bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection against 3,4‐methylenedioxymethamphetamine‐induced neurodegeneration produced by glutathione depletion in rats is mediated by attenuation of hyperthermia
Authors: O'Shea E. , Easton N. , Fry J. R. , Green A. R. , Marsden C. A.
Published: March 2003

3,4‐Methylenedioxymethamphetamine (MDMA) administration produces neurotoxic degeneration of serotonin terminals in rat brain. These effects occur only after systemic administration and not after central injection, suggesting that peripheral metabolism, possibly hepatic, is required for toxicity. Glutathione is one of the principal cellular defence mechanisms, but conjugation with glutathione can, on some occasions, increase the reactivity of certain molecules. Previous studies have shown that central administration of glutathione adducts of a MDMA metabolite produces a neurotoxicity profile similar to that of systemic MDMA. In the present study, depletion of peripheral (hepatic) glutathione by 43% with dl‐buthionine‐(S,R)‐sulfoximine (an inhibitor of glutathione synthesis) did not attenuate MDMA‐induced neurotoxicity as indicated by the 34% loss of [3H]paroxetine binding to the serotonin uptake sites in Dark Agouti rats treated with the inhibitor. However, a more profound depletion (92%) of glutathione by diethylmaleate (direct conjugation) administration significantly reduced the serotonergic neurotoxicity produced by MDMA. This depletion protocol also attenuated the hyperthermic response to MDMA. A combination protocol utilising both buthionine‐(S,R)‐sulfoximine and diethylmaleate that did not alter the hyperthermic response of the rats given MDMA also failed to attenuate the neurotoxicity. These findings indicate that glutathione depletion does not offer specific protection against MDMA‐induced serotonin neurotoxicity in Dark Agouti rats.
Tissue transglutaminase differentially modulates apoptosis in a stimuli‐dependent manner
Authors: Tucholski Janusz , Johnson Gail V. W.
Published: March 2003

Tissue transglutaminase is a unique member of the transglutaminase family as it not only catalyzes a transamidating reaction, but also binds and hydrolyzes GTP and ATP. Tissue transglutaminase has been reported to be pro‐apoptotic, however, conclusive evidence is still lacking. To elucidate the role of tissue transglutaminase in the apoptotic process human neuroblastoma SH‐SY5Y cells were stably transfected with vector only (SH/pcDNA), wild‐type tissue transglutaminase (SH/tTG) and tissue transglutaminase that has no transamidating activity but retains its other functions (SH/C277S). In these studies three different apoptotic stimuli were used osmotic stress, staurosporine treatment and heat shock to delineate the role of tissue transglutaminase as a transamidating enzyme in the apoptotic process. In SH/tTG cells, osmotic stress and staurosporine treatments resulted in significantly greater caspase‐3 activation and apoptotic nuclear changes then in SH/pcDNA or SH/C277S cells. This potentiation of apoptosis in SH/tTG cells was concomitant with a significant increase in thein situtransamidating activity of tissue transglutaminase. However, in the heat shock paradigm, which did not result in any increase in the transamidating activity in SH/tTG cells, there was a significant attenuation of caspase‐3 activity, LDH release and apoptotic chromatin condensation in SH/tTG and SH/C277S cells compared with SH/pcDNA cells. These findings indicate for the first time that the effect of tissue transglutaminase on the apoptotic process is highly dependent on the type of the stimuli and how the transamidating activity of the enzyme is affected. Tissue transglutaminase facilitates apoptosis in response to stressors that result in an increase in the transamidating activity of the enzyme. However, when the stressors do not result in an increase in the transamidating activity of tissue transglutaminase, than tissue transglutaminase can ameliorate the apoptotic response through a mechanism that is independent of its transamidating function. Further, neither the phosphatidylinositol‐3‐kinase pathway nor the extracellular‐regulated kinase pathway is downstream of the modulatory effects of wild‐type tissue transglutaminase or C277S‐tissue transglutaminase in the apoptotic cascade.
Patterns of gene expression are altered in the frontal and motor cortices of human alcoholics
Authors: Mayfield R. Dayne , Lewohl Joanne M. , Dodd Peter R. , Herlihy Amy , Liu Jianwen , Harris R. Adron
Published: March 2003

Alcoholism is a major health problem in Western countries, yet relatively little is known about the mechanisms by which chronic alcohol abuse causes the pathologic changes associated with the disease. It is likely that chronic alcoholism affects a number of signaling cascades and transcription factors, which in turn result in distinct gene expression patterns. These patterns are difficult to detect by traditional experiments measuring a few mRNAs at a time, but are well suited to microarray analyses. We used cDNA microarrays to analyze expression of approximately 10 000 genes in the frontal and motor cortices of three groups of chronic alcoholic and matched control cases. A functional hierarchy was devised for classification of brain genes and the resulting groups were compared based on differential expression. Comparison of gene expression patterns in these brain regions revealed a selective reprogramming of gene expression in distinct functional groups. The most pronounced differences were found in myelin‐related genes and genes involved in protein trafficking. Significant changes in the expression of known alcohol‐responsive genes, and genes involved in calcium, cAMP, and thyroid signaling pathways were also identified. These results suggest that multiple pathways may be important for neuropathology and altered neuronal function observed in alcoholism.
Age‐dependent decline of DNA repair activity for oxidative lesions in rat brain mitochondria
Authors: Chen Dexi , Cao Guodong , Hastings Teresa , Feng Yiqin , Pei Wei , O'Horo Cristine , Chen Jun
Published: March 2003

Endogenous oxidative damage to brain mitochondrial DNA and mitochondrial dysfunction are contributing factors in aging and in the pathogenesis of a number of neurodegenerative diseases. In this study, we characterized the regulation of base‐excision‐repair (BER) activity, the predominant repair mechanism for oxidative DNA lesions, in brain mitochondria as the function of age. Mitochondrial protein extracts were prepared from rat cerebral cortices at the ages of embryonic day 17 (E17) or postnatal 1‐, 2‐, and 3‐weeks, or 5‐ and 30‐months. The total BER activity and the activity of essential BER enzymes were examined in mitochondria using in vitro DNA repair assay employing specific repair substrates. Mitochondrial BER activity showed marked age‐dependent declines in the brain. The levels of overall BER activity were highest at E17, gradually decreased thereafter, and reached to the lowest at the age of 30‐month (∼80% reduction). The decline of overall BER activity with age was attributed to the decreased expression of repair enzymes such as 8‐OHdG glycosylase and DNA polymerase‐γ and, consequently, the reduced activity at the steps of lesion‐base incision, DNA repair synthesis and DNA ligation in the BER pathway. These results strongly suggest that the decline in BER activity may be an important mechanism contributing to the age‐dependent accumulation of oxidative DNA lesions in brain mitochondria.
The metabotropic glutamate receptor mGluR7b binds to the catalytic γ‐subunit of protein phosphatase 1
Authors: Enz Ralf
Published: March 2003

Correct targeting of enzymes represents an important biological mechanism to control post‐translational modifications of neurotransmitter receptors. The metabotropic glutamate receptor type 7 (mGluR7) exists in two splice variants (mGluR7a and mGluR7b), defined by different C‐termini that are phosphorylated by protein kinase C (PKC). Recently, the search for mGluR7a binding partners yielded several proteins that interacted with its C‐terminus. Here, a yeast two‐hybrid screen using the mGluR7b C‐terminus identified both variants of the catalytic γ‐subunit of protein phosphatase 1 (PP1γ1 and PP1γ2) as binding partners. The minimal interacting region of PP1γ1/2 contained the core domain and was homologous to a region of PP1α that is needed for functional expression. Although this core domain is highly conserved within the protein phosphatase family, PP1α1 and PP1β did not interact with mGluR7b. Binding between PP1γ1 and mGluR7b might be regulated by alternative splicing, as the variant‐specific distal part of the mGluR7b C‐terminus mediated the interaction. Within this domain, amino acids involved in the binding to PP1γ1 were mapped and biochemical assays using recombinant and native proteins verified the proposed interaction. Finally, the expression pattern of PP1γ1, PP1γ2 and mGluR7b was analysed in various CNS regions. In summary, these results suggest a regulation of mGluR7b by PP1γ.
Effects of desipramine treatment on norepinephrine transporter gene expression in the cultured SK‐N‐BE(2)M17 cells and rat brain tissue
Authors: Zhu Meng‐Yang , Kim Chun‐Hyung , Hwang Dong‐Youn , Baldessarini Ross J. , Kim Kwang‐Soo
Published: March 2003

The antidepressant desipramine (DMI) is a selective inhibitor of norepinephrine (NE) transport that down‐regulates the norepinephrine transporter (NET) protein in a concentration‐ and time‐dependent mannerin vitro. In this study, possible regulatory effects of DMI onNETmRNA and protein levels were investigated with the NET‐expressing SK‐N‐BE(2)M17 cell line and rat brain tissue. Northern blot analysis showed that incubation of the cultured cells with DMI (5–500 nm) for 3 days reduced levels ofNETmRNA in both its 5.8‐kb (by up to 58%) and 3.6‐kb forms (to 68%), whereas incubation for 14 days increased both levels (to 40% and 100%) in a concentration‐dependent manner. In contrast, NET protein levels decreased after 3–14 days of exposure of the cells to DMI, as determined by western blotting. Thein vitrofindings were supported byin vivotreatment of rats with DMI. Thus,in situhybridization demonstrated initially decreased, and later increased,NETmRNA levels in locus coeruleus (LC) tissue of rats treated with DMI; whereas NET protein levels in the LC were reduced after 14 days, but unchanged after three daily DMI treatments. Thus, DMI had similar effects on NET expressionin vitroandin vivo, with opposite changes inNETmRNA and protein levels, suggesting that the regulatory mechanisms involved are complex and non‐congruent.
Intranuclear localization of apoptosis‐inducing factor (AIF) and large scale dna fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite
Authors: Zhang Xiaopeng , Chen Jun , Graham Steven H. , Du Lina , Kochanek Patrick M. , Draviam Romesh , Guo Fengli , Nathaniel Paula D. , Szabó Csaba , Watkins Simon C. , Clark Robert S. B.
Published: March 2003

Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase‐independent pathway involving intranuclear translocation of mitochondrial apoptosis‐inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2–72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large‐scale DNA fragmentation (∼50 kbp), a signature event in AIF‐mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron‐enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large‐scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large‐scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury.
Regulation of regulators of G protein signaling mRNA expression in rat brain by acute and chronic electroconvulsive seizures
Authors: Gold Stephen J. , Heifets Boris D. , Pudiak Cindy M. , Potts Bryan W. , Nestler Eric J.
Published: March 2003

G protein‐coupled receptor (GPCR) signaling cascades may be key substrates for the antidepressant effects of chronic electroconvulsive seizures (ECS). To better understand changes in these signaling pathways, alterations in levels of mRNA's encoding regulators of G protein signaling (RGS) protein subtypes‐2, ‐4, ‐7, ‐8 and ‐10 were evaluated in rat brain using northern blotting andin situhybridization. In prefrontal cortex, RGS2 mRNA levels were increased several‐fold 2 h following an acute ECS. Increases in RGS8 mRNA were of lesser magnitude (30%), and no changes were evident for the other RGS subtypes. At 24 h following a chronic ECS regimen, RGS4, ‐7, and ‐10 mRNA levels were reduced by 20–30%; only RGS10 was significantly reduced 24 h after acute ECS. Levels of RGS2 mRNA were unchanged 24 h following either acute or chronic ECS. In hippocampus, RGS2 mRNA levels were markedly increased 2 h following acute ECS. More modest increases were seen for RGS4 mRNA expression, whereas levels of the other RGS subtypes were unaltered. At 24 h following chronic ECS, RGS7, ‐8 and ‐10 mRNA levels were decreased in the granule cell layer, and RGS7 and ‐8 mRNA levels were decreased in the pyramidal cell layers. Only RGS8 and ‐10 mRNA levels were significantly reduced in hippocampus 24 h following an acute ECS. Paralleling neocortex, RGS2 mRNA content was unchanged in hippocampus 24 h following either acute or chronic ECS. In ventromedial hypothalamus, RGS4 mRNA content was increased 24 h following chronic ECS, whereas RGS7 mRNA levels were only increased 24 h following an acute ECS. The increased RGS4 mRNA levels in hypothalamus were significant by 2 h following an acute ECS. These studies demonstrate subtype‐, time‐, and region‐specific regulation of RGS proteins by ECS, adaptations that may contribute to the antidepressant effects of this treatment.
Ceramide‐induced apoptosis of D283 medulloblastoma cells requires mitochondrial respiratory chain activity but occurs independently of caspases and is not sensitive to Bcl‐xL overexpression
Authors: Poppe Monika , Reimertz Claus , Münstermann Gudrun , Kögel Donat , Prehn Jochen H. M.
Published: March 2003

Ceramides are potent lipid second messengers that are involved in apoptotic and hypoxic/ischaemic neurone death. We investigated the role of mitochondria and the mitochondrial apoptosis pathway in ceramide‐induced cell death using human D283 medulloblastoma cells with a reduced mitochondrial DNA copy number (ρ– cells) and a corresponding defect in mitochondrial respiration. Treatment with the complex I inhibitor rotenone, C2‐ or C8‐ceramide induced cell death in D283 control cells, while ρ– cells were significantly protected. In contrast, activation of the mitochondrial apoptosis pathway by transient overexpression of the pro‐apoptotic Bax protein or exposure to the kinase inhibitor staurosporine induced apoptosis to a similar extent in control and ρ– cells. Overexpression of the antiapoptotic protein Bcl‐xL failed to inhibit the toxic effect of C2‐ceramide in D283 control cells, and no significant increase in caspase‐3‐like protease activity could be detected during the death process. Despite this, C2‐ceramide induced significant chromatin condensation and cell shrinkage in D283 control cells, reminiscent of apoptosis. These morphological alterations were associated with the activation of calpains. Both apoptotic morphology and calpain activation were attenuated in ρ– cells. Our data indicate that the apoptosis‐inducing effect of C2‐ceramide may require mitochondrial respiratory chain activity and can occur independently of the mitochondrial apoptosis pathway, but involves the activation of calpains.
Activity‐dependent NMDA receptor‐mediated activation of protein kinase B/Akt in cortical neuronal cultures
Authors: Sutton Greg , Chandler L. Judson
Published: March 2003

The serine/threonine protein kinase B (PKB)/Akt is a phosphoinositide 3‐kinase (PI3K) effector that is thought to play an important roll in a wide variety of cellular events. The present study examined whether PKB activation in cortical neuronal cultures is coupled with synaptic activity. A 1‐h incubation of neuronal cultures with tetrodotoxin (TTX), the PI3K inhibitor wortmannin, the NMDA receptor antagonist MK‐801 or removal of extracellular calcium significantly reduced basal levels of phospho(Ser473)‐PKB, indicating that activity‐dependent glutamate release maintains PKB activation through an NMDA receptor‐PI3K pathway. A 5‐min exposure to NMDA (50 µm) in the presence of TTX increased phospho‐PKB back to levels observed in the absence of TTX. NMDA stimulation of phospho‐PKB was blocked by wortmannin, the CaMKII inhibitor KN‐93, MK‐801, and removal of extracellular calcium. We have previously shown that NMDA receptors can bi‐directionally regulate activation of extracellular‐signal regulated kinase (ERK), and NMDA receptor stimulation of PKB in the present study appeared to mirror activation of ERK. These results suggest that in cultured cortical neurons, PKB activity is dynamically regulated by synaptic activity and is coupled to NMDA receptor activation. In addition, NMDA receptor activation of ERK and PKB may occur through overlapping signaling pathways that bifurcate at the level of Ras.
Peroxynitrite enhances astrocytic volume‐sensitive excitatory amino acid release via a src tyrosine kinase‐dependent mechanism
Authors: Haskew Renée E. , Mongin Alexander A. , Kimelberg Harold K.
Published: March 2003

Volume‐regulated anion channels (VRACs) are critically important for cell volume homeostasis, and under pathological conditions contribute to neuronal damage via excitatory amino (EAA) release. The precise mechanisms by which brain VRACs are activated and/or modulated remain elusive. In the present work we explored the possible involvement of nitric oxide (NO) and NO‐related reactive species in the regulation of VRAC activity and EAA release, using primary astrocyte cultures. The NO donors sodium nitroprusside and spermine NONOate did not affect volume‐activatedd‐[3H]aspartate release. In contrast, the peroxynitrite (ONOO–) donor 3‐morpholinosydnomine hydrochloride (SIN‐1) increased volume‐dependent EAA release by approx. 80–110% under identical conditions. Inhibition of ONOO–formation with superoxide dismutase completely abolished the effects of SIN‐1. Both the volume‐ and SIN‐1‐induced EAA release were sensitive to the VRAC blockers NPPB and ATP. Further pharmacological analysis ruled out the involvement of cGMP‐dependent reactions and modification of sulfhydryl groups in the SIN‐1‐inducedmodulation of EAA release. The src family tyrosine kinase inhibitor 4‐amino‐5‐(4‐chlorophenyl)‐7‐(t‐butyl)pyrazolo [3,4‐d]pyrimidine (PP2), but not its inactive analog PP3, abolished the effects of SIN‐1. A broader spectrum tyrosine kinase inhibitor tyrphostin A51, also completely eliminated the SIN‐1‐induced EAA release. Our data suggest that ONOO–up‐regulates VRAC activity via a src tyrosine kinase‐dependent mechanism. This modulation may contribute to EAA‐mediated neuronal damage in ischemia and other pathological conditions favoring cell swelling and ONOO–production.
Presenilin‐binding protein forms aggresomes in monkey kidney COS‐7 cells
Authors: Namekata Kazuhiko , Nishimura Noriyuki , Kimura Hideo
Published: March 2003

A novel presenilin‐binding protein (PBP) is specifically expressed in the brain, and its level in the soluble fraction of Alzheimer's disease (AD) brains is much less than that in the age‐matched controls. Recently, several proteins, including presenilin (PS), have been found to form structures of aggregated proteins, called aggresomes, when the production of the proteins exceeds their rate of degradation by proteasomes. Based on these observations it has been proposed that the aggresome may represent one of the mechanisms forthe formation of cytoplasmic deposits which are linked to the pathogenesis of neurodegenerative disorders including AD. It is shown here that the overexpression of PBP or the suppression of proteasome activity in monkey kidney COS‐7 cells leads to the accumulation of detergent‐insoluble and multiubiquitinated PBP aggregates. PBP also forms aggregates in primary cultures of neurons in the presence of a proteasome inhibitors. PBP aggregates have the characteristics of aggresomes, including the localization to microtubule organization centers and the disruption of intermediate filaments. These observations suggest that the malfunctioning of the proteasome can cause the formation of PBP aggresomes, which may lead to AD.
Inositol and higher inositol phosphates in neural tissues: homeostasis, metabolism and functional significance
Authors: Fisher Stephen K. , Novak James E. , Agranoff Bernard W.
Published: March 2003

Inositol phospholipids and inositol phosphates mediate well‐established functions in signal transduction and in Ca2+homeostasis in the CNS and non‐neural tissues. More recently, there has been renewed interest in other roles that bothmyo‐inositol and its highly phosphorylated forms may play in neural function. We review evidence thatmyo‐inositol serves as a clinically relevant osmolyte in the CNS, and that its hexakisphosphate and pyrophosphorylated derivatives may play roles in such diverse cellular functions as DNA repair, nuclear RNA export and synaptic membrane trafficking.
Selective loss of neurofilament expression in Cu/Zn superoxide dismutase (SOD1) linked amyotrophic lateral sclerosis
Authors: Menzies Fiona M. , Grierson Andrew J. , Cookson Mark R. , Heath Paul R. , Tomkins Janine , Figlewicz Denise A. , Ince Paul G. , Shaw Pamela J.
Published: March 2003

Neurofilament pathology is a hallmark of sporadic and familial amyotrophic lateral sclerosis (SALS and FALS). The disease mechanisms underlying this pathology are presently unclear, but recent evidence in SALS patients suggest that reductions in neurofilament light subunit (NFL) mRNA may contribute to the death of motor neurones. Mutations in the gene encoding Cu‐Zn superoxide dismutase (SOD1) represent the best‐studied cause of FALS, and a number of laboratory models of SOD1‐mediated disease exist. Here we have used microdissected lumbar spinal cord motor neurones from human SOD1 FALS patients as well as G93A SOD1 transgenic mice and demonstrated that reduced NFL mRNA levels are seen in both. To probe the molecular mechanisms underpinning these observations, we generated NSC34 motor neurone‐like cell lines expressing wild‐type and mutant SOD1. NSC34 cells expressing G37R or G93A SOD1 showed selective reductions in NFL and NFM mRNA and protein. These data suggest that NFL mRNA reductions are common to SALS and FALS patients, and that cells and mice expressing mutant SOD1 may enable us to characterize the molecular mechanism(s) responsible for the loss of neurofilament mRNA.
Activation of group II metabotropic glutamate receptors underlies microglial reactivity and neurotoxicity following stimulation with chromogranin A, a peptide up‐regulated in Alzheimer's disease
Authors: Taylor D. L. , Diemel L. T. , Cuzner M. L. , Pocock J. M.
Published: March 2003

Regulation of microglial reactivity and neurotoxicity is critical for neuroprotection in neurodegenerative diseases. Here we report that microglia possess functional group II metabotropic glutamate receptors, expressing mRNA and receptor protein for mGlu2 and mGlu3, negatively coupled to adenylate cyclase. Two different agonists of these receptors were able to induce a neurotoxic microglial phenotype which was attenuated by a specific antagonist. Chromogranin A, a secretory peptide expressed in amyloid plaques in Alzheimer's disease, activates microglia to a reactive neurotoxic phenotype. Chromogranin A‐induced microglial activation and subsequent neurotoxicity may also involve an underlying stimulation of group II metabotropic glutamate receptors since their inhibition reduced chromogranin A‐induced microglial reactivity and neurotoxicity. These results show that selective inhibition of microglial group II metabotropic glutamate receptors has a positive impact on neuronal survival, and may prove a therapeutic target in Alzheimer's disease.
The homeobox containing gene Lbx1 is required for correct dorsal–ventral patterning of the neural tube
Authors: Krüger Markus , Schäfer Konstanze , Braun Thomas
Published: March 2003

The specification of distinct classes of neurones in the neural tube is governed by extracellular signals and transcriptional mediators including neurogenic bHLH‐ and homeobox‐containing genes. Despite recent insights in the transcriptional code that controls generation of neuronal subtypes in the dorsal neural tube, the mechanisms that underlie regional identity in dorsal and lateral regions of the mammalian neural tube are not well understood. The homeobox gene Lbx1 is expressed in a subset of interneurons in the early neural tube and later in dorsal parts of the spinal cord. Here we show that Lbx1 is part of the machinery that controls formation of neurones in the dorsal horn. Targeted inactivation of Lbx1 leads to a severe reduction of neurones in the dorsal horn and to down‐regulation of dorsal markers such as Lmx1a from E11.5 onwards. In contrast, Pax3, another dorsally expressed homeobox gene, was up‐regulated in Lbx1 mutant mice at E18.5, suggesting the existence of a negative regulatory feedback loop. Lbx1/Pax3 double‐mutant mice showed an enhanced Pax3 phenotype characterized by a completely open neural tube. We suggest that Lbx1 plays a critical role in the specification of dorsal interneurons and that Lbx1 and Pax3 might act together synergistically to promote neural tube closure.
Proteasomal degradation of tau protein
Authors: David Della C. , Layfield Robert , Serpell Louise , Narain Yolanda , Goedert Michel , Spillantini Maria Grazia
Published: March 2003

Filamentous inclusions composed of the microtubule‐associated protein tau are a defining characteristic of a large number of neurodegenerative diseases. Here we show that tau degradation in stably transfected and non‐transfected SH‐SY5Y cells is blocked by the irreversible proteasome inhibitor lactacystin. Further, we find that in vitro, natively unfolded tau can be directly processed by the 20S proteasome without a requirement for ubiquitylation, and that a highly reproducible pattern of degradation intermediates is readily detectable during this process. Analysis of these intermediates shows that 20S proteasomal processing of tau is bi‐directional, proceeding from both N‐ and C‐termini, and that populations of relatively stable intermediates arise probably because of less efficient digestion of the C‐terminal repeat region. Our results are consistent with an in vivo role for the proteasome in tau degradation and support the existence of ubiquitin‐independent pathways for the proteasomal degradation of unfolded proteins.
D1 dopamine receptor stimulation increases GluR1 surface expression in nucleus accumbens neurons
Authors: Chao Steven Z. , Ariano Marjorie A. , Peterson Daniel A. , Wolf Marina E.
Published: March 2003

The goal of this study was to understand how dopamine receptors, which are activated during psychostimulant administration, might influence glutamate‐dependent forms of synaptic plasticity that are increasingly recognized as important to drug addiction. Regulation of the surface expression of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor subunit GluR1 plays a critical role in long‐term potentiation, a well‐characterized form of synaptic plasticity. Primary cultures of rat nucleus accumbens neurons were used to examine whether dopamine receptor stimulation influences cell surface expression of GluR1, detected using antibody to the extracellular portion of GluR1 and fluorescence microscopy. Surface GluR1 labeling on processes of medium spiny neurons and interneurons was increased by brief (5–15 min) incubation with a D1 agonist (1 µm SKF 81297). This effect was attenuated by the D1 receptor antagonist SCH 23390 (10 µm) and reproduced by the adenylyl cyclase activator forskolin (10 µm). Labeling was decreased by glutamate (10–50 µm, 15 min). These results are the first to demonstrate modulation of AMPA receptor surface expression by a non‐glutamatergic G protein‐coupled receptor. Normally, this may enable ongoing regulation of AMPA receptor transmission in response to changes in the activity of dopamine projections to the nucleus accumbens. When dopamine receptors are over‐stimulated during chronic drug administration, this regulation may be disrupted, leading to inappropriate plasticity in neuronal circuits governing motivation and reward.
Axin negatively affects tau phosphorylation by glycogen synthase kinase 3β
Authors: Stoothoff William H. , Bailey Craig D. C. , Mi Kaihong , Lin Sheng‐Cai , Johnson Gail V. W.
Published: March 2003

Glycogen synthase kinase 3β (GSK3β) is an essential protein kinase that regulates numerous functions within the cell. One critically important substrate of GSK3β is the microtubule‐associated protein tau. Phosphorylation of tau by GSK3β decreases tau–microtubule interactions. In addition to phosphorylating tau, GSK3β is a downstream regulator of the wnt signaling pathway, which maintains the levels of β‐catenin. Axin plays a central role in regulating β‐catenin levels by bringing together GSK3β and β‐catenin and facilitating the phosphorylation of β‐catenin, targeting it for ubiquitination and degradation by the proteasome. Although axin clearly facilitates the phosphorylation of β‐catenin, its effects on the phosphorylation of other GSK3β substrates are unclear. Therefore in this study the effects of axin on GSK3β‐mediated tau phosphorylation were examined. The results clearly demonstrate that axin is a negative regulator of tau phosphorylation by GSK3β. This negative regulation of GSK3β‐mediated tau phosphorylation is due to the fact that axin efficiently binds GSK3β but not tau and thus sequesters GSK3β away from tau, as an axin mutant that does not bind GSK3β did not inhibit tau phosphorylation by GSK3β. This is the first demonstration that axin negatively affects the phosphorylation of a GSK3β substrate, and provides a novel mechanism by which tau phosphorylation and function can be regulated within the cell.
The levels of mature glycosylated nicastrin are regulated and correlate with γ‐secretase processing of amyloid β‐precursor protein
Authors: Arawaka Shigeki , Hasegawa Hiroshi , Tandon Anurag , Janus Christopher , Chen Fusheng , Yu Gang , Kikuchi Kenji , Koyama Shingo , Kato Takeo , Fraser Paul E. , St George‐Hyslop Peter
Published: March 2003

Nicastrin, a type‐I transmembrane glycoprotein, is a necessary component of the high molecular weight presenilin (PS) complexes that mediate intramembranous cleavage of β‐amyloid precursor protein (βAPP) and Notch. Nicastrin undergoes trafficking‐dependent glycosylation maturation, and PS1 interacts preferentially with these maturely glycosylated forms of nicastrin. We investigated the effects of differing levels of the immature and mature endoglycosidase‐H‐resistant forms of nicastrin on Aβ40‐ and Aβ42‐peptide secretion in several cell lines stably expressing a mutant nicastrin (D336A/Y337A) that increases Aβ secretion. There was no correlation between Aβ secretion and the level of over‐expression of the immature forms of nicastrin. The total level of mature nicastrin remained constant, but mutant nicastrin replaced endogenous mature nicastrin in varying degrees. Differences in the levels of mature mutant nicastrin positively correlated with Aβ secretion, but did not influence either βAPP trafficking or processing by α‐ and β‐secretases. Proper trafficking and terminal maturation of nicastrin is therefore a necessary event for the regulated intramembranous proteolysis of βAPP.
Binge ethanol exposure decreases neurogenesis in adult rat hippocampus
Authors: Nixon Kimberly , Crews Fulton T.
Published: March 2003

Alcoholism is associated with cognitive deficits and loss of brain mass. Recent studies have indicated that neural progenitor cells proliferate throughout life forming neurons, astrocytes, and oligodendrocytes. The dentate gyrus is one neurogenic region of the adult brain containing neural progenitor cells. To determine if binge ethanol (EtOH) exposure alters neural progenitor cell proliferation and survival, bromodeoxyuridine was administered to adult male rats following an acute or chronic binge exposure paradigm. For an acute binge, rats were gavaged with a 5 g/kg dose of EtOH or vehicle, administered bromodeoxyuridine, and killed either 5 h or 28 days after EtOH treatment. In a 4‐day, chronic‐binge paradigm, rats were infused with EtOH three times per day (mean dose 9.3 g/kg/day) or isocaloric control diet. Rats were given bromodeoxyuridine once a day for the 4 days of chronic binge treatment, then perfused either immediately following the last dose of EtOH or 28 days later. In both EtOH treatment groups, binge EtOH decreased neural progenitor cell proliferation. Following the chronic four‐day binge, neural progenitor cell survival was decreased. These studies are the first to show EtOH inhibition of neural progenitor cell proliferation and survival in the adult, a possible new mechanism underlying alcoholic cognitive dysfunction.
Preservation of extracellular glutathione by an astrocyte derived factor with properties comparable to extracellular superoxide dismutase
Authors: Stewart Victoria C. , Stone Rebecca , Gegg Matthew E. , Sharpe Martyn A. , Hurst Roger D. , Clark John B. , Heales Simon J. R.
Published: March 2003

Cultured rat and human astrocytes and rat neurones were shown to release reduced glutathione (GSH). In addition, GSH oxidation was retarded by the concomitant release of a factor from the cells. One possibility is that this factor is extracellular superoxide dismutase (SOD). In support of this, the factor was found to bind heparin, have a molecular mass estimated to be between 50 and 100 kDa, and CuZn‐type SOD protein and cyanide sensitive enzyme activity were demonstrated in the cell‐conditioned medium. In addition, supplementation of native medium with exogenous CuZn‐type SOD suppressed GSH oxidation. We propose that preservation of released GSH is essential to allow for maximal up‐regulation of GSH metabolism in neurones. Furthermore, cytokine stimulation of astrocytes increased release of the extracellular SOD, and enhanced stability of GSH. This may be a protective strategy occurring in vivo under conditions of oxidative stress, and suggests that SOD mimetics may be of therapeutic use.
Prostaglandin A1 inhibits rotenone‐induced apoptosis in SH‐SY5Y cells
Authors: Wang Xiaoxia , Qin Zheng‐Hong , Leng Yan , Wang Yumei , Jin Xiannu , Chase Thomas N. , Bennett M. Catherine
Published: March 2003

The degeneration of nigral dopamine neurons in Parkinson's disease (PD) reportedly involves a defect in brain mitochondrial complex I in association with the activation of nuclear factor‐κB (NF‐κB) and caspase‐3. To elucidate molecular mechanisms possibly linking these events, as well as to evaluate the neuroprotective potential of the cyclopentenone prostaglandin A1 (PGA1), an inducer of heat shock proteins (HSPs), we exposed human dopaminergic SH‐SY5Y cells to the complex I inhibitor rotenone. Dose‐dependent apoptosis was preceded by the nuclear translocation of NF‐κB and then the activation of caspase‐3 over the ensuing 24 h. PGA1 increased the expression of HSP70 and HSP27 and protected against rotenone‐induced apoptosis, without increasing necrotic death. PGA1 blocked the rotenone‐induced nuclear translocation of NF‐κB and attenuated, but did not abolish, the caspase‐3 elevation. Unexpectedly, the caspase‐3 inhibitor, Ac‐DEVD.CHO (DEVD), at a concentration that completely prevented the caspase‐3 elevation produced by rotenone, failed to protect against apoptosis. These results suggest that complex I deficiency in dopamine cells can induce apoptosis by a process involving early NF‐κB nuclear translocation and caspase‐3 activation. PGA1 appears to protect against rotenone‐induced cell death by inducing HSPs and blocking nuclear translocation of NF‐κB in a process that attenuates caspase‐3 activation, but is not mediated by its inhibition.
Involvement of double‐stranded RNA‐dependent protein kinase and phosphorylation of eukaryotic initiation factor‐2α in neuronal degeneration
Authors: Chang Raymond Chuen‐Chung , Suen Ka‐Chun , Ma Chi‐Him , Elyaman Wassim , Ng Ho‐Keung , Hugon Jacques
Published: March 2003

Inhibition of protein translation plays an important role in apoptosis. While double‐stranded RNA‐dependent protein kinase (PKR) is named as it is activated by double‐stranded RNA produced by virus, its activation induces an inhibition of protein translation and apoptosis via the phosphorylation of the eukaryotic initiation factor 2α (eIF2α). PKR is also a stress kinase and its levels increase during ageing. Here we show that PKR activation and eIF2α phosphorylation play a significant role in apoptosis of neuroblastoma cells and primary neuronal cultures induced by the β‐amyloid (Aβ) peptides, the calcium ionophore A23187 and flavonoids. The phosphorylation of eIF2α and the number of apoptotic cells were enhanced in over‐expressed wild‐type PKR neuroblastoma cells exposed to Aβ peptide, while dominant‐negative PKR reduced eIF2α phosphorylation and apoptosis induced by Aβ peptide. Primary cultured neurons from PKR knockout mice were also less sensitive to Aβ peptide toxicity. Activation of PKR and eIF2α pathway by Aβ peptide are triggered by an increase in intracellular calcium because the intracellular calcium chelator BAPTA‐AM significantly reduced PKR phosphorylation. Taken together, these results reveal that PKR and eIF2α phosphorylation could be involved in the molecular signalling events leading to neuronal apoptosis and death and could be a new target in neuroprotection.
Effects on free radical generation by ligands of the peripheral benzodiazepine receptor in cultured neural cells
Authors: Jayakumar A. R. , Panickar K. S. , Norenberg M. D.
Published: March 2003

The effect of peripheral benzodiazepine receptor (PBR) ligands on free radical production was investigated in primary cultures of rat brain astrocytes and neurons as well as in BV‐2 microglial cell lines using the fluorescent dye dichlorofluorescein‐diacetate. Free radical production was measured at 2, 30, 60 and 120 min of treatment with the PBR ligands 1‐(2‐chlorophenyl‐N‐methylpropyl)‐3‐isoquinolinecarboxamide (PK11195), 7‐chloro‐5‐(4‐chlorophenyl)‐1,3‐dihydro‐1‐methyl‐2H‐1,4‐benzodiazepin‐2‐one (Ro5‐4864) and protoporphyrin IX (PpIX) (all at 10 nm). In astrocytes, all ligands showed a significant increase in free radical production at 2 min. The increase was short‐lived with PK11195, whereas with Ro5‐4864 it persisted for at least 2 h. PpIX caused an increase at 2 and 30 min, but not at 2 h. Similar results were observed in microglial cells. In neurons, PK11195 and PpIX showed an increase in free radical production only at 2 min; Ro5‐4864 had no effect. The central‐type benzodiazepine receptor ligand, clonazepam, was ineffective in eliciting free radical production in all cell types. As the PBR may be a component of the mitochondrial permeability transition (MPT) pore, and free radical production may occur following induction of the MPT, we further investigated whether cyclosporin A (CsA), an inhibitor of the MPT, could prevent free radical formation by PBR ligands. CsA (1 µm) completely blocked free radical production following treatment with PK11195 and Ro5‐4864 in all cell types. CsA was also effective in blocking free radical production in astrocytes following PpIX treatment, but it failed to do so in neurons and microglia. Our results indicate that exposure of neural cells to PBR ligands generates free radicals, and that the MPT may be involved in this process.
Chronic systemic complex I inhibition induces a hypokinetic multisystem degeneration in rats
Authors: Höglinger Günter U. , Féger Jean , Prigent Annick , Michel Patrick P. , Parain Karine , Champy Pierre , Ruberg Merle , Oertel Wolfgang H. , Hirsch Etienne C.
Published: March 2003

In Parkinson's disease, nigral dopaminergic neurones degenerate, whereas post‐synaptic striatal target neurones are spared. In some atypical parkinsonian syndromes, both nigral and striatal neurones degenerate. Reduced activity of complex I of the mitochondrial respiratory chain has been implicated in both conditions, but it remains unclear if this affects the whole organism or only the degenerating brain structures. We therefore investigated the differential vulnerability of various brain structures to generalized complex I inhibition. Male Lewis rats infused with rotenone, a lipophilic complex I inhibitor [2.5 mg/kg/day intraveneously (i.v.) for 28 days], were compared with vehicle‐infused controls. They showed reduced locomotor activity and loss of striatal dopaminergic fibres (54%), nigral dopaminergic neurones (28.5%), striatal serotoninergic fibres (34%), striatal DARPP‐32‐positive projection neurones (26.5%), striatal cholinergic interneurones (22.1%), cholinergic neurones in the pedunculopontine tegmental nucleus (23.7%) and noradrenergic neurones in the locus ceruleus (26.4%). Silver impregnation revealed pronounced degeneration in basal ganglia and brain stem nuclei, whereas the hippocampus, cerebellum and cerebral cortex were less affected. These data suggest that a generalized mitochondrial failure may be implicated in atypical parkinsonian syndromes but do not support the hypothesis that a generalized complex I inhibition results in the rather selective nigral lesion observed in Parkinson's disease.
Level of haem oxygenase does not obligatorily reflect the sensitivity of PC12 cells to an oxidative shock induced by glutathione depletion
Authors: Leon Albertine , Le Foll Isabelle , Charriault‐Marlangue Christiane , Leprince Jérome , Vaudry Hubert , Gabriel Cécilia , Duval Dominique
Published: March 2003

In order to investigate the function of haem oxygenase in neuronal cell death or survival, we have determined in PC12 cells whether induction of haem oxygenase mRNA and protein or inhibition of haem oxygenase activity may be able to modulate the cell response to an oxidative stress. Inhibition of glutathione biosynthesis by buthionine sulfoximine (BSO) has indeed been demonstrated, in this cell line, to decrease the intracellular content of glutathione and to trigger a gradual and programmed cell death. Inhibition of haem oxygenase by zinc protoporphyrin IX, a potent inhibitor of this enzyme, or by a recently described peptidic inhibitor, induced a significant decrease in the toxicity of BSO. This protective action was not due to an alteration in the metabolism of glutathione and was still observed when the protecting agent was added several hours after BSO treatment. Induction of haem oxygenase‐1 mRNA and protein by either haemin or pyrrolidine dithiocarbamate was associated with no protection or a significant reduction in the toxicity of BSO respectively. Our results indicate that induction of haem oxygenase‐1 is not obligatorily associated with an improved resistance towards oxidative stress and suggest that a byproduct of haem degradation may also become detrimental.
Human substance P receptor lacking the C‐terminal domain remains competent to desensitize and internalize
Authors: Richardson Mark D. , Balius Anastasia M. , Yamaguchi Keisuke , Freilich Emily R. , Barak Larry S. , Kwatra Madan M.
Published: March 2003

Substance P receptor (SPR) and its naturally occurring splice‐variant, lacking the C‐terminal tail, are found in brain and spinal cord. Whether C‐terminally truncated SPR desensitizes like full‐length SPR is controversial. We used a multivaried approach to determine whether human SPR (hSPR) and a C‐terminally truncated mutant, hSPRΔ325, differ in their desensitization and internalization. In HEK‐293 cells expressing either hSPRΔ325 or hSPR, SP‐induced desensitization of the two receptors was similar when measured by inositol triphosphate accumulation or by transient translocation of coexpressed PKCβII‐GFP to the plasma membrane. Moreover, translocation of β‐arrestin 1 or 2‐GFP (βarr1‐GFP or βarr2‐GFP) to the plasma membrane, and receptor internalization were also similar. However, hSPR and hSPRΔ325 differ in their phosphorylation and in their ability to form β‐arrestin‐containing endocytic vesicles. Unlike hSPR, hSPRΔ325 is not phosphorylated to a detectable level in intact HEK293 cells, and whereas hSPR forms vesicles containing either βarr1‐GFP or βarr2‐GFP, hSPRΔ325 does not form any vesicles with βarr1‐GFP, and forms fewer vesicles with βarr2‐GFP. We conclude that truncated hSPR undergoes agonist‐dependent desensitization and internalization without detectable receptor phosphorylation.
Differentiation of human retinal pigment epithelial cells into neuronal phenotype by N‐(4‐hydroxyphenyl)retinamide
Authors: Chen Shanyi , Samuel William , Fariss Robert N. , Duncan Todd , Kutty R. Krishnan , Wiggert Barbara
Published: March 2003

ARPE‐19, a human retinal pigment epithelial (RPE) cell line, has been widely used in studies of RPE function as well as gene expression. Here, we report the novel finding that N‐(4‐hydroxyphenyl)retinamide (fenretinide), a synthetic retinoic acid derivative and a potential chemopreventive agent against cancer, induced the differentiation of ARPE‐19 cells into a neuronal phenotype. The treated cells lost their epithelial phenotype and exhibited a typical neuronal shape with long processes (four to five times longer than the cell body). The onset of fenretinide‐induced neuronal differentiation was dose and time dependent, started within 1–2 days, and lasted at least 4 weeks. Immunohistochemical studies indicated that the expression of neurofilament proteins (NF160 and NF200), calretinin and neural cell adhesion molecule was increased in these differentiated cells. Western blot analysis indicated that cellular retinaldehyde‐binding protein, which is normally expressed in RPE cells, was decreased in treated cells. Protein analysis on a two‐dimensional gel followed by matrix‐assisted laser desorption ionization–time of flight mass spectrometric analysis demonstrated that heat‐shock protein 70 was increased after fenretinide treatment. Thus, fenretinide, a synthetic retinoid, is able to induce neuronal differentiation of human RPE cells in culture.