Prakash Gorroochurn Rudy Guerra Aaron Hamvas Sandy Hasstedt Simon Heath David Hinds Peter Holmans Steve Horvath Hsin-Ju Hsieh Mark Jobling Toby Johnson Tatiana Karafet Xiayi Ke Alison Klein Kathy Klos Michael Knapp Peter De Knijff Peter Kraft Aldi Kraja Karl Langefeld Mingyao Li Wentian Li Kung-Yee Liang Cecilia Lindgren Wenlei Liu Gregory Livshits Kathryn Lunetta Nancy Mandell Franz Manni Mary Marazita Jonathan Marchini Paul McKeigue Chantal Mérette Andrew Morris Valentina Moskvina Bertram Müller-Myhsok Ben Neale Omer Njajou Bernard North Michael Nothnagel Dale Nyholt Lyle Palmer Chi Pui Pang Palle Pedersen Bruce Pennington Thomas Perls François Pompanon Shaun Purcell Chris Amos Mariza de Andrade Nadine Andrieu Joan Bailey-Wilson Mathew Barber Michael Barmada Christopher Bartlett Anne Bassett Mark Beasley Tim Becker Elise Belle Joanna Biernacka Matthew Brown Shelley Bull Volker Burkart Steve Buyske Christopher Carlson Denise Carvalho-Silva Juliet Chapman Gary Chase Nilanjan Chatterjee Andrew Clark Andrew Collins Karen Conneely Heather Cordell Elizabeth Corder Robert Culverhouse Geesje M. Dallinga-Thie Francisco De La Vega Florence Demenais Anita DeStefano Giovanni Destro-Bisol Frank Dudbridge Isabelle Dupanloup Josee Dupuis Robert Elston Mary Emond Michael S. Esplin Eleanor Feingold Mary Feitosa Rui Feng Jose Fernandez Mark Fife Stephen Finch Sheila Fisher Guimin Gao Alisa Goldstein Harald Göring

M. Baehr G. Bagdy D. Bagnard C. Bailey G. Bakalkin N. L. Banik M. Bani-Yaghoub M. J. Bannon G. Barcelo-Coblijn S. Barger K. Barnham P. H. Barry R. P. Bazinet C. F. Bearer M. Beaulieu C.M. Becker J. Beckman D. Begley C. Benishin J. Bennett, Jr D. Ben-Shachar L. Berliocchi M. Bernaudin E. Berra I. Bezprozvanny U. Bickel T. Biederer L. Binder E. Blalock T. Blamk J. P. Blass R. Blitzer J. K. Blusztajn O. Bogler D. Boison J. P. Bolaños G. Bonanno E. R. Bongarzone V. Bonifati D. Borchelt R. Bordet R. Borges J. Borjigin C. Borner G. Bouche M. G. Boutelle P. Bovolenta W. D. Bowen N. Bowery M. T. Bowser R. Bowser J. Bowyer J. H. Brandstdtter R. Brandt E. G. Bremer J. P. Bressler S. Brimijoin C. Brodie S. Broeer C. Brosnan N. Brustovetsky R. Bujdoso R. W. Burgess A. L. Burlingame D. A. Butterfield

A. Baba J. Badaut S. Baekkeskov E. Bahraoui J. Balsinde M. J. Bannon R. A. Barker K. Barnham A. Barzilai E. C. Beattie M. Beinfeld E. Bellefroid R. Bendayan G. Bennett P. Bernardi B. Berra D. Bertrand T. Binz A. Bjorklund E. Blalock F. Blanchard N. Blau B. Bloch O. Bogler J. Bolaños G. Bolger P. Bonaventure V. Bonetto D. Boraschi E. Borrelli R. Bowser J. Boyes M. Brenner J. Bright W. S. Brimijoin F. Bronfman J. M. Bronstein P. Brophy R. Brosh E. P. M. Brouillet A. Brown M. D. Browning B. Brune R. Bruzzone N. J. Buckley G. Burdge D. A. Butterfield F. Bymaster

Amyloid β peptides generate oxidative stress in hippocampal astrocytes through a mechanism sensitive to inhibitors of the NADPH oxidase [diphenylene iodonium (DPI) and apocynin]. Seeking evidence for the expression and function of the enzyme in primary hippocampal astrocytes, we confirmed the expression of the subunits of the phagocyte NADPH oxidase by Western blot analysis and by immunofluorescence and coexpression with the astrocyte-specific marker glial fibrillary acidic protein both in cultures and in vivo. Functional assays using lucigenin luminescence, dihydroethidine, or dicarboxyfluorescein fluorescence to measure the production of reactive oxygen species (ROS) demonstrated DPI and apocynin-sensitive ROS generation in response to the phorbol ester PMA and to raised [Ca2+]c after application of ionomycin or P2u receptor activation. Stimulation by PMA but not Ca2+ was inhibited by the protein kinase C (PKC) inhibitors staurosporine and hispidin. Responses were absent in transgenic mice lacking gp91phox. Expression of gp91phox and p67phox was increased in reactive astrocytes, which showed increased rates of both resting and stimulated ROS generation. NADPH oxidase activity was modulated by intracellular pH, suppressed by intracellular alkalinization, and enhanced by acidification. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone suppressed basal ROS generation but markedly increased PMA-stimulated ROS generation. This was independent of mitochondrial ROS production, because it was unaffected by mitochondrial depolarization with rotenone and oligomycin. Thus, the NADPH oxidase is expressed in astrocytes and is functional, activated by PKC and intracellular calcium, modulated by pHi, and upregulated by astrocyte activation. The astrocytic NADPH oxidase is likely to play important roles in CNS physiology and pathology.

We looked at the possible interactions between astrocytes and neurones during reperfusion using an in vitro model of ischaemia-reperfusion injury, as a controlled environment that lends itself easily to manipulation of the numerous variables involved in such an insult. We constructed a chamber in which O-2 can be lowered to a concentration of 1 mu M and developed a primary cortical neuronal culture that is 99% pure and can survive to at least 10 days in vitro. We also established a novel system for the co-culture of astrocytes and neurones in order to study the communication between these cells in a manner that allows the complete separation of one cell type from another. Neurone cultures showed profound cell death following an ischaemic period of only 15 min. We co-cultured neurones that had been subjected to a 15-min ischaemic insult with either non-insulted astrocytes or astrocyte-conditioned medium during the reperfusion stage. Both astrocytes and astrocyte-conditioned medium enhanced neuronal survival. Our data also suggest that astrocyte-sourced neuronal glutathione synthesis may play a role in preventing neuronal death.

In Alzheimer's disease, amyloid beta (Abeta) peptide is deposited in neuritic plaques in the brain. The Abeta peptide 1-42 or the fragment 25-35 are neurotoxic. We here review our recent explorations of the mechanisms of Abeta toxicity in hippocampal cultures. Abeta had no effect on intracellular calcium in neurons but caused striking changes in nearby astrocytes. The [Ca(2+)](c) signals started approximately 5-15 min after Abeta application and consisted of sporadic [Ca(2+)](c) pulses. These were entirely dependent on extracellular Ca(2+), independent of ER Ca(2+) stores and resulted from Ca(2+) influx, probably through Abeta-induced membrane channels. The Ca(2+) signals were closely associated with transient, episodic acidification which may reflect displacement of protons from binding sites or Ca(2+)/2H(+) exchange. Abeta caused an increased rate of generation of reactive oxygen species (ROS), also seen in astrocytes and not in neurons. The increased ROS generation was blocked by inhibitors of the NADPH oxidase, strongly suggesting that this enzyme, normally associated with immune cells, is expressed in astrocytes. ROS generation was also Ca(2+)-dependent, suggesting that Abeta activation of the enzyme may be secondary to the increase in [Ca(2+)](c). Abeta caused delayed neuronal death despite the fact that all responses were seen only in astrocytes. Neurons could not be protected by glutamate receptor antagonists, but were rescued by inhibition of the NADPH oxidase, by antioxidants and by increasing glutathione. These data suggest that Abeta causes Ca(2+)-dependent oxidative stress by activating an astrocytic NADPH oxidase, and that neuronal death follows through a failure of antioxidant support.

beta-Amyloid (betaA) peptide is strongly implicated in the neurodegeneration underlying Alzheimer's disease, but the mechanisms of neurotoxicity remain controversial. This study establishes a central role for oxidative stress by the activation of NADPH oxidase in astrocytes as the cause of betaA-induced neuronal death. betaA causes a loss of mitochondrial potential in astrocytes but not in neurons. The mitochondrial response consists of Ca2+-dependent transient depolarizations superimposed on a slow collapse of potential. The slow response is both prevented by antioxidants and, remarkably, reversed by provision of glutamate and other mitochondrial substrates to complexes I and II. These findings suggest that the depolarization reflects oxidative damage to metabolic pathways upstream of mitochondrial respiration. Inhibition of NADPH oxidase by diphenylene iodonium or 4-hydroxy-3-methoxy-acetophenone blocks betaA-induced reactive oxygen species generation, prevents the mitochondrial depolarization, prevents betaA-induced glutathione depletion in both neurons and astrocytes, and protects neurons from cell death, placing the astrocyte NADPH oxidase as a primary target of betaA-induced neurodegeneration.

H. S. Bachelard P. Bagnoli G. Bakalkin M. Bakhiet D. Balfour G. Banhegyi E. Barker S. C. Barnett M. H. Baslow G. Battaglia M. Baudry K. Bauer A. Baumann T. A. Bayer N. Bazan R. P. Bazinet D. Begley D. Berg J. Bergquist L. Berliner L. Berliocchi M. Bernaudin G. Bewick U. Bickel L. Binder A. Bindoli Z. K. Binienda N. P. Birch O. Bizzozero I. Blair J. Blasi J. P. Blass W. Blenau B. Bloch T. Boettcher O. Bogler K. M. Boje K. Borges R. Borges F. Bosetti R. M. Botting N. Brakch R. Brambilla T. A. Branchek J. H. Brandstätter D. W. Brann J. T. Brenna G. J. Brewer J. J. Bright R. Brinton S. Broeer K. J. Brooks J. R. Brorson G. Brown I. R. Brown T. Brown R. Browning A. J. Bruce-Keller M. Bruess B. Brune J.-F. Brunet N. J. Buckley L. Buée R. W. Burgess J. Burke A. Bush D. A. Butterfield J. H. Byrne

The hph-1 mice have defective tetrahydrobiopterin biosynthesis and share many neurochemical similarities with l-dopa-responsive dystonia (DRD) in humans. In both, there are deficiencies in GTP cyclohydrolase I and low brain levels of dopamine (DA). Striatal tyrosine hydroxylase (TH) levels are decreased while the number of DA neurones in substantia nigra (SN) appears normal. The hph-1 mouse is therefore a useful model in which to investigate the biochemical mechanisms underlying dystonia in DRD. In the present study, the density of striatal DA terminals and DA receptors and the expression of D-1, D-2, and D-3 receptors, preproenkephalin (PPE-A), preprotachykinin (PPT), and nitric oxide synthase (NOS) mRNAs in the striatum and nucleus accumbens and nigral TH mRNA expression were examined. Striatal DA terminal density as judged by specific [3H]mazindol binding was not altered while the levels of TH mRNA were elevated in the SN of hph-1 mice compared to control (C57BL) mice. Total and subregional analysis of the striatum and nucleus accumbens showed that D-2 receptor ([3H]spiperone) binding density was increased while D-1 receptor ([3H]SCH 23390) and D-3 receptor ([3H]7-OH-DPAT) binding density was not altered. In the striatum and nucleus accumbens, expression of PPT mRNA was elevated but PPE-A mRNA, D-1, D-2 receptor, and nNOS mRNA were not changed in hph-1 mice compared to controls. These findings suggest that an imbalance between the direct strionigral and indirect striopallidal output pathways may be relevant to the genesis of DRD. However, the pattern of changes observed is not that expected as a result of striatal dopamine deficiency and suggests that other effects of GTP cyclohydrolase I deficiency may be involved.

Although the accumulation of the neurotoxic peptide β amyloid (βA) in the CNS is a hallmark of Alzheimer's disease, the mechanism of βA neurotoxicity remains controversial. In cultures of mixed neurons and astrocytes, we found that both the full-length peptide βA (1–42) and the neurotoxic fragment (25–35) caused sporadic cytoplasmic calcium [intracellular calcium ([Ca 2 + ] c )] signals in astrocytes that continued for hours, whereas adjacent neurons were completely unaffected. Nevertheless, after 24 hr, although astrocyte cell death was marginally increased, ∼50% of the neurons had died. The [Ca 2 + ] c signal was entirely dependent on Ca 2 + influx and was blocked by zinc and by clioquinol, a heavy-metal chelator that is neuroprotective in models of Alzheimer's disease. Neuronal death was associated with Ca 2 + -dependent glutathione depletion in both astrocytes and neurons. Thus, astrocytes appear to be the primary target of βA, whereas the neurotoxicity reflects the neuronal dependence on astrocytes for antioxidant support.

Mitochondrial cytochrome oxidase is competitively and reversibly inhibited by inhibitors that bind to ferrous heme, such as carbon monoxide and nitric oxide. In the case of nitric oxide, nanomolar levels inhibit cytochrome oxidase by competing with oxygen at the enzyme's heme–copper active site. This raises the Km for cellular respiration into the physiological range. This effect is readily reversible and may be a physiological control mechanism. Here we show that a number of in vitro and in vivo conditions result in an irreversible increase in the oxygen Km. These include: treatment of the purified enzyme with peroxynitrite or high (μM) levels of nitric oxide; treatment of the endothelial-derived cell line, b.End5, with NO; activation of astrocytes by cytokines; reperfusion injury in the gerbil brain. Studies of cell respiration that fail to vary the oxygen concentration systematically are therefore likely to significantly underestimate the degree of irreversible damage to cytochrome oxidase.

Objectives: Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation. by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. Methods: We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. Results: GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent. eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS-GFP fusion protein were significantly increased following GTPCH gene transfer. Conclusions: These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be it rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states. (C) 2002 Elsevier Science B.V. All rights reserved.

Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post-mortem Alzheimer's disease brain. beta-Amyloid is strongly implicated in Alzheimer's pathology and can be formed intracellularly in neurones. We have investigated the possibility that beta-amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the beta-amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimer's brain. Mitochondrial respiration, electron transport chain complex activities, alpha-ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. Beta-amyloid caused a significant reduction in state 3 and state 4 mitochondrial respiration that was further diminished by the addition of nitric oxide. Cytochrome oxidase, alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by beta-amyloid. The K(m) of cytochrome oxidase for reduced cytochrome c was raised by beta-amyloid. We conclude that beta-amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimer's disease.

β-Amyloid deposition and compromised energy metabolism both occur in vulnerable brain regions in Alzheimer's disease. It is not known whether β-amyloid is the cause of impairment of energy metabolism, nor whether impaired energy metabolism is specific to neurons. Our results, using primary neuronal cultures, show that 24-h incubation with Aβ25–35 caused a generalized decrease in the specific activity of mitochondrial enzymes per milligram of cellular protein, induced mitochondrial swelling, and decreased total mitochondrial number. Incubation with Aβ25–35 decreased ATP concentration to 58% of control in neurons and 71% of control in astrocytes. Levels of reduced glutathione were also lowered by Aβ25–35 in both neurons (from 5.1 to 2.9 nmol/mg protein) and astrocytes (from 25.2 to 14.9 nmol/mg protein). We conclude that 24-h treatment with extracellular Aβ25–35 causes mitochondrial dysfunction in both astrocytes and neurons, the latter being more seriously affected. In astrocytes mitochondrial impairment was confined to complex I inhibition, whereas in neurons a generalized loss of mitochondria was seen.