bluuebunny/crossref_metadata_2025 huggingface dataset:
Life Without Huntingtin
Authors: Metzler Martina , Chen Nansheng , Helgason Cheryl D , Graham Rona K. , Nichol Kerrie , McCutcheon Krista , Nasir Jamal , Humphries R. Keith , Raymond Lynn A. , Hayden Michael R.
Published: March 2003

Abstract : Huntington disease (HD) is a neurodegenerative disorder
associated with polyglutamine expansion in a recently identified protein,
huntingtin. Huntingtin is widely expressed and plays a crucial role in
development, because gene‐targeted HD‐/‐ mouse embryos die early in
embryogenesis. To analyze the function of normal huntingtin, we have generated
HD‐/‐ embryonic stem (ES) cells and used an in vitro model of ES
cell differentiation to analyze their ability to develop into neuronal cells.
Expression analysis of wild‐type ES cells revealed that huntingtin is
expressed at all stages during ES cell differentiation with high expression in
neurons. Expression levels increased with the maturation of differentiating
neurons, demonstrating that expression of huntingtin is developmentally
regulated in cell culture and resembles the pattern of expression observed in
differentiating neurons in the mouse brain. It is interesting that
HD‐/‐ ES cells could differentiate into mature postmitotic neurons that expressed functional voltage‐ and neurotransmittergated ion channels. Moreover, both excitatory and inhibitory spontaneous postsynaptic currents were observed, indicating the establishment of functional synapses in the absence of huntingtin. These results demonstrate that huntingtin is not required for the generation of functional neurons with features characteristic of postmitotic neurons in the developing mouse brain.
Increased Vulnerability of Hippocampal Neurons from Presenilin‐1 Mutant Knock‐In Mice to Amyloid β‐Peptide Tox
Authors: Guo Qing , Sebastian Lois , Sopher Bryce L. , Miller Miles W. , Ware Carol B. , Martin George M. , Mattson Mark P.
Published: March 2003

Abstract : Many cases of early‐onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin‐1 (PS1) gene. Overexpression of PS1 mutations in cultured PC12 cells increases their vulnerability to apoptosis‐induced trophic factor withdrawal and oxidative insults. We now report that primary hippocampal neurons from PS1 mutant knock‐in mice, which express the human PS1M146V mutation at normal levels, exhibit increased vulnerability to amyloid β‐peptide toxicity. The endangering action of mutant PS1 was associated with increased superoxide production, mitochondrial membrane depolarization, and caspase activation. The peroxynitrite‐scavenging antioxidant uric acid and the caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl ketone protected hippocampal neurons expressing mutant PS1 against cell death induced by amyloid β‐peptide. Increase oxidative stress may contribute to the pathogenic action of PS1 mutations, and antioxidants may counteract the adverse property of such AD‐linked mutations.
Activation of Stress‐Activated Protein Kinases Correlates with Neurite Outgrowth Induced by Protease Inhibition in PC12 Cells
Authors: Giasson Benoit I , Bruening Wendy , Durham Heather D , Mushynski Walter E
Published: March 2003

Abstract : PC12 cells are well characterized for their ability to
differentiate into neuronal‐like cells when challenged with nerve growth
factor. It has been reported that the calpain and proteasome inhibitor
N‐acetyl‐Leu‐Leu‐norleucinal (CI) is also able to induce neurite
outgrowth in PC12 cells. In this study, we report that the inhibitor of
proteasomal chymotrypsin‐like activity,
carbobenzoxy‐Ile‐Glu‐(O‐tert‐butyl)‐Ala‐Leu‐aldehyde (PSI), can also induce differentiation of PC12 cells. Induction of neurite outgrowth with PSI, CI, or its close analogue, carbobenzoxy‐Leu‐Leu‐leucinal (MG132), was associated with stress‐activated protein kinase (SAPK) activation. Neurite formation induced by protease inhibition was independent of mitogen‐activated protein kinase/extracellular signal‐regulated kinase, p38/reactivating kinase, or phosphatidylinositol 3‐kinase activities. The exact mechanism by which protease inhibition activates SAPKs remains to be elucidated ; however, our results suggest that the SAPK signal transduction cascade may be an alternative and/or parallel pathway in the regulation of neuronal differentiation.
Biochemical and Autoradiographic Measurements of Brain Serotonin Synthesis Rate in the Freely Moving Rat
Authors: Gharib Abdallah , Balende Cédar , Sarda Nicole , Weissmann Dinah , Plenevaux Alain , Luxen André , Bobillier Pierre , Pujol Jean‐François
Published: March 2003

Abstract: Biochemical approaches were used in freely moving rats to
determine, under steady‐state conditions, the brain/arterial plasma partition
coefficients of L‐tryptophan and α‐[3H]methyl‐L‐tryptophan, from which the lumped constant for the α‐methyl‐L‐tryptophan method of estimating the rate of brain serotonin synthesis is calculated. The lumped constants were significantly different in the various structures examined: 0.149 ± 0.003 in the raphe dorsalis, 0.103 ± 0.002 in the raphe centralis, 0.087 ± 0.003 in the reticular formation, and 0.62 ± 0.08 in the pineal gland. From these data we proposed a two‐compartment model to calculate the rate of serotonin synthesis by quantitative autoradiography using a three‐time point experiment. Rates of synthesis for the raphe dorsalis and the reticular formation (620 ± 57 and 80 ± 35 pmol/g of tissue/min, respectively) were similar to those measured simultaneously by biochemical means, but rates were 50% higher for the raphe centralis (568 ± 90 vs. 381 ± 31 pmol/g of tissue/min). The lack of dynamic equilibrium of the tracer between plasma and tissue pools may explain the discrepancy between the two methods. Our findings did not confirm previous data, indicating that the application of the autoradiographic method to measure the rate of brain serotonin synthesis using α‐methyl‐L‐tryptophan as tracer has limitations.
Receptor‐Mediated Transcytosis of Cyclophilin B Through the Blood—Brain Barrier
Authors: Carpentier Mathieu , Descamps Laurence , Allain Fabrice , Denys Agnès , Durieux Sandrine , Fenart Laurence , Kieda Claudine , Cecchelli Roméo , Spik Geneviève
Published: March 2003

Abstract: Cyclophilin B (CyPB) is a cyclosporin A (CsA)‐binding protein mainly located in intracellular vesicles and secreted in biological fluids. In previous works, we demonstrated that CyPB interacts with T lymphocytes and enhances in vitro cellular incorporation and activity of CsA. In addition to its immunosuppressive activity, CsA is able to promote regeneration of damaged peripheral nerves. However, the crossing of the drug from plasma to neural tissue is restricted by the relative impermeability of the blood—brain barrier. To know whether CyPB might also participate in the delivery of CsA into the brain, we have analyzed the interactions of CyPB with brain capillary endothelial cells. First, we demonstrated that CyPB binds to two types of binding sites present at the surface of capillary endothelial cells from various species of tissues. The first type of binding sites (KD = 300 nM; number of sites = 3 × 106) is related to interactions with negatively charged compounds such as proteoglycans. The second type of binding sites, ∼50,000 per cell, exhibits a higher affinity for CyPB (KD = 15 nM) and is involved in an endocytosis process, indicating it might correspond to a functional receptor. Finally, the use of an in vitro model of blood—brain barrier allowed us to demonstrate that CyPB is transcytosed by a receptor‐mediated pathway (flux = 16.5 fmol/cm2/h). In these conditions, CyPB did not significantly modify the passage of CsA, indicating that it is unlikely to provide a pathway for CsA brain delivery.
Alterations in Ionized and Total Blood Magnesium After Experimental Traumatic Brain Injury
Authors: Bareyre Florence M. , Saatman Kathryn E. , Helfaer Mark A. , Sinson Grant , Weisser Justin D. , Brown Adrienne L. , McIntosh Tracy K.
Published: March 2003

Abstract: Experimental evidence suggests that magnesium plays a role in the pathophysiological sequelae of brain injury. The present study examined the variation of blood ionized and total magnesium, as well as potassium, sodium, and ionized calcium, after experimental fluid percussion brain injury in rats. Blood ionized magnesium concentration significantly declined from 0.45 ± 0.02 to 0.32 ± 0.02 mM by 30 min postinjury and stayed depressed for the 24‐h study period in vehicle‐treated rats. Blood total magnesium concentration was 0.59 ± 0.01 mM and remained stable over time in brain‐injured vehicle‐treated animals. When magnesium chloride (125 μmol/rat) was administered 1 h postinjury, ionized magnesium levels were restored by 2 h postinjury and remained at normal values up to 24 h following brain trauma. Magnesium treatment also significantly reduced posttraumatic neuromotor impairments 1 and 2 weeks after the insult, but failed to attenuate spatial learning deficits. A significant positive and linear correlation could be established between ionized magnesium levels measured 24 h postinjury and neuromotor outcome at 1 and 2 weeks. We conclude that acute ionized magnesium measurement may be a predictor of long‐term neurobehavioral outcome following head injury and that delayed administration of magnesium chloride can restore blood magnesium concentration and attenuate neurological motor deficits in brain‐injured rats.
Glycosylation Sites Flank Phosphorylation Sites on Synapsin I
Authors: Cole Robert N. , Hart Gerald W.
Published: March 2003

Abstract: Synapsin I is concentrated in nerve terminals, where it appears to anchor synaptic vesicles to the cytoskeleton and thereby ensures a steady supply of fusion‐competent synaptic vesicles. Although phosphorylation‐dependent binding of synapsin I to cytoskeletal elements and synaptic vesicles is well characterized, little is known about synapsin I’s O‐linked N‐acetylglucosamine (O‐GlcNAc) modifications. Here, we identified seven in vivo O‐GlcNAcylation sites on synapsin I by analysis of HPLC‐purified digests of rat brain synapsin I. The seven O‐GlcNAcylation sites (Ser55, Thr56, Thr87, Ser516, Thr524, Thr562, and Ser576) in synapsin I are clustered around its five phosphorylation sites in domains B and D. The proximity of phosphorylation sites to O‐GlcNAcylation sites in the regulatory domains of synapsin I suggests that O‐GlcNAcylation may modulate phosphorylation and indirectly affect synapsin I interactions. With use of synthetic peptides, however, the presence of an O‐GlcNAc at sites Thr562 and Ser576 resulted in only a 66% increase in the Km of calcium/calmodulin‐dependent protein kinase II phosphorylation of site Ser566 with no effect on its Vmax. We conclude that O‐GlcNAcylation likely plays a more direct role in synapsin I interactions than simply modulating the protein’s phosphorylation.
Induction of the Manganese Superoxide Dismutase Gene by Sphingomyelinase and Ceramide
Authors: Pahan Kalipada , Dobashi Kazushige , Ghosh Bidyut , Singh Inderjit
Published: March 2003

Abstract : The present study reports the effect of ceramide

generated by hydrolysis of membrane sphingomyelin with bacterial

sphingomyelinase (SMase) and of cell‐permeable ceremide analogues on the

expression of manganese superoxide dismutase (MnSOD). Incubation of the rat

primary astrocytes with SMase led to a time‐and dose‐dependent increase in

MnSOD activity. The increase in MnSOD activity was accompanied by an increase

in MnSOD protein and mRNA. A similar effect on the expression of MnSOD was

observed with the addition of cell‐permeable ceramide analogues (C2

and C6). On the other hand, C2‐dihydroceramide

(N‐acetylsphinganine), which lacks the functional critical double

bond, was ineffective in inducing the expression of MnSOD. Nuclear run‐on

analysis showed that SMase and ceramide increased the rate of transcription of

the MnSOD gene. Besides astrocytes, SMase was also found to induce the

expression of MnSOD in rat mesangial cells, C6 glial cells, PC12 cells, and human skin fibroblasts. Markedly higher expression of mRNA, protein, and activity of MnSOD in skin fibroblasts from patients with Farber disease, a human disorder with pathognomonic accumulation of ceramide due to a deficiency of ceramidase, than in normal skin fibroblasts indicate that ceramide may act as a physiological inducer of MnSOD gene expression. However, stimulation of ceramide‐mediated DNA fragmentation by antisense knockdown of MnSOD suggests that induction of MnSOD by ceramide is a protective response of the cell.
Brains of Aged Apolipoprotein E‐Deficient Mice Have Increased Levels of F2‐Isoprostanes, In Vivo Markers of Lipid Peroxidation
Authors: Praticò Domenico , Rokach Joshua , Tangirala Rajendra K.
Published: March 2003

Abstract : Apolipoprotein E (apoE) is the major apolipoprotein of

the CNS. Differential expression of apoE isoforms has been linked to longevity

and to the pathogenesis of Alzheimer's disease. Several studies have

demonstrated that this glycoprotein is important in mature as well as in aging

CNS, where it may serve neurotrophic and/or neuroprotective functions. Some

reports have shown that apoE‐deficient mice have age‐dependent

neurodegeneration and cognitive impairment ; others have not confirmed these

observations. ApoE‐deficient mice also develop hypercholesterolemia on a chow

diet and have in vivo increased plasma lipid peroxidation products.

F2‐isoprostanes are prostaglandin F2α isomers and

chemically stable peroxidation products of arachidonic acid. Both isoprostane

F2α‐III and isoprostane F2α‐VI were markedly elevated in the brains of aged apoE‐deficient mice compared with either wild‐type C57 B1/6 mice or a distinct mouse model of hypercholesterolemia, the low‐density lipoprotein receptor‐deficient mouse. By contrast, no difference in isoprostane levels was observed in young apoE‐deficient mice compared with age‐matched wild‐type control mice. Our findings indicate that disorder of lipid metabolism in the absence of apoE can induce an age‐dependent increase in brain lipid peroxidation products.
Glutathione and Signal Transduction in the Mammalian CNS
Authors: Janáky R. , Ogita K. , Pasqualotto B. A. , Bains J. S. , Oja S. S. , Yoneda Y. , Shaw C. A.
Published: March 2003

Abstract : The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its γ‐glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose‐dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)‐2‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.
Light Exposue Activates Retina Ganglion Cell Lysophosphatidic Acid Acyl Transferase and Phosphatidic Acid Phosphatase by a c‐fos‐Dependent Mechanism
Authors: Zerpa G. A. de Arriba , Guido M. E. , Bussolino D. F. , Pasquare S. J. , Castagnet P. I. , Giusto N. M. , Caputto B. L.
Published: March 2003

Abstract : We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in anglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos‐dependent mechanism because an oligonucleotide antisense to c‐fos mRNA substantially blocked the light‐dark differences. We also studied in viro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light ; no increase was observed in preparations obtained from light‐exposed animals reated with the c‐fos antisense oligonucleotide. No light‐dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos‐like proteins.
Noradrenaline Release from Cultured Mouse Postganglionic Sympathetic Neurons
Authors: Trendelenburg Anne‐Ulrike , Cox Sandra Leagh , Gaiser Eugen Gerhard , Meyer Angelika , Starke Klaus
Published: March 2003

Abstract : The possible existence of

α2‐autoreceptors, P2‐autoreceptors, and adenosine

A1‐ or A2A‐receptors was studied in cultured

thoracolumbar postganglionic sympathetic neurons from mice. The cells were

preincubated with [3H]noradrenaline and then superfused. The

selective α2‐adrenoceptor agonist UK 14,304 reduced the

electrically evoked overflow of tritium. When the cultures were stimulated by

trains of increasing pulse number, ranging from a single pulse to 72 pulses at

3 Hz, the concentration‐inhibition curve of UK 14,304 was shifted

progressively to the right and the maximal inhibition obtainable became

progressively smaller. Six α‐adrenoceptor antagonists shifted the

concentration‐inhibition curve of UK 14,304 in a parallel manner to the right.

Neither ATP (3‐300 μM), adenosine (0.01‐100 μM), the

selective A1‐receptor agonist cyclopentyladenosine (1‐1,000

nM), nor the selective A2A‐receptor agonist CGS‐21680

(1‐10,000 nM) changed the basal or the electrically evoked overflow

of tritium. It is concluded that the cultured neurons possess presynaptic,

release‐inhibiting α2‐autoreceptors. As in intact tissues,

the effectiveness of presynaptic α2‐adrenergic inhibition

depends on the “strength” of the releasing stimulus. The

pKD values of the six antagonists against UK 14,304

indicate that the autoreceptors belong to the pharmacological

α2D and hence the genetic α2A/D subtype of

α2‐adrenoceptor. Neither P2‐autoreceptors nor receptors for adenosine, the degradation product of ATP, were detected.
In Vivo Development of Brain Phosphocreatine in Normal and Creatine‐Treated Rabbit Pups
Authors: Holtzman D. , Khait I. , Mulkern R. , Allred E. , Rand T. , Jensen F. , Kraft R.
Published: March 2003

Abstract : To study the effects of creatine (Cr) on brain energy

metabolism and on hypoxia‐induced seizures, 5‐to 30‐day‐old rabbit pups were

given subcutaneous Cr (3 g/kg) for 3 days before exposure to 4% O2

for 8 min. In saline‐treated controls, hypoxic seizures were most frequent at

15 days (80% of pups) and 20 days (60%) of age. Seizures were prevented at 15

days and reduced 60% at 20 days in Cr‐treated pups. In surface coil‐localized

brain 31P nuclear magnetic resonance spectra, with signal from both cerebral gray (GM) and white (WM) matter, the phosphocreatine (PCr)/nucleoside triphosphate (NTP) ratio doubled between 5 and 30 days of age in controls. In all Cr‐injected pups, brain PCr/NTP increased to values seen in 30‐day‐old controls. When spectra were acquired in predominantly GM and WM slices in vivo, the PCr/NTP ratio was very low in GM at 5 days but reached adult levels by 15 days in controls. In WM, the ratio increased steadily from 5 to 30 days of age. In Cr‐injected pups, PCr/NTP increased to mature levels in WM and in GM at all ages. In conclusion, hypoxic seizures occur midway in the time course of brain PCr/NTP increase in rabbit pups as previously described in rat pups. In both altricial pups, systemic Cr increases brain PCr/NTP ratio and prevents hypoxic seizures. These results suggest that mature levels of PCr and/or Cr in brain limit EEG activation either directly or indirectly by preventing hypoxic metabolic changes.
Tyrosine Phosphorylation and Association of BIT with SHP‐2 Induced by Neurotrophins
Authors: Ohnishi Hiroshi , Yamada Masashi , Kubota Misae , Hatanaka Hiroshi , Sano Shin‐ichiro
Published: March 2003

Abstract: BIT (brain immunoglobulin‐like molecule with tyrosine‐based activation motifs) is a membrane glycoprotein that has two cytoplasmic TAMs (tyrosine‐based activation motifs). We previously reported that tyrosine‐phosphorylated TAMs of BIT interact with the Src homology 2 domain‐containing protein tyrosine phosphatase SHP‐2 both in vitro and in transfected cells, and this association results in a potent stimulation of the phosphatase activity of SHP‐2. Both BIT and SHP‐2 are highly expressed in the mammalian brain, and they may play important roles in the regulation of synaptic function. In this study, we found that nerve growth factor (NGF) treatment of PC12 cells leads to the tyrosine phosphorylation of BIT and a subsequent complex formation between BIT and SHP‐2. Furthermore, brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) also induced the tyrosine phosphorylation of BIT and the association with SHP‐2 in primary cultured rat neurons. Our results suggest that the BIT‐SHP‐2 signaling pathway is a novel signal transduction mechanism of neurons that acts in response to neurotrophic factors such as NGF, BDNF, and NT‐3.
RGS mRNA Expression in Rat Striatum
Authors: Burchett Scott A. , Bannon Michael J. , Granneman James G.
Published: March 2003

Abstract: Single injections of cocaine, amphetamine, or methamphetamine increased RGS2 mRNA levels in rat striatum by two‐ to fourfold. The D1 dopamine receptor‐selective antagonist SCH‐23390 had no effect by itself but strongly attenuated RGS2 mRNA induction by amphetamine. In contrast, the D2 receptor‐selective antagonist raclopride induced RGS2 mRNA when administered alone and greatly enhanced stimulation by amphetamine. To examine the effects of repeated amphetamine on RGS2 expression, rats were treated with escalating doses of amphetamine (1.0‐7.5 mg/kg) for 4 days, followed by 8 days of multiple daily injections (7.5 mg/kg/2 h × four injections). Twenty hours after the last injection the animals were challenged with amphetamine (7.5 mg/kg) or vehicle and killed 1 h later. In drug‐naive animals, acute amphetamine induced the expression of RGS2, 3, and 5 and the immediate early genes c‐fos and zif/268. RGS4 mRNA levels were not affected. Prior repeated treatment with amphetamine strongly suppressed induction of immediate early genes and RGS5 to a challenge dose of amphetamine. In sharp contrast, prior exposure to amphetamine did not reduce the induction of RGS2 and RGS3 mRNAs to a challenge dose of amphetamine, indicating that control of these genes is resistant to amphetamine‐induced tolerance. These data establish a role for dopamine receptors in the regulation of RGS2 expression and suggest that RGS2 and 3 might mediate some aspects of amphetamine‐induced tolerance.
α‐Conotoxin ImI Inhibits the α‐Bungarotoxin‐Resistant Nicotinic Response in Bovine Adrenal Chromaffin Cells
Authors: Broxton N. M. , Down J. G. , Gehrmann J. , Alewood P. F. , Satchell D. G. , Livett B. G.
Published: March 2003

Abstract: The activity of α‐conotoxin (α‐CTX), ImI, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic α‐CTX ImI was a potent inhibitor of the neuronal nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 μM, log IC50 = 0.4 ± 0.07), showing competitive inhibition of nicotine‐evoked catecholamine secretion. α‐CTX ImI also inhibited nicotine‐evoked 45Ca2+ uptake but not 45Ca2+ uptake stimulated by 56 mM K+. In contrast, α‐CTX ImI had no effect at the neuromuscular junction over the concentration range 1‐20 μM. Bovine chromaffin cells are known to contain the α3β4, α7, and (possibly) α3β4α5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by α‐bungarotoxin, indicating that α7 nicotinic receptors are not involved. We propose that α‐CTX ImI interacts selectively with the functional (α3β4 or α3β4α5) nicotinic acetylcholine receptor to inhibit the neuronal‐type nicotinic response in bovine chromaffin cells.
Autoradiographic Reevaluation of the Binding Properties of 125I‐[Leu31,Pro34]Peptide YY and 125I‐Peptide YY3‐36 to Neuropeptide Y Receptor Subtypes in Rat Forebrain
Authors: Gobbi M. , Mennini T. , Vezzani A.
Published: March 2003

Abstract:
125I‐[Leu31,Pro34]peptide YY (PYY) and 125I‐PYY3‐36, initially described as selective neuropeptide Y Y1 and Y2 receptor ligands, respectively, were recently shown to label also Y4 and Y5 receptors. We used receptor autoradiography to assess whether these ligands can be reliably used to investigate the various neuropeptide Y receptors in rat forebrain. In most of the brain regions examined (in coronal sections at the level of dorsal hippocampus), specific 125I‐[Leu31,Pro34]PYY binding was completely inhibited by 1 μM BIBP‐3226, a selective Y1 receptor ligand, but unaffected by 10 nM rat pancreatic polypeptide, selectively inhibiting Y4 receptors, suggesting that Y4 receptors are present in negligible numbers compared with Y1 receptors in the areas examined. Significant numbers of BIBP‐3226‐insensitive 125I‐[Leu31,Pro34]PYY binding sites were measured in the CA3 subfield of the hippocampus only, possibly representing Y5 receptors. 125I‐PYY3‐36 binding was unchanged by 1 μM BIBP‐3226, whereas a population of 125I‐PYY3‐36 binding sites was sensitive to 100 nM [Leu31,Pro34]neuropeptide Y, likely representing Y5 receptors. The possibility of distinguishing between Y2 and Y5 receptors using 125I‐PYY3‐36 as radioligand was validated by their different regional distribution and their distinct changes 24 h after kainate seizures, i.e., binding to Y5 receptors was selectively decreased in the outer cortex, whereas binding to Y2 receptors was enhanced in the hippocampus. Thus, the use of selective unlabeled compounds is required for distinguishing the various receptor subtypes labeled by 125I‐[Leu31,Pro34]PYY and 125I‐PYY3‐36 in rat brain tissue.
The AMP‐Activated Protein Kinase Is Involved in the Regulation of Ketone Body Production by Astrocytes
Authors: Blázquez Cristina , Woods Angela , De Ceballos María L , Carling David , Guzmán Manuel
Published: March 2003

Abstract : The possible role of the AMP‐activated protein kinase (AMPK), a highly conserved stress‐activated kinase, in the regulation of ketone body production by astrocytes was studied. AMPK activity in rat cortical astrocytes was three times higher than in rat cortical neurons. AMPK in astrocytes was shown to be functionally active. Thus, incubation of astrocytes with 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR), a cellpermeable activator of AMPK, stimulated both ketogenesis from palmitate and carnitine palmitoyltransferase I. This was concomitant to a decrease of intracellular malonyl‐CoA levels and an inhibition of acetyl‐CoA carboxylase/fatty acid synthesis and 3‐hydroxy‐3‐methylglutaryl‐CoA reductase/cholesterol synthesis. Moreover, in microdialysis experiments AICAR was shown to stimulate brain ketogenesis markedly. The effect of chemical hypoxia on AMPK and the ketogenic pathway was studied subsequently. Incubation of astrocytes with azide led to a remarkable drop of fatty acid β‐oxidation. However, activation of AMPK during hypoxia compensated the depression of β‐oxidation, thereby sustaining ketone body production. This effect seemed to rely on the cascade hypoxia → increase of the AMP/ATP ratio → AMPK stimulation → acetyl‐CoA carboxylase inhibition → decrease of malonyl‐CoA concentration → carnitine palmitoyltransferase I deinhibition → enhanced ketogenesis. Furthermore, incubation of neurons with azide blunted lactate oxidation, but not 3‐hydroxybutyrate oxidation. Results show that (a) AMPK plays an active role in the regulation of ketone body production by astrocytes, and (b) ketone bodies produced by astrocytes during hypoxia might be a substrate for neuronal oxidative metabolism.
Metabolic Impairment Elicits Brain Cell Type‐Selective Changes in Oxidative Stress and Cell Death in Culture
Authors: Park Larry C. H. , Calingasan Noel Y. , Uchida Koji , Zhang Hui , Gibson Gary E.
Published: March 2003

Abstract: Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases. Thiamine deficiency (TD) is an animal model in which chronic oxidative stress and inflammation lead to selective neuronal death, whereas other cell types show an inflammatory response. Therefore, the current studies determined the response of different brain cell types to TD and/or inflammation in vitro and tested whether their responses reflect inherent properties of the cells. The cells that have been implicated in TD‐induced neurotoxicity, including neurons, microglia, astrocytes, and brain endothelial cells, as well as neuroblastoma and BV‐2 microglial cell lines, were cultured in either thiamine‐depleted media or in normal culture media with amprolium, a thiamine transport inhibitor. The activity levels of a key mitochondrial enzyme, α‐ketoglutarate dehydrogenase complex (KGDHC), were uniquely distributed among different cell types: The highest activity was in the endothelial cells, and the lowest was in primary microglia and neurons. The unique distribution of the activity did not account for the selective response to TD. TD slightly inhibited general cellular dehydrogenases in all cell types, whereas it significantly reduced the activity of KGDHC exclusively in primary neurons and neuroblastoma cells. Among the cell types tested, only in neurons did TD induce apoptosis and cause the accumulation of 4‐hydroxy‐2‐nonenal, a lipid peroxidation product. On the other hand, chronic lipopolysaccharide‐induced inflammation significantly inhibited cellular dehydrogenase and KGDHC activities in microglia and astrocytes but not in neurons or endothelial cells. The results demonstrate that the selective cell changes during TD in vivo reflect inherent properties of the different brain cell types.
Involvement of Activated Caspase‐3‐Like Proteases in N‐Methyl‐D‐Aspartate‐Induced Apoptosis in Cerebrocortical Neurons
Authors: Tenneti Lalitha , Lipton Stuart A.
Published: March 2003

Abstract: Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity‐mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase‐3 in NMDA‐induced neuronal apoptosis, we used an affinity‐labeling technique: Biotinylated N‐acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde (DEVD.CHO) preferentially labels conformationally active caspase‐3‐like proteases, allowing us to visualize affinity‐labeled caspases with streptavidin‐fluorescein isothiocyanate under confocal microscopy. NMDA‐induced apoptosis of cerebrocortical neurons was associated with a time‐dependent increase in conformationally active caspase‐3‐like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18‐24 h, conformationally active caspase‐3‐like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity‐labeled caspase‐3‐like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase‐3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA‐induced apoptosis. Our results suggest that caspase‐3‐like proteases play a major role in excitotoxin‐induced neuronal apoptosis.
Purification and Characterization of Bovine Brain Lysosomal Pepstatin‐Insensitive Proteinase, the Gene Product Deficient in the Human Late‐Infantile Neuronal Ceroid Lipofuscinosis
Authors: Junaid Mohammed A. , Wu Guoxin , Pullarkat Raju K.
Published: March 2003

Abstract: A lysosomal pepstatin‐insensitive proteinase (CLN2p) deficiency is the underlying defect in the classical late‐infantile neuronal ceroid lipofuscinosis (LINCL, CLN2). The natural substrates for CLN2p and the causative factors for the neurodegeneration in this disorder are still not understood. We have now purified the CLN2p from bovine brain to apparent homogeneity. The proteinase has a molecular mass of 46 kDa and an aminoterminal sequence, L‐H‐L‐G‐V‐T‐P‐S‐V‐I‐R‐K, that is identical to the human enzyme. Peptide:N‐glycosidase F and endoglycosidase H treatment of the CLN2p reduced its molecular mass to 39.5 and 40.5 kDa, respectively, suggesting the presence of as many as five N‐glycosylated residues. The CLN2p activity was not affected by common protease inhibitors, and thiol reagents, metal chelators, and divalent metal ions had no significant effect on the proteolytic activity of the CLN2p. Among the naturally occurring neuropeptides, angiotensin II, substance P, and β‐amyloid were substrates for the CLN2p, whereas angiotensin I, Leu‐enkephalin, and γ‐endorphin were not. Peptide cleavage sites indicated that the CLN2p is a tripeptidyl peptidase that cleaves peptides having free amino‐termini. Synthetic amino‐ and carboxyl‐terminal peptides from the subunit c sequence, which is the major storage material in LINCL, are hydrolyzed by the CLN2p, suggesting that the subunit c may be one of the natural substrates for this proteinase and its accumulation in LINCL is the direct result of the proteinase deficiency.
Differential Effects of Corticostriatal and Thalamostriatal Deafferentation on Expression of the Glutamate Transporter GLT1 in the Rat Striatum
Authors: Liévens J.‐C. , Salin P. , Had‐Aissouni L. , Mahy N. , Goff L. Kerkerian‐Le
Published: March 2003

Abstract: This study compared the effects of the disruption of the two main presumably glutamatergic striatal inputs, the corticostriatal and thalamostriatal pathways, on GLT1 expression in the rat striatum, using in situ hybridization and immunohistochemistry. Unilateral ibotenate‐induced thalamic lesion produced no significant changes in striatal GLT1 mRNA labeling and immunostaining as assessed at 5 and 12 days postlesion. In contrast, significant increases in both parameters were measured after bilateral cortical lesion by superficial thermocoagulation. GLT1 mRNA levels increased predominantly in the dorsolateral part of the striatum; there, the increases were significant at 5 (+84%), 12 (+101%), and 21 (+45%) but not at 35 days postlesion. GLT1 immunostaining increased significantly and homogeneously by 17‐26% at 12 and 21 days postlesion. The increase in GLT1 expression at 12 days postlesion was further confirmed by western blot analysis; in contrast, a 36% decrease in glutamate uptake activity was measured at the same time point. These data indicate that striatal GLT1 expression depends on corticostriatal but not thalamostriatal innervation. Comparison of our results with previous data showing that cortical lesion by aspiration down‐regulates striatal GLT1 expression further suggests that differential changes in GLT1 expression, and thus presumably in glial cell function, may occur in the target striatum depending on the way the cortical neurons degenerate.
Amyloid β‐Peptide Effects on Synaptosomes from Apolipoprotein E‐Deficient Mice
Authors: Keller Jeffrey N. , Lauderback Christopher M. , Butterfield D. Allan , Kindy Mark S. , Yu Jin , Markesbery William R.
Published: March 2003

Abstract: Apolipoprotein E (apoE) is present in the brain and may contribute to neurophysiologic or neuropathologic events, depending on environmental and genetic influences. Recent studies indicate a role for apoE in synaptic plasticity and maintenance of synaptic membrane symmetry, suggesting that apoE may be involved in regulating synaptic homeostasis. In the present study, cerebrocortical synaptosomes were prepared from transgenic mice lacking apoE (apoE KO) to analyze the possible contribution of apoE toward maintaining homeostasis in synaptosomes. Synaptosomal preparations from apoE KO and wild‐type mice exhibited similar basal levels of reactive oxygen species, mitochondrial function, and caspase activity; however, following application of amyloid β‐peptide [Aβ(1‐40)], apoE KO synaptosomes displayed increased levels of oxidative stress, mitochondrial dysfunction, and caspase activation compared with synaptosomes from wild‐type mice. Synaptosomal membranes from apoE KO mice were more fluid than wild‐type synaptosomes and contained higher levels of thiobarbituric acid‐reactive substances, consistent with elevated levels of lipid peroxidation occurring in the synapses of apoE KO mice. Together, these data are consistent with a role for apoE in maintaining homeostasis by attenuating oxidative stress, caspase activation, and mitochondrial homeostasis in synapses.
Visualization and Trafficking of the Vesicular Acetylcholine Transporter in Living Cholinergic Cells
Authors: Santos Magda S. , Barbosa José , Kushmerick Christopher , Gomez Marcus V. , Prado Vania F. , Prado Marco A. M.
Published: March 2003

Abstract: The present experiments investigated the trafficking of the vesicular acetylcholine transporter (VAChT) tagged with the enhanced green fluorescent protein (EGFP) in living cholinergic cells (SN56). The EGFP‐VAChT chimera was located in endosomal‐like compartments in the soma of SN56 cells, and it was also targeted to varicosities of neurites. In contrast, EGFP alone in cells was soluble in the cytoplasm. The C‐terminal cytoplasmic tail of VAChT has been implicated in targeting of VAChT to synaptic vesicles; thus, we have examined the role of the C‐terminal region in the trafficking to varicosities. A C‐terminal fragment tagged with EGFP appeared to be selectively accumulated in varicosities when expressed in SN56 cells. Interestingly, the protein was not freely soluble in the cytosol, and it presented a punctate pattern of expression. However, EGFP‐C terminus did not present this peculiar pattern of expression in a nonneuronal cell line (HEK 293). Moreover, the C‐terminal region of VAChT did not seem to be essential for VAChT trafficking, as a construct that lacks the C‐terminal tail was, similar to EGFP‐VAChT, partially targeted to endocytic organelles in the soma and sorted to varicosities. These experiments visualize VAChT for the first time in living cells and suggest that there might be multiple signals that participate in trafficking of VAChT to sites of synaptic vesicle accumulation.
Accumulation of N‐Acetyl‐L‐Aspartate in the Brain of the Tremor Rat, a Mutant Exhibiting Absence‐Like Seizure and Spongiform Degeneration in the Central Nervous System
Authors: Kitada Kazuhiro , Akimitsu Tomohide , Shigematsu Yosuke , Kondo Akira , Maihara Toshiro , Yokoi Norihide , Kuramoto Takashi , Sasa Masashi , Serikawa Tadao
Published: March 2003

Abstract: The tremor rat is a mutant that exhibits absence‐like seizure and spongiform degeneration in the CNS. By positional cloning, a genomic deletion was found within the critical region in which the aspartoacylase gene is located. Accordingly, no aspartoacylase expression was detected in any of the tissues examined, and abnormal accumulation of N‐acetyl‐L‐aspartate (NAA) was shown in the mutant brain, in correlation with the severity of the vacuole formation. Therefore, the tremor rat may be regarded as a suitable animal model of human Canavan disease, characterized by spongy leukodystrophy that is caused by aspartoacylase deficiency. Interestingly, direct injection of NAA into normal rat cerebroventricle induced 4‐ to 10‐Hz polyspikes or spikewave‐like complexes in cortical and hippocampal EEG, concomitantly with behavior characterized by sudden immobility and staring. These results suggested that accumulated NAA in the CNS would induce neuroexcitation and neurodegeneration directly or indirectly.
A Synthetic Peptide Ligand of Neural Cell Adhesion Molecule (NCAM) IgI Domain Prevents NCAM Internalization and Disrupts Passive Avoidance Learning
Authors: Foley Andrew G. , Hartz Barbara P. , Gallagher Helen C. , Rønn Lars C. B. , Berezin Vladimir , Bock Elisabeth , Regan Ciaran M.
Published: March 2003

Abstract: The neural cell adhesion molecule (NCAM) mediates cell adhesion and signal transduction through trans‐homophilic‐ and/or cis‐heterophilic‐binding mechanisms. Intraventricular infusions of anti‐NCAM have revealed a functional requirement of NCAM for the consolidation of memory in rats and chicks in a specific interval 6‐8 h after training. We have now extended these studies to a synthetic peptide ligand of NCAM (C3) with an affinity for the IgI domain and the capability of inhibiting NCAM‐mediated neurite outgrowth in vitro. Intraventricular administration of a single 5 μg bolus of C3 strongly inhibited recall of a passive avoidance response in adult rats, when given during training or in the 6‐8‐h posttraining period. The effect of C3 on memory consolidation was similar to that obtained with anti‐NCAM as the amnesia was not observed until the 48‐h recall time. The unique amnesic action of C3 during training could be related to disrupted NCAM internalization following training. In the 3‐4‐h posttraining period NCAM 180, the synapse‐associated isoform, was down‐regulated in the hippocampal dentate gyrus. This effect was mediated by ubiquitination and was prevented by C3 administration during training. These findings indicate NCAM to be involved in both the acquisition and consolidation of a passive avoidance response in the rat. Moreover, the study provides the first in vivo evidence for NCAM internalization in learning and identifies a synthetic NCAM ligand capable of modulating memory processes in vivo.
A Systemic Administration of NMDA Induces Immediate Early Gene pip92 in the Hippocampus
Authors: Chung Kwang Chul , Shin Song Woo , Yoo Min , Lee Min Young , Lee Hyun Woo , Choe Byung Kil , Ahn Young Soo
Published: March 2003

Abstract : In the mammalian CNS, aspartate and glutamate are major

excitatory amino acids, and their receptors are believed to mediate a wide

range of physiological and pathological processes, including

neurotransmission, plasticity, excitotoxicity, and various forms of

neurodegeneration. The immediate early gene pip92 has been identified

in serum‐stimulated BALB/c 3T3 fibroblasts, activated T lymphocytes treated

with cycloheximide, and fibroblast growth factor‐stimulated hippocampal cells

during neuronal differentiation. In this study we have demonstrated that

pip92 is expressed in the mouse brain after a single intraperitoneal

injection of NMDA. The distribution of pip92 mRNA levels in the

NMDA‐treated mouse brain was investigated using in situ RT‐PCR. The

region‐specific activation of pip92 in the CNS was observed 3 h after

NMDA injection, and high levels of pip92 mRNA were detected in the

hippocampal dentate gyrus and piriform cortex regions. In addition, the

activation of pip92 by NMDA was mediated by activation of

mitogen‐activated protein kinases (MAPKs), such as c‐Jun N‐terminal kinase

(JNK) and p38 kinase, but not extracellular signal‐regulated kinase (ERK) in

the mouse hippocampus and immortalized rat hippocampal progenitor cells. This

study suggests that pip92 is likely to play an important role in neuronal cell death induced by excitotoxic NMDA injury in the CNS.
The Transcription Factor E2F1 Modulates Apoptosis of Neurons
Authors: Hou Sheng T. , Callaghan Debbie , Fournier Marie‐Christine , Hill Irene , Kang Liping , Massie Bernard , Morley Paul , Murray Christine , Rasquinha Ingrid , Slack Ruth , MacManus John P.
Published: March 2003

Abstract : The transcription factor E2F1 is known to mediate apoptosis in isolated quiescent and postmitotic cardiac myocytes, and its absence decreases the size of brain infarction following cerebral ischemia. To demonstrate directly that E2F1 modulates neuronal apoptosis, we used cultured cortical neurons to show a temporal association of the transcription and expression of E2F1 in neurons with increased neuronal apoptosis. Cortical neurons lacking E2F1 expression (derived from E2F1 ‐/‐ mice) were resistant to staurosporine‐induced apoptosis as evidenced by the significantly lower caspase 3‐like activity and a lesser number of cells with apoptotic morphology in comparison with cortical cultures derived from wild‐type mice. Furthermore, overexpressing E2F1 alone using replication‐deficient recombinant adenovirus was sufficient to cause neuronal cell death by apoptosis, as evidenced by the appearance of hallmarks of apoptosis, such as the threefold increase in caspase 3‐like activity and increased laddered DNA fragmentation, in situ endlabeled DNA fragmentation, and numbers of neuronal cells with punctate nuclei. Taken together, we conclude that E2F1 plays a key role in modulating neuronal apoptosis.
Regulation of Eukaryotic Initiation Factor 4E Phosphorylation in the Nervous System of Aplysia californica
Authors: Dyer John R , Sossin Wayne S
Published: March 2003

We have used an antibody that specifically recognizes eukaryotic initiation factor 4E (eIF4E) when it is phosphorylated at Ser207 to

characterize eIF4E phosphorylation in the nervous system of Aplysia.

The level of phosphorylated eIF4E, but not the level of total eIF4E, was

significantly correlated with the basal rate of translation measured from

different animals. Serotonin (5‐HT), a transmitter that regulates the rate of

translation in Aplysia neurons, had mixed effects on eIF4E

phosphorylation. 5‐HT decreased eIF4E phosphorylation in sensory cell clusters

through activation of protein kinase C. 5‐HT increased eIF4E phosphorylation

in the whole pleural ganglia. In the Aplysia nervous system, eIF4E phosphorylation correlated with phosphorylation of the p38 MAP kinase, but not the p42 MAP kinase (ERK). Furthermore, an inhibitor of the p38 MAP kinase significantly decreased basal eIF4E phosphorylation, but an inhibitor of the MAP or ERK kinase (MEK) did not. Despite the correlation of eIF4E phosphorylation with the basal rate of translation, inhibition of eIF4E phosphorylation by an inhibitor of the p38 MAP kinase did not significantly decrease the rate of translation.