bluuebunny/crossref_metadata_2025 huggingface dataset:
Contrasting Patterns and Cellular Specificity of Transcriptional Regulation of the Nuclear Receptor Nerve Growth Factor‐Inducible B by Haloperidol and Clozapine in the Rat Forebrain
Authors: Beaudry Geneviève , Langlois Marie‐Claire , Weppe Isabelle , Rouillard Claude , Lévesque Daniel
Published: March 2003

Abstract: This study was designed to investigate the possible involvement of members of the nuclear receptor family of transcription factors in the effects of antipsychotic drugs used in the treatment of schizophrenia. We have identified, using RT‐PCR screening, an important modulation of nerve growth factor‐inducible B (NGFI‐B) mRNA levels by typical and atypical neuroleptics in the rat forebrain. NGFI‐B, a member of the nuclear receptor family, can be observed in target structures of dopaminergic pathways. Using in situ hybridization, we also demonstrate that typical and atypical antipsychotics induced contrasting patterns of expression of NGFI‐B after both acute and chronic administration. An acute treatment with clozapine or haloperidol induces high NGFI‐B mRNA levels in the prefrontal and cingulate cortices and in the nucleus accumbens shell. However, haloperidol, but not clozapine, dramatically increases NGFI‐B expression in the dorsolateral striatum. In contrast, chronic treatment with clozapine reduces NGFI‐B expression below basal levels in the rat forebrain, whereas haloperidol still induces high NGFI‐B mRNA levels in the dorsolateral striatum. Finally, using a double in situ hybridization technique, we show that acute administration of both neuroleptics increases NGFI‐B expression in neurotensin‐containing neurons in the nucleus accumbens shell, whereas the effects of haloperidol in the dorsolateral striatum are mainly observed in enkephalin‐containing neurons. These results are the first demonstration that members of the nuclear receptor family of transcription factors could play an important role in the effects of antipsychotic drugs.
Behavioral Deficits Associated with Dietary Induction of Decreased Brain Docosahexaenoic Acid Concentration
Authors: Moriguchi Toru , Greiner Rebecca Sheaff , Salem Norman
Published: March 2003

Abstract: Docosahexaenoic acid (DHA), an n‐3 fatty acid, is rapidlydeposited during the period of rapid brain development. The influence of n‐3fatty acid deficiency on learning performance in adult rats over twogenerations was investigated. Rats were fed either an n‐3 fatty acid‐adequate(n‐3 Adq) or ‐deficient (n‐3 Def) diet for three generations (F1‐F3). Levelsof total brain n‐3 fatty acids were reduced in the n‐3 Def group by 83 and 87%in the F2 and F3 generations, respectively. In the Morris water maze, the n‐3Def group showed a longer escape latency and delayed acquisition of this taskcompared with the n‐3 Adq group in both generations. The acquisition andmemory levels of the n‐3 Def group in the F3 generation seemed to be lowerthan that of the F2 generation. The 22:5n‐6/22:6n‐3 ratio in the frontalcortex and dams' milk was markedly increased in the n‐3 Def group, and thisratio was significantly higher in the F3 generation compared with the F2generation. These results suggest that learning and cognitive behavior arerelated to brain DHA status, which, in turn, is related to the levels of themilk/dietary n‐3 fatty acids.
Monoamine release by neurons of a primitive nervous system: an amperometric study
Authors: Gillis Marc‐Antoine , Anctil Michel
Published: March 2003

We measured monoamine release from dissociated neurons of the sea pansy Renilla koellikeri, a representative of the most evolutionarily ancient animals with nervous systems, by real‐time monitoring of exocytosis using the amperometric method with carbon‐fiber microelectrodes. Depolarization‐induced, as well as spontaneously active, neurons exhibited calcium‐dependent exocytotic events at both the soma and the terminal bulb of neuritic processes. All spontaneously active neurons exhibited a bursting activity pattern in which amplitudes of exocytotic events appeared to be distributed in a quantal‐like fashion. Fast Fourier transform analysis of bursting activity in 20 such neurons revealed burst harmonics with a major frequency of 8 Hz and a dominant rate of 95 Hz for individual exocytotic events within bursts. The results suggest that exocytotic transmitter release is as ancient as neurons and that endogenously bursting neurons in the sea pansy are as complex as those of higher animals. In addition, the observation that both soma and neuritic terminals of the same neuron can release transmitter suggests that local release sites in these cnidarian neurons are not critical for nerve net function.
Hydrogen peroxide removal and glutathione mixed disulfide formation during metabolic inhibition in mesencephalic cultures
Authors: Ehrhart Julie , Zeevalk Gail D.
Published: March 2003

Compromised mitochondrial energy metabolism and oxidative stress have been associated with the pathophysiology of Parkinson's disease. Our previous experiments exemplified the importance of GSH in the protection of neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. This study further defines the role of oxidative stress during energy inhibition and begins to unravel the mechanisms by which GSH and other antioxidants may contribute to cell survival. Treatment of mesencephalic cultures with 10 µmbuthionine sulfoximine for 24 h depleted total GSH by 60%, whereas 3 h exposure to 5 mm3‐amino‐1,2,4‐triazole irreversibly inactivated catalase activity by 90%. Treatment of GSH‐depleted cells with malonate (40 mm) for 6, 12 or 24 h both potentiated and accelerated the time course of malonate toxicity, however, inhibition of catalase had no effect. In contrast, concomitant treatment with buthionine sulfoximine plus 3‐amino‐1,2,4‐triazole in the presence of malonate significantly potentiated toxicity over that observed with malonate plus either inhibitor alone. Consistent with these findings, GSH depletion enhanced malonate‐induced reactive oxygen species generation prior to the onset of toxicity. These findings demonstrate that early generation of reactive oxygen species during mitochondrial inhibition contributes to cell damage and that GSH serves as a first line of defense in its removal. Pre‐treatment of cultures with 400 µmascorbate protected completely against malonate toxicity (50 mm, 12 h), whereas treatment with 1 mmTrolox provided partial protection. Protein–GSH mixed disulfide formation during oxidative stress has been suggested to either protect vulnerable protein thiols or conversely to contribute to toxicity. Malonate exposure (50 mm) for 12 h resulted in a modest increase in mixed disulfide formation. However, exposure to the protective combination of ascorbate plus malonate increased membrane bound protein–GSH mixed disulfides three‐fold. Mixed disulfide levels returned to baseline by 72 h of recovery indicating the reversible nature of this formation. These results demonstrate an early role for oxidative events during mitochondrial impairment and stress the importance of the glutathione system for removal of reactive oxygen species. Catalase may serve as a secondary defense as the glutathione system becomes limiting. These findings also suggest that protein–GSH mixed disulfide formation under these circumstances may play a protective role.
Analysis of the mutant Drosophila N‐ethylmaleimide sensitive fusion‐1 protein in comatose reveals molecular correlates of the behavioural paralysis
Authors: Mohtashami Mahmood , Stewart Bryan A. , Boulianne Gabrielle L. , Trimble William S.
Published: March 2003

NEM‐sensitive fusion protein (NSF) is an ATPase required for many intracellular membrane trafficking steps. Recent studies have suggested that NSF alters the conformation of the SNAP receptors (SNAREs) to permit their interaction, or to uncouple them after they interact. Most organisms have a single NSF gene product but Drosophila express two highly related isoforms, dNSF‐1 and dNSF‐2. dNSF‐1 is encoded by the gene comatose (comt), first identified as the locus of a temperature‐sensitive paralytic mutation. Here we show that dNSF‐1 is most abundant in the nervous system and can be detected in larval and adult CNS. Subcellular fractionation revealed that dNSF‐1 was enriched in a vesicle fraction along with the synaptic vesicle protein synaptotagmin. comt flies maintained at the non‐permissive temperature rapidly accumulate sodium dodecyl sulfate (SDS)‐resistant SNARE complexes at the restrictive temperature, with concomitant translocation of dNSF‐1 from cytosol and membrane fractions into a Triton X‐100 insoluble fraction. The long recovery of comt flies after heat shock induced paralysis correlated with the irreversibility of this translocation. Interestingly, while dNSF‐1 also translocates in comtTP7 larvae, there is no associated neurophysiological phenotype at the neuromuscular junction (nmj) or accumulation of SDS‐resistant complexes in the CNS. Together, these results suggest that dNSF‐1 is required for adult neuronal function, but that in the larval nmj function may be maintained by other isoforms.
Molecular cloning and characterization of neural activity‐related RING finger protein (NARF): a new member of the RBCC family is a candidate for the partner of myosin V
Authors: Ohkawa Noriaki , Kokura Kenji , Matsu‐ura Toru , Obinata Takashi , Konishi Yoshiyuki , Tamura Taka‐aki
Published: March 2003

Activity‐dependent synaptic plasticity has been thought to be a cellular basis of memory and learning. The late phase of long‐term potentiation (L‐LTP), distinct from the early phase, lasts for up to 6 h and requires de novo synthesis of mRNA and protein. Many LTP‐related genes are enhanced in the hippocampus during pentyrenetetrazol (PTZ)‐ and kainate (KA)‐mediated neural activation. In this study, mice were administered intraperitoneal injections of PTZ 10 times, once every 48 h, and showed an increase in seizure indexes. Genes related to plasticity were efficiently induced in the mouse hippocampus. We used a PCR‐based cDNA subtraction method to isolate genes that are expressed in the hippocampus of repeatedly PTZ‐treated mice. One of these genes, neural activity‐related RING finger protein (NARF), encodes a new protein containing a RING finger, B‐box zinc finger, coiled‐coil (RBCC domain) and β‐propeller (NHL) domain, and is predominantly expressed in the brain, especially in the hippocampus. In addition, KA up‐regulated the expression of NARF mRNA in the hippocampus. This increase correlated with the activity of the NMDA receptor. By analysis using GFP‐fused NARF, the protein was found to localize in the cytoplasm. Enhanced green fluorescent protein‐fused NARF was also localized in the neurites and growth cones in neuronal differentiated P19 cells. The C‐terminal β‐propeller domain of NARF interacts with myosin V, which is one of the most abundant myosin isoforms in neurons. The NARF protein increases in hippocampal and cerebellar neurons after PTZ‐induced seizure. These observations indicated that NARF expression is enhanced by seizure‐related neural activities, and NARF may contribute to the alteration of neural cellular mechanisms along with myosin V.
Nuclear factor κB is a critical determinant in N‐methyl‐d‐aspartate receptor‐mediated neuroprotection
Authors: Lipsky Robert H. , Xu Ke , Zhu Daming , Kelly Charles , Terhakopian Artin , Novelli Antonello , Marini Ann M.
Published: March 2003

The role of a nuclear factor κB (NF‐κB) in NMDA receptor‐mediated neuroprotection is not known. A candidate sequence from the 5′ flanking region of exon 3 of the rat brain‐derived neurotrophic factor (BDNF) gene was used to show that exposure of rat cerebellar granule cells to 100 μm NMDA activated a specific DNA binding activity that was blocked by the NMDA receptor antagonist MK‐801. Anti‐p65 antibody or anti‐p50 antibody ‘supershifted’ the DNA binding activity, suggesting that the DNA–protein complex was composed of p65 and p50 subunits. NMDA receptor‐mediated neuroprotection was blocked when cerebellar neurons were transfected with a double‐stranded oligonucleotide containing the BDNF gene NF‐κB sequence. Furthermore, nuclear extracts prepared from neurons treated with NMDA and the double‐stranded NF‐κB oligonucleotide showed reduced DNA binding activity to the target sequence, supporting the idea that NF‐κB may be involved in the transcriptional activation of the BDNF gene. To address this issue, we quantified the level of exon 3‐specific BDNF mRNA. Relative to GAPDH mRNA levels and compared with untreated neurons, NMDA increased exon 3‐specific BDNF mRNA twofold. In contrast, pretreatment of neurons with the NF‐κB target DNA abolished the increase in BDNF mRNA following addition of NMDA. We also determined that BDNF itself induced an NF‐κB DNA binding activity. Taken together, these data support a mechanism where NF‐κB plays a critical role in NMDA‐mediated neuroprotection.
Different action on dopamine catabolic pathways of two endogenous 1,2,3,4‐tetrahydroisoquinolines with similar antidopaminergic properties
Authors: Antkiewicz‐Michaluk Lucyna , Michaluk Jerzy , Mokrosz Maria , Romanska Irena , Lorenc‐Koci Elbieta , Ohta Shigeru , Vetulani Jerzy
Published: March 2003

The effect of single and multiple 1‐methyl‐1,2,3,4‐tetrahydroisoquinoline (1MeTIQ) and 1‐benzyl‐1,2,3,4‐tetrahydroisoquinoline (1BnTIQ) administration on concentrations of dopamine and its metabolites: homovanillic acid (HVA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) and 3‐methoxytyramine (3MT) in three brain areas was studied HPLC with electrochemical detection in Wistar rats. The rate of dopamine catabolism in the striatum along the N‐oxidative and O‐methylation pathways was assessed by calculation of the ratio of appropriate metabolites to dopamine concentration. In addition, the spontaneous and apomorphine‐stimulated locomotor activity, and muscle rigidity was studied after acute administration of 1MeTIQ and 1BnTIQ. We have found that 1MeTIQ did not change the level of dopamine and HVA in all investigated structures both after a single and chronic administration. However, the levels of intermediary dopamine metabolites, DOPAC and 3MT, were distinctly affected. The level of DOPAC was strongly depressed (by 60–70%) while the level of extraneuronal matabolite 3MT was significantly elevated (by 170–200%). In contrast to 1MeTIQ, 1BnTIQ depressed the level of dopamine (by approximately 60%) and increased the level of total metabolite, HVA, (by 40%) especially in the striatum, but the levels of DOPAC and 3MT remained unchanged. The paper has shown that 1MeTIQ and 1BnTIQ produced different effects on dopamine catabolism. Potential neuroprotective compound 1MeTIQ did not change the rate of total dopamine catabolism, it strongly inhibited the monoamine oxidase (MAO)‐dependent catabolic pathway and significantly activated the catechol‐O‐methyltransferase (COMT)‐dependent O‐methylation. In contrast 1BnTIQ, a compound with potential neurotoxic activity, produced the significant increase of the rate of dopamine metabolism with strong activation of the oxidative MAO‐dependent catabolic pathway. Interestingly, both compounds produced similar antidopaminergic functional effects: antagonism of apomorphine hyperactivity and induction of muscle rigidity. The results may explain the biochemical basis of the neuroprotective and of the neurotoxic properties endogenous brain tetrahydroisoquinoline derivatives.
Hypo‐osmotic swelling‐activated release of organic osmolytes in brain slices: implications for brain oedema in vivo
Authors: Bothwell J. H. , Rae C. , Dixon R. M. , Styles P. , Bhakoo K. K.
Published: March 2003

A decrease in the intracellular levels of osmotically active species has invariably been seen after swelling of mammalian brain tissue preparations. The exact identity of the species, and the manner of their decrease, remain to be described. We investigated the swelling‐activated decrease of organic osmolytes in rat cortical brain slices using 1H‐ and 31P‐magnetic resonance spectroscopy. We found that acute hypo‐osmotic shock causes decreases in the levels of a range of intracellular amino acids and amino acid derivatives, N‐acetyl‐aspartate, creatine, GABA, glutamate, hypotaurine, and also in the levels of the methylamines glycerol‐phosphorylcholine, phosphorylcholine and choline. Incubation of cortical slices with the anion channel blockers niflumic acid and tamoxifen caused inhibition of organic osmolyte efflux, suggesting that such osmolyte efflux occurs through anion channels. Intracellular phosphocreatine was also seen to decrease during acute hypo‐osmotic superfusion, although intracellular ATP remained constant. In addition, the acidification of an intracellular compartment was observed during hypo‐osmotic superfusion. Our results suggest a link between brain energy reserve and brain osmoregulation.
The non‐peptidyl fungal metabolite L‐783,281 activates TRK neurotrophin receptors
Authors: Wilkie Neil , Wingrove Peter B. , Bilsland James G. , Young Lisa , Harper Sarah J. , Hefti Franz , Ellis Semantha , Pollack Scott J.
Published: March 2003

Neurotrophin binding to the extracellular surface of the Trk family of tyrosine kinase receptors leads to the activation of multiple signalling cascades, culminating in neuroregenerative effects, including neuronal survival and neurite outgrowth. Since neurotrophins themselves are not ideal drug candidates due to their poor pharmacokinetic behaviour and bioavailability, small molecule neurotrophin mimetics may be beneficial in treating a number of neurodegenerative disorders. The present study demonstrates that L‐783,281, a non‐peptidyl fungal metabolite, is capable of stimulating TrkA, B and C phosphorylation to various extents in CHO cells stably expressing human Trk receptors. L‐783,281 also stimulated Trk phosphorylation in a number of rat and human primary neuronal cultures, whereas the highly similar compound, L‐767,827, was without effect. Mechanistic studies utilizing transiently transfected PDGF/TrkA and TrkA/PDGF chimeras, demonstrated that L‐783,281 is likely to interact with the intracellular domain of the TrkA receptor. Further investigations suggested that L‐783,281 was nevertheless able to instigate receptor dimerization by binding in a non‐covalent manner. Although the cytotoxicity of the compound was shown to preclude its effects in neuronal survival and neurite outgrowth assays, it is a prototype for a small molecule neurotrophin mimetic that activates Trk by interacting at a site different from the neurotrophin‐binding site.
Immunoseparation of sphingolipid‐enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG‐1 by anti‐GD3 ganglioside monoclonal antibody
Authors: Prinetti Alessandro , Prioni Simona , Chigorno Vanna , Karagogeos Domna , Tettamanti Guido , Sonnino Sandro
Published: March 2003

Rat cerebellar granule cells differentiated in culture were fed [1–3H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent‐insoluble sphingolipid‐enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1–3H]sphingosine‐fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c‐Src, Lyn and Fyn and in the GPI‐anchored neuronal adhesion molecule TAG‐1. The cell lysate and the sphingolipid‐enriched membrane fraction were subjected to immunoprecipitation with anti‐GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid‐enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid‐enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c‐Src, Lyn, Fyn and TAG‐1 were associated with the anti‐GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti‐GD3 antibody immunoprecipitation, and that the association of c‐Src, Fyn, Lyn, and TAG‐1 with the sphingolipid‐enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.
Internalization of mammalian fluorescent cellular prion protein and N‐terminal deletion mutants in living cells
Authors: Lee Kil S. , Magalhães Ana C. , Zanata Silvio M. , Brentani Ricardo R. , Martins Vilma R. , Prado Marco A. M.
Published: March 2003

The cellular prion protein (PrPc) is a glycosylphosphatidylinositol (GPI)‐anchored plasma membrane protein whose conformational altered forms (PrPsc) are known to cause neurodegenerative diseases in mammals. In order to investigate the intracellular traffic of mammalian PrPcin living cells, we have generated a green fluorescent protein (GFP) tagged version of PrPc. The recombinant protein was properly anchored at the cell surface and its distribution pattern was similar to that of the endogenous PrPc, with labeling at the plasma membrane and in an intracellular perinuclear compartment. Comparison of the steady‐state distribution of GFP‐PrPcand two N‐terminal deletion mutants (Δ32‐121 and Δ32‐134), that cause neurological symptoms when expressed in PrP knockout mice, was carried out. The mutant proteins accumulated in the plasma membrane at the expense of decreased labeling in the perinuclear region when compared with GFP‐PrPc. In addition, GFP‐PrPc, but not the two mutants, internalized from the plasma membrane in response to Cu2+treatment and accumulated at a perinuclear region in SN56 cells. Our data suggest that GFP‐PrPccan be used to follow constitutive and induced PrPctraffic in living cells.
Role of polyamine metabolism in kainic acid excitotoxicity in organotypic hippocampal slice cultures
Authors: Liu Wei , Liu Ruolan , Schreiber Steve S. , Baudry Michel
Published: March 2003

Polyamines are ubiquitous cations that are essential for cell growth, regeneration and differentiation. Increases in polyamine metabolism have been implicated in several neuropathological conditions, including excitotoxicity. However, the precise role of polyamines in neuronal degeneration is still unclear. To investigate mechanisms by which polyamines could contribute to excitotoxic neuronal death, the present study examined the role of the polyamine interconversion pathway in kainic acid (KA) neurotoxicity using organotypic hippocampal slice cultures. Treatment of cultures with N1,N(2)‐bis(2,3‐butadienyl)‐1,4‐butanediamine (MDL 72527), an irreversible inhibitor of polyamine oxidase, resulted in a partial but significant neuronal protection, especially in CA1 region. In addition, this pre‐treatment also attenuated KA‐induced increase in levels of lipid peroxidation, cytosolic cytochrome C release and glial cell activation. Furthermore, pre‐treatment with a combination of cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) and MDL 72527 resulted in an additive and almost total neuronal protection against KA toxicity, while the combination of MDL 72527 and EUK‐134 (a synthetic catalase/superoxide dismutase mimetic) did not provide additive protection. These data strongly suggest that the polyamine interconversion pathway partially contributes to KA‐induced neurodegeneration via the production of reactive oxygen species.
Overexpression of manganese superoxide dismutase attenuates neuronal death in human cells expressing mutant (G37R) Cu/Zn‐superoxide dismutase
Authors: Flanagan Shawn W. , Anderson Richard D. , Ross Mark A. , Oberley Larry W.
Published: March 2003

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor function and eventual death as a result of degeneration of motor neurons in the spinal cord and brain. The discovery of mutations in SOD1, the gene encoding the antioxidant enzyme Cu/Zn‐superoxide dismutase (CuZnSOD), in a subset of ALS patients has led to new insight into the pathophysiology of ALS. Utilizing a novel adenovirus gene delivery system, our laboratory has developed a human cell culture model using chemically differentiated neuroblastoma cells to investigate how mutations in SOD1 lead to neuronal death. Expression of mutant SOD1 (G37R) resulted in a time and dose‐related death of differentiated neuroblastoma cells. This cell death was inhibited by overexpression of the antioxidant enzyme manganese superoxide dismutase (MnSOD). These observations support the hypothesis that mutant SOD1‐ associated neuronal death is associated with alterations in oxidative stress, and since MnSOD is a mitochondrial enzyme, suggest that mitochondria play a key role in disease pathogenesis. Our findings in this model of inhibition of mutant SOD1‐associated death by MnSOD represent an unique approach to explore the underlying mechanisms of mutant SOD1 cytotoxicity and can be used to identify potential therapeutic agents for further testing.
Altered glycosylation of acetylcholinesterase in APP (SW) Tg2576 transgenic mice occurs prior to amyloid plaque deposition
Authors: Fodero Lisa R. , Sáez‐Valero Javier , McLean Catriona A. , Martins Ralph N. , Beyreuther Konrad , Masters Colin L. , Terry A. Robertson  , Small David H.
Published: March 2003

Previous studies have shown that a minor glycoform of acetylcholinesterase (AChE) is increased in Alzheimer's disease brain and cerebrospinal fluid. This glycoform can be distinguished from other AChE species by its lack of binding to concanavalin A (Con A). In this study, the temporal relationship between AChE glycosylation and Aβ deposition was examined in Tg2576 mice. There was a significant (p < 0.05) difference in AChE glycosylation in Tg2576 mice compared with age‐matched background strain control mice at 4 months of age. This difference in glycosylation was also observed in 8‐ and 12‐month‐old Tg2576 mice. In contrast, Aβ plaques were only seen in the Tg2576 mice at 12 months of age, and were not detected at 4 and 8 months of age. Soluble human‐sequence Aβ was detected as early as 4 months of age in the transgenic mice. The altered AChE glycosylation was due to an increase in a minor AChE isoform, which did not bind Con A, similar to that previously observed to be increased in Alzheimer's disease brain and cerebrospinal fluid. The results demonstrate that in transgenic mice altered AChE glycosylation is associated with very early events in the development of AD‐like pathology. The study supports the possibility that glycosylation may also be a useful biomarker of AD.
Expression and characterization of the Drosophila X11‐like/Mint protein during neural development
Authors: Hase Momoko , Yagi Yoshimasa , Taru Hidenori , Tomita Susumu , Sumioka Akio , Hori Kazuya , Miyamoto Kanako , Sasamura Takeshi , Nakamura Makoto , Matsuno Kenji , Suzuki Toshiharu
Published: March 2003

The X11‐like (X11L) protein was originally isolated as a protein bound to the cytoplasmic domain of the β‐amyloid precursor protein (APP), which is associated with Alzheimer's disease. In mammals, X11L is believed to play an important role in the regulation of APP metabolism. Here we isolated and characterized the Drosophila X11L (dX11L) protein, also may be referred to this protein as Drosophila Mint (dMint), Lin 10 (dLin10) or X11 (dX11), is thought to be expressed in neuronal tissues from late embryonic through to the adult stages of the fly. The phosphotyrosine interaction domain of dX11L interacts with the cytoplasmic domain of the Drosophila amyloid precursor protein‐like (APPL) similar to the way human X11L (hX11L) interacts with APP. Overexpression of dX11L on post‐mitotic neurons had a lethal effect on flies and, when it was localized to the eye imaginal disc, disruption of compound eye morphology due to enhanced apoptosis of neuronal cells was observed. Overexpression of hX11L and the PDZ domain of dX11L resulted in identical eye phenotypes. The PDZ domain is highly conserved between Drosophila and human, and appears to be responsible for this phenotype. Our findings suggest that the X11L family may be involved with the regulation of apoptosis during neural cell development and that aberrant X11L function could be contribute in this way to the neuronal degeneration observed in Alzheimer's disease.
Altered arachidonic acid biosynthesis and antioxidant protection mechanisms in Schwann cells grown in elevated glucose
Authors: Mîinea Cristinel , Kuruvilla Rejji , Merrikh Houra , Eichberg Joseph
Published: March 2003

In cultured Schwann cells, elevated glucose induces alterations in arachidonic acid metabolism that cause a decrease in the content of glycerophospholipid arachidonoyl‐containing molecular species (ACMS). This could result from decreased de novo arachidonic acid biosynthesis, or increased arachidonic acid release from phospholipids. Incorporation of radioactive 8,11,14‐eicosatrienoic acid into ACMS was lower for cells grown in 30 mm versus 5 mm glucose, consistent with a decrease in Δ5 desaturase activity. However, neither basal arachidonic acid release from prelabeled cells nor stimulated generation of arachidonic acid in the presence of the reacylation inhibitor, thimerosal, the phosphotyrosine phosphatase inhibitor, bipyridyl peroxovanadium, or both together, were altered by varying the glucose concentrations, indicating that arachidonic acid turnover did not contribute to ACMS depletion. Free cytosolic NAD+/NADH decreased, whereas NADP+/NADPH remained unchanged for cells grown in elevated glucose, implying that decreased desaturase activity is a result of metabolic changes other than cofactor availability. Schwann cells in elevated glucose were susceptible to oxidative stress, as shown by increased malondialdehyde, depleted glutathione levels, and reduced cytosolic superoxide dismutase activity. Glutathione‐altering compounds had no effect on ACMS levels, in contrast to N‐acetylcysteine and α‐lipoic acid, which partly corrected ACMS depletion in phosphatidylcholine. These findings suggest that in the Schwann cell cultures, a high glucose level elicits oxidative stress and weakens antioxidant protection mechanisms which could decrease arachidonic acid biosynthesis and that this deficit can be partly corrected by treatment with exogenous antioxidants.
Agonists activate Gi1α or Gi2α fused to the human mu opioid receptor differently
Authors: Massotte Dominique , Brillet Karl , Kieffer Brigitte , Milligan Graeme
Published: March 2003

As preferential coupling of opioid receptor to various inhibitory Gα subunits is still under debate, we have investigated the selectivity of the human mu opioid receptor fused to a pertussis toxin insensitive C351I Gi1α or C352I Gi2α in stably transfected HEK 293 cells. Overall agonist binding affinities were increased for both fusion constructs when compared to the wild type receptor. [35S]GTPγS binding was performed on pertussis toxin treated cells to monitor coupling efficiency of the fusion constructs. Upon agonist addition hMOR‐C351I Gi1a exhibited an activation profile similar to the non‐fused receptor while hMOR‐C352I Gi2α was poorly activated. Interestingly no correlation could be drawn between agonist binding affinity and efficacy. Upon agonist addition, forskolin‐stimulated cAMP production, as measured using a reporter gene assay, was inhibited by signals transduced via the fused Gi1α and Gi2α mainly. In contrast both fusion constructs were able to initiate ERK‐MAPK phosphorylation via coupling to endogenous G proteins only. In conclusion our data indicate that hMOR couples more efficiently to Gi1α than Gi2α and that the coupling efficacy is clearly agonist‐dependent.
Alterations in metabolism and gene expression in brain regions during cuprizone‐induced demyelination and remyelination
Authors: Jurevics Helga , Largent Carrie , Hostettler Janell , Sammond Deanne W. , K. Matsushima Glenn ,   Amber Kleindienst,, Toews Arrel D. , Morell Pierre
Published: March 2003

Exposure of mice to the copper chelator, cuprizone, results in CNS demyelination. There is remyelination after removal of the metabolic insult. We present brain regional studies identifying corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6 weeks of exposure. We examined recovery of cerebroside and ability to synthesize cerebroside and cholesterol following removal of the toxicant. The temporal pattern for concentration of myelin basic protein resembled that of cerebroside. We applied Affymetrix GeneChip technology to corpus callosum to identify temporal changes in levels of mRNAs during demyelination and remyelination. Genes coding for myelin structural components were greatly down‐regulated during demyelination and up‐regulated during remyelination. Genes related to microglia/macrophages appeared in a time‐course (peaking at 6 weeks) correlating with phagocytosis of myelin and repair of lesions. mRNAs coding for many cytokines had peak expression at 4 weeks, compatible with intercellular signaling roles. Of interest were other genes with temporal patterns correlating with one of the three above patterns, but of function not obviously related to demyelination/remyelination. The ability to correlate gene expression with known pathophysiological events should help in elucidating further function of such genes as related to demyelination/remyelination.
Cerebral dicarboxylate transport and metabolism studied with isotopically labelled fumarate, malate and malonate
Authors: Hassel Bjørnar , Bråthe Anders , Petersen Dirk
Published: March 2003

Transport and metabolism of dicarboxylates may be important in the glial‐neuronal metabolic interplay. Further, exogenous dicarboxylates have been suggested as cerebral energy substrates. After intrastriatal injection of [14C]fumarate or [14C]malate, glutamine attained a specific activity 4.1 and 2.6 times higher than that of glutamate, respectively, indicating predominantly glial uptake of these four‐carbon dicarboxylates. In contrast, the three‐carbon dicarboxylate [14C]malonate gave a specific activity in glutamate which was approximately five times higher than that of glutamine, indicating neuronal uptake of malonate. Therefore, neurones and glia take up different types of dicarboxylates, probably by different transport mechanisms. Labelling of alanine from [14C]fumarate and [14C]malate demonstrated extensive malate decarboxylation, presumably in glia. Intravenous injection of 75 µmol [U‐13C]fumarate rapidly led to high concentrations of [U‐13C]fumarate and [U‐13C]malate in serum, but neither substrate labelled cerebral metabolites as determined by 13C NMR spectroscopy. Only after conversion of [U‐13C]fumarate into serum glucose was there 13C‐labelling of cerebral metabolites, and only at <10% of that obtained with 75 µmol [3‐13C]lactate or [2‐13C]acetate. These findings suggest a very low transport capacity for four‐carbon dicarboxylates across the blood–brain barrier and rule out a role for exogenous fumarate as a cerebral energy substrate.
Role of the 5′ untranslated region (UTR) in the tissue‐specific regulation of rat tryptophan hydroxylase gene expression by stress
Authors: Chamas Firas , Sabban Esther L.
Published: March 2003

There are tissue specific discrepancies in expression of tryptophan hydroxylase (TPH) between the pineal gland and brainstem. TPH mRNA levels in the pineal are much higher than in the brainstem, however, the two tissues contain comparable protein levels. This discrepancy could result from different translation efficiency of two of the TPH mRNA isoforms. Using PCR‐based methods, we analyzed the relative expression, in pineal and brainstem, of two TPH mRNA isoforms differing in the length of their untranslated region (5′UTR). The levels of the TPHα were found to be 960‐fold more abundant than the 51‐nucleotide longer TPHβ, in the pineal. TPHβ was also detected for the first time in the brainstem, where TPHβ/TPHα was about five‐fold higher than in the pineal. To study the role of the different 5′UTRs, each was cloned in‐frame upstream of luciferase, and transfected into PC12 cells. Both 5′UTRs enhanced luciferase activity, with TPHβ 5′UTR being more effective than TPHα 5′UTR, indicating selective regulation of translation efficiency. We also examined whether physiological manipulations alter the distribution of the TPH mRNA isoforms. Repeated stress had no effect in pineal, but led to a marked preferential induction of TPHβ in brainstem. Modulation of TPH gene expression in serotonergic neurons could result from selective and tissue specific regulation of its mRNA isoforms.
Activation of stress signaling molecules in bat brain during arousal from hibernation
Authors: Lee Moonyong , Choi Inho , Park Kyoungsook
Published: March 2003

Induction of glucose‐regulated proteins (GRPs) is a ubiquitous intracellular response to stresses such as hypoxia, glucose starvation and acidosis. The induction of GRPs offers some protection against these stressesin vitro, but the specific role of GRPsin vivoremains unclear. Hibernating bats present a goodin vivomodel to address this question. The bats must overcome local high oxygen demand in tissue by severe metabolic stress during arousal thermogenesis. We used brain tissue of a temperate batRhinolopus ferrumequinumto investigate GRP induction by high metabolic oxygen demand and to identify associated signaling molecules. We found that during 30 min of arousal, oxygen consumption increased from nearly zero to 11.9/kg/h, which was about 8.7‐fold higher than its active resting metabolic rate. During this time, body temperature rose from 7°C to 35°C, and levels of TNF‐α and lactate in brain tissue increased 2–2.5‐fold, indicating a high risk of oxygen shortage. Concomitantly, levels of GRP75, GRP78 and GRP94 increased 1.5–1.7‐fold. At the same time, c‐Jun N‐terminal protein kinase (JNK) activity increased 6.4‐fold, and extracellular signal‐regulated protein kinase (ERK) activity decreased to a similar degree (6.1‐fold). p38 MAPK activity was very low and remained unchanged during arousal. In addition, survival signaling molecules protein kinase B (Akt) and protein kinase C (PKC) were activated 3‐ and 5‐fold, respectively, during arousal. Taken together, our results showed that bat brain undergoes high oxygen demand during arousal from hibernation. Up‐regulation of GRP proteins and activation of JNK, PKCγ and Akt may be critical for neuroprotection and the survival of bats during the repeated process.
Calcium/calmodulin‐dependent kinase II phosphorylation of the GABAA receptor α1 subunit modulates benzodiazepine binding
Authors: Churn Severn B. , Rana Aniruddha , Lee Kangmin , Parsons J. Travis , De Blas Angel , Delorenzo Robert J.
Published: March 2003

γ‐Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post‐translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin‐dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric‐modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor α1 subunit. The data suggest that the CaM kinase II‐dependent phosphorylation of the GABAA receptor α1 subunit modulated allosteric modulator binding to the GABAA receptor.
Distinct responses of basal ganglia substance P systems to low and high doses of methamphetamine
Authors: Hanson Glen R. , Bush Lloyd , Keefe Kristen A. , Alburges Mario E.
Published: March 2003

Substance P (SP) is a neuropeptide closely associated with basal ganglia dopaminergic neurons. Because some neuropeptide systems in the basal ganglia (i.e. neurotensin and metenkephalin) are differentially affected by treatment with low or high doses of methamphetamine, we determined if basal ganglia SP pathways were also differentially influenced in a dose‐dependent manner by this psychostimulant. Employing in vivo microdialysis, it was observed that the lowdose (0.5 mg/kg) of methamphetamine increased the extracellular concentration of SP in the substantia nigra, but not in globus pallidus or striatum. In contrast, the high dose (10 mg/kg) of methamphetamine did not increase extracellular SP content in any of these structures. The effect of the low‐dose methamphetamine treatment on nigral extracellular SP levels was blocked by pre‐treatment with either a D1 or D2 antagonist. In addition, 12 h after similar methamphetamine treatments, a dose‐dependent differential response in SP tissue levels occurred in some of the regions examined. When these changes occurred, the low dose of methamphetamine usually reduced, whereas the high dose increased, SP tissue content. This study demonstrated opposite responses of the basal ganglia SP system to low and high doses of methamphetamine and suggested that a combination of dopamine D1 and D2 receptor activity contributed to these effects.
Characterization of a tyramine receptor from Caenorhabditis elegans
Authors: Rex Elizabeth , Komuniecki Richard W.
Published: March 2003

Octopamine (OA) plays an important role in the regulation of a number of key processes in nematodes, including pharyngeal pumping, locomotion and egg‐laying. However, while putative OA receptors can be tentatively identified in the Caenorhabditis elegans database, no OA receptors have been functionally characterized from any nematode. We have isolated two cDNAs, ser‐2 and ser‐2a, encoding putative C.elegans serotonin/OA receptors (C02D4.2, ser‐2). The sequences of these cDNAs differ from that predicted by GeneFinder and lack 42 bp of exon 2. In addition, ser‐2a appears to be alternatively spliced and lacks a predicted 23 amino acids in the third intracellular loop. COS‐7 cells expressing SER‐2 bind [3H]LSD in the low nM range and exhibit Kis for tyramine, octopamine and serotonin of 0.07, 2, and 13.7 µm, respectively. Significantly, tyramine reduces forskolin‐stimulated cAMP levels in HEK293 cells stably expressing SER‐2 with an IC50 of about 360 nm, suggesting that SER‐2 is a tyramine receptor.
Up‐regulation of the PACAP type‐1 receptor (PAC1) promoter by neurotrophins in rat PC12 cells and mouse cerebellar granule cells via the Ras/mitogen‐activated protein kinase cascade
Authors: Jamen Françoise , Bouschet Tristan , Laden Jean‐Charles , Bockaert Joël , Brabet Philippe
Published: March 2003

The pituitary adenylate cyclase‐activating polypeptide type‐1 receptor (PAC1) has been involved in the survival and differentiation of neuroblasts during development. This study examined the effects of various neurotrophins on the activity of the mouse PAC1 promoter/luciferase reporter constructs in rat PC12 cells and in 8‐day‐old mouse cerebellar granule cells. In PC12 cells, both differentiating factors such as nerve growth factor (NGF) and mitogens such as epidermal growth factor (EGF) and insulin growth factor‐1 (IGF‐1) up‐regulated PAC1 promoter activity by 2–4‐fold in a concentration‐dependent manner. Although PACAP differentiated the PC12 cells, it had no effect on the PAC1 promoter and antagonized the stimulatory effect of NGF. In cerebellar granule cells, IGF‐1 and brain‐derived neurotrophic factor (BDNF) also stimulated the activity of the PAC1 promoter. NGF and IGF‐1 increased endogenous PAC1 mRNA levels, and the NGF‐induced up‐regulation is the result of an increase in transcription from PAC1 promoter instead of an increase in mRNA stability. The mitogen‐activated protein kinase (MAPK) kinase inhibitor, PD98059, prevented the transcriptional effects both in PC12 and cerebellar granule cells. Moreover, expression of dominant‐negative Ras protein in PC12 cells also prevented the NGF effect. Our results show that the PAC1 promoter can be up‐regulated by diverse neurotrophins via an MAPK‐dependent pathway and suggest a role for the Ras protein.
Matrix metalloproteinase mediated degradation of basement membrane proteins in Trembler J neuropathy nerves
Authors: Misko A. , Ferguson T. , Notterpek L.
Published: March 2003

A single point mutation in peripheral myelin protein 22 (pmp22) of the Trembler‐J (TrJ) mouse models the human peripheral neuropathy, Charcot–Marie–Tooth disease type 1 A (CMT1A). An unexplored aspect of this disease is the gradual remodeling of the extracellular matrix in affected nerves. To elucidate the mechanism responsible for these changes, the levels of the extracellular matrix molecules laminin, collagen IV, and fibronectin were determined. In TrJ nerves, laminin is modestly increased while full‐length forms of collagen IV and fibronectin are decreased. Matrix metalloproteinases (MMPs) are known to degrade multiple matrix molecules; therefore, nerves were assayed for MMP‐2 and MMP‐9 proteins. In neuropathy nerves, elevated levels of MMP‐2 and MMP‐9 were detected on western blots, and gelatin zymography confirmed the up‐regulation of gelatinalytic activity in affected samples. Immunostaining studies revealed an increase in the numbers of MMP‐2‐ and MMP‐9‐expressing cells in TrJ nerves. Cell type‐specific immunolabeling showed that infiltrating macrophages are a significant source of both MMP‐2 and MMP‐9. Finally, the degradation of exogenous collagen IV by TrJ nerve lysates was prevented with a specific MMP inhibitor. Together these observations suggest that infiltration by MMP‐expressing macrophages contributes to the remodeling of the TrJ nerve matrix.
Heterogeneity of the calcium‐induced permeability transition in isolated non‐synaptic brain mitochondria
Authors: Kristián Tibor , Weatherby Tina M. , Bates Timothy E. , Fiskum Gary
Published: March 2003

Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium‐induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non‐synaptosomal rat brain mitochondria were incubated in a potassium‐based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron‐opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large‐amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron‐dense precipitates that contained calcium, as determined by electron‐energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.