bluuebunny/crossref_metadata_2025 huggingface dataset:
JKK1, an upstream activator of JNK/SAPK, is activated in Alzheimer's disease
Authors: Zhu Xiongwei , Ogawa Osamu , Wang Yang , Perry George , Smith Mark A.
Published: July 2010

JNK/SAPK has been implicated in the pathogenesis of Alzheimer's disease, but the upstream cascade leading to JNK/SAPK activation has not been elucidated in the disease. In this study, we focused on one of the physiological activators of JNK/SAPK, JNK kinase 1 (JKK1). Although there was no significant difference in the level and distribution of total JKK1 between Alzheimer's disease (AD) and age‐matched control cases, increased levels of activated phospho‐JKK1 were specifically localized to neurofibrillary pathology including neurofibrillary tangles, senile plaque neurites, granulovaualar degenerations and neuropil threads in severe AD (Braak stage V–VI), considerably overlapping with its downstream effector, phospho‐JNK/SAPK, suggesting both a functional and mechanistic link. Nuclear localization of phospho‐JKK1 was also found in mild (Braak stage III–IV) but not in severe AD cases (Braak stage V–VI), suggesting a possible re‐distribution correlating with the progress of the disease. By immunoblot analyses, phospho‐JKK1 was significantly increased in AD over control cases. Together, these findings lend further credence to the notion that the JNK/SAPK pathway is dysregulated in AD and also indicate an active role for this pathway in disease pathogenesis.
Human group IIA secretory phospholipase A2potentiates Ca2+influx through L‐type voltage‐sensitive Ca2+channels in cultured rat cortical neurons
Authors: Yagami Tatsurou , Ueda Keiichi , Asakura Kenji , Nakazato Hitoshi , Hata Satoshi , Kuroda Takayuki , Sakaeda Toshiyuki , Sakaguchi Gaku , Itoh Naohiro , Hashimoto Yutaka , Hori Yozo
Published: July 2010

Mammalian group IIA secretory phospholipase A2(sPLA2‐IIA) generates prostaglandin D2(PGD2) and triggers apoptosis in cortical neurons. However, mechanisms of PGD2generation and apoptosis have not yet been established. Therefore, we examined how second messengers are involved in the sPLA2‐IIA‐induced neuronal apoptosis in primary cultures of rat cortical neurons. sPLA2‐IIA potentiated a marked influx of Ca2+into neurons before apoptosis. A calcium chelator and a blocker of the L‐type voltage‐sensitive Ca2+channel (L‐VSCC) prevented neurons from sPLA2‐IIA‐induced neuronal cell death in a concentration‐dependent manner. Furthermore, the L‐VSCC blocker ameliorated sPLA2‐IIA‐induced morphologic alterations and apoptotic features such as condensed chromatin and fragmented DNA. Other blockers of VSCCs such as N type and P/Q types did not affect the neurotoxicity of sPLA2‐IIA. Blockers of L‐VSCC significantly suppressed sPLA2‐IIA‐enhanced Ca2+influx into neurons. Moreover, reactive oxygen species (ROS) were generated prior to apoptosis. Radical scavengers reduced not only ROS generation, but also the sPLA2‐IIA‐induced Ca2+influx and apoptosis. In conclusion, we demonstrated that sPLA2‐IIA potentiates the influx of Ca2+into neurons via L‐VSCC. Furthermore, the present study suggested that eicosanoids and ROS generated during arachidonic acid oxidative metabolism are involved in sPLA2‐IIA‐induced apoptosis in cooperation with Ca2+.
Lithium enhances long‐term potentiation independently of hippocampal neurogenesis in the rat dentate gyrus
Authors: Son Hyeon , Yu In Tag , Hwang Se‐Jin , Kim Jin Seuk , Lee Sang‐Hum , Lee Yong‐Sung , Kaang Bong‐Kiun
Published: July 2010

We measured the temporal and spatial profiles of neural precursor cells, hippocampal long‐term potentiation (LTP), and signaling molecules in neurogenesis‐induced adult rats. Chronic lithium treatment produced a significant 54% and 40% increase in the numbers of bromodeoxyuridine [BrdU(+)] cells after 12 h and 28 days, respectively, after treatment completion in the dentate gyrus (DG). Both LTP obtained from slices perfused with artificial cerebrospinal fluid (ACSF‐LTP) and LTP recorded in the presence of bicuculline (bicuculline‐LTP) were significantly greater in the lithium group than in the saline controls. Although the number of BrdU(+) cells, approximately 90% of which were double‐labeled with a neural marker neuronal nuclear protein, were markedly increased in the granule cell layer (GCL) 28 days after the completion of the 28‐day lithium treatment, the magnitude of LTP observed at this time was similar to that observed 12 h after completing the 28‐day lithium treatment. However, protein levels of calcium and calmodulin‐dependent protein kinase II, p‐Elk and TrkB were highly elevated until 28 days after the 28‐day lithium treatment. Acute lithium treatment for 2 days also enhanced LTP, which was accompanied by the elevated expression of p‐CREB, but not by neurogenesis. Our results suggest that the enhancement of LTP is independent of the increased number of neurons per se and it is more closely associated with key molecules, which are probably involved in neurogenesis.
Subcellular segregation of distinct heteromeric NMDA glutamate receptors in the striatum
Authors: Dunah Anthone W. , Standaert David G.
Published: July 2010

FunctionalN‐methyl‐d‐aspartate (NMDA) glutamate receptors are composed of heteromeric complexes of NR1, the obligatory subunit for channel activity, and NR2 or NR3 family members, which confer variability in the properties of the receptors. Recent studies have provided evidence for the existence of both binary (containing NR1 and either NR2A or NR2B) and ternary (containing NR1, NR2A, and NR2B) receptor complexes in the adult mammalian brain. However, the mechanisms regulating subunit assembly and receptor localization are not well understood. In the CNS, NMDA subunits are present both at intracellular sites and the post‐synaptic membrane of neurons. Using biochemical protein fractionation and co‐immunoprecipitation approaches we have found that in rat striatum binary NMDA receptors are widely distributed, and can be identified in the light membrane, synaptosomal membrane, and synaptic vesicle‐enriched subcellular compartments. In contrast, ternary receptors are found exclusively in the synaptosomal membranes. When striatal proteins are chemically cross‐linked prior to subcellular fractionation, ternary NMDA receptors can be precipitated from the light membrane and synaptic vesicle‐enriched fractions where this type of receptor complex is not detectable under normal conditions. These findings suggest differential targeting of distinct types of NMDA receptor assemblies between intracellular and post‐synaptic sites based on subunit composition. This targeting may underlie important differences in the regulation of the transport pathways involved in both normal as well as pathological receptor functions.
Metabotropic glutamate receptor 5‐regulated Elk‐1 phosphorylation and immediate early gene expression in striatal neurons
Authors: Mao Limin , Wang John Q.
Published: July 2010

The Gαq protein‐coupled metabotropic glutamate receptor subtype‐5 (mGluR5) is densely expressed in medium spiny projection neurons of striatum. Emerging evidence suggests a significant role of mGluR5 in the addictive plasticity of striatal neurons that is likely derived from inducible cellular gene expression related to stimulation of mGluR5 and associative signaling proteins. In this study, we found that activation of mGluR5 with a selective agonist (RS)‐2‐chloro‐5‐hydroxy‐phenylglycine (CHPG) induced a rapid and transient phosphorylation of a transcription regulator Elk‐1 in cultured striatal neurons from rat E19 embryos or neonatal day‐1 pups. The Elk‐1 phosphorylation was dose‐dependent and occurred in neurochemically identified GABAergic neurons, but not glia. A series of experiments further demonstrated that the CHPG‐stimulated Elk‐1 phosphorylation was mediated through selective activation of mGluR5‐regulated phospholipase C and associative second messenger system, i.e. 1,4,5,‐triphosphate‐sensitive Ca2+ release. Moreover, the Elk‐1 phosphorylation was partially dependent on mGluR5‐mediated co‐activation of NMDA, but not kainate/AMPA receptors and L‐type voltage‐operated Ca2+ channels. Using an immediate early gene c‐fos as a report of inducible gene expression, we found that CHPG induced marked c‐fos mRNA expression. The c‐fos induction kinetically corresponded to the Elk‐1 phosphorylation and was attenuated by antisense oligonucleotides that selectively knocked down Elk‐1 proteins. These results indicate that glutamatergic tone on mGluR5 is positively coupled to Elk‐1 phosphorylation in striatal neurons via multiple signaling mechanisms involving Ca2+ release and NMDA activation, and the mGluR5‐mediated Elk‐1 phosphorylation facilitates gene transcription.
Facilitation of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor transmission in the suprachiasmatic nucleus by aniracetam enhances photic responses of the biological clock in rodents
Authors: Moriya Takahiro , Ikeda Masayuki , Teshima Koji , Hara Reiko , Kuriyama Koji , Yoshioka Tohru , N. Allen Charles , Shibata Shigenobu
Published: July 2010

This study was designed to test whether the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor‐facilitating drug, aniracetam, could potentiate photic responses of the biological clock in the suprachiasmatic nucleus (SCN) of rodents. Using the whole‐cell patch technique, we first demonstrated that AMPA currents elicited by either local AMPA application or optic chiasm stimulation were augmented by aniracetam in the neurons of the SCN. The AMPA application‐elicited increase of intracellular Ca2+ concentration in SCN slices was also enhanced by aniracetam treatment. The systemic injection of aniracetam dose‐dependently (10–100 mg/kg) potentiated the phase delay in behavioral rhythm induced by brief light exposure of low intensity (3 lux) but not high intensity (10 or 60 lux) during early subjective night. Under the blockade of NMDA receptors by (+) MK801, aniracetam failed to potentiate a light (3 lux)‐induced phase delay in behavioral rhythm. Aniracetam increased the photic induction of c‐Fos protein in the SCN that was elicited by low intensity light exposure (3 lux). These results suggest that AMPA receptor‐mediated responses facilitated by aniracetam can explain enhanced photic responses of the biological clock in the SCN of rodents.
Neuronal uptake and metabolism of glycerol and the neuronal expression of mitochondrial glycerol‐3‐phosphate dehydrogenase
Authors: Nguyen Nga Huynh Tran , Bråthe Anders , Hassel Bjørnar
Published: July 2010

Glycerol is effective in the treatment of brain oedema but it is unclear if this is due solely to osmotic effects of glycerol or whether the brain may metabolize glycerol. We found that intracerebral injection of [14C]glycerol in rat gave a higher specific activity of glutamate than of glutamine, indicating neuronal metabolism of glycerol. Interestingly, the specific activity of GABA became higher than that of glutamate. NMR spectroscopy of brains of mice given 150 µmol [U‐13C]glycerol (0.5 m i.v.) confirmed this predominant labelling of GABA, indicating avid glycerol metabolism in GABAergic neurones. Uptake of [14C]glycerol into cultured cerebellar granule cells was inhibited by Hg2+, suggesting uptake through aquaporins, whereas Hg2+ stimulated glycerol uptake into cultured astrocytes. The neuronal metabolism of glycerol, which was confirmed in experiments with purified synaptosomes and cultured cerebellar granule cells, suggested neuronal expression of glycerol kinase and some isoform of glycerol‐3‐phosphate dehydrogenase. Histochemically, we demonstrated mitochondrial glycerol‐3‐phosphate dehydrogenase in neurones, whereas cytosolic glycerol‐3‐phosphate dehydrogenase was three to four times more active in white matter than in grey matter, reflecting its selective expression in oligodendroglia. The localization of mitochondrial and cytosolic glycerol‐3‐phosphate dehydrogenases in different cell types implies that the glycerol‐3‐phosphate shuttle is of little importance in the brain.
Regulation of the glutamate transporter EAAT1 by the ubiquitin ligase Nedd4‐2 and the serum and glucocorticoid‐inducible kinase isoforms SGK1/3 and protein kinase B
Authors: Boehmer Christoph , Henke Guido , Schniepp Roman , Palmada Monica , Rothstein Jeffrey D. , Bröer Stefan , Lang Florian
Published: July 2010

Surface expression of the glial glutamate transporter EAAT1 is stimulated by insulin‐like growth factor 1 through activation of phosphatidylinositol‐3‐kinase. Downstream targets include serum and glucocorticoid‐sensitive kinase isoforms SGK1, SGK2 and SGK3, and protein kinase B. SGK1 regulates Nedd4‐2, a ubiquitin ligase that prepares cell membrane proteins for degradation. To test whether Nedd4‐2, SGK1, SGK3 and protein kinase B regulate EAAT1, cRNA encoding EAAT1 was injected into Xenopus oocytes with or without additional injection of wild‐type Nedd4‐2, constitutively active S422DSGK1, inactive K127NSGK1, wild‐type SGK3 and/or constitutively active T308D,S473DPKB. Glutamate induces a current in Xenopus oocytes expressing EAAT1, but not in water‐injected oocytes, which is decreased by co‐expression of Nedd4‐2, an effect reversed by additional co‐expression of S422DSGK1, SGK3 and T308D,S473DPKB, but not K127NSGK1. Site‐directed mutagenesis of the SGK1 phosphorylation sites in the Nedd4‐2 protein (S382A,S468ANedd4‐2) and in the EAAT1 protein (T482AEAAT1, T482DEAAT1) significantly blunts the effect of S422DSGK1. Moreover, the current is significantly larger in T482DEAAT1‐ than in T482AEAAT1‐expressing oocytes, indicating that a negative charge mimicking phosphorylation at T482 increases transport. The experiments reveal a powerful novel mechanism that regulates the activity of EAAT1. This mechanism might participate in the regulation of neuronal excitability and glutamate transport in other tissues.
Tumor necrosis factor‐α at the crossroads of neuronal life and death during HIV‐associated dementia
Authors: Saha Ramendra N. , Pahan Kalipada
Published: July 2010

Human immunodeficiency type‐1 (HIV‐1) infection is known to cause disorders of the CNS, including HIV‐associated dementia (HAD). It is suspected that tumor necrosis factor‐α (TNF‐α) released by infected microglia and macrophages play a role in neuronal injury seen in HAD patients. Accordingly, studies suggest that the level of TNF‐α mRNA increases with increasing severity of dementia in patients, and that inhibitors of TNF‐α release reduces neuronal injury in murine model of HAD. However, the exact role of TNF‐α in relation to neuronal dysfunction is a matter of ongoing debate. One school of thought hails TNF‐α as the inducer and mediator of neurodegeneration and their evidence suggest that TNF‐α kill neurons directly by recruiting caspases or may kill indirectly by various means. In sharp contrast to this, another concept theory envisages a role for TNF‐α in negotiating neuroprotection during HAD. The current compilation examines these contradictory concepts, and evaluates their efficacy in the light of TNF‐α signaling. It also attempts to elaborate the current consensus outlook of TNF‐α's role during HAD.
Erasure of kinase phosphorylation in astrocytes during oxygen–glucose deprivation is controlled by ATP levels and activation of phosphatases
Authors: Yung Hong Wa , Tolkovsky Aviva M.
Published: July 2010

We have examined the relationship between adenosine triphosphate (ATP) concentration and loss of maintenance of kinase‐signalling cascades in primary cortical astrocytes during oxygen–glucose deprivation (OGD) as this may constitute an irreversible step that commits astrocytes to cell death. We report that the phosphorylation of Akt, ERK, JNK and p38 kinases, whose activities depend on serine, threonine and tyrosine phosphorylation, were all increased during OGD. All these phosphorylations were reduced to below detection limits when ATP levels were less than 10% of normal levels. Using ERK and Akt as representative examples, we show that this erasure is not irreversible as both ERK and Akt phosphorylations can be partially restored by addition of glucose under anoxic conditions. We further investigated whether OGD caused any change in phosphatase activity. The PP1/PP2A phosphatase inhibitors okadaic acid and caliculyn A, but not cyclosporine A, delayed the removal of ERK and Akt phosphorylation under OGD. By comparing the extent of phosphorylation increase under OGD and normoxic conditions, we calculate that phosphatase activity was increased by approximately 3.6‐fold during OGD. These data show that ATP levels control an important checkpoint in kinase function, and that ATP levels may need to be considered when studies of kinase function in relation to OGD are conducted.
Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines
Authors: Demeuse P. , Fragner P. , Leroy‐Noury C. , Mercier C. , Payen L. , Fardel O. , Couraud P. ‐O. , Roux F.
Published: July 2010

The blood–brain barrier (BBB) plays an important role in controlling the passage of molecules from blood to brain extracellular fluid. The multidrug efflux pump P‐glycoprotein (P‐gp) is highly expressed in the luminal membrane of brain endothelium and contributes to the formation of a functional barrier to lipid‐soluble drugs such as anticancer agents. The mdr1a P‐gp‐encoding gene is exclusively expressed in the rodent BBB. Primary cultures of rat brain endothelial cells and GP8.3 cells showed a dramatic decrease in mdr1a mRNA level and some expression of mdr1b mRNA. GPNT cells, derived from GP8.3 cells after transfection with a puromycin resistance gene, were chronically treated with 5 µg/mL puromycin, a P‐gp substrate. Compared with rat brain endothelial cells and GP8.3 cells, GPNT cells exhibited a very high level of expression of mdr1a mRNA together with a moderate level of mdr1b mRNA expression. Accordingly, P‐gp expression and activity were strongly increased. When GP8.3 and puromycin‐starved GPNT cells were treated with puromycin, mdr1a expression was selectively increased. High expression of mdr1a mRNA in GPNT cells may thus be related to the chronic treatment with puromycin. We conclude that GPNT cells may be used as a valuable ratin vitromodel for studying the regulation of mdr1a expression at the BBB level.
Determination of the oxidation states of manganese in brain, liver, and heart mitochondria
Authors: Gunter Thomas E. , Miller Lisa M. , Gavin Claire E. , Eliseev Roman , Salter Jason , Buntinas Linas , Alexandrov Andrei , Hammond Sean , Gunter Karlene K.
Published: July 2010

Excess brain manganese can produce toxicity with symptoms that resemble those of Parkinsonism and causes that remain elusive. Manganese accumulates in mitochondria, a major source of superoxide, which can oxidize Mn2+to the powerful oxidizing agent Mn3+. Oxidation of important cell components by Mn3+has been suggested as a cause of the toxic effects of manganese. Determining the oxidation states of intramitochondrial manganese could help to identify the dominant mechanism of manganese toxicity. Using X‐ray absorbance near edge structure (XANES) spectroscopy, we have characterized the oxidation state of manganese in mitochondria isolated from brain, liver, and heart over concentrations ranging from physiological to pathological. Results showed that (i) spectra from different model manganese complexes of the same oxidation state were similar to each other and different from those of other oxidation states and that the position of the absorption edge increases with oxidation state; (ii) spectra from intramitochondrial manganese in isolated brain, heart and liver mitochondria were virtually identical; and (iii) under these conditions intramitochondrial manganese exists primarily as a combination of Mn2+complexes. No evidence for Mn3+was detected in samples containing more than endogenous manganese levels, even after incubation under conditions promoting reactive oxygen species (ROS) production. While the presence of Mn3+complexes cannot be proven in the spectrum of endogenous mitochondrial manganese, the shape of this spectrum could suggest the presence of Mn3+near the limit of detection, probably as MnSOD.
Ceramide pathways modulate ethanol‐induced cell death in astrocytes
Authors: Pascual María , Valles Soraya L. , Renau‐Piqueras Jaime , Guerri Consuelo
Published: July 2010

We showed previously that alcohol exposure duringin vivobrain development induced astroglial damage and caused cell death. Because ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, we now investigate whether ethanol‐induced cell death in astrocytes is mediated by ceramide signalling pathways triggering apoptosis. Here we show that both ethanol and ceramide are able to induce apoptotic death in cultured astrocytes, in a dose‐dependent manner, and that C2‐ceramide addition potentiates the apoptotic effects of ethanol. Cell death induced by ethanol is associated with stimulation of neutral and acidic sphingomyelinase (SMase) and ceramide generation, as well as with activation of stress‐related kinases, c‐Jun N‐terminal kinase (JNK), p38 mitogen‐activated protein kinase (p38) and extracellular signal‐regulated kinase (ERK) pathways. We also provide evidence for the participation of JNK and p38 in ethanol‐induced cell death, because pharmacological inhibitors of these kinases largely prevent the apoptosis induced by ethanol or by ethanol and C2‐ceramide. Furthermore, we show that ethanol‐induced ERK activation triggers the stimulation of cyclo‐oxygenase‐2 (COX‐2) and the release of prostaglandin E2, and that blockade of the mitogen‐activated protein kinase kinase (MEK)/ERK pathway by PD98059 abolishes the up‐regulation of COX‐2 induced by ethanol plus ceramide, and decreases the ethanol‐induced apoptosis. These results strongly suggest that ethanol is able to stimulate the SMase–ceramide pathway, leading to the activation of signalling pathways implicated in cell death. These findings provide an insight into the mechanisms involved in ethanol‐induced astroglial cell death during brain development.
Oligomerization and neurotoxicity of the amyloid ADan peptide implicated in familial Danish dementia
Authors: Gibson Gillian , Gunasekera Nicola , Lee Maria , Lelyveld Victor , El‐Agnaf Omar M. A. , Wright Andrew , Austen Brian
Published: July 2010

Familial Danish dementia (FDD) is a rare neurodegenerative disorder, which is pathologically characterized by widespread cerebral amyloid angiopathy, parenchymal protein deposits and neurofibrillary degeneration. FDD is associated with mutation in the BRI gene. In FDD a decamer duplication between codons 265 and 266 in the 3′ region of the BRI gene originates an amyloid peptide named ADan, 11 residues longer than the wild‐type peptide produced from the normal BRI gene. ADan deposits have been found widely distributed in the CNS of FDD cases. The deposits of ADan are predominantly non‐fibrillar aggregates. We show here that synthetic ADan forms oligomers in vitro, seen by Tricine–PAGE and gel filtration, and higher aggregates, which are seen by atomic force spectroscopy and electron microscopy as carrot‐shaped objects that bunch together. Here we report that oligomeric ADan is toxic to neuronal cell lines. We find that the soluble non‐fibrillar oligomeric species of both the reduced and oxidized forms of ADan are toxic. These results support the idea that the non‐fibrillar soluble aggregates are the pathogenic species, which may play a central role in the pathogenesis of FDD, and imply that similar mechanism may also be involved in other neurodegenerative diseases associated with amyloid deposits.
Subunit composition and quantitative importance of GABAAreceptor subtypes in the cerebellum of mouse and rat
Authors: Pöltl Angelika , Hauer Birgit , Fuchs Karoline , Tretter Verena , Sieghart Werner
Published: July 2010

In cerebellum, 13 different GABAAreceptor subunits are expressed. The number of different receptor subtypes formed in this tissue, their subunit composition and their quantitative importance so far has not been determined. In the present study, immunodepletion by immunoaffinity chromatography, as well as immunoprecipitation and western blot analysis was performed using 13 different subunit‐specific antibodies to provide an overview on the subunit composition and abundance of GABAAreceptor subtypes in mouse and rat cerebellum. Results obtained indicate that α1βxγ2, α1α6βxγ2, α6βxγ2, α6βxδ and α1α6βxδ are the major GABAAreceptor subtypes present in the cerebellum. In addition, small amounts of α1βxδ receptors and a series of minor receptor subtypes containing α2, α3, α4, α5, γ1or γ3subunits are also present in the cerebellum. Whereas the abundance of α1α6βxγ2, α6βxδ and α1α6βxδ receptors is different in mouse and rat cerebellum, that of other receptors is quite similar in these tissues. Data obtained for the first time provide an overview on the GABAAreceptor subtypes present in the cerebellum and represent the basis for further studies investigating changes in receptor expression and composition under pathological conditions.
Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells
Authors: Fornai Francesco , Lenzi Paola , Gesi Marco , Soldani Paola , Ferrucci Michela , Lazzeri Gloria , Capobianco Loredana , Battaglia Giuseppe , De Blasi Antonio , Nicoletti Ferdinando , Paparelli Antonio
Published: July 2010

Mice treated with the psychostimulant methamphetamine (MA) showed the appearance of intracellular inclusions in the nucleus of medium sized striatal neurones and cytoplasm of neurones of the substantia nigra pars compacta but not in the frontal cortex. All inclusions contained ubiquitin, the ubiquitin activating enzyme (E1), the ubiquitin protein ligase (E3‐like, parkin), low and high molecular weight heat shock proteins (HSP 40 and HSP 70). Inclusions found in nigral neurones stained for α‐synuclein, a proteic hallmark of Lewy bodies that are frequently observed in Parkinson's disease and other degenerative disorders. However, differing from classic Lewy bodies, MA‐induced neuronal inclusions appeared as multilamellar bodies resembling autophagic granules. Methamphetamine reproduced this effect in cultured PC12 cells, which offered the advantage of a simple cellular model for the study of the molecular determinants of neuronal inclusions. PC12 inclusions, similar to those observed in nigral neurones, were exclusively localized in the cytoplasm and stained for α‐synuclein. Time‐dependent experiments showed that inclusions underwent a progressive fusion of the external membranes and developed an electrodense core. Inhibition of dopamine synthesis by α‐methyl‐p‐tyrosine (αMpT), or administering the antioxidant S‐apomorphine largely attenuated the formation of inclusions in PC12 cells exposed to MA. Inclusions were again observed when αMpT‐treated cells were loaded with l‐DOPA, which restored intracellular dopamine levels.
Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal neurodegeneration in rats: possible relevance for atypical parkinsonism in Guadeloupe
Authors: Champy Pierre , Höglinger Günter U. , Féger Jean , Gleye Christophe , Hocquemiller Reynald , Laurens Alain , Guérineau Vincent , Laprévote Olivier , Medja Fadia , Lombès Anne , Michel Patrick P. , Lannuzel Annie , Hirsch Etienne C. , Ruberg Merle
Published: July 2010

In Guadeloupe, epidemiological data have linked atypical parkinsonism with fruit and herbal teas from plants of the Annonaceae family, particularly Annona muricata. These plants contain a class of powerful, lipophilic complex I inhibitors, the annonaceous acetogenins. To determine the neurotoxic potential of these substances, we administered annonacin, the major acetogenin of A. muricata, to rats intravenously with Azlet osmotic minipumps (3.8 and 7.6 mg per kg per day for 28 days). Annonacin inhibited complex I in brain homogenates in a concentration‐dependent manner, and, when administered systemically, entered the brain parenchyma, where it was detected by matrix‐associated laser desorption ionization – time of flight mass spectrometry, and decreased brain ATP levels by 44%. In the absence of evident systemic toxicity, we observed neuropathological abnormalities in the basal ganglia and brainstem nuclei. Stereological cell counts showed significant loss of dopaminergic neurones in the substantia nigra (− 31.7%), and cholinergic (− 37.9%) and dopamine and cyclic AMP‐regulated phosphoprotein (DARPP‐32)‐immunoreactive GABAergic neurones (− 39.3%) in the striatum, accompanied by a significant increase in the number of astrocytes (35.4%) and microglial cells (73.4%). The distribution of the lesions was similar to that in patients with atypical parkinsonism. These data are compatible with the theory that annonaceous acetogenins, such as annonacin, might be implicated in the aetiology of Guadeloupean parkinsonism and support the hypothesis that some forms of parkinsonism might be induced by environmental toxins.
Increased expression and processing of caspase‐12 after traumatic brain injury in rats
Authors: Larner Stephen F. , Hayes Ronald L. , McKinsey Deborah M. , Pike Brian R. , Wang Kevin K. W.
Published: July 2010

Traumatic brain injury (TBI) disrupts tissue homeostasis resulting in pathological apoptotic activation. Recently, caspase‐12 was reported to be induced and activated by the unfolded protein response following excess endoplasmic reticulum (ER) stress. This study examined rat caspase‐12 expression using the controlled cortical impact TBI model. Immunoblots of fractionalized cell lysates found elevated caspase‐12 proform (∼60 kDa) and processed form (∼12 kDa), with peak induction observed within 24 h post‐injury in the cortex (418% and 503%, respectively). Hippocampus caspase‐12 proform induction peaked at 24 h post‐injury (641%), while processed form induction peaked at 6 h (620%). Semi‐quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis confirmed elevated caspase‐12 mRNA levels after TBI. Injury severity (1.0, 1.2 or 1.6 mm compression) was associated with increased caspase‐12 mRNA expression, peaking at 5 days in the cortex (657%, 651% and 1259%, respectively) and 6 h in the hippocampus (435%, 451% and 460%, respectively). Immunohistochemical analysis revealed caspase‐12 induction in neurons in both the cortex and hippocampus as well as in astrocytes at the contusion site. This is the first report of increased expression of caspase‐12 following TBI. Our results suggest that the caspase‐12‐mediated ER apoptotic pathway may play a role in rat TBI pathology independent of the receptor‐ or mitochondria‐mediated apoptotic pathways.
Developmental changes in expression and subcellular localization of the DNA repair glycosylase, MYH, in the rat brain
Authors: Lee Heung‐Man , Hu Zhaoyong , Ma Huaxian , Greeley George H. , Wang Cheng , Englander Ella W.
Published: July 2010

Mammalian cells employ a network of DNA repair pathways. DNA repair is required during development to ensure accuracy of DNA replication in the rapidly dividing embryonic cells and to maintain genomic integrity in the mature organism. An enzyme involved in repair of replication errors generated on either normal or oxidatively damaged DNA templates, is the mammalian ortholog of the Escherichia coli MutY DNA glycosylase (MYH). We show that levels of MYH isoform, detected at the E14 embryonic stage, decrease during embryonic and neonatal rat development, while new isoforms appear and gradually increase in the neonate and adult brain. The temporally declining expression of embryonic MYH resembles the pattern of proliferating cell nuclear antigen (PCNA) decline during this period. Immunohistochemical analyses of the embryonic brain show that cells staining for MYH initially coincide with cells staining for PCNA. At later stages PCNA declines, while MYH is detected primarily outside the nucleus. MutY‐like glycosylase activity for adenines misincorporated opposite oxidized guanines is detected in both, embryonic and adult brain extracts. Together, these findings suggest that in proliferating embryonic cells, MYH might be primarily involved in post replicative repair of nuclear DNA, whereas in post mitotic neurons, in the repair of mitochondrial DNA.
Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies
Authors: Trimmer Patricia A. , Borland M. Kathleen , Keeney Paula M. , Bennett James P. , Parker W. Davis
Published: July 2010

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or α‐synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long‐term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to α‐synuclein, ubiquitin, parkin, synphilin‐1, neurofilament, β‐tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.
An ALS mouse model with a permeable blood–brain barrier benefits from systemic cyclosporine A treatment
Authors: Kirkinezos Ilias G. , Hernandez Dayami , Bradley Walter G. , Moraes Carlos T.
Published: July 2010

To test potentially beneficial drugs to amyotrophic lateral sclerosis (ALS), we created an ALS mouse model with a permeable blood–brain barrier, by crossing the G93A‐SOD1 transgenic mouse with a multiple drug resistance type 1a/b (mdr1a/b) gene knockout mouse. To validate the model, we administered cyclosporine A intraperitoneally to the mice. Cyclosporine A accumulated in the brain and spinal cord of this mouse model, whereas it was unable to penetrate the CNS of mdr1a/b wild‐type animals. Systemic administration of cyclosporine A extended the life of the double‐mutant male mice by approximately 12%. Surprisingly, the effect was more robust in male mice and only marginal in female mice. These results demonstrate the usefulness of this combined mouse model for the testing of potentially therapeutic drugs and support the role of mitochondrial‐mediated apoptosis in the pathway to motor neuron death in SOD1‐associated ALS.
Neuron‐glia communication: metallothionein expression is specifically up‐regulated by astrocytes in response to neuronal injury
Authors: Chung Roger S. , Adlard Paul A. , Dittmann Justin , Vickers James C. , Chuah Meng Inn , West Adrian K.
Published: July 2010

Recent data suggests that metallothioneins (MTs) are major neuroprotective proteins within the CNS. In this regard, we have recently demonstrated that MT‐IIA (the major human MT‐I/‐II isoform) promotes neural recovery following focal cortical brain injury. To further investigate the role of MTs in cortical brain injury, MT‐I/‐II expression was examined in several different experimental models of cortical neuron injury. While MT‐I/‐II immunoreactivity was not detectable in the uninjured rat neocortex, by 4 days, following a focal cortical brain injury, MT‐I/‐II was found in astrocytes aligned along the injury site. At latter time points, astrocytes, at a distance up to several hundred microns from the original injury tract, were MT‐I/‐II immunoreactive. Induced MT‐I/‐II was found both within the cell body and processes. Using a cortical neuron/astrocyte co‐culture model, we observed a similar MT‐I/‐II response following in vitro injury. Intriguingly, scratch wound injury in pure astrocyte cultures resulted in no change in MT‐I/‐II expression. This suggests that MT induction was specifically elicited by neuronal injury. Based upon recent reports indicating that MT‐I/‐II are major neuroprotective proteins within the brain, our results provide further evidence that MT‐I/‐II plays an important role in the cellular response to neuronal injury.
Brain capillary endothelium and choroid plexus epithelium regulate transport of transferrin‐bound and free iron into the rat brain
Authors: Deane Rashid , Zheng Wei , Zlokovic Berislav V.
Published: July 2010

Iron transport into the CNS is still not completely understood. Using a brain perfusion technique in rats, we have shown a significant brain capillary uptake of circulating transferrin (Tf)‐bound and free 59Fe (1 nm) at rates of 136 ± 26 and 182 ± 23 μL/g/min, respectively, while their respective transport rates into brain parenchyma were 1.68 ± 0.56 and 1.52 ± 0.48 μL/g/min. Regional Tf receptor density (Bmax) in brain endothelium determined with 125I‐holo‐Tf correlated well with 59Fe‐Tf regional brain uptake rates reflecting significant vascular association of iron. Tf‐bound and free circulating 59Fe were sequestered by the choroid plexus and transported into the CSF at low rates of 0.17 ± 0.01 and 0.09 ± 0.02 μL/min/g, respectively, consistent with a 10‐fold brain‐CSF concentration gradient for 59Fe, Tf‐bound or free. We conclude that transport of circulating Tf‐bound and free iron could be equally important for its delivery to the CNS. Moreover, data suggest that entry of Tf‐bound and free iron into the CNS is determined by (i) its initial sequestration by brain capillaries and choroid plexus, and (ii) subsequent controlled and slow release from vascular structures into brain interstitial fluid and CSF.
Acetylcholine turnover rates in rat brain regions during cocaine self‐administration
Authors: Smith James E. , Vaughn Tina C. , Co Conchita
Published: July 2010

The involvement of cholinergic neurons in the brain processes underlying reinforcement has been recently demonstrated. This experiment assessed the potential role of cholinergic neurons in cocaine reinforcement by measuring the turnover rates of acetylcholine in brain regions of rats self‐administering cocaine and in yoked cocaine and yoked vehicle‐infused controls. The activity of cholinergic innervations of and/or interneurons in the olfactory tubercle, caudate putamen, diagonal band‐pre‐optic region, ventral pallidum, lateral and medial hypothalamus, hippocampus, ventral tegmental area and visual cortices reflected by the turnover rates of acetylcholine were significantly altered in rats self‐administering cocaine compared to yoked cocaine infused controls. These changes implicate the involvement of cholinergic neurons with cell bodies in the diagonal band‐pre‐optic region, the medial septum and several brainstem nuclei and interneurons in the caudate‐putamen and ventral pallidum in the processes underlying cocaine self‐administration. The identified cholinergic neuronal systems may have a broader role in the brain processes for natural reinforcers (i.e. food, water, etc.) since drugs of abuse are believed to produce reinforcing effects through these systems.
Noradrenaline transporter blockers raise extracellular dopamine in medial prefrontal but not parietal and occipital cortex: differences with mianserin and clozapine
Authors: Valentini V. , Frau R. , Di Chiara G.
Published: July 2010

This study compared the interaction between noradrenaline (NA) and dopamine (DA) mechanisms in the prefrontal (PFCX) and in the parietal (ParCX) and occipital (OccCX) cortex. The effect of reboxetine and desipramine, two NA transporter blockers, of mianserin, an antagonist of α2and 5‐HT2 receptors, and of clozapine, an atypical antipsychotic, on dialysate DA in the medial PFCX, ParCX and OccCX was studied. We also assessed the influence of a prior 6‐hydroxydopamine (6‐OHDA) lesion of the dorsal noradrenergic bundle (DNAB) on the effect of reboxetine and clozapine on dialysate DA in the PFCX and ParCX. Systemic administration of reboxetine and desipramine dose‐dependently increased dialysate DA in the PFCX but not in the ParCX and OccCX. In contrast, mianserin and clozapine raised dialysate DA in the ParCX and OccCX to an even larger extent than in the PFCX. 6‐OHDA lesions of DNAB abolished the increase of dialysate DA elicited by reboxetine in the PFCX and by clozapine both in the PFCX and in the ParCX. It is concluded that, although PFCX and ParCX/OccCX share the presence of a strong control of DA transmission by NA through α2receptors, they differ in the extent to which DA is cleared from the extracellular compartment by uptake through the NA transporter. This process, although extensive in the PFCX, appears insignificant in the ParCX and OccCX, probably as a result of the higher ratio of NA to DA resulting in exclusion of DA from NA transporter.
Alpha2‐adrenoceptor mediated co‐release of dopamine and noradrenaline from noradrenergic neurons in the cerebral cortex
Authors: Devoto Paola , Flore Giovanna , Pira Luigi , Longu Giorgio , Gessa Gian Luigi
Published: July 2010

Previous results suggest that extracellular dopamine (DA) in the rat cerebral cortex originates from dopaminergic and noradrenergic terminals. To further clarify this issue, dialysate DA, dihydroxyphenylacetic acid (DOPAC) and noradrenaline (NA) were measured both in the medial prefrontal cortex (mPFC) and in the occipital cortex (OCC), with dense and scarce dopaminergic projections, respectively. Moreover, the effect of the α2‐adrenoceptor antagonist RS 79948 and the D2‐receptor antagonist haloperidol on extracellular DA, DOPAC and NA was investigated. Extracellular DA and DOPAC concentrations in the OCC were 43% and 9%, respectively, those in the mPFC. Haloperidol (0.1 mg/kg i.p.) increased DA and DOPAC (by 35% and 150%, respectively) in the mPFC, but was ineffective in the OCC. In contrast, RS 79948 (1.5 mg/kg i.p.) increased NA, DA and DOPAC, both in the mPFC (by approximately 50%, 60% and 130%, respectively) and the OCC (by approximately 50%, 80% and 200%, respectively). Locally perfused, the DA transporter blocker GBR 12909 (10 µm) was ineffective in either cortex, whereas desipramine (DMI, 100 µm) markedly increased extracellular NA and DA in both cortices. The weak haloperidol effect on DA efflux was not enhanced after DA‐ and NA‐transporter blockade, whereas after DMI, RS 79948 markedly increased extracellular NA, and especially DA and DOPAC in both cortices. The results support the hypothesis that most extracellular DA in the cortex is co‐released with NA from noradrenergic terminals, such co‐release being primarily controlled by α2‐adrenoceptors.
In vivo efflux of serotonin in the dorsal raphe nucleus of 5‐HT1Areceptor knockout mice
Authors: Bortolozzi Analía , Amargós‐Bosch Mercè , Toth Miklos , Artigas Francesc , Adell Albert
Published: July 2010

In the dorsal raphe nucleus (DR), extracellular serotonin (5‐HT) regulates serotonergic transmission through 5‐HT1Aautoreceptors. In this work we usedin vivomicrodialysis to examine the effects of stressful and pharmacological challenges on DR 5‐HT efflux in 5‐HT1Areceptor knockout (5‐HT1A–/–) mice and their wild‐type counterparts (5‐HT1A+/+). Baseline 5‐HT concentrations did not differ between both lines of mice, which is consistent with a lack of tonic control of 5‐HT1Aautoreceptors on DR 5‐HT release. (R)‐(+)‐8‐Hydroxy‐2‐(di‐n‐propylamino)tetralin hydrobromide (8‐OH‐DPAT, 0.5 mg/kg) reduced 5‐HT levels to 30% of basal values in 5‐HT1A+/+ mice, but not in 5‐HT1A–/– mice. The selective 5‐HT1Breceptor agonist 1,4‐dihydro‐3‐(1,2,3,6‐tetrahydro‐4‐pyridinyl)‐5H‐pyrrolo[3,2‐b]pyridin‐5‐one dihydrochloride (CP 93129, 300 µm) reduced dialysate 5‐HT to the same extent (30–40% of baseline) in the two genotypes, which suggests a lack of compensatory changes in 5‐HT1Breceptors in the DR of such mutant mice. Both a saline injection and handling for 3 min increased DR dialysate 5‐HT in mutants, but not in 5‐HT1A+/+ mice. Fluoxetine (5 and 20 mg/kg) elevated 5‐HT in a dose‐dependent manner in both genotypes. However, this effect was markedly more pronounced in the 5‐HT1A–/– mice. The increased responsiveness of the extracellular 5‐HT in the DR of 5‐HT1Areceptor knockout mice reflects a lack of the autoinhibitory control exerted by 5‐HT1Aautoreceptors.
Dopamine neurons in culture express VGLUT2 explaining their capacity to release glutamate at synapses in addition to dopamine
Authors: Dal Bo Gregory , St‐Gelais Fannie , Danik Marc , Williams Sylvain , Cotton Mathieu , Trudeau Louis‐Eric
Published: July 2010

Dopamine neurons have been suggested to use glutamate as a cotransmitter. To identify the basis of such a phenotype, we have examined the expression of the three recently identified vesicular glutamate transporters (VGLUT1‐3) in postnatal rat dopamine neurons in culture. We found that the majority of isolated dopamine neurons express VGLUT2, but not VGLUT1 or 3. In comparison, serotonin neurons express only VGLUT3. Single‐cell RT‐PCR experiments confirmed the presence of VGLUT2 mRNA in dopamine neurons. Arguing for phenotypic heterogeneity among axon terminals, we find that only a proportion of terminals established by dopamine neurons are VGLUT2‐positive. Taken together, our results provide a basis for the ability of dopamine neurons to release glutamate as a cotransmitter. A detailed analysis of the conditions under which DA neurons gain or loose a glutamatergic phenotype may provide novel insight into pathophysiological processes that underlie diseases such as schizophrenia, Parkinson's disease and drug dependence.
Progressive sensorimotor impairment is not associated with reduced dopamine and high energy phosphate donors in a model of ataxia‐telangiectasia
Authors: Mount Howard T. J. , Martel Jean‐Claude , Fluit Paul , Wu Yingji , Gallo‐Hendrikx Eleanor , Cosi Cristina , Marien Marc R.
Published: July 2010

Ataxia‐telangiectasia (A‐T) is a genetic disease, associated with progressive motor impairment and a lack of functional ATM protein. It has been reported that immunoreactive tyrosine hydroxylase and dopamine transporter are reduced in anAtm–/–mouse model of A‐T. These observations led to a hypothesis that A‐T is associated with loss of nigrostriatal dopamine and prompted the launch of clinical trials to evaluate a therapeutic utility of the anti‐parkinsonian drug,l‐DOPA. To test for dopamine depletion more directly, we measured regional levels of monoamines and their metabolites in theAtm–/–mouse brain. We also measured levels of NAD+, a cofactor for dopamine biosynthesis and substrate of the DNA damage surveillance enzyme, poly(ADP‐ribose) polymerase (PARP). Constitutive activation of PARP has been posited to cause NAD+depletion. We observed no reduction in monoamine transmitters and no depletion of NAD+, or other high energy phosphate donors in the adultAtm–/–cerebellum, striatum, or ventral mesencephalon. However, our studies did reveal a progressive sensorimotor impairment inAtm–/–mice that may serve as a relevant proxy for progressive neurological impairment in the human disease. Our results call into question the involvement of dopamine in A‐T and the therapeutic strategy of enhancing dopaminergic function withl‐DOPA.