Assessing neurodegenerative phenotypes in Drosophila dopaminergic neurons by climbing assays and whole brain immunostaining.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's (5). Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human α-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function (2). Accordingly, this model has been successfully used to identify potential therapeutic targets in PD (8). Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line (3, 10).

Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Oxidative stress has been associated with the etiology of both sporadic and monogenic forms of PD. The transcription factor Nrf2, a conserved global regulator of cellular antioxidant responses, has been implicated in neuroprotection against PD pathology. However, direct evidence that upregulation of the Nrf2 pathway is sufficient to confer neuroprotection in genetic models of PD is lacking. Expression of the PD-linked gene encoding α-synuclein in dopaminergic neurons of Drosophila results in decreased locomotor activity and selective neuron loss in a progressive age-dependent manner, providing a genetically accessible model of PD. Here we show that upregulation of the Nrf2 pathway by overexpressing Nrf2 or its DNA-binding dimerization partner, Maf-S, restores the locomotor activity of α-synuclein-expressing flies. Similar benefits are observed upon RNA-interference-mediated downregulation of the prime Nrf2 inhibitor, Keap1, as well as in conditions of keap1 heterozygosity. Consistently, the α-synuclein-induced dopaminergic neuron loss is suppressed by Maf-S overexpression or keap1 heterozygosity. Our data validate the sustained upregulation of the Nrf2 pathway as a neuroprotective strategy against PD. This model provides a genetically accessible in vivo system in which to evaluate the potential of additional Nrf2 pathway components and regulators as therapeutic targets.

Pin1 promotes cell death in NGF-dependent neurons through a mechanism requiring c-Jun activity.

Developing neurons deprived of trophic support undergo apoptosis mediated by activation of c-Jun N-terminal kinases (JNK) and c-Jun, induction of the Bcl-2 homology 3-only protein Bim(EL), Bax-dependent loss of mitochondrial cytochrome c, and caspase activation. However, the mechanisms that regulate each of these events are only partially understood. Here we show that the prolyl isomerase Pin1 functions as a positive regulator of neuronal death through a c-Jun-dependent mechanism. Ectopic Pin1 promoted caspase-dependent death of NGF-maintained neurons that was associated with an accumulation of Ser(63)-phosphorylated c-Jun in neuronal nuclei and was partially dependent on Bax. Downregulating Pin1 prior to NGF withdrawal suppressed the accumulation of phosphorylated c-Jun, inhibited the release of cytochrome c, and significantly delayed cell death. Pin1 knockdown inhibited NGF deprivation-induced death to a similar extent in Bim (+/+) and Bim (-/-) neurons. The protective effect of Pin1 knockdown was significantly greater than that caused by loss of Bim and nearly identical to that caused by a dominant negative form of c-Jun. Finally, cell death induced by ectopic Pin1 was largely blocked by expression of dominant negative c-Jun. These results suggest a novel mechanism by which Pin1 promotes cell death involving activation of c-Jun.

Targeting hypoxia-inducible factor (HIF) as a therapeutic strategy for CNS disorders.

Hypoxia occurs when oxygen availability drops below the levels necessary to maintain normal rates of metabolism. Because of its high metabolic activity, the brain is highly sensitive to hypoxia. Severe or prolonged oxygen deprivation in the brain contributes to the damage associated with stroke and a variety of other neuronal disorders. Conversely, the extreme hypoxic environment found in the core of many brain tumors supports the growth of the tumor and the survival of tumor cells. Normal cells exposed to transient or moderate hypoxia are generally able to adapt to the hypoxic conditions largely through activation of the hypoxia-inducible transcription factor HIF. HIF-regulated genes encode proteins involved in energy metabolism, cell survival, erythropoiesis, angiogenesis, and vasomotor regulation. In many instances of hypoxia or hypoxia and ischemia, the induction of HIF target genes may be beneficial. When these same insults occur in tissues that are normally poorly vascularized, such as the retina and the core of solid tumors, induction of the same HIF target genes can promote disease. Major new insights into the molecular mechanisms that regulate the oxygen-sensitivity of HIF, and in the development of compounds with which to manipulate HIF activity, are forcing serious consideration of HIF as a therapeutic target for diverse CNS disorders associated with hypoxia.

Control of cytochrome c oxidase activity by nitric oxide.

Over the past decade it was discovered that, over-and-above multiple regulatory functions, nitric oxide (NO) is responsible for the modulation of cell respiration by inhibiting cytochrome c oxidase (CcOX). As assessed at different integration levels (from the purified enzyme in detergent solution to intact cells), CcOX can react with NO following two alternative reaction pathways, both leading to an effective, fully reversible inhibition of respiration. A crucial finding is that the rate of electron flux through the respiratory chain controls the mechanism of inhibition by NO, leading to either a "nitrosyl" or a "nitrite" derivative. The two mechanisms can be discriminated on the basis of the differential photosensitivity of the inhibited state. Of relevance to cell pathophysiology, the pathway involving the nitrite derivative leads to oxidative degradation of NO, thereby protecting the cell from NO toxicity. The aim of this work is to review the information available on these two mechanisms of inhibition of respiration.

Control of mitochondrial respiration by NO*, effects of low oxygen and respiratory state.

Nitric oxide (NO.) inhibits mitochondrial respiration by binding to the binuclear heme a3/CuB center in cytochrome c oxidase. However, the significance of this reaction at physiological O2 levels (5-10 microM) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O2], and [NO.] were measured simultaneously at physiological O2 levels with constant O2 delivery, to model in vivo respiratory dynamics. Under these conditions NO. inhibited mitochondrial respiration with an IC50 of 0.14 +/- 0.01 microm in state 3 versus 0.31 +/- 0.04 microM in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO. is due to greater control over respiration by an NO.-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO. in vivo and its implications for the control of vessel-parenchymal O2 gradients.

Reversible inhibition of cytochrome c oxidase by peroxynitrite proceeds through ascorbate-dependent generation of nitric oxide.

Reversible inhibition of cytochrome c oxidase (CcOX) by nitric oxide (NO*) has potential physiological roles in the regulation of mitochondrial respiration, redox signaling, and apoptosis. However peroxynitrite (ONOO-), an oxidant formed from the reaction of NO* and superoxide, appears mostly detrimental to cell function. This occurs both through direct oxidant reactions and by decreasing the availability of NO* for interacting with CcOX. When isolated CcOX respires with ascorbate as a reducing substrate, the conversion of ONOO- to NO* is observed. It is not known whether this can be ascribed to a direct interaction of the enzyme with ONOO-. In this investigation, the role of ascorbate in this system was examined using polarographic methods to measure NO* production and CcOX activity simultaneously in both the purified enzyme and isolated mitochondria. It was found that ascorbate alone accounts for >90% of the NO* yield from ONOO- in the presence or absence of purified CcOX in turnover. The yield of NO was CcOX-independent but was dependent on ascorbate and ONOO- concentrations and was not affected by metal chelators. Consistent with this, the interaction of ONOO- with CcOX in respiring isolated mitochondria only yielded NO* when ascorbate was also present in the incubation. These observations are discussed in the context of ONOO-/ascorbate reactivity and the interaction of CcOX with reactive nitrogen species.

Nitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell.

The aim of this work is to review the information available on the molecular mechanisms by which the NO radical reversibly downregulates the function of cytochrome c oxidase (CcOX). The mechanisms of the reactions with NO elucidated over the past few years are described and discussed in the context of the inhibitory effects on the enzyme activity. Two alternative reaction pathways are presented whereby NO reacts with the catalytic intermediates of CcOX populated during turnover. The central idea is that at "cellular" concentrations of NO (

Nitric oxide and mitochondrial complex IV.

Micromolar nitric oxide (NO) rapidly (ms) inhibits cytochrome c oxidase in turnover with physiological substrates. Two reaction mechanisms have been identified leading, respectively, to formation of a nitrosyl- [a3(2+) -NO] or a nitrite- [a3(3+) -NO2-] derivative of the enzyme. In the presence of O2, the nitrosyl adduct recovers activity slowly, following NO displacement at k' approximately equal to 0.01 s(-1) (37 degrees C); the recovery of the nitrite adduct is much faster. Relevant to pathophysiology, the enzyme does not degrade NO by following the first mechanism, whereas by following the second one it promotes NO oxidation and disposal as nitrite/nitrate. The reaction between NO and cytochrome c oxidase has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. Oxygraphic measurements show that cultured cells or isolated state 4 mitochondria exposed to micromolar (or less) NO recover from NO inhibition rapidly, as if the nitrite reaction was predominant. Mitochondria in state 3 display a slightly more persistent inhibition than in state 4, possibly due to a higher accumulation of the nitrosyl adduct. Among a number of parameters that appear to control the switch over between the two mechanisms, the concentration of reductants (reduced cytochrome c) at the cytochrome c oxidase site has been proved to be the most relevant one.

Mitochondria, nitric oxide, and cardiovascular dysfunction.

Cardiovascular diseases encompass a wide spectrum of abnormalities with diverse etiologies. The molecular mechanisms underlying these disorders include a variety of responses such as changes in nitric oxide- (NO) dependent cell signaling and increased apoptosis. An interesting aspect that has received little or no attention is the role mitochondria may play in the vascular changes that occur in both atherosclerosis and hypertension. With the changing perspective of the organelle from simply a role in metabolism to a contributor to signal transduction pathways, the role of mitochondria in cells with relatively low energy demands such as the endothelium has become important to understand. In this context, the definition of the NO-cytochrome c oxidase signaling pathway and the influence this has on cytochrome c release is particularly important in understanding apoptotic mechanisms involving the mitochondrion. This review examines the role of compromised mitochondrial function in a variety of vascular pathologies and the modulation of these effects by NO. The interaction of NO with the various mitochondrial respiratory complexes and the role NO plays in modulating mitochondrial-mediated apoptosis in these systems will be discussed.

Redox-linked protonation of cytochrome c oxidase: the effect of chloride bound to CuB.

The effect of bound Cl- on the redox-linked protonation of soluble beef heart cytochrome c oxidase (CcOX) has been investigated at pH 7.3-7.5 by multiwavelength stopped-flow spectroscopy, using phenol red as the pH indicator in an unbuffered medium. Reduction by Ru-II hexamine of the Cl-bound enzyme is associated with an overall apparent uptake of 1.40 +/- 0.21 H+/aa3, whereas 2.28 +/- 0.36 H+/aa3 is taken upon reduction of the Cl-free enzyme. Bound Cl- has no effect on the extent of H+ uptake coupled to heme a reduction (0.59 +/- 0.06 H+/aa3), but significantly decreases (by approximately 0.9 H+/aa3) the apparent stoichiometry of H+ uptake coupled to heme a3-Cu(B) reduction, by eliminating the net H+ uptake linked to Cu(B) reduction. To account for these results, we propose that, after the transfer of the first electron to the active site, reduction of Cu(B) is associated with Cl- dissociation, addition of a H+, and diffusion into the bulk (with subsequent dissociation) of HCl. In the physiologically competent Cl--free enzyme, an OH- likely bound to oxidized Cu(B) is protonated upon arrival of the first electron, and dissociates as H2O. The relevance of this finding to the understanding of the enzyme mechanism is discussed.

Nitric oxide reacts with the single-electron reduced active site of cytochrome c oxidase.

The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa(3)) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a(3) reduction, which is extremely slow in the K354M mutant (k(1) = 0.09 +/- 0.01 s(-1); k(2) = 0.005 +/- 0.001 s(-1)) but much faster in the D124N aa(3) (k(1) = 21 +/- 6 s(-1); k(2) = 2.2 +/- 0.5 s(-1)). NO causes a very large increase (>100-fold) in the rate constant of heme a(3) reduction in the K354M mutant but only a approximately 5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O(2) when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a(3) reduction is approximately 100-fold faster than the extremely low turnover rate, we conclude that, contrary to O(2), NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H(+)-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site.