 yields shorter translation products which arise from an alternative initiator methionine located within exon 3. The resulting proteins would lack a signal peptide sequence, which would likely alter their cellular trafficking and processing.
Single GABAergic synaptic terminals from rat midbrain exhibit functional P2X and dinucleotide receptors, able to induce GABA secretion
Authors: Gómez‐Villafuertes Rosa , Gualix Javier , Miras‐Portugal M. Teresa
Published: March 2003

GABAergic terminals from rat midbrain characterized by immunolocalization of glutamic acid decarboxylase and/or the vesicular inhibitory amino acid transporter respond to ATP or P1,P5‐di(adenosine‐5′) pentaphosphate (Ap5A) with an increase in the intrasynaptosomal calcium concentration measured by a microfluorimetric technique in single synaptic terminals. The ATP response is mediated through the activation of P2X receptors with an abundant presence of P2X3 subunits. Ap5A, however, exerts its effects by acting through a different receptor termed the dinucleotide receptor. Both receptors, once activated in the presence of extrasynaptosomal calcium, induce a concentration‐dependent GABA release from synaptosomal populations with EC50 values of 16 and 20 µm for ATP and Ap5A, respectively. Specific inhibition of GABA release is obtained with pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (80 µm) on the ATP effect and with P1,P5‐di(inosine‐5′) pentaphosphate (100 nm) on the dinucleotide receptor.