bluuebunny/crossref_metadata_2025 huggingface dataset:
Focal neurometabolic alterations in mice deficient for succinate semialdehyde dehydrogenase
Authors: Gibson K. M. , Schor D. S. M. , Gupta M. , Guerand W. S. , Senephansiri H. , Burlingame T. G. , Bartels H. , Hogema B. M. , Bottiglieri T. , Froestl W. , Snead O. C. , Grompe M. , Jakobs C.
Published: March 2003

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1–/–) deficient mice previously revealed elevated γ‐hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified β‐alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of β‐alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5‐dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH‐deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.
Injury‐induced ‘switch’ from GTP‐regulated to novel GTP‐independent isoform of tissue transglutaminase in the rat spinal cord
Authors: Festoff Barry W. , SantaCruz Karen , Arnold Paul M. , Sebastian Cyril T. , Davies Peter J. A. , Citron Bruce A.
Published: March 2003

We recently found that alternative transcripts of tissue transglutaminase (tTG or TG2) were present in hippocampal brain regions of Alzheimer's disease (AD), but not in control, non‐demented, age‐matched brains. Since antecedent non‐severe trauma has been implicated in AD and other neurodegenerative diseases, such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), we were interested in whether alternative transcripts might be detected in a model of neurotrauma, controlled‐contusion spinal cord injury (SCI) in the rat. Implicated in diverse roles from growth and differentiation to apoptotic cell death, only bifunctional tTG, of the nine member TG family, has dual catalytic activities: guanine trinucleotide (GTP) hydrolyzing activity (GTPase), as well as protein cross‐linking. These functions imply two physiological functions: programmed cell life and death. These may have profound roles in the nervous system since studies in cultured astrocytes found tTG short (S) mRNA transcripts induced by treatment with injury‐related cytokines. In the developing rat spinal cord, tTG activity is concentrated in ventral horn alpha motoneurons, but neither studies of spinal cord tTG gene expression, nor evaluation of the GTP‐regulated isoforms in tissues, have been reported. We now report increased tTG protein and gene expression occurring rapidly after SCI. In parallel, novel appearance of a second, short form transcript, in addition to the normal long (L) isoform, occurs by 8 h of injury. Up‐regulation of tTG message and activity following neural injury. with appearance of a truncated GTP‐unregulated S form, may represent new approaches to drug targets in neurotrauma.
Regulation of DARPP‐32 dephosphorylation at PKA‐ and Cdk5‐sites by NMDA and AMPA receptors: distinct roles of calcineurin and protein phosphatase‐2A
Authors: Nishi Akinori , Bibb James A. , Matsuyama Seiichiro , Hamada Miho , Higashi Hideho , Nairn Angus C. , Greengard Paul
Published: March 2003

Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP‐32 (dopamine‐ and cAMP‐regulated phosphoprotein of Mr 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP‐32 at Thr34 by cAMP‐dependent protein kinase, and antagonized by phosphorylation of DARPP‐32 at Thr75 by cyclin‐dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP‐32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP‐32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca2+‐dependent activation of the Ca2+‐/calmodulin‐dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca2+‐dependent activation of dephosphorylation by protein phosphatase‐2A. The results provide support for a complex effect of glutamate on dopaminergic signaling through the regulation of dephosphorylation of different sites of DARPP‐32 by different protein phosphatases.
The translocation of focal adhesion kinase in brain synaptosomes is regulated by phosphorylation and actin assembly
Authors: Bongiorno‐Borbone Lucilla , Onofri Franco , Giovedì Silvia , Ferrari Rosaria , Girault Jean‐Antoine , Benfenati Fabio
Published: March 2003

Focal adhesion kinase (FAK) and the related proline‐rich tyrosine kinase 2 (PYK2) are non‐receptor protein tyrosine kinases that transduce extracellular signals through the activation of Src family kinases and are highly enriched in neurones. To further elucidate the regulation of FAK and PYK2 in nervous tissue, we investigated their distribution in brain subcellular fractions and analysed their translocation between membrane and cytosolic compartments. We have found that FAK and PYK2 are present in a small membrane‐associated pool and a larger cytosolic pool in various neuronal compartments including nerve terminals. In intact nerve terminals, inhibition of Src kinases inhibited the membrane association of FAK, but not of PYK2, whereas tyrosine phosphatase inhibition sharply increased the membrane association of both FAK and PYK2. Disruption of the actin cytoskeleton was followed by a decrease in the membrane‐associated pool of FAK, but not of PYK2. For both kinases, a significant correlation was found between autophosphorylation and membrane association. The data indicate that FAK and PYK2 are present in nerve terminals and that the membrane association of FAK is regulated by both phosphorylation and actin assembly, whereas that of PKY2 is primarily dependent on its phosphorylation state.
Characterization of [125I]epibatidine binding and nicotinic agonist‐mediated 86Rb+ efflux in interpeduncular nucleus and inferior colliculus of β2 null mutant mice
Authors: Marks Michael J. , Whiteaker Paul , Grady Sharon R. , Picciotto Marina R. , McIntosh J. Michael , Collins Allan C.
Published: March 2003

The β2 nicotinic acetylcholine receptor subunit null mutation eliminated most high affinity [3H]epibatidine binding in mouse brain, but significant binding remained in accessory olfactory nucleus, medial habenula, inferior colliculus and interpeduncular nucleus. Residual [125I]epibatidine binding sites in the inferior colliculus and interpeduncular nucleus were subsequently characterized. Inhibition of [125I]epibatidine binding by 12 agonists and six antagonists was very similar in these regions. Most acetylcholine‐stimulated 86Rb+ efflux is eliminated in thalamus and superior colliculus of β2 null mutants, but significant activity remained in inferior colliculus and interpeduncular nucleus. This residual activity was subsequently characterized. The 12 nicotinic agonists tested elicited concentration‐dependent 86Rb+ efflux. Epibatidine was the most potent agonist. Cytisine was also potent and efficacious. EC50 values for quaternary agonists were relatively high. Cytisine‐stimulated 86Rb+ efflux was inhibited by six classical nicotinic antagonists. Mecamylamine and d‐tubocurarine were most potent, while decamethonium was the least potent. Agonists and antagonists exhibited similar potency in both brain regions. α‐Bungarotoxin (100 nm) did not significantly inhibit cytisine‐stimulated 86Rb+ efflux, while the α3β4 selective antagonist, αConotoxinAuIB, inhibited a significant fraction of the response in both brain regions. Thus, β2 null mutant mice express residual nicotinic activity with properties resembling those of α3β4*‐nAChR.
Alternative non‐coding exons support serotonin transporter mRNA expression in the brain and gut
Authors: Ozsarac N. , Santha E. , Hoffman B. J.
Published: March 2003

A number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5′ rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5′ untranslated region. To determine whether the three alternatively spliced mRNA species that contain one of the following untranslated regions (i) exon 1A, 63 bp (ii) exon 1A + 1B, 125 bp or (iii) exon 1C, 101 bp, were expressed in a tissue‐specific manner, we used RT–PCR and exon‐specific oligonucleotide hybridization. Our results suggest two of the variants (1A + 1B and 1A) may utilize the same promoter; however, they are not equally expressed. While in the adult CNS and adrenal medulla, the shorter mRNA consisting of exon 1A was considerably more abundant, in the stomach and heart, the two variants were equally expressed. The third splice variant exon 1C is only expressed in the gut and to a lesser extent in the heart. The data from this study suggest the splice variant consisting of exon 1C may utilize a distinct promoter compared to the other two.
Different pathways for iNOS‐mediated toxicity in vitro dependent on neuronal maturation and NMDA receptor expression
Authors: Golde Sabine , Chandran Siddharthan , Brown Guy C. , Compston Alastair
Published: March 2003

Co‐localization of activated microglia and damaged neurones seen in brain injury suggests microglia‐induced neurodegeneration. Activated microglia release two potential neurotoxins, excitatory amino acids and nitric oxide (NO), but their contribution to mechanisms of injury is poorly understood. Using co‐cultures of rat microglia and embryonic cortical neurones, we show that inducible NO synthase (iNOS)‐derived NO aloneis responsible for neuronal death from interferon γ(IFNγ) +lipopolysaccharide (LPS)‐activated microglia. Neurones remain sensitive to NO irrespective of maturation state but, whereas blocking NMDA receptor activation with MK801 has no effect on NO‐mediated toxicity to immature neurones, MK801 rescues 60–70% of neurones matured in culture for 12 days. Neuronal expression of NMDA receptors increases with maturation in culture, accounting for increased susceptibility to excitotoxins seen in more mature cultures. We show that MK801 delays the death of more mature neurones caused by the NO‐donor DETA/NO indicating that NO elicits an excitotoxic mechanism, most likely through neuronal glutamate release. Thus, similar concentrations of nitric oxide cause neuronal death by two distinct mechanisms: NO acts directly upon immature neurones but indirectly, via NMDA receptors, on more mature neurones. Our results therefore extend existing evidence for NO‐mediated toxicity and show a complex interaction between inflammatory and excitotoxic mechanisms of injury in mature neurones.
Insulin‐like growth factor (IGF)‐I binding to a cell membrane associated IGF binding protein‐3 acid‐labile subunit complex in human anterior pituitary gland
Authors: Wilczak Nadine , Kühl Nicole , Chesik Daniel , Geerts Albert , Luiten Paul , De Keyser Jacques
Published: March 2003

The binding characteristics of [125I]insulin‐like growth factor (IGF)‐I were studied in human brain and pituitary gland. Competition binding studies with DES(1‐3)IGF‐I and R3‐IGF‐I, which display high affinity for the IGF‐I receptor and low affinity for IGF binding proteins (IGFBPs), were performed to distinguish [125I]IGF‐I binding to IGF‐I receptors and IGFBPs. Specific [125I]IGF‐I binding in brain regions and the posterior pituitary was completely displaced by DES(1‐3)IGF‐I and R3‐IGF‐I, indicating binding to IGF‐I receptors. In contrast, [125I]IGF‐I binding in the anterior pituitary was not displaced by DES(1‐3)IGF‐I and R3‐IGF‐I, suggesting binding to an IGF‐binding site that is different from the IGF‐I receptor. Binding affinity of IGF‐I to this site was about 10‐fold lower than for the IGF‐I receptor. Using western immunoblotting we were also unable to detect IGF‐I receptors in human anterior pituitary. Instead, western immunoblotting and immunoprecipitation experiments showed a 150‐kDa IGFBP‐3‐acid labile subunit (ALS) complex in the anterior pituitary and not in the posterior pituitary and other brain regions. RT‐PCR experiments showed the expression of ALS mRNA in human anterior pituitary indicating that the anterior pituitary synthesizes ALS. In the brain regions and posterior pituitary, IGFBP‐3 was easily washed away during pre‐incubation procedures as used in the [125I]IGF‐I binding experiments. In contrast, the IGFBP‐3 complex in the anterior pituitary could not be removed by these washing procedures. Our results indicate that the human anterior pituitary contains a not previously described tightly cell membrane‐bound 150‐kDa IGFBP‐3–ALS complex that is absent in brain and posterior pituitary.
Muscarinic receptor‐mediated phosphorylation of cyclic AMP response element binding protein in human neuroblastoma cells
Authors: Greenwood Jeffrey M. , Dragunow Michael
Published: March 2003

This study describes the effect of signalling through muscarinic acetylcholine receptors on two transcription factors implicated in long‐term synaptic plasticity and memory formation, EGR1 and the cyclic AMP response element binding protein (CREB). In SK‐N‐SH neuroblastoma cells, treatment with the cholinergic agonist carbachol led to maximal induction of EGR1 1 h after stimulation. This was preceded by the phosphorylation of CREB, which peaked as early as 5 minutes after carbachol treatment. The levels of both EGR1 and phosphorylated CREB (pCREB) slowly decayed over 4–8 h. CREB phosphorylation and EGR1 induction showed similar sensitivity to carbachol concentration, with EC50 values in the range of 1–10 µM, and the changes in both transcription factors were blocked by the muscarinic antagonist atropine. As has been described elsewhere, EGR1 induction was dependent on activation of p42/44 MAP kinase, as it was blocked by the MEK inhibitor U0126. However, CREB phosphorylation by carbachol was largely unaffected by MAP kinase blockade. As both CREB phosphorylation and EGR1 induction have been linked to long‐term potentiation and some forms of memory consolidation, these results may implicate CREB and EGR1 in independent or partially independent cholinergic signalling pathways involved in memory processes.
Protective effects of docosahexaenoic acid in staurosporine‐induced apoptosis: involvement of phosphatidylinositol‐3 kinase pathway
Authors: Akbar Mohammed , Kim Hee‐Yong
Published: March 2003

Docosahexaenoic acid (22:6n‐3, DHA) is highly enriched in neuronal membranes and is considered to be essential for proper brain function. We have previously demonstrated in Neuro 2A cells that DHA as a membrane component protects cells from apoptotic death induced by serum deprivation (Kimet al. 2000). In the present study we demonstrate that staurosporine (ST) induces apoptosis in Neuro 2A cells and DHA enrichment prior to the ST treatment significantly inhibits the apoptotic cell death, as evidenced by the reduction of caspase‐3 activity, cleavage of pro‐caspase‐3 to active caspase‐3, DNA strand‐breaking and laddering. Enrichment of cells with other fatty acids such as oleic and arachidonic acids did not exert such an effect, indicating that the antiapoptotic effect was specific to DHA enrichment. Among the several protein kinase inhibitors, only phosphatidylinositol 3‐kinase (PI3‐K) inhibitors, wortmanin, and LY‐294002 abolished the protective effect of DHA in ST‐induced apoptosis. Concurrently, ST‐treatment significantly decreased the phosphorylation status of Akt at Ser‐473 and Thr‐308 as well as Akt activity, and this reduction was partially prevented by DHA enrichment. The extent of the antiapoptotic effect of DHA correlated with a time‐dependent increase in the phosphatidylserine (PS) content upon DHA enrichment. When cells were enriched with DHA in serine‐free medium, the PS increase diminished and the DHA effect on caspase‐3 activation as well as Akt phosphorylation in ST‐induced apoptosis was no longer apparent, suggesting that DHA's role in accumulating membrane PS is an important component for the observed protection. In summary, DHA enrichment uniquely protects ST‐induced apoptosis in a PS‐ and PI3‐K‐dependent manner. From these data, we suggest that the antiapoptotic effect of DHA is mediated at least in part through the PI3‐K/Akt pathway, facilitated by DHA‐induced PS accumulation.
Direct visualization of the gamma secretase‐generated carboxyl‐terminal domain of the amyloid precursor protein: association with Fe65 and translocation to the nucleus
Authors: Kinoshita A. , Whelan C. M. , Smith C. J. , Berezovska O. , Hyman B. T.
Published: March 2003

Amyloid‐β, the peptide that deposits as senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by a gamma secretase‐mediated intramembranous cleavage. In addition to amyloid‐β, this cleavage produces a carboxyl‐terminal intracellular fragment which has an unknown function. The carboxyl‐terminal domain of APP interacts in the cytoplasm with an adapter protein, Fe65. We demonstrate by laser scanning confocal microscopy that a gamma secretase generated APP carboxyl‐terminal domain, tagged with green fluorescent protein (GFP), translocates to the nucleus in a manner dependent upon stabilization by the adapter protein Fe65; APP which has been mutated to block interactions with Fe65 cannot be detected in the nucleus. The APP‐CT domain continues to interact with Fe65 in the nucleus, as determined by both colocalization and fluorescence resonance energy transfer (FRET). Visualization of the APP‐CT‐Fe65 complex in the nucleus may serve as a readout for processes that modify gamma secretase release of APP‐CT.
Erucylphosphocholine‐induced apoptosis in glioma cells: involvement of death receptor signalling and caspase activation
Authors: Kugler Wilfried , Erdlenbruch Bernhard , Jünemann Anja , Heinemann Dagmar , Eibl Hansjörg , Lakomek Max
Published: March 2003

Erucylphosphocholine (ErPC) is a promising anti‐neoplastic drug for the treatment of malignant brain tumours. It exerts strong anti‐cancer activity in vivo and in vitro and induces apoptosis even in chemoresistant glioma cell lines. The purpose of this study was to expand on our previous observations on the potential mechanisms of ErPC‐mediated apoptosis with a focus on death receptor activation and the caspase network. A172 and T98G glioma cells were treated with ErPC for up to 48 h. ErPC effects on the expression of the tumour necrosis factor (TNF) and TNF‐related apoptosis‐inducing ligand (TRAIL) receptor system, and on caspase activation were determined. ErPC had no effect on the expression of TNFα or TRAIL. Inhibition of the TNF or TRAIL signalling pathway with antagonistic antibodies or fusion proteins did not affect apoptosis induced by ErPC, and a dominant‐negative FADD construct did not abolish ErPC‐induced effects. Western blot analysis indicated that ErPC‐triggered apoptosis resulted in a time‐dependent processing of caspases‐3, ‐7, ‐8 and ‐9 into their respective active subunits. Co‐treatment of A172 cells with different caspase inhibitors prevented apoptosis but did not abrogate cell death. These data suggest that A172 cells might have an additional caspase‐independent pathway that insures cell death and guarantees killing of those tumour cells whose caspase pathway is incomplete.
Numb isoforms containing a short PTB domain promote neurotrophic factor‐induced differentiation and neurotrophic factor withdrawal‐induced death of PC12 Cells
Authors: Pedersen Ward A. , Chan Sic L. , Zhu Haiyan , Abdur‐Rahman Lilanie A. , Verdi Joseph M. , Mattson Mark P.
Published: March 2003

The development of the nervous system is regulated by trophic signals that control cell proliferation, differentiation, and survival. Numb is an evolutionarily conserved protein identified by its ability to control cell fate in the nervous system ofDrosophila. Mammals express four isoforms of Numb that differ in the length of a phosphotyrosine‐binding (PTB) domain and a proline‐rich region (PRR). Using PC12 cells stably expressing each of the human isoforms, we show that Numb regulates sensitivity of the cells to neurotrophic factor‐induced differentiation and neurotrophic factor withdrawal‐induced death in an isoform‐specific manner. Numb isoforms containing a short PTB domain enhance the differentiation response to NGF and enhance apoptosis upon NGF withdrawal; Numb isoforms containing a long PTB domain exhibit the same sensitivity to NGF as vector‐transfected cells. These effects of Numb were found to be independent of the length of the PRR. In undifferentiated conditions, the levels of full‐length TrkA and of phosphorylated p44/p42 mitogen‐activated protein kinase (MAPK) are increased in cells expressing Numb isoforms with a short PTB domain, indicating an up‐regulation of NGF signaling pathways. Furthermore, we provide evidence that the mechanism whereby short PTB domain Numb isoforms sensitize cells to trophic factor deprivation‐induced apoptosis involves elevations in intracellular calcium concentrations. Our results suggest that Numb sensitizes cells to neurotrophin responses in an isoform‐specific manner, an effect that may play an important role in the development and plasticity of the nervous system.
Suppression of the morphine‐induced rewarding effect in the rat with neuropathic pain: implication of the reduction in µ‐opioid receptor functions in the ventral tegmental area
Authors: Ozaki Satoru , Narita Minoru , Narita Michiko , Iino Masahiko , Sugita Junichi , Matsumura Yumiko , Suzuki Tsutomu
Published: March 2003

The present study was designed to investigate the rewarding effect, G‐protein activation and dopamine (DA) release following partial sciatic nerve ligation in the rat. Here we show for the first time that morphine failed to produce a place preference in rats with nerve injury. Various studies provide arguments to support that the mesolimbic dopaminergic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc), is critical of the motivational effects of opioids. In the present study, there were no significant differences between sham‐operated and sciatic nerve‐ligated rats in the increases in guanosine‐5′‐o‐(3‐[35S]thio)triphosphate ([35S]GTPγS) binding to membranes of the N.Acc stimulated by either DA, the D1 receptor agonist SKF81297, the D2 receptor agonist N‐propylnoraporphine or the D3 receptor agonist 7‐hydroxy‐2‐dipropylaminotetralin (7‐OH DPAT). In contrast, the increases in [35S]GTPγS binding to membranes of the VTA induced by either morphine or a selective µ‐opioid receptor agonist [d‐Ala2, NMePhe4, Gly(ol)5]enkephalin were significantly attenuated in nerve‐ligated rats as compared with sham‐ operated rats. Furthermore, the enhancement of DA release in the N.Acc stimulated by morphine was significantly suppressed by sciatic nerve ligation. These findings suggest that attenuation of the morphine‐induced place preference under neuropathic pain may result from a decrease in the morphine‐induced DA release in the N.Acc with reduction in the µ‐opioid receptor‐mediated G‐protein activation in the VTA.
Kinetics of nitric oxide‐cyclic GMP signalling in CNS cells and itspossible regulation by cyclic GMP
Authors: Wykes Victoria , Bellamy Tomas C. , Garthwaite John
Published: March 2003

Physiologically, nitric oxide (NO) signal transduction occurs through soluble guanylyl cyclase (sGC), which catalyses cyclic GMP (cGMP) formation. Knowledge of the kinetics of NO‐evoked cGMP signals is therefore critical for understanding how NO signals are decoded. Studies on cerebellar astrocytes showed that sGC undergoes a desensitizing profile of activity, which, in league with phosphodiesterases (PDEs), was hypothesized to diversify cGMP responses in different cells. The hypothesis was tested by examining the kinetics ofcGMP in rat striatal cells, in which cGMP accumulated in neurones in response to NO. Based on the effects of selective PDE inhibitors, cGMP hydrolysis following exposure to NO was attributed to a cGMP‐stimulated PDE (PDE 2). Analysis of NO‐induced cGMP accumulation in the presence of a PDE inhibitor indicated that sGC underwent marked desensitization. However, the desensitization kinetics determined under these conditions described poorly the cGMP profile observed in the absence of the PDE inhibitor. An explanation shown plausible theoretically was that cGMP determines the level of sGC desensitization. In support, tests in cerebellar astrocytes indicated an inverse relationship between cGMP level and recovery of sGC from its desensitized state. We suggest that the degree of sGC desensitization is related to the cGMP concentration and that this effect is not mediated by (de)phosphorylation.
Survival‐promoting activity of nimodipine and nifedipine in rat motoneurons: implications of an intrinsic calcium toxicity in motoneurons
Authors: Arakawa Yoshihiro , Nishijima Chiaki , Shimizu Nao , Urushidani Tetsuro
Published: March 2003

L‐type calcium channel antagonists, nimodipine and nifedipine, were tested for effects on the survival of purified rat motoneurons in culture. They showed significant activity, with maximum survival at 30 µm after 3 days in culture as high as 75%, which was comparable to the maximum effect obtained with brain‐derived neurotrophic factor, a potent neurotrophic factor for rat motoneurons. It was also found that depolarizing conditions with a high potassium concentration (30 mm) were toxic to motoneurons. This toxicity was blocked by co‐treatment with nimodipine. These results implicate a pre‐existing calcium burden through calcium channels in motoneurons; they may offer further insights into understanding the selective death of motoneurons and have therapeutic implications in amyotrophic lateral screlosis.
Synphilin‐1 degradation by the ubiquitin‐proteasome pathway and effects on cell survival
Authors: Lee Gwang , Junn Eunsung , Tanaka Mikiei , Kim Yong Man , Lee Sang Seop , Mouradian M. Maral
Published: March 2003

Parkinson's disease is characterized by loss of nigral dopaminergic neurons and the presence of cytoplasmic inclusions known as Lewy bodies. α‐Synuclein and its interacting partner synphilin‐1 are among constituent proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these inclusions supports a role for this protein degradation pathway in the processing of proteins involved in this disease. To begin elucidating the kinetics of synphilin‐1 in cells, we studied its degradation pathway in HEK293 cells that had been engineered to stably express FLAG‐tagged synphilin‐1. Pulse‐chase experiments revealed that this protein is relatively stable with a half‐life of about 16 h. Treatment with proteasome inhibitors resulted in attenuation of degradation and the accumulation of high molecular weight ubiquitinated synphilin‐1 in immunoprecipitation/immunoblot experiments. Additionally, proteasome inhibitors stimulated the formation of peri‐nuclear inclusions which were immunoreactive for synphilin‐1, ubiquitin and α‐synuclein. Cell viability studies revealed increased susceptibility of synphilin‐1 over‐expressing cells to proteasomal dysfunction. These observations indicate that synphilin‐1 is ubiquitinated and degraded by the proteasome. Accumulation of ubiquitinated synphilin‐1 due to impaired clearance results in its aggregation as peri‐nuclear inclusions and in poor cell survival.
Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin
Authors: Lanthier Julie , Bouthillier Alain , Lapointe Marjolaine , Demeule Michel , Béliveau Richard , Desrosiers Richard R.
Published: March 2003

Protein l‐isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down‐regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post‐translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component β‐tubulin, one of the major PIMT substrates, had an increased amount (two‐fold) of l‐isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down‐regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy.
Mechanism of plasminogen activator inhibitor‐1 regulation by oncostatin M and interleukin‐1 in human astrocytes
Authors: Kasza Aneta , Kiss Daniel L. , Gopalan Sunita , Xu Weili , Rydel Russell E. , Koj Aleksander , Kordula Tomasz
Published: March 2003

Glial cells that produce and respond to various cytokines mediate inflammatory processes in the brain. Here, we show that oncostatin M (OSM) and interleukin‐1 (IL‐1) regulate the expression of plasminogen activator inhibitor‐1 (PAI‐1) and urokinase‐type plasminogen activator (uPA) in human astrocytes. Using the PAI‐1 reporter constructs we show that the −58 to −51 proximal element mediates activation by both cytokines. This element is already bound by c‐fos/c‐jun heterodimers in unstimulated astrocytes, and treatment with cytokine strongly stimulates both expression of c‐fos and binding of c‐fos/c‐jun heterodimers. In addition, IL‐1 activates an inhibitory mechanism that down‐regulates PAI‐1 expression after longer exposure to this cytokine. Overexpression of dominant‐negative signal transducer and activator of transcription‐1 (STAT1), STAT3, STAT5 and inhibitor of nuclear factor‐κB (IκB) suppressed OSM/IL‐1‐induced expression of the PAI‐1 reporter construct. We conclude that OSM and IL‐1 regulate the PAI‐1 gene expression via up‐regulating c‐fos levels and subsequent binding of c‐fos/c‐jun heterodimers to the proximal element of the PAI‐1 gene.
Increased CNS uptake and enhanced antinociception of morphine‐6‐glucuronide in rats after inhibition of P‐glycoprotein
Authors: Lötsch Jörn , Schmidt Rodolfo , Vetter Gregor , Schmidt Helmut , Niederberger Ellen , Geisslinger Gerd , Tegeder Irmgard
Published: March 2003

Morphine‐6‐glucuronide (M6G) is a substrate of P‐glycoprotein (P‐gp), which forms an outward transporter at the blood–brain barrier. Inhibition of P‐gp may therefore be expected to cause increased CNS uptake of M6G. We directly assessed the spinal concentrations of M6G and its antinociceptive effects in rats following pharmacological inhibition of P‐gp. Spinal cord tissue concentrations of M6G were assessed by microdialysis with probes transversally implanted through the dorsal horns of the spinal cord at level L4. Ten rats received M6G intravenously (0.018 mg/kg loading dose plus 0.00115 mg/kg/min for an 8‐h infusion), five of them together with PSC833 to inhibit P‐gp (32‐h infusion, starting 24 h before the addition of M6G). Antinociceptive effects were explored by means of formalin tests. After having obtained evidence for enhanced CNS uptake and antinociception of M6G in the presence of PSC833, additional behavioural experiments were performed in another 32 rats to assess the dose dependency of the antinociceptive effects of M6G either with or without PSC833 in comparison with both PSC833 alone and placebo. Inhibition of P‐gp increased the M6G concentrations in the spinal cord approximately three‐fold whereas the plasma concentrations were increased only by a factor of 1.4, which resulted in a more than doubled spinal cord/plasma concentration ratio (from 0.08 ± 0.03 for M6G alone to 0.17 ± 0.08 for M6G plus PSC833). Antinociceptive effects of M6G were significantly enhanced by inhibition of P‐gp. Inhibition of P‐gp alters the transport of M6G across the blood–brain barrier, resulting in enhanced spinal cord uptake and enhanced antinociception.
Pleiotrophin, an angiogenic and mitogenic growth factor, is expressed in human gliomas
Authors: Mentlein Rolf , Held‐Feindt Janka
Published: March 2003

Pleiotrophin (PTN) is a mitogenic/angiogenic, 15.3 kDa heparin‐binding peptide that is found in embryonic or early postnatal, but rarely in adult, tissues. Since developmentally regulated factors often re‐appear in malignant cells, we examined PTN expression in human glioma cell lines, cell cultures derived from solid gliomas and glioma sections. PTN mRNA or protein was detected by reverse transcriptase‐polymerase chain reaction, immunohistochemistry, western blot or enzyme‐linked immunoassay in all WHO III and IV grade gliomas and cells analyzed in vitro or in situ. One WHO II grade glioma investigated was PTN negative. In vitro, PTN was synthesized in perinuclear regions of glioma cells, secreted into the cultivation medium, but its production varied considerably between glioma cells cultivated from different solid gliomas or glioma cell lines. In situ, PTN expression was restricted to distinct parts/cells of the tumour. PTN did not influence the proliferation of glioma cells themselves, but stimulated [3H]thymidine incorporation into DNA of microglial cells. Furthermore, in Boyden chamber assays, PTN showed a strong chemotactic effect on murine BV‐2 microglial cells. PTN is supposed to be a paracrine growth/angiogenic factor that is produced by gliomas and contributes to their malignancy by targeting endothelial and microglial cells.
Serotonin synthesis and its light–dark variation in the rat retina
Authors: Chanut Evelyne , Nguyen‐Legros Jeanine , Labarthe Benoît , Trouvin Jean‐Hugues , Versaux‐Botteri Claudine
Published: March 2003

Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by photoreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To demonstrate local synthesis of serotonin in the rat retina, substrates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5‐hydroxytryptophan levels, whereas α‐methyltryptophan, a competitive substrate inhibitor, was hydroxylated into α‐methyl‐5‐hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT–PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5‐HT is used for melatonin synthesis.
Modulation of astrocyte inducible nitric oxide synthase and cytokine expression by interferon β is associated with induction and inhibition of interferon γ‐activated sequence binding activity
Authors: Hua Liwei L. , Kim Mee‐Ohk , Brosnan Celia F. , Lee Sunhee C.
Published: March 2003

Although interferon (IFN)‐β is firmly established as a therapeutic agent for multiple sclerosis, information regarding its role in astrocyte cytokine production is limited. In primary cultures of human astrocytes, we determined the effects of IFN‐β on astrocyte cytokine [tumor necrosis factor‐alpha (TNF‐α) and interleukin (IL)‐6] and inducible nitric oxide synthase (iNOS) expression by ribonuclease protection assay and ELISA. We found that IFN‐β inhibited astrocyte cytokine/iNOS induced by IL‐1 plus IFN‐γ, but in the absence of IFN‐γ, IFN‐β enhanced IL‐1‐induced cytokine/iNOS expression. Electrophoretic mobility shift analysis (EMSA) demonstrated that IFN‐γ induced sustained IFN‐γ‐activated sequence (GAS) binding, while IFN‐β induced transient GAS binding. When used together, IFN‐β inhibited IFN‐γ‐induced GAS binding activity. Nuclear factor‐kappa B (NF‐κB) activation was not altered by either IFNs, whereas IFN stimulated response element (ISRE) was only activated by IFN‐β and not IFN‐γ. These results suggest that IFN‐β can both mimic and antagonize the effect of IFN‐γ by modulating induction of nuclear GAS binding activity. Our results demonstrating differential regulation of astrocyte cytokine/iNOS induction by IFN‐β are novel and have implications for inflammatory diseases of the human CNS.
Ontogeny and the possible function of a novel epidermal growth factor‐like repeat domain‐containing protein, NELL2, in the rat brain
Authors: Kim Hyun , Ha Chang Man , Choi Jungil , Choi Eun Jung , Jeon Jongrye , Kim Changmee , Park Sang Kyu , Kang Sang Soo , Kim Kyungjin , Lee Byung Ju
Published: March 2003

In this study we investigated the mRNA expression of NELL2, a neural tissue‐specific epidermal growth factor (EGF)‐like repeat domain‐containing protein, in the developing and adult rat CNS using in situ hybridization histochemistry and northern blot analysis. The possible candidates that interact with or be regulated by NELL2 were screened with a cDNA expression array in antisense (AS) NELL2 oligodeoxynucleotide (ODN)‐injected rat hypothalami. NELL2 mRNA was detected as early as embryonic day 10, and was predominant in the CNS throughout the pre‐natal stages. Its expression gradually increased during embryonic development and its strong expression was observed throughout the CNS until embryonic day 20. It was detected in the ventricular zone of the spinal cord, medulla and pons in 12‐day‐old‐embryos, suggesting that NELL2 plays a role in the neurogenesis of these areas. After birth its expression gradually decreased, but high levels of expression could be observed in the tenia tecta, piriform cortex, hippocampus, dentate gyrus, cerebellar cortex, ambiguus nucleus, and inferior olivary nucleus of adult rat brains. The analysis of cDNA expression arrays revealed that the administration of AS NELL2 ODN markedly decreased the expression of several Ca2+‐binding proteins and those involved in the transport and release of vesicles such as EF‐hand Ca2+‐binding protein p22 and rab7. This finding was confirmed by relative reverse transcription‐polymerase chain reaction. The effect of NELL2 on synaptic vesicle content in median eminence (ME) nerve terminals was determined with synaptophysin levels as a marker protein in the AS NELL2 ODN‐injected rat. It was significantly decreased by the AS ODN. These data suggest that NELL2 may play an important role in the development of the CNS as well as maintenance of neural functions, by regulating the intracellular machinery involving Ca2+ signaling, synaptic transport and/or release of vesicles.
Astrocytic but not neuronal increased expression and redistribution of parkin during unfolded protein stress
Authors: Ledesma Maria Dolores , Galvan Cristian , Hellias Bianca , Dotti Carlos , Jensen Poul Henning
Published: March 2003

Parkin is a ubiquitin ligase that facilitates proteasomal protein degradation and is involved in a common autosomal recessive form of Parkinson's disease. Its expression is part of the unfolded protein response in cell lines where its overexpression protects against unfolded protein stress. How parkin expression is regulated in brain primary cells under stress situations is however, less well established. Here, the cellular and subcellular localization of parkin under basal conditions and during unfolded protein stress was investigated in primary cultures of rat astrocytes and hippocampal neurons. Immunofluorescense microscopy and biochemical analysis demonstrated that parkin is mainly associated with the endoplasmic reticulum (ER) in hippocampal neurons while it is associated with Golgi membranes, the nuclei and light vesicles in astrocytes. The constitutive parkin expression was high in neurons as compared with astrocytes. However, unfolded protein stress elicited a selective increase in astrocytic parkin expression and a change in distribution, whereas neuronal parkin remained largely unmodified. The cell specific differences argue in favour of different cellular binding sites and substrates for the protein and a pathogenic role for astrocytes in Parkinson's disease caused by parkin dysfunction.
Type‐specific inositol 1,4,5‐trisphosphate receptor localization in the vomeronasal organ and its interaction with a transient receptor potential channel, TRPC2
Authors: Brann Jessica H. , Dennis John C. , Morrison Edward E. , Fadool Debra A.
Published: March 2003

The vomeronasal organ (VNO) is the receptor portion of the accessory olfactory system and transduces chemical cues that identify social hierarchy, reproductive status, conspecifics and prey. Signal transduction in VNO neurons is apparently accomplished via an inositol 1,4,5‐trisphosphate (IP3)‐activated calcium conductance that includes a different set of G proteins than those identified in vertebrate olfactory sensory neurons. We used immunohistochemical (IHC) and SDS–PAGE/western analysis to localize three IP3 receptors (IP3R) in the rat VNO epithelium. Type‐I IP3R expression was weak or absent. Antisera for type‐II and ‐III IP3R recognized appropriate molecular weight proteins by SDS–PAGE, and labeled protein could be abolished by pre‐adsorption of the respective antibody with antigenic peptide. In tissue sections, type‐II IP3R immunoreactivity was present in the supporting cell zone but not in the sensory cell zone. Type‐III IP3R immunoreactivity was present throughout the sensory zone and overlapped that of transient receptor potential channel 2 (TRPC2) in the microvillar layer of sensory epithelium. Co‐immunoprecipitation of type‐III IP3R and TRPC2 from VNO lysates confirmed the overlapping immunoreactivity patterns. The protein–protein interaction complex between type‐III IP3R and TRPC2 could initiate calcium signaling leading to electrical signal production in VNO neurons.
Histamine activates tyrosine hydroxylase in bovine adrenal chromaffin cells through a pathway that involves ERK1/2 but not p38 or JNK
Authors: Cammarota Martín , Bevilaqua Lia R. M. , Rostas John A. P. , Dunkley Peter R.
Published: March 2003

In bovine adrenal chromaffin cells (BACC) histamine promotes a rapid increase in the intracellular levels of Ca2+ together with the release of catecholamines and the phosphorylation of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). In this study we investigated the role of the mitogen‐activated protein kinases (MAPK) extracellular signal‐regulated kinases (ERK1/2), stress activated protein kinase (p38) and Jun N‐terminal kinases (JNK) on the histamine‐induced activation and phosphorylation of TH. We found that in BACC histamine produced a rapid, long lasting and histamine type‐1 (H1) receptor‐dependent increase in the phosphorylation levels of ERK1/2, p38 and JNK which was accompanied by a H1 receptor‐dependent increase in TH activity. This increase in TH activity was partially blocked by the MEK1/2 inhibitor U0126 but was unaffected by the p38 antagonist SB203580 or the JNK blocker JNKI1. To study the effect of MAPK inhibition on histamine‐induced TH phosphorylation, we generated phospho‐specific antibodies against the different phosphorylated forms of TH. Treatment with U0126 totally inhibited the histamine‐induced phosphorylation of TH at Ser31, without affecting the phosphorylation of either Ser40 or Ser19. Neither SB203580 nor JNKI1 treatments produced any significant modification of the histamine‐induced TH phosphorylation. Our data support the hypothesis that the up‐regulation of the ERK1/2 pathway, but not that of p38 or JNK, promoted by histamine is involved in the phosphorylation of TH at Ser31 and that this phosphorylation event is required for the full activation of this enzyme.
Rac and Cdc42‐dependent regulation of c‐Jun N‐terminal kinases by the δ‐opioid receptor
Authors: Kam Angel Y. F. , Chan Anthony S. L. , Wong Yung H.
Published: March 2003

Heptahelical opioid receptors utilize Gi proteins to regulate a multitude of effectors including the classical adenylyl cyclases and the more recently discovered mitogen‐activated protein kinases (MAPKs). The c‐Jun NH2‐terminal kinases (JNKs) belong to one of three subgroups of MAPKs. In NG108‐15 neuroblastoma × glioma hybrid cells that endogenously express δ‐opioid receptors, δ‐agonist dose‐dependently stimulated JNK activity in a pertussis toxin‐sensitive manner. By using COS‐7 cells transiently transfected with the cDNAs of δ‐opioid receptor and hemagglutinin (HA)‐tagged JNK, we delineated the signaling components involved in this pathway. Sequestration of Gβγ subunits by transducin suppressed the opioid‐induced JNK activity. The possible involvement of the small GTPases was also examined. Expression of dominant negative mutants of Rac and Cdc42 blocked the opioid‐induced JNK activation, and a partial inhibition was observed in the presence of the dominant negative mutant of Ras. In contrast, the dominant negative mutant of Rho did not affect the opioid‐induced JNK activation. In addition, the receptor‐mediated JNK activation was dependent on Src family tyrosine kinases, but independent of phosphatidylinositol‐3 kinase and EGF receptor tyrosine kinases. Collectively, these results demonstrate functional regulation of JNK by the δ‐opioid receptor, and this pathway requires Gβγ, Src kinases and the small GTPases Rac and Cdc42.
Regulation of α‐synuclein by bFGF in cultured ventral midbrain dopaminergic neurons
Authors: Rideout Hardy J. , Dietrich Paula , Savalle Magali , Dauer William T. , Stefanis Leonidas
Published: March 2003

α‐Synuclein is a neuronal protein that is implicated in the control of synaptic vesicle function and in Parkinson's disease (PD). Consequently, alterations of α‐synuclein levels may play a role in neurotransmission and in PD pathogenesis. However, the factors that regulate α‐synuclein levels are unknown. Growth factors mediate neurotrophic and plasticity effects in CNS neurons, and may play a role in disease states. Here we examine the regulation of α‐synuclein levels in primary CNS neurons, with particular emphasis on dopaminergic neurons. E18 rat cortical neurons and dopaminergic neurons of E14 rat ventral midbrain showed an induction of α‐synuclein protein levels with maturation in culture. Application of basic Fibroblast growth factor (bFGF) promoted α‐synuclein expression selectively within dopaminergic, and not GABAergic or cortical neurons. This induction was blocked by actinomycin D, but not by inhibition of bFGF‐induced glial proliferation. α‐Synuclein levels were not altered by glial‐derived neurotrophic factor (GDNF), or by apoptotic stimuli. We conclude that bFGF promotes α‐synuclein expression in cultured ventral midbrain dopaminergic neurons through a direct transcriptional effect. These results suggest that distinct growth factors may thus mediate plasticity responses or influence disease states in ventral midbrain dopaminergic neurons.