bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron (III) induces aggregation of hyperphosphorylated τ and its reduction to iron (II) reverses the aggregation: implications in the formation of neurofibrillary tangles of Alzheimer's disease
Authors: Yamamoto Akira , Shin Ryong‐Woon , Hasegawa Kazuhiro , Naiki Hironobu , Sato Hiroyuki , Yoshimasu Fumio , Kitamoto Tetsuyuki
Published: July 2010

Iron as well as aluminum is reported to accumulate in neurons with neurofibrillary tangles (NFTs) of Alzheimer's disease (AD) brain. Previously we demonstrated that aluminum (III) shows phosphate‐dependent binding with hyperphosphorylated τ (PHFτ), the major constituent of NFTs, thereby inducing aggregation of PHFτ. Herein we report that iron (III) can also induce aggregation of soluble PHFτ. Importantly, for the aggregation of PHFτ to occur, iron in the oxidized state (III) is essential since iron in the reduced state (II) lacks such ability. Furthermore, iron (III)‐induced aggregation is reversed by reducing iron (III) to iron (II). Thus the iron‐participating aggregation is mediated not only by τ phosphorylation but also by the transition of iron between reduced (II) and oxidized (III) states. Further incubation of insoluble PHFτ aggregates isolated from AD brain with reducing agents produced liberation of solubilized PHFτ and iron (II), indicating that PHFτ in association with iron (III) constitutes the insoluble pool of PHFτ. These results indicate that iron might play a role in the aggregation of PHFτ leading to the formation of NFTs in AD brain.
Proteasome inhibition by paired helical filament‐tau in brains of patients with Alzheimer's disease
Authors: Keck Susi , Nitsch Robert , Grune Tilman , Ullrich Oliver
Published: July 2010

Alzheimer's disease (AD) is characterized neuropathologically by intracellular neurofibrillary tangles (NFTs) formed of tau‐based paired helical filaments (PHFs) and extracellular β‐amyloid plaques. The degree of Alzheimer dementia correlates with the severity of PHFs and NFTs. As an intraneuronal accumulation of oxidatively damaged proteins has been found in the brains of patients with AD, a dysfunction of the proteasomal system, which degrades damaged proteins, has been assumed to cause protein aggregation and therefore neurodegeneration in AD. In this study, we revealed that such proteasome dysfunction in AD brain results from the inhibitory binding of PHF‐tau to proteasomes. We analysed the proteasome activity in brains from patients with AD and age‐matched controls, and observed a significant decrease to 56% of the control level in the straight gyrus of patients with AD. This loss of activity was not associated with a decrease in the proteasome protein. PHF‐tau co‐precipitated during proteasome immunoprecipitation and proteasome subunits could be co‐isolated during isolation of PHFs from AD brain. Furthermore, the proteasome activity in human brains strongly correlated with the amount of co‐precipitated PHF‐tau during immunoprecipitation of proteasome. Incubation of isolated proteasomes with PHF‐tau isolated from AD brain, and with PHFs after in vitro assembly from human recombinant tau protein, resulted in a distinct inhibition of proteasome activity by PHF‐tau. As this inhibition of proteasome activity was sufficient to induce neuronal degeneration and death, we suggest that PHF‐tau is able directly to induce neuronal damage in the AD brain.
The adhesion protein TAG‐1 has a ganglioside environment in the sphingolipid‐enriched membrane domains of neuronal cells in culture
Authors: Loberto Nicoletta , Prioni Simona , Prinetti Alessandro , Ottico Elena , Chigorno Vanna , Karagogeos Domna , Sonnino Sandro
Published: July 2010

We studied the interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid‐enriched membrane domains by ganglioside photolabeling combined with cell surface biotin labeling. After cell photolabeling with radioactive photoactivable derivatives of GM3, GM1 and GD1b gangliosides, followed by cell surface biotin labeling, sphingolipid‐enriched domains were prepared and immunoprecipitated with streptavidin‐coupled beads, under experimental conditions preserving the integrity of the lipid domain. About 50% of the total radioactivity linked to proteins was associated with acylated tubulin, about 10% with a 135‐kDa protein present as a series of species with pI ranging from 6.5 to 8.0, about 5% with a protein of about 70 kDa and with pI near to 6.5. By immunoprecipitation with streptavidin‐coupled beads under conditions disrupting the integrity of the lipid domain, the 135 kDa protein was recovered in the immunoprecipitate, that did not contain tubulin. Thus, the 135 kDa protein has an exoplasmic domain, and it was then identified as the GPI‐anchored neural cell adhesion molecule TAG‐1. Remarkably, TAG‐1 was cross‐linked in a similar extent by the photoactivated ganglioside GM3, GM1 and GD1b. The three gangliosides bear different oligosaccharide chains, suggesting that the ganglioside/TAG−1 interaction is not specifically associated with the ganglioside oligosaccharide structure.
Intranigral infusion of interleukin‐1β activates astrocytes and protects from subsequent 6‐hydroxydopamine neurotoxicity
Authors: Saura Josep , Parés Mireia , Bové Jordi , Pezzi Susana , Alberch Jordi , Marin Concepció , Tolosa Eduardo , Martí María José
Published: July 2010

Activation of glial cells is a prevalent response to neuronal damage in brain disease and ageing, with potential neuroprotective and neurotoxic consequences. We were interested in studying the role of glial activation on dopaminergic neurons of the substantia nigra in an animal model of Parkinson's disease. Thus, we evaluated the effect of a pre‐existing glial activation on the dopaminergic neuronal death induced by striatal infusion of 6‐hydroxydopamine. We established a model of local glial activation by stereotaxic infusion of interleukin‐1β in the substantia nigra of adult rats. Interleukin‐1β (20 ng) induced a marked activation of astrocytes at days 2, 5 and 10, revealed by heat‐shock protein 27 and glial fibrillary acid protein immunohistochemistry, but did not affect the microglial markers OX‐42 and heat‐shock proteins 32 or 47. Intranigral infusion of interleukin‐1β 5 days before a striatal injection of 6‐hydroxydopamine significantly protected nigral dopaminergic cell bodies, but not striatal terminals from the 6‐hydroxydopamine lesion. Also, in the animals pre‐treated with interleukin‐1β, a significant prevention of 6‐hydroxydopamine‐induced reduction of adjusting steps, but not of 6‐hydroxydopamine‐induced amphetamine rotations, were observed. These data show the characterization of a novel model of local astroglial activation in the substantia nigra and support the hypothesis of a neuroprotective role of activated astrocytes in Parkinson's disease.
Mice transgenic for exon 1 of Huntington's disease: properties of cholinergic and dopaminergic pre‐synaptic function in the striatum
Authors: Vetter J. M. , Jehle T. , Heinemeyer J. , Franz P. , Behrens P. F. , Jackisch R. , Landwehrmeyer G. B. , Feuerstein T. J.
Published: July 2010

In Huntington's disease (HD), neuronal loss is most prominent in the striatum leading to emotional, cognitive and progressive motor dysfunction. The R6/2 mice, transgenic for exon 1 of the HD gene, develop a neurological phenotype with similarities to these features of HD. In striatal tissue, electrically evoked release of tritiated acetylcholine (ACh) and dopamine (DA) were compared in wild‐type (WT) and R6/2 mice. In R6/2 mice, the evoked release of ACh, its M2 autoreceptor‐mediated maximum inhibition and its dopamine D2 heteroreceptor‐mediated maximum inhibition was diminished to 51%, 74% and 87% of controls, respectively. Also, the activities of choline acetyltransferase and of synaptosomal high‐affinity choline uptake decreased progressively with age in these mice. In the DA release model, however, electrical stimulation elicited equal amounts of [3H]‐DA both in WT and R6/2 mice. Moreover, high‐affinity DA uptake into striatal slices was similar in WT and R6/2 mice. In order to confirm these findings in vivo, intrastriatal levels of extracellular DA were measured by intracerebral microdialysis in freely moving mice: striatal DA levels were found to be equal in WT and R6/2 mice. In conclusion, in the transgenic R6/2 mice changes occur mainly in striatal cholinergic neurones and their pre‐synaptic modulation, but not in the dopaminergic afferent terminals. Whether similar events also contribute to the pathogenesis of HD in humans has to be established.
Temporal–spatial characterization of chicken clock genes: circadian expression in retina, pineal gland, and peripheral tissues
Authors: Chong Nelson W. , Chaurasia Shyam S. , Haque Rashidul , Klein David C. , Iuvone P. Michael
Published: July 2010

The molecular core of the vertebrate circadian clock is a set of clock genes, whose products interact to control circadian changes in physiology. These clock genes are expressed in all tissues known to possess an endogenous self‐sustaining clock, and many are also found in peripheral tissues. In the present study, the expression patterns of two clock genes, cBmal1 and cMOP4, were examined in the chicken, a useful model for analysis of the avian circadian system. In two tissues which contain endogenous clocks – the pineal gland and retina – circadian fluctuations of both cBmal1 and cMOP4 mRNAs were observed to be synchronous; highest levels occurred at Zeitgeber time 12. Expression of these genes is also rhythmic in several peripheral tissues; however, the phases of these rhythms differ from those in the pineal gland and retina: in the liver the peaks of cMOP4 and cBmal1 mRNAs are delayed 4–8 h and in the heart they are advanced by 4 h, relative to those in the pineal gland and retina. These results provide the first temporal characterization of cBmal1 and cMOP4 mRNAs in avian tissues: their presence in avian peripheral tissues indicates they may influence temporal features of daily rhythms in biochemical, physiological, and behavioral functions at these sites.
ASPP2 inhibits APP‐BP1‐mediated NEDD8 conjugation to cullin‐1 and decreases APP‐BP1‐induced cell proliferation and neuronal apoptosis
Authors: Chen Yuzhi , Liu Wenyun , Naumovski Louie , Neve Rachael L.
Published: July 2010

APP‐BP1, first identified as a protein that interacts with the carboxyl (C) terminus of the amyloid precursor protein (APP), is one‐half of the bipartite activating enzyme for the ubiquitin‐like protein NEDD8. We report here that APP‐BP1 also specifically interacts with apoptosis stimulating protein of p53 ASPP2 in non‐transfected cells through the functional predominant N‐terminal domain ASPP2(332–483). ASPP2 inhibits the ability of APP‐BP1 to rescue the ts41 cell cycle mutation and inhibits APP‐BP1 induced apoptosis in primary neurons. ASPP2 reduces the ability of NEDD8 to conjugate to Cullin‐1, inhibits APP‐BP1‐dependent ts41 cell proliferation, and blocks the ability of APP‐BP1 to cause apoptosis and to cause DNA synthesis in neurons. We also show that ASPP2 activates nuclear factor‐κB (NF‐κB) transcriptional activity, which seems to be inhibited by the neddylation pathway since the dominant negative NEDD8 activating enzyme causes enhanced NF‐κB activity. Our data provide the first in vivo evidence that ASPP2 is a negative regulator of the neddylation pathway through specific interaction with APP‐BP1 and suggest that dysfunction of the APP–BP1 interaction with APP may be one cause of Alzheimer's disease.
Neuronal nitric oxide synthase proteolysis limits the involvement of nitric oxide in kainate‐induced neurotoxicity in hippocampal neurons
Authors: Araújo Inês M. , Ambrósio António F. , Leal Ermelindo C. , Santos Paulo F. , Carvalho Arsélio P. , Carvalho Caetana M.
Published: July 2010

In this work, we investigated the role of nitric oxide (NO) in neurotoxicity triggered by α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor activation in cultured hippocampal neurons. In the presence of cyclothiazide (CTZ), short‐term exposures to kainate (KA; 5 and 15 min, followed by 24‐h recovery) decreased cell viability. Both NBQX and d‐AP‐5 decreased the neurotoxicity caused by KA plus CTZ. Long‐term exposures to KA plus CTZ (24 h) resulted in increased toxicity. In short‐, but not in long‐term exposures, the presence of NO synthase (NOS) inhibitors (l‐NAME and 7‐NI) decreased the toxicity induced by KA plus CTZ. We also found that KA plus CTZ (15‐min exposure) significantly increased cGMP levels. Furthermore, short‐term exposures lead to decreased intracellular ATP levels, which was prevented by NBQX, d‐AP‐5 and NOS inhibitors. Immunoblot analysis revealed that KA induced neuronal NOS (nNOS) proteolysis, gradually lowering the levels of nNOS according to the time of exposure. Calpain, but not caspase‐3 inhibitors, prevented this effect. Overall, these results show that NO is involved in the neurotoxicity caused by activation of non‐desensitizing AMPA receptors, although to a limited extent, since AMPA receptor activation triggers mechanisms that lead to nNOS proteolysis by calpains, preventing a further contribution of NO to the neurotoxic process.
Bcl‐2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome c translocation and caspase‐3 activity
Authors: Zhao Heng , Yenari Midori A. , Cheng Danye , Sapolsky Robert M. , Steinberg Gary K.
Published: July 2010

Bcl‐2 protects against both apoptotic and necrotic death induced by several cerebral insults. We and others have previously demonstrated that defective herpes simplex virus vectors expressing Bcl‐2 protect against various insults in vitro and in vivo, including cerebral ischemia. Because the infarct margin may be a region that is most amenable to treatment, we first determined whether gene transfer to the infarct margin is possible using a focal ischemia model. Since ischemic injury with and without reperfusion may occur by different mechanisms, we also determined whether Bcl‐2 protects against focal cerebral ischemic injury either with or without reperfusion in rats. Bax expression, cytochrome c translocation and activated caspase‐3 expression were also assessed. Viral vectors overexpressing Bcl‐2 were delivered to the infarct margin. Reperfusion resulted in larger infarcts than permanent occlusion. Bcl‐2 overexpression significantly improved neuron survival in both ischemia models. Bcl‐2 overexpression did not alter overall Bax expression, but inhibited cytosolic accumulation of cytochrome c and caspase‐3 activation. Thus, we provide the first evidence that gene transfer to the infarct margin is feasible, that overexpression of Bcl‐2 protects against damage to the infarct margin induced by ischemia with and without reperfusion, and that Bcl‐2 overexpression using gene therapy attenuates apoptosis‐related proteins. This suggests a potential therapeutic strategy for stroke.
Potassium channel gene therapy can prevent neuron death resulting from necrotic and apoptotic insults
Authors: Lee Angela L. , Dumas Theodore C. , Tarapore Phiroz E. , Webster Brian R. , Ho Dora Y. , Kaufer Daniela , Sapolsky Robert M.
Published: July 2010

Necrotic insults such as seizure are excitotoxic. Logically, membrane hyperpolarization by increasing outwardly conducting potassium channel currents should attenuate hyperexcitation and enhance neuron survival. Therefore, we overexpressed a small‐conductance calcium‐activated (SK2) or voltage‐gated (Kv1.1) channel via viral vectors in cultured hippocampal neurons. We found that SK2 or Kv1.1 protected not only against kainate or glutamate excitotoxicity but also increased survival after sodium cyanide or staurosporine.In vivooverexpression of either channel in dentate gyrus reduced kainate‐induced CA3 lesions. In hippocampal slices, the kainate‐induced increase in granule cell excitability was reduced by overexpression of either channel, suggesting that these channels exert their protective effects during hyperexcitation. It is also important to understand any functional disturbances created by transgene overexpression alone. In the absence of insult, overexpression of Kv1.1, but not SK2, reduced baseline excitability in dentate gyrus granule cells. Furthermore, while no behavioral disturbances during spatial acquisition in the Morris water maze were observed with overexpression of either channel, animals overexpressing SK2, but not Kv1.1, exhibited a memory deficit post‐training. This difference raises the possibility that the means by which these channel subtypes protect may differ. With further development, potassium channel vectors may be an effective pre‐emptive strategy against necrotic insults.
Lipopolysaccharide‐induced dopaminergic cell death in rat midbrain slice cultures: role of inducible nitric oxide synthase and protection by indomethacin
Authors: Shibata Haruki , Katsuki Hiroshi , Nishiwaki Mayumi , Kume Toshiaki , Kaneko Shuji , Akaike Akinori
Published: July 2010

Glial cell activation associated with inflammatory reaction may contribute to pathogenic processes of neurodegenerative disorders, through production of several cytotoxic molecules. We investigated the consequences of glial activation by interferon‐γ (IFN‐γ)/lipopolysaccharide (LPS) in rat midbrain slice cultures. Application of IFN‐γ followed by LPS caused dopaminergic cell death and accompanying increases in nitrite production and lactate dehydrogenase release. Aminoguanidine, an inhibitor of inducible nitric oxide synthase (iNOS), or SB203580, an inhibitor of p38 mitogen‐activated protein kinase, prevented dopaminergic cell loss as well as nitrite production. SB203580 also suppressed expression of iNOS and cyclooxygenase‐2 (COX‐2) induced by IFN‐γ/LPS. A COX inhibitor indomethacin protected dopaminergic neurons from IFN‐γ/LPS‐induced injury, whereas selective COX‐2 inhibitors such as NS‐398 and nimesulide did not. Notably, indomethacin was able to attenuate neurotoxicity of a nitric oxide (NO) donor. Neutralizing antibodies against tumour necrosis factor‐α and interleukin‐1β did not inhibit dopaminergic cell death caused by IFN‐γ/LPS, although combined application of these antibodies blocked lactate dehydrogenase release and decrease in the number of non‐dopaminergic neurons. These results indicate that iNOS‐derived NO plays a crucial role in IFN‐γ/LPS‐induced dopaminergic cell death, and that indomethacin exerts protective effect by mechanisms probably related to NO neurotoxicity rather than through COX inhibition.
Expression of death‐associated protein kinase and recruitment to the tumor necrosis factor signaling pathway following brief seizures
Authors: Henshall David C. , Araki Tomohiro , Schindler Clara K. , Shinoda Sachiko , Lan Jing‐Quan , Simon Roger P.
Published: July 2010

Death‐associated protein (DAP) kinase is calcium‐regulated and known to function downstream of death receptors, prompting us to examine its role in the mechanism of seizure‐induced neuronal death. Brief seizures were focally evoked in rats, eliciting neuronal death within the CA3 subfield of the hippocampus, and to a lesser extent, cortex. Western blotting confirmed expression of DAP kinase within hippocampus and cortex at the predicted weight of ∼160 kDa. Immunohistochemistry revealed seizures triggered a significant increase in numbers of DAP kinase‐expressing cells within CA3 and cortex, without affecting cell counts within seizure‐resistant CA2 or the dentate gyrus. Numbers of DAP kinase‐expressing cells were increased in relation to specific patterns of injury‐causing seizure activity, electrographically defined. Seizures caused an early increase in DAP kinase binding to actin, and association with calmodulin. Co‐immunoprecipitation studies also revealed seizures triggered binding of DAP kinase to the tumor necrosis factor receptor 1 and the Fas‐associated death domain protein, commensurate with caspase‐8 proteolysis. In contrast, within surviving fields of the hippocampus, DAP kinase interacted with the molecular chaperone 14–3−3. These data suggest DAP kinase is involved in the molecular pathways activated during seizure‐induced neuronal death.
Identification of members of the Bex gene family as olfactory marker protein (OMP) binding partners
Authors: Behrens Maik , Margolis Joyce W. , Margolis Frank L.
Published: July 2010

Olfactory marker protein (OMP) expression is a hallmark of mature vertebrate olfactory receptor neurons (ORNs). Evidence for OMP function derives from altered behavioral and electrophysiological activities of OMP‐KO mice. The molecular basis for the altered phenotype following the deletion of OMP is still unclear. Recent structural studies predict the involvement of OMP in protein–protein interaction. Here we report the identification of an OMP partner, Bex2, by phage‐display screening of an olfactory mucosal cDNA‐library. In situ hybridization demonstrates cellular co‐localization of OMP mRNA with mRNAs for Bex1, Bex2, and Bex3 in ORNs of olfactory tissue of the mouse. The OMP/Bex interaction has been confirmed by demonstrating the chemical cross‐linking of recombinant rat OMP with a synthetic peptide derived from the Bex amino acid sequence. The subcellular localization of Bex and OMP proteins was evaluated in transfected HEK293 cells. Bex is visualized in the nucleus and cytoplasm. Following co‐transfection we observed the unexpected presence of some OMP in the nucleus along with Bex. Together, these data argue convincingly that we have identified Bex as an OMP partner whose further characterization will provide insight to the role of OMP and to the mechanism of theOMP/Bex interaction in ORN differentiation and function.
Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease
Authors: Höglinger Günter U. , Carrard Géraldine , Michel Patrick P. , Medja Fadia , Lombès Anne , Ruberg Merle , Friguet Bertrand , Hirsch Etienne C.
Published: July 2010

Two biochemical deficits have been described in the substantia nigra in Parkinson's disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1‐methyl‐4‐phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6‐hydroxydopamine, but not of inhibitors of mitochondrial complex II–V or excitotoxins [N‐methyl‐d‐aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6‐hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6‐hydroxydopamine at concentrations at which they increased free radical levels ≥ 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.
Ebselen inhibits NO‐induced apoptosis of differentiated PC12 cells via inhibition of ASK1‐p38 MAPK‐p53 and JNK signaling and activation of p44/42 MAPK and Bcl‐2
Authors: Sarker Krishna P. , Biswas Kamal K. , Rosales Jesusa L. , Yamaji Kazuyo , Hashiguchi Teruto , Lee Ki‐Young , Maruyama Ikuro
Published: July 2010

Ebselen, a selenium‐containing heterocyclic compound, prevents ischemia‐induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO‐induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO‐induced phosphatidyl Serine exposure, cytochrome c release, and caspase‐3 activation by ebselen. Analysis of key apoptotic regulators during NO‐induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling‐regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen‐activated protein kinase (MAPK) and c‐jun N‐terminal protein kinase (JNK). Moreover, ebselen inhibits NO‐induced p53 phosphorylation at Ser15 and c‐Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol‐redox‐dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl‐2 in SNP‐treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.
Lactate and glucose as energy substrates during, and after, oxygen deprivation in rat hippocampal acute and cultured slices
Authors: Cater Heather L. , Chandratheva Arvind , Benham Christopher D. , Morrison Barclay , Sundstrom Lars E.
Published: July 2010

The effects of raised brain lactate levels on neuronal survival following hypoxia or ischemia is still a source of controversy among basic and clinical scientists. We have sought to address this controversy by studying the effects of glucose and lactate on neuronal survival in acute and cultured hippocampal slices. Following a 1‐h hypoxic episode, neuronal survival in cultured hippocampal slices was significantly higher if glucose was present in the medium compared with lactate. However, when the energy substrate during the hypoxic period was glucose and then switched to lactate during the normoxic recovery period, the level of cell damage in the CA1 region of organotypic cultures was significantly improved from 64.3 ± 2.1 to 74.6 ± 2.1% compared with cultures receiving glucose during and after hypoxia. Extracellular field potentials recorded from the CA1 region of acute slices were abolished during oxygen deprivation for 20 min, but recovered almost fully to baseline levels with either glucose (82.6 ± 10.0%) or lactate present in the reperfusion medium (108.1 ± 8.3%). These results suggest that lactate alone cannot support neuronal survival during oxygen deprivation, but a combination of glucose followed by lactate provides for better neuroprotection than either substrate alone.
Pharmacologic suppression of neuronal oxidative damage and dendritic degeneration following direct activation of glial innate immunity in mouse cerebrum
Authors: Milatovic Dejan , Zaja‐Milatovic Snjeanna , Montine Kathleen S. , Horner Philip J. , Montine Thomas J.
Published: July 2010

Activation of glial innate immunity is widely proposed to contribute to a number of degenerative and destructive diseases of brain. However, the precise role of activated innate immunity has been difficult to define in vivo because of multiple simultaneous pathogenic processes and responses to injury that confound interpretation of results from complex models of disease. Here, we used the model of intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) to test the hypothesis that directly activated glial innate immunity leads to neurodegeneration in cerebrum and to establish the molecular determinants of and neuroprotectants from such innate immunity‐mediated neuronal damage. Our results showed that ICV LPS induced delayed, reversible oxidative damage to cerebral neuronal membranes as measured by F4‐neuroprostanes that was coincident with degeneration of the hippocampal pyramidal neuron dendritic system, but not neuron death, in adult mice. Both neuronal oxidative damage and dendritic degeneration were NF‐κB and iNOS dependent and were completely suppressed by ibuprofen and α‐tocopherol, but not naproxen or γ‐tocopherol. These results prove that activation of glial innate immunity can lead to neurodegeneration independent of other pathologic processes, closely associate oxidative damage to neuronal membranes with degeneration of the dendritic system, and provide a possible explanation for the varying efficacy of neuroprotectants that have been suggested in observational studies of dementia.
Glycogen in the brain of Drosophila melanogaster: diurnal rhythm and the effect of rest deprivation
Authors: Zimmerman John E. , Mackiewicz Miroslaw , Galante Raymond J. , Zhang Lin , Cater Jacqueline , Zoh Christine , Rizzo Wendy , Pack Allan I.
Published: July 2010

One function of sleep is thought to be the restoration of energy stores in the brain depleted during wakefulness. One such energy store found in mammalian brains is glycogen. Many of the genes involved in glycogen regulation in mammals have also been found in Drosophila melanogaster and rest behavior in Drosophila has recently been shown to have the characteristics of sleep. We therefore examined, in the fly, variation in the glycogen contents of the brain, the whole head and the body throughout the rest/activity cycle and after rest deprivation. Glycogen in the brain varies significantly throughout the day (p = 0.001) and is highest during rest and lowest while flies are active. Glycogen levels in the whole head and body do not show diurnal variation. Brain glycogen drops significantly when flies are rest deprived for 3 h (p = 0.034) but no significant differences are observed after 6 h of rest deprivation. In contrast, glycogen is significantly depleted in the body after both 3 and 6 h of rest deprivation (p < 0.0001 and p < 0.0001, respectively). Glycogen in the fly brain changes in relationship to rest and activity and demonstrates a biphasic response to rest deprivation similar to that observed in mammalian astrocytes in culture.
Small peptides patterned after the N‐terminus domain of SNAP25 inhibit SNARE complex assembly and regulated exocytosis
Authors: Blanes‐Mira Clara , Merino Jaime M. , Valera Elvira , Fernández‐Ballester Gregorio , Gutiérrez Luis M. , Viniegra Salvador , Pérez‐Payá Enrique , Ferrer‐Montiel Antonio
Published: July 2010

Synthetic peptides patterned after the C‐terminus of synaptosomal associated protein of 25 kDa (SNAP25) efficiently abrogate regulated exocytosis. In contrast, the use of SNAP25 N‐terminal‐derived peptides to modulate SNAP receptors (SNARE) complex assembly and neurosecretion has not been explored. Here, we show that the N‐terminus of SNAP25, specially the segment that encompasses 22Ala‐44Ile, is essential for the formation of the SNARE complex. Peptides patterned after this protein domain are potent inhibitors of SNARE complex formation. The inhibitory activity correlated with their propensity to adopt an α‐helical secondary structure. These peptides abrogated SNARE complex formation only when added previous to the onset of aggregate assembly. Analysis of the mechanism of action revealed that these peptides disrupted the binary complex formed by SNAP25 and syntaxin. The identified peptides inhibited Ca2+‐dependent exocytosis from detergent‐permeabilized excitable cells. Noteworthy, these amino acid sequences markedly protected intact hippocampal neurones against hypoglycaemia‐induced, glutamate‐mediated excitotoxicity with a potency that rivaled that displayed by botulinum neurotoxins. Our findings indicate that peptides patterned after the N‐terminus of SNAP25 are potent inhibitors of SNARE complex formation and neuronal exocytosis. Because of their activity in intact neurones, these cell permeable peptides may be hits for antispasmodic and analgesic drug development.
The mechanism of membrane‐translocation of regulator of G‐protein signaling (RGS) 8 induced by Gα expression
Authors: Masuho Ikuo , Itoh Masayuki , Itoh Hiroshi , Saitoh Osamu
Published: July 2010

RGS (regulators of G‐protein signaling) proteins comprise a large family that modulates heterotrimeric G‐protein signaling. This protein family has a common RGS domain and functions as GTPase‐activating proteins for the α‐subunits of heterotrimeric G‐proteins located at the plasma membrane. RGS8 was identified as a neuron‐specific RGS protein, which belongs to the B/R4 subfamily. We previously showed that RGS8 protein was translocated to the plasma membrane from the nucleus on coexpression of GTPase‐deficient Gαo (GαoQL). Here, we first examined which subtypes of Gα can induce the translocation of RGS8. When the Gαi family was expressed, the translocation of RGS8 did occur. To investigate the mechanism of this translocation, we generated a mutant RGS8 with reduced affinity to Gαo and an RGS‐insensitive (RGS‐i) mutant of GαoQL. Co‐expression experiments with both mutants revealed that disruption of the Gα–RGS8 interaction abolished the membrane‐translocation of RGS8 despite the apparent membrane localization of RGS‐i GαoQL. These results demonstrated that RGS8 is recruited to the plasma membrane where G‐proteins are activated mainly by direct association with Gα.
The significance of the mutated divalent metal transporter (DMT1) on iron transport into the Belgrade rat brain
Authors: Moos Torben , Morgan Evan H.
Published: July 2010

Brain iron transport and distributional pattern of divalent metal transporter I (DMT1) were studied in homozygous Belgrade rats (b/b) which suffer from a mutation in the DMT1 gene. In adult rats, brain uptake of transferrin‐bound iron injected intravenously (i.v.) was significantly lower compared with that in heterozygous Belgrade (+/b) and Wistar rats, whereas transferrin uptake was identical. The difference in iron uptake was not apparent until 30 min after injection. The brain iron concentration was lower, and neuronal transferrin receptor‐immunoreactivity higher, in adult b/b rats, thus confirming their iron‐deficient stage. Antibodies targeting different sites on the DMT1 molecule consistently detected DMT1 in neurones and choroid plexus at the same level irrespective of strain, but failed to detect DMT1 in brain capillary endothelial cells (BCECs), or macro‐ or microglial cells. The absence of DMT1 in BCECs was confirmed in immunoblots of purified BCECs. DMT1 was virtually undetectable in neurones of rats aged 18 post‐natal days irrespective of strain. Neuronal expression of transferrin receptors and DMT1 in adult rats implies that neurones at this age acquire iron by receptor‐mediated endocytosis of transferrin followed by iron transport out of endosomes mediated by DMT1. The existence of the mutated DMT1 molecule in neurones suggests that the low cerebral iron uptake in b/b rats derives from a reduced neuronal uptake rather than an impaired iron transport through the blood–brain barrier.
Enhanced detection and retrograde axonal transport of PrPc in peripheral nerve
Authors: Moya Kenneth L. , Hässig Raymonde , Créminon Christophe , Laffont Isabelle , Di Giamberardino Luigi
Published: July 2010

Neuroinvasion of the CNS during orally acquired transmissible spongiform encephalopathies (TSEs) may involve the transport of the infectious agent from the periphery to the CNS via the peripheral nerves. If this occurs within axons, the mechanism of axonal transport may be fundamental to the process. In studies of peripheral nerve we observed that the cellular prion protein (PrPc) is highly resistant to detergent extraction. The implication of this is an underestimation of the abundance of PrPc in peripheral nerve. We have developed nerve extraction conditions that enhance the quantification of the protein in nerve 16‐fold. Application of these conditions to evaluate the accumulation of PrPc distal to a cut nerve now reveals that PrPc is retrogradely transported from the axon ending. These results provide a potential cellular mechanism for TSE infectivity to gain entry to the CNS from the periphery.
Axonal transport of human α‐synuclein slows with aging but is not affected by familial Parkinson's disease‐linked mutations
Authors: Li Wenxue , Hoffman Paul N. , Stirling Wanda , Price Donald L. , Lee Michael K.
Published: July 2010

Biochemical and genetic abnormalities of α‐synuclein (α‐Syn) are implicated in the pathogenesis of Parkinson's disease (PD) and other α‐synucleinopathies. The abnormal intraneuronal accumulations of α‐Syn in Lewy bodies (LBs) and Lewy neurites (LNs) have implicated defects in axonal transport of α‐Syn in the α‐synucleinopathies. Using human (Hu) α‐Syn transgenic (Tg) mice, we have examined whether familial PD (FPD)‐linked mutations (A30P and A53T) alter axonal transport of Huα‐Syn. Our studies using peripheral nerves show that Huα‐Syn and Moα‐Syn are almost exclusively transported in the slow component (SC) of axonal transport and that the FPD‐linked α‐Syn mutations do not have obvious effects on the axonal transport of α‐Syn. Moreover, older pre‐symptomatic A53T Huα‐Syn Tg mice do not show gross alterations in the axonal transport of α‐Syn and other proteins in the SC, indicating that the early stages of α‐synucleinopathy in A53T α‐Syn Tg mice are not associated with gross alterations in the slow axonal transport. However, the axonal transport of α‐Syn slows significantly with aging. Because the rate of axonal transport affects the stability and accumulation of proteins in axons, age‐dependent‐slowing α‐Syn is a likely contributor to axonal aggregation of α‐Syn in α‐synucleinopathy.
Trk is a calmodulin‐binding protein: implications for receptor processing
Authors: Llovera Marta , De Pablo Yolanda , Egea Joaquim , Encinas Mario , Peiró Sandra , Martín‐Zanca Dionisio , Rocamora Nativitat , Comella Joan X.
Published: July 2010

The tyrosine kinase receptors for the neurotrophins (Trk) are a family of transmembrane receptors that regulate the differentiation and survival of different neuronal populations. Neurotrophin binding to Trk leads to the activation of several signalling pathways including a rapid, but moderate, increase in intracellular calcium levels. We have previously described the role of calcium and its sensor protein, calmodulin, in Trk‐activated intracellular pathways. Here we demonstrate that calmodulin is able to precipitate TrkA from PC12 cell lysates. Using recombinant GST‐fusion proteins containing the complete intracellular domain of TrkA, or fragments of this region, we show that calmodulin binds directly to the C‐terminal domain of TrkA in a Ca2+‐dependent manner. We have also co‐immunoprecipitated endogenous Trk and calmodulin in primary cultures of cortical neurones. Moreover, we provide evidence that calmodulin is involved in the regulation of TrkA processing in PC12 cells. Calmodulin inhibition results in the generation of a TrkA‐derived p41 fragment from the cytosolic portion of the protein. This fragment is autophosphorylated in tyrosines and can recruit PLCγ and Shc adaptor proteins. These results suggest that calmodulin binding to Trk may be important for the regulation of Trk intracellular localization and cleavage.
Topiramate modulation of kainate‐induced calcium currents is inversely related to channel phosphorylation level
Authors: Ängehagen Mikael , Ben‐Menachem Elinor , Shank Richard , Rönnbäck Lars , Hansson Elisabeth
Published: July 2010

Topiramate (TPM) is a structurally novel broad‐spectrum anticonvulsant known to modulate the activity of several ligand‐ and voltage‐gated ion channels in neurons. These include an inhibitory effect on the AMPA and kainate subtypes of glutamate receptors, mixed modulatory effects (usually positive) on some types of GABAA receptors, negative modulatory effects on some types of voltage‐gated Na+ and Ca2+ channels, and a positive modulatory effect on at least one type of K+ channel. The nature of these effects at the molecular level has not been established, but two previous studies have implicated the phosphorylation state of these receptor/channel complexes as an influencing factor in the activity of TPM. Here, we report that the ability of TPM to inhibit a kainate‐induced accumulation of free Ca2+ in cultured neurons from rat cerebral cortex is inversely related to the level of cAMP‐dependent protein kinase (cAPK) mediated phosphorylation of kainate‐activated receptors/channels. Specifically, when cell cultures were pre‐treated with forskolin or dibutyryl cAMP, indirect activators of cAPK, the activity of TPM was abolished, whereas when the cells were pre‐treated with H89, an inhibitor of cAPK, the relative activity of TPM was enhanced. The results of this study support the hypothesis that TPM binds to phosphorylation sites on AMPA and kainate receptors, but only in the dephosphorylated state and thereby exerts an allosteric modulatory effect on channel conductance.
PrPc on the road: trafficking of the cellular prion protein
Authors: Prado Marco A. M. , Alves‐Silva Juliana , Magalhães Ana C. , Prado Vania F. , Linden Rafael , Martins Vilma Regina , Brentani Ricardo Renzo
Published: July 2010

The glycosylphosphatidylinositol (GPI)‐anchored cellular prion protein (PrPc) has a fundamental role in prion diseases. Intracellular trafficking of PrPc is important in the generation of protease resistant PrP species but little is known of how endocytosis affects PrPc function. Here, we discuss recent experiments that have illuminated how PrPc is internalized and what are the possible destinations taken by the protein. Contrary to what would be expected for a GPI‐anchored protein there is increasing evidence that clathrin‐mediated endocytosis and classical endocytic organelles participate in PrPc trafficking. Moreover, the N‐terminal domain of PrPc may be involved in sorting events that can direct the protein during its intracellular journey. Indeed, the concept that the GPI‐anchor determines PrPc trafficking has been challenged. Cellular signaling can be triggered or be regulated by PrPc and we suggest that endocytosis of PrPc may influence signaling in several ways. Definition of the processes that participate in PrPc endocytosis and intracellular trafficking can have a major impact on our understanding of the mechanisms involved in PrPc function and conversion to protease resistant conformations.
Expression of SOD1 G93A or wild‐type SOD1 in primary cultures of astrocytes down‐regulates the glutamate transporter GLT‐1: lack of involvement of oxidative stress
Authors: Tortarolo Massimo , Crossthwaite Andrew J. , Conforti Laura , Spencer Jeremy P. , Williams Robert J. , Bendotti Caterina , Rattray Marcus
Published: July 2010

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate‐induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down‐regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS‐linked mutant hSOD1G93A and wild‐type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT‐PCR it was shown that expression of either hSOD1G93A or hSOD1wt in astrocytes produced down‐regulation of the levels of a glutamate transporter GLT‐1, without alterations in its mRNA level. hSOD1G93A or hSOD1wt expression caused a decrease of the monomeric form of GLT‐1 without increasing oxidative multimers of GLT‐1. The effects were selective to GLT‐1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT‐1 protein, [3H]d‐aspartate uptake was reduced in cultures expressing hSOD1G93A or hSOD1wt. The hSOD1‐induced decline in GLT‐1 protein and [3H]d‐aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2′,7′‐dichlorofluorescein (DCF)‐induced fluorescence revealed that expression of hSOD1G93A or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT‐1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild‐type and G93A mutant form, but does not involve the production of intracellular oxidative stress.
X11α impairs γ‐ but not β‐cleavage of amyloid precursor protein
Authors: King Gwendalyn D. , Cherian Kay , Turner R. Scott
Published: July 2010

The phosphotyrosine binding domain of the neuronal protein X11α/mint‐1 binds to the C‐terminus of amyloid precursor protein (APP) and inhibits catabolism to β‐amyloid (Aβ), but the mechanism of this effect is unclear. Coexpression of X11α or its PTB domain with APPswe inhibited secretion of Aβ40 but not APPsβswe, suggesting inhibition of γ‐ but not β‐secretase. To further probe cleavage(s) inhibited by X11α, we coexpressed β‐secretase (BACE‐1) or a component of the γ‐secretase complex (PS‐1Δ9) with APP, APPswe, or C99, with and without X11α, in HEK293 cells. X11α suppressed the PS‐1Δ9‐induced increase in Aβ42 secretion generated from APPswe or C99. However, X11α did not impair BACE‐1‐mediated proteolysis of APP or APPswe to C99. In contrast to impaired γ‐cleavage of APPswe, X11α or its PTB domain did not inhibit γ‐cleavage of NotchΔE to NICD (the Notch intracellular domain). The X11α PDZ–PS.1Δ9 interaction did not affect γ‐cleavage activity. In a cell‐free system, X11α did not inhibit the catabolism of APP C‐terminal fragments. These data suggest that X11α may inhibit Aβ secretion from APP by impairing its trafficking to sites of active γ‐secretase complexes. By specifically targeting substrate instead of enzyme X11α may function as a relatively specific γ‐secretase inhibitor.
Glucocorticoids modulate neurotransmitter‐induced glycogen metabolism in cultured cortical astrocytes
Authors: Allaman Igor , Pellerin Luc , Magistretti Pierre J.
Published: July 2010

Glucocorticoids (GC) are considered as key modulators of glycogen homeostasis in peripheral tissues, but their role in the central nervous system has only partially been characterized. Exposure of primary cultures of cortical astrocytes to dexamethasone (DEX), a synthetic glucocorticoid, results in the reduction of noradrenaline (NA)‐induced glycogen synthesis in a concentration‐dependent manner with a IC50 of 4.88 nm and a maximum inhibition of 51%. Such an effect is mediated via glucocorticoid receptors (GRs), since it is mimicked by the glucocorticoid analogue RU28362 (100 nm) and prevented by the GR antagonist RU38486 (1 µm). DEX does not act through alteration of signal transduction mechanisms, as cAMP formation induced by noradrenergic stimulation was unchanged. Moreover, glycogen synthesis was inhibited to the same extent when DEX was applied either together or only after a brief NA application. Neither [3H]2‐deoxyglucose uptake nor lactate release was altered by DEX in the presence of NA, demonstrating that inhibition of glycogen synthesis is not a consequence of reduced glucose utilization or availability. Interestingly, enhancement of glycogen synthase activity induced by NA was reduced in the presence of DEX (−27%). These results suggest that GC could have a significant influence on neuroenergetics as they could modulate activity‐related changes in brain glycogen metabolism.