bluuebunny/crossref_metadata_2025 huggingface dataset:
CCAAT Displacement Protein (CDP/Cut) Binds a Negative Regulatory Element in the Human Tryptophan Hydroxylase Gene
Authors: Teerawatanasuk Nongnit , Skalnik David G , Carr Lucinda G
Published: March 2003

Abstract : Tryptophan hydroxylase (TPH) is the rate‐limiting enzyme
in the biosynthesis of serotonin, a neurotransmitter that has been implicated
in many psychiatric illnesses. The mechanism of transcriptional regulation of
the human TPH gene is largely unknown. We have identified a negative
regulatory element located between nucleotides ‐310 and ‐220 in the human
TPH (hTPH) gene. Electromobility shift analyses performed
with the ‐310/‐220 hTPH probe and nuclear extract from P815‐HTR (a
TPH‐expressing cell line) revealed two slow migrating protein‐DNA complexes,
designated I and II. CCAAT displacement protein (CDP/Cut) is involved in
complex I formation as shown in electromobility shift analysis, using
consensus oligonucleotide competitor and antibody. Mutations in the CDP/Cut
binding site not only disrupted the CDP‐DNA complex but also disrupted the
second complex, suggesting that the core binding sequences of the two proteins
are overlapping. The functional importance of these protein‐DNA interactions
was assessed by transiently transfecting wild‐type and mutant
pTPH/luciferase reporter constructs into P815‐HTR cells. Mutations in
the core CDP/Cut site resulted in an approximately fourfold increase in
relative luciferase activities. Because CDP/Cut has been shown to repress
transcription of many target genes, we speculate that disruption of the
CDP/Cut binding was responsible, at least in part, for the activation of
hTPH gene.
GTP Cyclohydrolase I Gene Expression in the Brains of Male and Female hph‐1 Mice
Authors: Shimoji Mika , Hirayama Kei , Hyland Keith , Kapatos Gregory
Published: March 2003

Abstract : The hph‐1 mouse is characterized by low levels
of GTP cyclohydrolase I (GTPCH) and tetrahydrobiopterin. A quantitative
double‐lable in situ hybridization technique was used to examine CNS GTPCH
mRNA expression within serotonin, dopamine, and norepinephrine neurons of male
and female wild‐type and hph‐1 mice. In wild‐type male and female
animals the highest levels of GTPCH mRNA expression were observed within
serotonin neurons, followed by norepinephrine and then dopamine neurons.
Wild‐type female animals were found to express lower levels of GTPCH mRNA in
each cell type when compared with levels seen in wild‐type males. GTPCH mRNA
abundance in all three cell types was lower in hph‐1 male than in
wild‐type male mice, with the greatest reduction in serotonin neurons. GTPCH
mRNA levels were also lower in hph‐1 female than in wild‐type female
mice, again with the greatest reduction occurring in serotonin neurons.
Comparison of hph‐1 male and hph‐1 female mice revealed that
the sex‐linked difference in GTPCH mRNA expression observed in wild‐type
neurons was only present within female dopamine neurons. Overall, these
results indicate that not only are basal levels of GTPCH mRNA expression
heterogeneous across wild‐type murine monoamine cell types but that gene
expression is also modified in a sex‐linked and cell‐specific fashion by the
hph‐1 gene locus. The hph‐1 mutation does not lie within the
GT‐PCH mRNA coding region. The 5′ flanking region of the GTPCH gene was
cloned and sequenced and shown to be identical for both wild‐type and
hph‐1 genomic DNA. Transient transfection assays performed in PC12
cells demonstrated that this 5′ flanking region was sufficient to
initiate transcription of a luciferase reporter gene. Although the
hph‐1 mutation does not lie within the 5′ flanking region of the GTPCH gene, this region of the gene can function as a core promoter and is thus crucial to the control of GTPCH gene expression.
The Extracellular Calcium‐Sensing Receptor Is Expressed in Rat Microglia and Modulates an Outward K+ Channel
Authors: Chattopadhyay Naibedya , Ye Chianping , Yamaguchi Toru , Nakai Masamichi , Kifor Olga , Vassilev Peter M. , Nishimura Robert N. , Brown Edward M.
Published: March 2003

Abstract: The calcium‐sensing receptor (CaR) is a G protein‐coupled receptor that “senses” extracellular calcium ions (Ca2+o) as an extracellular first messenger. In this report, we have shown that the CaR is expressed in primary cultures of microglial cells derived from rat brain as assessed by RT‐PCR using four CaR‐specific primer pairs followed by sequencing of the amplified products, by northern blot analysis using a CaR‐specific probe, as well as by immunocytochemistry and western analysis utilizing a specific polyclonal anti‐CaR antiserum. In addition, raising Ca2+, from 0.75 to 3.0 mM or addition of the polycationic CaR agonist neomycin or a “calcimimetic” CaR activator (R‐467; NPS Pharmaceuticals) increased the open state probability (Po) of a Ca+‐activated Kf channel having a unitary conductance of 84 ± 4 pS, indicating that the channel is modulated by the CaR. Therefore, our data strongly suggest that a functional CaR is expressed in cultured rat microglia, similar to that in parathyroid gland and kidney, which could potentially play an important role(s) in regulating microglial function.
Expression of the SM‐20 Gene Promotes Death in Nerve Growth Factor‐Dependent Sympathetic Neurons
Authors: Lipscomb Elizabeth A. , Sarmiere Patrick D. , Crowder Robert J. , Freeman Robert S.
Published: March 2003

Abstract: Sympathetic neurons undergo apoptosis when deprived of nerve growth factor (NGF). Inhibitors of RNA or protein synthesis block this death, suggesting that gene expression is important for apoptosis in this system. We have identified SM‐20 as a new gene that increases in expression in sympathetic neurons after NGF withdrawal. Expression of SM‐20 also increases during neuronal death caused by cytosine arabinoside or the phosphatidylinositol 3‐kinase inhibitor LY294002. In addition, SM‐20 protein synthesis is elevated in NGF‐deprived neurons compared with neurons maintained with NGF. Importantly, expression of SM‐20 in sympathetic neurons causes cell death in the presence of NGF. These results suggest that SM‐20 may function to regulate cell death in neurons.
Selective Blocking Effects of Tropisetron and Atropine on Recombinant Glycine Receptors
Authors: Maksay Gábor , Laube Bodo , Betz Heinrich
Published: March 2003

Abstract : Some serotonin 5‐HT3 receptor ligands of

tropeine structure have been recently shown to modulate ionophore function and

binding of glycine receptors. This led us to study the effects of the

tropeines tropisetron and atropine on recombinant human glycine receptors

transiently expressed in Xenopus oocytes by using whole‐cell

voltage‐clamp electrophysiology. Glycine currents were inhibited by atropine

in an apparently competitive manner and with considerable selectivity of the

tropeines for α2 versus α1 subunits.

Coexpression of β with α subunits and replacement of the N‐terminal

region of the α1 subunits by the corresponding β segment

resulted in similar increases in the inhibitory potencies. Our data suggest

common sites of the tropeines for inhibition on the N‐terminal region of

glycine receptors. The point mutations R271K and R271L of the

α1 subunit decreased, whereas a T112A substitution increased,

the inhibition constants (Ki) of the tropeines. These

changes in the Ki values of the tropeines were associated

with opposite changes in the EC50 of glycine. Selectivities for the

tropeines versus glycine (EC50/Ki) varied within three orders of magnitude. These results, when expressed in terms of free energy changes, can be interpreted according to a two‐state receptor model.
Presynaptic k‐Opioid and Muscarinic Receptors Inhibit the Calcium‐Dependent Component of Evoked Glutamate Release from Striatal Synaptosomes
Authors: Rawls Scott M , McGinty Jacqueline F , Terrian David M
Published: March 2003

Abstract : In addition to cytosolic efflux, reversal of excitatory amino acid (EAA) transporters evokes glutamate exocytosis from the striatum in vivo. Both k‐opioid and muscarinic receptor agonists suppress this calcium‐dependent response. These data led to the hypothesis that the calcium‐independent efflux of striatal glutamate evoked by transporter reversal may activate a transsynaptic feedback loop that promotes glutamate exocytosis from thalamo‐ and/or corticostriatal terminals in vivo and that this activation is inhibited by presynaptic k and muscarinic receptors. Corollaries to this hypothesis are the predictions that agonists for these putative presynaptic receptors will selectively inhibit the calcium‐dependent component of glutamate released from striatal synaptosomes, whereas the calcium‐independent efflux evoked by an EAA transporter blocker, L‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (L‐trans‐PDC), will be insensitive to such receptor ligands. Here we report that a muscarinic agonist, oxotremorine (0.01‐10 μM), and a k‐opioid agonist, U‐69593 (0.1‐100 μM), suppressed the calcium‐dependent release of glutamate that was evoked by exposing striatal synaptosomes to the potassium channel blocker 4‐aminopyridine. The presynaptic inhibition produced by these ligands was concentration dependent, blocked by appropriate receptor antagonists, and not mimicked by the δ‐opioid agonist [D‐Pen2,5]‐enkephalin. The finding that glutamate efflux evoked by L‐trans‐PDC from isolated striatal nerve endings was entirely calcium independent supports the notion that intact basal ganglia circuitry mediates the calcium‐dependent effects of this agent on glutamate efflux in vivo. Furthermore, because muscarinic or k‐opioid receptor activation inhibits calcium‐dependent striatal glutamate release in vitro as it does in vivo, it is likely that both muscarinic and k receptors are inhibitory presynaptic heteroceptors expressed by striatal glutamatergic terminals.
Rapsyn Variants in Ciliary Ganglia and Their Possible Effects on Clustering of Nicotinic Receptors
Authors: Conroy William G. , Berg Darwin K.
Published: March 2003

Abstract : Nicotinic acetylcholine receptors (nAChRs) containing the α7 gene product can influence a range of cellular events in neurons, depending on receptor location. On chick ciliary neurons, the receptors are concentrated on somatic spines, but little is known about mechanisms responsible for sequestering them there. Rapsyn is 43‐kDa protein essential for clustering nicotinic receptors at the vertebrate neuromuscular junction. RT‐PCR confirmed previous studies showing that the chick ciliary ganglion expresses rapsyn transcripts, including several splice variants lacking part or all of exon 2. Heterologous expression of rapsyn constructs, together with nicotinic receptor constructs, shows that chicken full‐length rapsyn can induce clustering of both muscle and neuronal nicotinic receptors. Splice variants lacking one or both leucine zipper motifs of exon 2 are unable to cluster the receptors, though, like full‐length rapsyn, they cluster themselves. Immunological analysis demonstrates the presence of full‐length rapsyn in chick muscle extracts but fails to detect either full‐length or splice‐variant versions of rapsyn at significant levels in ganglion extracts. The results suggest that rapsyn does not cluster α7‐nAChRs on ciliary neurons in any way similar to that of receptors at the neuromuscular junction where rapsyn and the receptors are present in approximately equimolar amounts.
Brain‐Derived Neurotrophic Factor Exerts Opposing Effects on β2‐Adrenergic Receptor According to Depolarization Status of Cerebellar Neurons
Authors: Gaiddon Christian , Larmet Yves , Trinh Emmanuelle , Boutillier Anne‐Laurence , Sommer Bernd , Loeffler Jean‐Philippe
Published: March 2003

Abstract : To investigate the molecular mechanisms underlying

brain‐derived neurotrophic factor (BDNF)‐controlled synaptic plasticity, we

studied β2‐adrenergic receptor (β2‐AR)

expression in cultured cerebellar granule cells. We show that, depending on

the state of depolarization, BDNF exerts opposite effects on

β2‐AR expression. In neurons maintained in low K+

medium (5 mM K+) that will enter apoptosis, BDNF increases

β2‐AR and β2‐AR transcripts. In contrast, in

depolarized neurons (high K+ medium, 25 mM K+)

BDNF represses β2‐AR expression. The use of reporter genes

(driven by the β2‐AR promoter or restricted regulatory

elements) revealed that BDNF exerts its opposite effects at the

transcriptional level by recruiting a cyclic AMP response element (CRE) and

the trans‐acting factor CRE binding protein. These results provide

the first evidence that a neurotrophin, e.g., BDNF, may exert an opposite

effect on receptor expression and function (β2‐AR) according to the depolarization status of the neuron. Based on this finding, we propose that BDNF not only mediates neuronal survival, but is also involved in the modulation of the general sensitivity of the neuron to external signals, thus maintaining its optimal functional integration within the neuronal network.
Glutathione Efflux Induced by NMDA and Kainate
Authors: Wallin Camilla , Weber Stephen G. , Sandberg Mats
Published: March 2003

Abstract : Neurotoxicity in acute as well as chronic neurological diseases may be partly mediated by oxidative stress caused by overactivation of glutamate receptors. A key component of the cellular defense against oxidative stress is reduced glutathione. In our earlier work, we have shown that ischemia in brain induces increased efflux, elevated metabolism, and decreased tissue concentrations of glutathione. In this study, we have evaluated the effect of glutamate receptor activation on the efflux of glutathione from hippocampus in vitro. NMDA and kainate induced a delayed increase in glutathione, taurine, and phosphoethanolamine efflux. Extracellular glutathione was recovered mainly in the reduced form (85‐95%) ; the efflux was dependent on extracellular calcium but unrelated to dantrolene‐sensitive intracellular calcium release and independent of glutathione or NO synthesis. The NMDA‐induced efflux of glutathione was enhanced by blockage of γ‐glutamyl transpeptidase, indicating an increased transpeptidation of glutathione after NMDA receptor activation. Our results suggest that increased efflux of glutathione could be a factor in initiating nerve cell death via a change in intracellular redox potential and/or a decrease in the intracellular capacity for inactivation of reactive oxygen species.
α4 but Not α3 and α7 Nicotinic Acetylcholine Receptor Subunits Are Lost from the Temporal Cortex in Alzheimer's Disease
Authors: Martin‐Ruiz C. M. , Court J. A. , Molnar E. , Lee M. , Gotti C. , Mamalaki A. , Tsouloufis T. , Tzartos S. , Ballard C. , Perry R. H. , Perry E. K.
Published: March 2003

Abstract : Neuronal nicotinic acetylcholine receptors labelled with

tritiated agonists are reduced in the cerebral cortex in Alzheimer's disease

(AD), but to date it has not been demonstrated which nicotinic receptor

subunits contribute to this deficit. In the present study, autopsy tissue from

the temporal cortex of 14 AD cases and 15 age‐matched control subjects was

compared using immunoblotting with antibodies against recombinant peptides

specific for α3, α4, and α7 subunits, in conjunction with

[3H]epibatidine binding. Antibodies to α3, α4, and

α7 produced one major band on western blots at 59, 51, and 57 kDa,

respectively. [3H]Epibatidine binding and α4‐like

immunoreactivity (using antibodies against the extracellular domain and

cytoplasmic loop of the α4 subunit) were reduced in AD cases compared

with control subjects (p <0.02) and with a subgroup of control

subjects (n = 9) who did not smoke prior to death (p <0.05) for

the former two parameters. [3H]Epibatidine binding and cytoplasmic

α4‐like immunoreactivity were significantly elevated in a subgroup of

control subjects (n = 4) known to have smoked prior to death (p <0.05). There were no significant changes in α3‐ or α7‐like immunoreactivity associated with AD or tobacco use. The selective involvement of α4 has implications for understanding the role of nicotinic receptors in AD and potential therapeutic targets.
The Stimulation of Ketogenesis by Cannabinoids in Cultured Astrocytes Defines Carnitine Palmitoyltransferase I as a New Ceramide‐Activated Enzyme
Authors: Blázquez Cristina , Sánchez Cristina , Daza Andrés , Galve‐Roperh Ismael , Guzmán Manuel
Published: March 2003

Abstract: The effects of cannabinoids on ketogenesis in primary cultures of rat astrocytes were studied. ▵9‐Tetrahydrocannabinol (THC), the major active component of marijuana, produced a malonyl‐CoA‐independent stimulation of carnitine palmitoyltransferase I (CPT‐I) and ketogenesis from [14C]palmitate. The THC‐induced stimulation of ketogenesis was mimicked by the synthetic cannabinoid HU‐210 and was prevented by pertussis toxin and the CB1 cannabinoid receptor antagonist SR141716. Experiments performed with different cellular modulators indicated that the THC‐induced stimulation of ketogenesis was independent of cyclic AMP, Ca2+, protein kinase C, and mitogen‐activated protein kinase (MAPK). The possible involvement of ceramide in the activation of ketogenesis by cannabinoids was subsequently studied. THC produced a CB1 receptor‐dependent stimulation of sphingomyelin breakdown that was concomitant to an elevation of intracellular ceramide levels. Addition of exogenous sphingomyelinase to the astrocyte culture medium led to a MAPK‐independent activation of ketogenesis that was quantitatively similar and not additive to that exerted by THC. Furthermore, ceramide activated CPT‐I in astrocyte mitochondria. Results thus indicate that cannabinoids stimulate ketogenesis in astrocytes by a mechanism that may rely on CB1 receptor activation, sphingomyelin hydrolysis, and ceramide‐mediated activation of CPT‐I.
L‐Glutamate Suppresses Amyloid β‐Protein‐Induced Stellation of Cultured Rat Cortical Astrocytes
Authors: Abe Kazuho , Saito Hiroshi
Published: March 2003

Abstract: Alzheimer’s amyloid β‐protein (Aβ) has been reported to potentiate glutamate toxicity in neurons, but very little is known about interaction between Aβ and glutamate in astrocytes. Therefore, in the present study, we investigated the effects of Aβ and glutamate on morphology of astrocytes. Cultured rat cortical astrocytes exhibited polygonal morphology in the absence of stimulation and differentiated into process‐bearing stellate cells following exposure to Aβ (20 μM). L‐Glutamate (30‐1,000 μM) had no effect on astrocyte morphology in the absence of stimulation but strongly suppressed Aβ‐induced stellation. The suppressive effect of L‐glutamate on Aβ‐induced stellation was not mimicked by glutamate receptor agonists and not blocked by glutamate receptor antagonists. In contrast, the suppressive effect of L‐glutamate was mimicked by D‐ and L‐aspartate and transportable glutamate uptake inhibitors. These results suggest that Aβ‐induced astrocyte stellation is suppressed by a mechanism related to glutamate transporters.
Overexpression of the Neuritotrophic Cytokine S100β Precedes the Appearance of Neuritic β‐Amyloid Plaques in APPV717F Mice
Authors: Sheng J. G. , Mrak R. E. , Bales K. R. , Cordell B. , Paul S. M. , Jones R. A. , Woodward S. , Zhou X. Q. , McGinness J. M. , Griffin W. S. T.
Published: March 2003

Abstract: Homozygous APPV717F transgenic mice overexpress a human β‐amyloid precursor protein (βAPP) minigene encoding a familial Alzheimer’s disease mutation. These mice develop Alzheimer‐type neuritic β‐amyloid plaques surrounded by astrocytes. S100β is an astrocyte‐derived cytokine that promotes neurite growth and promotes excessive expression of βAPP. S100β overexpression in Alzheimer’s disease correlates with the proliferation of βAPP‐immunoreactive neurites in β‐amyloid plaques. We found age‐related increases in tissue levels of both βAPP and S100β mRNA in transgenic mice. Neuronal βAPP overexpression was found in cell somas in young mice, whereas older mice showed βAPP overexpression in dystrophic neurites in plaques. These age‐related changes were accompanied by progressive increases in S100β expression, as determined by S100β load (percent immunoreactive area). These increases were evident as early as 1 and 2 months of age, months before the appearance of β‐amyloid deposits in these mice. Such precocious astrocyte activation and S100β overexpression are similar to our earlier findings in Down’s syndrome. Accelerated age‐related overexpression of S100β may interact with age‐associated overexpression of mutant βAPP in transgenic mice to promote development of Alzheimer‐like neuropathological changes.
Correlation Between Structure of Bcl‐2 and Its Inhibitory Function of JNK and Caspase Activity in Dopaminergic Neuronal Apoptosis
Authors: Choi Won‐Seok , Yoon So‐Young , Chang In Ick , Choi Eui‐Ju , Rhim Hyewhon , Jin Byung K. , Oh Tae H. , Krajewski Stanislaw , Reed John C. , Oh Young J.
Published: March 2003

Abstract: To examine the correlation between the structure of Bcl‐2
and its inhibitory function of c‐Jun N‐terminal kinase (JNK) and caspase
activity, we established a dopaminergic neuronal cell line, MN9D
overexpressing Bcl‐2 (MN9D/Bcl‐2) or its structural mutants. The mutants
comprised a point mutation in the BH1 (G145A; MN9D/BH1) or BH2 (W188A;
MN9D/BH2) domain and a deletion mutation in the C‐terminal (MN9D/C22), BH3
(MN9D/BH3), or BH4 (MN9D/BH4) domain. As determined by the TUNEL (terminal
deoxynucleotidyltransferase nick end‐labeling) and MTT
[3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] reduction
assay, apoptotic death of MN9D/Neo cells reached 80‐90% within 24 h in
response to 1 μM staurosporine. Upon staurosporine treatment, JNK activity increased six‐ to sevenfold over the basal level within 2‐4 h. Treatment of MN9D/Neo with both staurosporine and a caspase inhibitor, Z‐VAD, attenuated cell death without suppressing JNK activation. Both staurosporine‐induced cell death and JNK activation were attenuated in MN9D/Bcl‐2. As determined by cleavage of poly(ADP‐ribose) polymerase into 85 kDa, Bcl‐2 blocked caspase activity as well. When cells overexpressing one of the Bcl‐2 mutants were treated with staurosporine, death was attenuated in MN9D/BH1, MN9D/BH2, and MN9D/C22 but not in MN9D/BH3 and MN9D/BH4. Similarly, both JNK and caspase activation were blocked in MN9D/BH1, MN9D/BH2, and MN9D/C22, whereas they were not suppressed in MN9D/BH3 and MN9D/BH4. Taken together, our data indicate that there exists a close structural and functional correlation of Bcl‐2 to JNK and caspase activity in staurosporine‐induced dopaminergic neuronal cell death.
p‐Ethynylphenylalanine
Authors: Stokes Alan H. , Xu Yimei , Daunais James A. , Tamir Hadassah , Gershon Michael D. , Butkerait Paul , Kayser Bernd , Altman Janina , Beck Wolfgang , Vrana Kent E.
Published: March 2003

Abstract: Tryptophan hydroxylase (TPH) is the initial and rate‐limiting enzyme in serotonin biosynthesis. The enzyme activity is dependent on molecular oxygen, a tetrahydropterin cosubstrate, and ferrous iron. The present study demonstrates that TPH is inhibited by a novel compound, p‐ethynylphenylalanine (pEPA), produced by the Heck reaction of trimethylsilylacetylene with N‐tert‐butyloxycarbonyl‐4‐iodo‐L‐phenylalanine methyl ester. pEPA is a more potent and specific inhibitor of TPH than p‐chlorophenylalanine (pCPA). In the present study, pEPA was demonstrated to inhibit competitively and reversibly TPH in vitro (Ki = 32.6 ± 6.2 μM vs. tryptophan). pEPA displayed little inhibitory activity toward tyrosine hydroxylase (EC 1.14.16.2), the initial and rate‐limiting enzyme for catecholamine biosynthesis, and no inhibition of phenylalanine hydroxylase or tyrosinase. In addition, pEPA was a poor ligand for the serotonin transporter and several serotonin receptors. Administration of pEPA (30 mg/kg) to rats produced a 95 ± 5% decrease in TPH activity in brain homogenates and a concomitant decrease in serotonin and 5‐hydroxyindole‐3‐acetic acid levels (85%) at 24 h after injection. In contrast, pCPA produced a similar effect (87 ± 5% decrease in TPH activity) only at 10 times the concentration (300 mg/kg). These results suggest that pEPA is a selective, reversible, and potent inhibitor of TPH both in vitro and in vivo. The potential for pEPA to inhibit selectively and reversibly the biosynthesis of serotonin may contribute to the characterization of the role of serotonin in behavioral and physiological activities.
A Monoclonal Antibody to Amyloid Precursor Protein Induces Neuronal Apoptosis
Authors: Rohn Troy T. , Ivins Kathryn J. , Bahr Ben A. , Cotman Carl W. , Cribbs David H.
Published: March 2003

Abstract: Although there is considerable evidence suggesting that altered metabolism of β‐amyloid precursor protein (APP) and accumulation of its β‐amyloid fragment are key features of Alzheimer’s disease (AD), the normal physiological function of APP remains elusive. We investigated the potential role of APP in neurons using the monoclonal antibody 22C11, which binds to the extracellular domain of the human, rat, or mouse APP. Exposure of cortical neurons to 22C11 induced morphological changes including neurite degeneration, nuclear condensation, and internucleosomal DNA cleavage that were consistent with neurons dying by apoptosis. Supporting a role for 22C11‐mediated apoptosis occurring by binding to APP were data demonstrating that preincubation of 22C11 with either purified APP or a synthetic peptide (APP66‐81) that contains the epitope for 22C11 significantly attenuated neuronal damage induced by 22C11. The specificity of 22C11 was further supported by data showing no apparent effects of either mouse IgG or the monoclonal antibody P2‐1, which is specific for the aminoterminal end of human but not rat APP. In addition, biochemical features indicative of apoptosis were the formation of 120‐ and 150‐kDa breakdown products of fodrin following treatment of cortical neurons with 22C11. Both the morphological and the biochemical changes induced by 22C11 were prevented following pretreatment of neurons with the general caspase inhibitor N‐benzyloxycarbonyl‐Val‐Ala‐Asp(O‐methyl)‐fluoromethyl ketone. Prior incubation of cortical neurons with GSH ethyl ester (GEE), a cell‐permeable form of GSH, resulted in complete protection from the 22C11 insult, thus implicating an oxidative pathway in 22C11‐mediated neuronal degeneration. This was further supported by the observation that prior treatment of neurons with buthionine sulfoximine, an inhibitor of γ‐glutamylcysteinyl synthetase, potentiated the toxic effects of 22C11. Finally, with use of compartmented cultures of hippocampal neurons, it was also demonstrated that selective application of 22C11 caused local neuritic degeneration that was prevented by the addition of GEE to the neuritic compartment. Thus, the binding of a monoclonal antibody to APP initially triggers neurite degeneration that is followed by caspase‐dependent apoptosis in neuronal cultures and illustrates a novel property of this protein in neurons that may contribute to the profound neuronal cell death associated with AD.
Light‐Induced Tyrosine Phosphorylation of BIT in the Rat Suprachiasmatic Nucleus
Authors: Nakahata Yasukazu , Okumura Nobuaki , Shima Takaki , Okada Masato , Nagai Katsuya
Published: March 2003

Abstract: Circadian changes of protein tyrosine phosphorylation in the hypothalamic suprachiasmatic nucleus have been studied using rats maintained under 12‐h light/ 12‐h dark cycles as well as constant dark conditions. We found that tyrosine phosphorylation of BIT (brain immunoglobulin‐like molecule with tyrosine‐based activation motifs), a transmembrane glycoprotein of 90‐95 kDa, was higher in the light period than in the dark period and was increased after light exposure in the dark period. Similar changes in tyrosine phosphorylation were observed under constant dark conditions, but its amplitude was weaker than that in 12‐h light/12‐h dark cycles. As the tyrosine‐phosphorylated form of BIT is able to bind to the Src homology 2 domain of a protein tyrosine phosphatase, SHP‐2, we examined association of these proteins in suprachiasmatic nucleus extracts and found that SHP‐2 was coprecipitated with BIT in parallel with its tyrosine phosphorylation. These results suggest that tyrosine phosphorylation of BIT might be involved in light‐induced entrainment of the circadian clock.
Synergetic Activation of p38 Mitogen‐Activated Protein Kinase and Caspase‐3‐Like Proteases for Execution of Calyculin A‐Induced Apoptosis but Not N‐Methyl‐d‐Aspartate‐Induced Necrosis in Mouse Cortical Neurons
Authors: Ko Hyuk Wan , Han Kong Sook , Kim Eun Young , Ryu Bo Rum , Yoon Won Joo , Jung Yong‐Keun , Kim Seong Up , Gwag Byoung Joo
Published: March 2003

Abstract: We examined the possibility that p38 mitogen‐activated protein kinase and caspase‐3 would be activated for execution of apoptosis and excitotoxicity, the two major types of neuronal death underlying hypoxicischemic and neurodegenerative diseases. Mouse cortical cell cultures underwent widespread neuronal apoptosis 24 h following exposure to 10‐30 nM calyculin A, a selective inhibitor of Ser/Thr phosphatase I and IIA. Activity of p38 was increased 2‐4 h following exposure to 30 nM calyculin A. Addition of 3‐10 μM PD169316, a selective p38 inhibitor, partially attenuated calyculin A neurotoxicity. Activity of caspase‐3‐like proteases was increased in cortical cell cultures exposed to 30 nM calyculin A for 8‐16 h as shown by cleavage of DEVD‐p‐nitroanilide and phosphorylated tau. Proteolysis of tau was completely blocked by addition of 100 μM N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl ketone (z‐VAD‐fmk), a broad‐spectrum inhibitor of caspases, but incompletely by 10 μM PD169316. Calyculin A neurotoxicity was partially sensitive to 100 μM z‐VAD‐fmk. Cotreatment with 10 μM PD169316 and 100 μM z‐VAD‐fmk showed additive neuroprotection against calyculin A. Neither PD169316 nor z‐VAD‐fmk showed a beneficial effect against excitotoxic neuronal necrosis induced by exposure to 20 μM NMDA. Thus, caspase‐3‐like proteases and p38 likely contribute to calyculin A‐induced neuronal apoptosis but not NMDA‐induced neuronal necrosis.
Beneficial Effects of Dietary Restriction on Cerebral Cortical Synaptic Terminals
Authors: Guo ZhiHong , Ersoz Arzu , Butterfield D. Allan , Mattson Mark P.
Published: March 2003

Abstract : Recent studies have shown that rats and mice maintained

on a dietary restriction (DR) regimen exhibit increased resistance of neurons

to excitotoxic, oxidative, and metabolic insults in experimental models of

Alzheimer's, Parkinson's, and Huntington's diseases and stroke. Because

synaptic terminals are sites where the neurodegenerative process may begin in

such neurodegenerative disorders, we determined the effects of DR on synaptic

homeostasis and vulnerability to oxidative and metabolic insults. Basal levels

of glucose uptake were similar in cerebral cortical synaptosomes from rats

maintained on DR for 3 months compared with synaptosomes from rats fed ad

libitum. Exposure of synaptosomes to oxidative insults (amyloid β‐peptide

and Fe2+) and a metabolic insult (the mitochondrial toxin 3‐nitropropionic acid) resulted in decreased levels of glucose uptake. Impairment of glucose uptake following oxidative and metabolic insults was significantly attenuated in synaptosomes from rats maintained on DR. DR was also effective in protecting synaptosomes against oxidative and metabolic impairment of glutamate uptake. Loss of mitochondrial function caused by oxidative and metabolic insults, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated in synaptosomes from rats maintained on DR. Levels of the stress proteins HSP‐70 and GRP‐78 were increased in synaptosomes from DR rats, consistent with previous data suggesting that the neuroprotective mechanism of DR involves a “preconditioning” effect. Collectively, our data provide the first evidence that DR can alter synaptic homeostasis in a manner that enhances the ability of synapses to withstand adversity.
Trimerization of Cell Adhesion Molecule L1 Mimics Clustered L1 Expression on the Cell Surface
Authors: Hall Heike , Bozic Damir , Fauser Charlotte , Engel Jürgen
Published: March 2003

Abstract : Several studies indicate that cell adhesion molecules have to be clustered on the cell surface to engage in adhesive functions. We investigated adhesive functions of clustered versus monomeric L1 extracellular parts in vitro to distinguish how clustering affects ligand binding and promotion of neurite outgrowth. Trimeric L1 was recombinantly expressed and covalently assembled by the cartilage matrix protein's coiled‐coil domain. Trimeric L1 has an apparent molecular mass of ~380 kDa in the nonreduced form and ~130 kDa in the reduced form. Rotary shadowing electron micrographs of trimeric L1 revealed a rod‐like shape terminating in three globular domains. Monomeric L1 assumes a horseshoe shape of domains Ig I‐IV followed by a rod‐like structure consisting of Ig V and VI and fibronectin type III 1‐5. Circular dichroism measurements showed that the secondary structure consists of β‐sheets. Trimeric L1 binds to itself, to monomeric L1, to laminin‐1, and to α5β1 integrin in a concentration‐dependent manner. In contrast, binding of monomeric L1 could only be saturated with itself but not with laminin‐1 and with α5β1 integrin. Promotion of neurite outgrowth from PC12 cells cultured on adsorbed trimeric L1 was increased by 100%, whereas on monomeric L1 the increase was only 50% over the control value. Promotion of neurite outgrowth from PC12 cells was specifically inhibited in a concentration‐dependent manner by a polyclonal antibody against L1. These findings show that clustering of only three extracellular domains increases considerably L1's binding affinity to different ligands and enhances neurite outgrowth, suggesting that adhesive functions of L1 on the cell surface depend on cluster formation.
Protective Effect of Harmalol and Harmaline on MPTP Neurotoxicity in the Mouse and Dopamine‐Induced Damage of Brain Mitochondria and PC12 Cells
Authors: Lee Chung Soo , Han Eun Sook , Jang Yoon Young , Han Jeong Ho , Ha Hyun Wook , Kim Doo Eung
Published: March 2003

The present study elucidated the protective effect of β‐carbolines (harmaline, harmalol, and harmine) on oxidative neuronal damage. MPTP treatment increased activities of total superoxide dismutase, catalase, and glutathione peroxidase and levels of malondialdehyde and carbonyls in the basal ganglia, diencephalon plus midbrain of brain compared with control mouse brain. Coadministration of harmalol (48 mg/kg) attenuated the MPTP effect on the enzyme activities and formation of tissue peroxidation products. Harmaline, harmalol, and harmine attenuated both the 500 μM

MPP+‐induced inhibition of electron flow and membrane potential

formation and the 100 μM dopamine‐induced thiol oxidation and

carbonyl formation in mitochondria. The scavenging action of β‐carbolines

on hydroxyl radicals was represented by inhibition of

2‐deoxy‐d‐ribose degradation. Harmaline and harmalol (100

μM) attenuated 200 μM dopamine‐induced viability loss

in PC12 cells. The β‐carbolines (50 μM) attenuated 50

μM dopamine‐induced apoptosis in PC12 cells. The compounds alone

did not exhibit significant cytotoxic effects. The results indicate that

β‐carbolines attenuate brain damage in mice treated with MPTP and

MPP+‐induced mitochondrial damage. The compounds may prevent dopamine‐induced mitochondrial damage and PC12 cell death through a scavenging action on reactive oxygen species and inhibition of monoamine oxidase and thiol oxidation.
Neurite Outgrowth Induced by a Synthetic Peptide Ligand of Neural Cell Adhesion Molecule Requires Fibroblast Growth Factor Receptor Activation
Authors: Rønn Lars C B , Doherty Patrick , Holm Arne , Berezin Vladimir , Bock Elisabeth
Published: March 2003

The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM‐NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons from wild‐type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant‐negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant‐negative form of the mouse FGFR1, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule‐stimulated neurite outgrowth. The C3 peptide appears to stimulate neurite outgrowth by specifically activating an NCAM‐FGFR‐dependent signaling cascade and may therefore be of considerable interest as a tool for the determination of NCAM‐dependent neurite outgrowth as well as a potential drug capable of promoting outgrowth and regeneration of NCAM‐responsive axons.
Determination of the Phospholipid Precursor of Anandamide and Other N‐Acylethanolamine Phospholipids Before and After Sodium Azide‐Induced Toxicity in Cultured Neocortical Neurons
Authors: Hansen Henrik H , Hansen Steen Honoré , Schousboe Arne , Hansen Harald S
Published: March 2003

Phospholipase d‐mediated hydrolysis of

N‐acylethanolamine phospholipids (NAPEs) releases anandamide and

other N‐acylethanolamines, resulting in different actions at cellular

targets in the CNS. Recently, we have demonstrated that these N‐acyl

lipids accumulate in cultured neocortical neurons subjected to sodium

azide‐induced cell injury. We here extend the information on the NAPE

response, reporting on the composition of N‐acylspecies of NAPE,

employing a new methodological approach of HPLC‐coupled electrospray

ionization mass spectrometry. Exposure to sodium azide (5 mM)

increased the total amount of NAPE threefold over control levels ; however, no

alteration of the relative composition of NAPE species was detected. The

anandamide precursor (20 : 4‐NAPE) constituted only 0.1% of all NAPEs detected

in the neurons. Total NAPE species in control cells amounted to 956‐1,060

pmol/107 cells. Moreover, we detected the presence of an unknown

NAPE species with molecular weight identical to 20 : 4‐NAPE. This may suggest

the presence of a putative stereoisomer of the anandamide precursor with at

least one trans‐configured double bond in the N‐arachidonoyl

moiety. These results show that with the present method, neuronal NAPE species

can be identified and quantified with respect to N‐acyl composition,

including a trans‐isomer of the anandamide precursor. The anandamide precursor is up‐regulated to the same extent as other NAPEs upon neuronal injury.
Identification and Characterization of Nuclear Factor κB Binding Sites in the Murine bcl‐χ Promoter
Authors: Glasgow J. N. , Wood T. , Perez‐Polo J. R.
Published: March 2003

Abstract: Signal transduction pathways that mediate neuronal
commitment to apoptosis involve the nuclear factor κB (NF‐κB)
transcription factor. Bcl‐XL is a potent regulator of apoptosis in
the CNS and is highly expressed in the developing and adult brain. We
identified three putative NF‐κB DNA binding sequences clustered upstream
of the brain‐specific transcription start site in the upstream promoter
region. Recombinant p50/p50 and NF‐κB proteins from nuclear extracts
bound to these sites as determined by electrophoretic mobility shift assay and
biotin‐oligonucleotide/streptavidin affinity assays. NF‐κB
overexpression, coupled with bcl‐x promoter/reporter assays using a
series of murine bcl‐x promoter and deletion mutants, has identified
the downstream 1.1 kb of the bcl‐x promoter as necessary for basal
promoter activity and induction by NF‐κB. The mutagenic removal of
NF‐κB binding sites individually or in combination revealed altered
response patterns to p49/p65 and p50/p65 overexpression. These results support
the hypothesis that NF‐κB can act to enhance Bcl‐XL
expression via highly selective interactions, where NF‐κB binding and
bcl‐x promoter activation are dependent on both DNA binding site sequence and NF‐κB subunit composition. Our data suggest that molecular events associated with NF‐κB promote regulation of neuronal apoptosis in the developing or injured CNS.
Plasticin, a Type III Neuronal Intermediate Filament Protein, Assembles as an Obligate Heteropolymer
Authors: Asch William S. , Schechter Nisson
Published: March 2003

Abstract: The assembly characteristics of the neuronal intermediate
filament protein plasticin were studied in SW13 cells in the presence and
absence of a cytoplasmic filament network. Full‐length plasticin cannot
polymerize into homopolymers in filament‐less SW13c1.2Vim‐ cells
but efficiently coassembles with vimentin in SW13c1.1Vim‐ cells. By
cotransfecting plasticin and vimentin in SW13c1.1Vim‐ cells, we
show that plasticin assembly requires vimentin in noncatalytic amounts.
Differing effects on assembly were seen with point mutations of plasticin
monomers that were analogous to the keratin mutations that cause epidermolysis
bullosa simplex (EBS). In particular, plasticin monomers with point mutations
analogous to those in EBS do not uniformly inhibit neurofilament (NF) network
formation. A point mutation in the helix termination sequence resulted in
complete filament aggregation when coexpressed with vimentin but showed
limited coassembly with low‐ and medium‐molecular‐weight NF proteins (NF‐L and
NF‐M, respectively). In transfected SW13c1.1Vim+ cells, a point mutation in the first heptad of the α‐helical coil region formed equal amounts of filaments, aggregates, and a mixture of filaments and aggregates. Furthermore, coexpression of this point mutation with NF‐L and NF‐M was associated with a shift toward increased numbers of aggregates. These results suggest that there are important structural differences in assembly properties between homologous fish and mammalian intermediate filament proteins. These structural differences may contribute to the distinctive growth characteristics of the teleost visual pathway.
Progressive Segregation of Unmyelinated Axons in Peripheral Nerves, Myelin Alterations in the CNS, and Cyst Formation in the Kidneys of Myelin and Lymphocyte Protein‐Overexpressing Mice
Authors: Frank Marcus , Atanasoski Suzana , Sancho Sara , Magyar Josef P. , Rülicke Thomas , Schwab Martin E. , Suter Ueli
Published: March 2003

Abstract: Myelin and lymphocyte protein (MAL) is a putative tetraspan proteolipid that is highly expressed by Schwann cells and oligodendrocytes as a component of compact myelin. Outside of the nervous system, MAL is found in apical membranes of epithelial cells, mainly in the kidney and stomach. Because MAL is associated with glycosphingolipids, it is thought to be involved in the organization, transport, and maintenance of glycosphingolipid‐enriched membrane microdomains. In this report, we describe the generation and analysis of transgenic mice with increased MAL gene dosage. Immunohistochemical analysis revealed that the localization of MAL overexpression in the transgenic animals corresponded closely to the MAL expression pattern observed in wildtype animals, indicating correct spatial regulation of the transgene. Phenotypically, MAL overexpression led to progressive dissociation of unmyelinated axons from bundles in the PNS, a tendency to hypomyelination and aberrant myelin formation in the CNS, and the formation of large cysts in the tubular region of the kidney. Thus, increased expression of MAL appears to be deleterious to membranous structures in the affected tissues, indicating a requirement for tight control of endogenous MAL expression in Schwann cells, oligodendrocytes, and kidney epithelial cells.
Ischemia‐Induced Phosphorylation of Initiation Factor 2 in Differentiated PC12 Cells
Authors: Muñoz F. , Martín M. E. , Tomico J. Manso‐ , Berlanga J. , Salinas M. , Fando J. L.
Published: March 2003

Abstract: An in vitro model of ischemia was obtained by subjectingPC12 cells differentiated with nerve growth factor to a combination of glucosedeprivation plus anoxia. Immediately after the ischemic period, the proteinsynthesis rate was significantly inhibited (80%) and western blots of cellextracts revealed a significant accumulation of phosphorylated eukaryoticinitiation factor 2, α subunit, eIF2(αP) (42%). Upon recovery,eIF2(αP) levels returned to control values after 30 min, whereas proteinsynthesis was still partially inhibited (33%) and reached almost controlvalues within 2 h. The activities of the mammalian eIF2α kinases,double‐stranded RNA‐activated protein kinase, mammalian GCN2 homologue, andendoplasmic reticulum‐resident kinase, were determined. None of theeIF2α kinases studied showed increased activity in ischemic cells ascompared with controls. Exposure of cells to cell‐permeable inhibitors ofprotein phosphatases 1 and 2A, calyculin A or tautomycin, induced dose‐ andtime‐dependent accumulation of eIF2(αP), mimicking an ischemic effect.Protein phosphatase activity, as measured with [32P]phosphorylasea as a substrate, diminished during ischemia and returned to controllevels upon 30‐min recovery. In addition, the rate of eIF2(αP)dephosphorylation was significantly lower in ischemic cells, paralleling boththe greatest translational inhibition and the highest eIF2(αP) levels.The endogenous phosphatase activity from control and ischemic extracts showeddifferent sensitivity to inhibitor 2 and fostriecin in in vitro assays,inhibitor‐2 effect in ischemic cells being lower than in control cells.Together these results indicate that an eIF2α phosphatase, probablyprotein phosphatase 1, is implicated in the ischemia‐induced eIF2(αP)accumulation in PC12 cells.
Inhibition of Aberrant and Constitutive Phosphorylation of the High‐Molecular‐Mass Neurofilament Subunit by CEP‐1347 (KT7515), an Inhibitor of the Stress‐Activated Protein Kinase Signaling Pathway
Authors: O'Ferrall Erin K. , Robertson Janice , Mushynski Walter E.
Published: March 2003

Abstract: Previous studies have implicated stress‐activated proteinkinases (SAPKs) in aberrant phosphorylation of the high‐molecular‐massneurofilament subunit (NFH). We now present direct evidence for thisinvolvement using CEP‐1347, a specific inhibitor of SAPK activation.Inhibition by this drug of stress‐induced phosphorylation of NFH and themiddle‐molecular‐mass neurofilament subunit in the perikaryon of dorsal rootganglion (DRG) neurons paralleled the decrease in levels of activated SAPKsand was essentially complete at 1 μM CEP‐1347. In addition, a rolefor SAPKs in the constitutive phosphorylation of NFH was demonstrated.Longterm treatment of unstressed DRG neurons with CEP‐1347 lowered thesteady‐state phosphorylation level of NFH in neurites. No such effect was seenin neurons treated with PD 098059, which blocks activation of extracellularsignal‐regulated kinase 1/2. DRG neurites were shown to contain high basallevels of activated SAPKs. These included a 55‐kDa SAPK whose activation wascompletely abolished at 0.05 μM CEP‐1347 and a 45‐kDa SAPK thatwas not affected by the drug. These results indicate that SAPKs are involvedin both stress‐induced and constitutive phosphorylation of NFH. The differingresponses of SAPKs in neurites and cell bodies to CEP‐1347 inhibition furthersuggest the presence of different signaling pathways in the two neuronalcompartments.