α-Synuclein Attenuates Maneb Neurotoxicity through the Modulation of Redox-Sensitive Transcription Factors.

The accumulation and aggregation of α-synuclein is a pathognomonic sign of Parkinson's disease (PD). Maneb (MB) exposure has also been reported as one environmental triggering factor of this multifactorial neurodegenerative disease. In our laboratory, we have previously reported that mild overexpression of α-synuclein (200% increase with respect to endogenous neuronal levels) can confer neuroprotection against several insults. Here, we tested the hypothesis that α-synuclein can modulate the neuronal response against MB-induced neurotoxicity. When exposed to MB, cells with endogenous α-synuclein expression displayed increased reactive oxygen species (ROS) associated with diminished glutamate-cysteine ligase catalytic subunit (GCLc) and hemeoxygenase-1 (HO-1) mRNA expressions and upregulation of the nuclear factor erythroid 2-related factor 2 (NRF2) repressor, BTB domain and CNC homolog 1 (BACH1). We found that α-synuclein overexpression (wt α-syn cells) attenuated MB-induced neuronal damage by reducing oxidative stress. Decreased ROS found in MB-treated wt α-syn cells was associated with unaltered GCLc and HO-1 mRNA expressions and decreased BACH1 expression. In addition, the increased SOD2 expression and catalase activity were associated with forkhead box O 3a (FOXO3a) nuclear compartmentalization. Cytoprotective effects observed in wt α-syn cells were also associated with the upregulation of silent information regulator 1 (SIRT1). In control cells, MB-treatment downregulated glutathione peroxidase 4 mRNA levels, which was coincident with increased ROS content, lipid peroxidation, and mitochondrial alterations. These deleterious effects were prevented by ferrostatin-1, an inhibitor of ferroptosis, under conditions of endogenous α-synuclein expression. The overexpression of α-synuclein attenuated MB toxicity by the activation of the same mechanisms as ferrostatin-1. Overall, our findings suggest that mild overexpression of α-synuclein attenuates MB-induced neurotoxicity through the modulation of NRF2 and FOXO3a transcription factors and prevents cell death probably by intervening in mechanisms associated with ferroptosis. Thus, we postulate that early stages of α-synuclein overexpression could be potentially neuroprotective against MB neurotoxicity.

Zinc and the modulation of Nrf2 in human neuroblastoma cells.

Zinc plays a key role in the modulation of neuronal redox homeostasis. A decreased zinc availability is associated with neuronal NADPH oxidase and nitric oxide synthase activation, deregulation of redox signaling, and impaired glutathione synthesis. The present work tested the hypothesis that zinc is necessary in the neuronal defense response against dopamine (DA)-induced oxidative stress, in particular through heme oxygenase-1 (HO-1) upregulation. DA showed higher cytotoxicity when zinc availability was low. Human IMR-32 neuroblastoma cells responded to high DA concentrations (100 µM) by upregulating HO-1. This upregulation involved Nrf2 translocation to the nucleus, degradation of the Bach-1 repressor, and Nrf2-DNA binding, but it was independent of ERK1/2 activation. DA-mediated induction of HO-1 expression was dependent on the concentration of zinc in the medium. IMR-32 cells incubated in zinc deficient medium showed an impaired response to DA, with lower HO-1 mRNA and protein levels than control DA-challenged cells. This altered HO-1 upregulation was reversed by zinc supplementation. In the presence of DA, Nrf2 nuclear translocation and Bach-1 degradation were lower in zinc deficient cells. The mechanisms involved include: i) impaired Nrf2-tubulin interactions and ii) alterations in the proteasome-mediated degradation of Bach-1 secondary to a decreased ubiquitylation. Results suggest that zinc is crucial in the neuronal response to DA-induced oxidative stress in part through its role in the modulation of the Nrf2-and Bach-1-driven upregulation of HO-1 expression.

Specific roles for Group V secretory PLA2 in retinal iron-induced oxidative stress. Implications for age-related macular degeneration.

Iron accumulation and oxidative stress are hallmarks of retinas from patients with age-related macular degeneration (AMD). We have previously demonstrated that iron-overloaded retinas are a good in vitro model for the study of retinal degeneration during iron-induced oxidative stress. In this model we have previously characterized the role of cytosolic phospholipase A2 (cPLA2) and calcium-independent isoform (iPLA2). The aim of the present study was to analyze the implications of Group V secretory PLA2 (sPLA2), another member of PLA2 family, in cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-κB) regulation. We found that sPLA2 is localized in cytosolic fraction in an iron concentration-dependent manner. By immunoprecipitation (IP) assays we also demonstrated an increased association between Group V sPLA2 and COX-2 in retinas exposed to iron overload. However, COX-2 activity in IP assays was observed to decrease in spite of the increased protein levels observed. p65 (RelA) NF-κB levels were increased in nuclear fractions from retinas exposed to iron. In the presence of ATK (cPLA2 inhibitor) and YM 26734 (sPLA2 inhibitor), the nuclear localization of both p65 and p50 NF-κB subunits was restored to control levels in retinas exposed to iron-induced oxidative stress. Membrane repair mechanisms were also analyzed by studying the participation of acyltransferases in phospholipid remodeling during retinal oxidation stress. Acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylserine (PS), were observed to show an inhibited acylation profile in retinas exposed to iron while phosphatidylethanolamine (PE) showed the opposite. The use of PLA2 inhibitors demonstrated that PS is actively deacylated during iron-induced oxidative stress. Results from the present study suggest that Group V sPLA2 has multiple intracellular targets during iron-induced retinal degeneration and that the specific role of sPLA2 could be related to inflammatory responses by its participation in NF-κB and COX-2 regulation.

Differential participation of phospholipase A2 isoforms during iron-induced retinal toxicity. Implications for age-related macular degeneration.

Both elevated iron concentrations and the resulting oxidative stress condition are common signs in retinas of patients with age-related macular degeneration (AMD). The role of phospholipase A(2) (PLA(2)) during iron-induced retinal toxicity was investigated. To this end, isolated retinas were exposed to increasing Fe(2+) concentrations (25, 200 or 800 µM) or to the vehicle, and lipid peroxidation levels, mitochondrial function, and the activities of cytosolic PLA(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) were studied. Incubation with Fe(2+) led to a time- and concentration-dependent increase in retinal lipid peroxidation levels whereas retinal cell viability was only affected after 60 min of oxidative injury. A differential release of arachidonic acid (AA) and palmitic acid (PAL) catalyzed by cPLA(2) and iPLA(2) activities, respectively, was also observed in microsomal and cytosolic fractions obtained from retinas incubated with iron. AA release diminished as the association of cyclooxygenase-2 increased in microsomes from retinas exposed to iron. Retinal lipid peroxidation and cell viability were also analyzed in the presence of cPLA(2) inhibitor, arachidonoyl trifluoromethyl ketone (ATK), and in the presence of iPLA(2) inhibitor, bromoenol lactone (BEL). ATK decreased lipid peroxidation levels and also ERK1/2 activation without affecting cell viability. BEL showed the opposite effect on lipid peroxidation. Our results demonstrate that iPLA(2) and cPLA(2) are differentially regulated and that they selectively participate in retinal signaling in an experimental model resembling AMD.

Effect of transition metals in synaptic damage induced by amyloid beta peptide.

The amyloid beta-peptide (Abeta), which is thought to be the major cause of Alzheimer's disease (AD), is known to be capable of aggregating in different states: soluble monomers and oligomers, and insoluble aggregates. The Abeta aggregation state as well as its toxicity has been related to the interaction between the peptide and transition metals such as iron and copper. However, this relationship, as well as the effects of Abeta on the synaptic endings, is not fully understood. The aggregation states of Abeta in the presence of iron and copper, as well as their effects on synaptic viability and signaling were investigated in this work. During acute incubation treatments (5 min-4 h), Abeta/metal impaired mitochondrial function to the same extent as has been observed with the metal alone. However, in the presence of Abeta/iron (10 and 50 muM), plasma membrane integrity was disrupted to a greater extent than when generated by either iron or Abeta alone, indicating that the membrane constitutes the first target of synaptic injury. Akt activation by Abeta/iron was evident after 5 min of incubation and was higher than that observed in the presence of the metal alone. This activation was barely detected after 4 h of incubation, demonstrating that there is no correlation between the extent of synaptic damage and the activation of this kinase. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation profile was different from that observed for Akt. Accordingly, the presence of Abeta/metal could differentially modulate the activity of these kinases. This work shows evidence of the initial events locally triggered at the synapse by Abeta and transition metals. As synapses have been proposed as the starting point of Abeta/metal-triggered events, the characterization of early mechanisms occurring in models that mimic AD could be important for the search of unexplored therapeutics tools.

Selective localization of phosphatidylcholine-derived signaling in detergent-resistant membranes from synaptic endings.

Detergent-resistant membranes (DRMs) are a class of specialized microdomains that compartmentalize several signal transduction processes. In this work, DRMs were isolated from cerebral cortex synaptic endings (Syn) on the basis of their relative insolubility in cold Triton X-100 (1%). The lipid composition and marker protein content were analyzed in DRMs obtained from adult and aged animals. Both DRM preparations were enriched in Caveolin, Flotillin-1 and c-Src and also presented significantly higher sphingomyelin (SM) and cholesterol content than purified Syn. Total phospholipid-fatty acid composition presented an increase in 16:0 (35%), and a decrease in 20:4n-6 (67%) and 22:6n-3 (68%) content in DRM from adults when compared to entire synaptic endings. A more dramatic decrease was observed in the 20:4n-6 and 22:6n-3 content in DRMs from aged animals (80%) with respect to the results found in adults. The coexistence of phosphatidylcholine-specific-phospholipase C (PC-PLC) and phospholipase D (PLD) in Syn was previously reported. The presence of these signaling pathways was also investigated in DRMs isolated from adult and aged rats. Both PC-PLC and PLD pathways generate the lipid messenger diacylglycerol (DAG) by catalyzing PC hydrolysis. PC-PLC and PLD1 localization were increased in the DRM fraction. The increase in DAG generation (60%) in the presence of ethanol, confirmed that PC-PLC was also activated when compartmentalized in DRMs. Conversely, PLD2 was excluded from the DRM fraction. Our results show an age-related differential fatty acid composition and a selective localization of PC-derived signaling in synaptic DRMs obtained from adult and aged rats.

Age-associated changes of insulin action on the hydrolysis of diacylglycerol generated from phosphatidic acid.

Age-related changes in insulin action on diacylglycerol (DAG) degradation was studied in rat cerebral cortex synaptosomes. The generation of monoacylglycerol (MAG) and water soluble products (WSP, glycerol plus glycerol-3-phosphate) from DAG was studied in cerebral cortex (CC) synaptosomes from adult (4-month-old) and aged (28-month-old) rats. Additionally, the effect of porcine insulin and tyrosine phosphorylation was evaluated in the same group of animals. In this study we demonstrate that the age-related increase in WSP generation was accompanied by unmodified MAG levels. In the presence of diacylglycerol lipase (DAG lipase) inhibitor, RHC-80267, a lower inhibitory effect on MAG production was observed in CC synaptosomes from aged rats with respect to that in adult membranes. Under these experimental conditions, WSP formation was only diminished in aged membranes. Insulin stimulated MAG and WSP formation at long incubation times (30 min) in adult animals, while it had an inhibitory effect in aged animals. Insulin plus vanadate (as tyrosine-phosphatase inhibitor) inhibited MAG production at short incubation times whereas the same effect was observed in aged animals at long times of incubation. WSP formation was stimulated by insulin plus vanadate both in adult and aged animals at 30 min of incubation. Our results show that insulin differentially modulates MAG and WSP production from exogenous PA in CC synaptosomes from aged rats compared with adult rats.

Phospholipase D and phosphatidate phosphohydrolase activities in rat cerebellum during aging.

Aging is a process that affects different organs, of which the brain is particularly susceptible. PA and DAG are central intermediates in the phosphoglyceride as well as in the neutral lipid biosynthetic pathway, and they have also been implicated in signal transduction. Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAP) are the enzymes that generate PA and DAG. The latter can be transformed into MAG by diacylglycerol lipase (DGL). In the present study, we examine how aging modulates the PLD, PAP, and DGL isoforms in cerebellar subcellular fractions from 4- (adult), 28-, and 33-mon-old (aged) rats. PI-4,5-bisphosphonate (PIP2)-dependent PLD, PAP1, and DGL1 were distributed in different percentages in all cerebellum subcellular fractions. On the other hand, PAP2 and DGL2 activities were observed in all subcellular fractions except in the cytosolic fraction. Aging modified the enzyme distribution pattern. In addition, aging decreased nuclear (45%), mitochondrial-synaptosomal (55%), and cytosolic (71%) PAP1 activity and increased (28%) microsomal PAP1 activity. DGL1 activity was decreased in nuclear (85%) and mitochondrial-synaptosomal (63%) fractions by aging. On the other hand, PIP2-dependent PLD activities were increased in the mitochondrial-synaptosomal fraction. PAP2 and DGL2 were increased in the microsomal fraction by 87 and 114%, respectively, and they were decreased in the nuclear fraction. The changes observed in cerebellum PAP1 and DGL1 activities from aged rats with respect to adult rats could be related to modifications in lipid metabolism. Differential PA metabolization during aging through PIP2-dependent PLD/PAP2/DGL2 activities could be related to alterations in the neural signal transduction mechanisms.

Differential modulation of phospholipase D and phosphatidate phosphohydrolase during aging in rat cerebral cortex synaptosomes.

Phosphatidylcholine (PC) hydrolysis generates two important second messengers: phosphatidic acid (PA) and diacylglycerol (DAG). Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAPase) are involved in their generation and therefore are key enzymes in signal transduction. Specific isoforms of these enzymes are activated by receptor occupancy in brain. Phosphatidylinositol 4,5-bisphosphate-dependent PLD (PIP2-PLD) and N-ethylmaleimide-insensitive PAPase (PAP2) have been suggested to act in series to generate the biologically active lipids PA and DAG. In the present study we examine age-induced changes mainly in PIP2-PLD and PAP2 activities in cerebrocortical synaptosomes from adult (4 months) and aged (28 months) Wistar rats. Aging increases the activity of both enzymes. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and cytosol (from cerebral cortex) stimulate PLD activity in adult and senescent synaptosomal membranes, the effect being greater in the latter. Under the same experimental conditions PAP2 activity was stimulated in aged membranes whereas in adult membranes GTPgammaS had no effect and cytosol showed a slight inhibitory effect. Diacylglycerol lipase (DGL) activity differs from that of PAP2 in aged rats and it was 21% inhibited with respect to synaptosomal membranes from adult rats. Increased sinaptosomal PLD activity in aged membranes appears to be independent of G protein regulation, whereas PAP2 activity is differentially regulated by GTPgammaS in aged membranes with respect to adult membranes. Our results suggest that under G-protein activation conditions, DAG production by the serial activation of PLD and PAP2 activities is increased in synaptosomal membranes from aged brain. The present paper demonstrates that PA generation (PLD activity) and degradation (PAPase activity) are differentially modulated during the aging process.

Age-associated changes in central nervous system glycerolipid composition and metabolism.

In this review, changes in brain lipid composition and metabolism due to aging are outlined. The most striking changes in cerebral cortex and cerebellum lipid composition involve an increase in acidic phospholipid synthesis. The most important changes with respect to fatty acyl composition involve a decreased content in polyunsaturated fatty acids (20:4n-6, 22:4n-6, 22:6n-3) and an increased content in monounsaturated fatty acids (18:1n-9 and 20:1n-9), mainly in ethanolamine and serineglycerophospholipids. Changes in the activity of the enzymes modifying the phospholipid headgroup occur during aging. Serine incorporation into phosphatidylserine through base-exchange reactions and phosphatidylcholine synthesis through phosphatidylethanolamine methylation increases in the aged brain. Phosphatidate phosphohydrolase and phospholipase D activities are also altered in the aged brain thus producing changes in the lipid second messengers diacylglycerol and phosphatidic acid.

Alternative pathways for phospholipid synthesis in different brain areas during aging.

Morphological and biochemical changes take place in the membrane of aged brain. In particular, studies on aged rats report alterations in brain phospholipid synthesis and in phospholipid-specific fatty acid composition. However, no significant changes in main phospholipid class content have been reported in aged brain, possibly owing to alterations in the alternative pathways for phospholipid synthesis during aging. Therefore, the present study was designed to determine the effect of aging on the enzyme activities responsible for phospholipid synthesis by alternative pathways. Indifferent brain areas of adult (3.5-month-old) and aged (28.5-month-old) rats we examined: 1) the activity of base exchange enzymes, which is a calcium-dependent, energy-independent and calcium stimulated enzymatic pathway; 2) phosphatidylethanolamine (PE) synthesis by phosphatidylserine decarboxylase activity (PSD); 3) phosphatidylcholine (PC) synthesis by transfer of methyl groups to endogenous PE by phosphatidylethanolamine N-methyltransferase activity (PEMT); 4) the synthesis of phosphatidylglycerol (PG) through phospholipase D (PLD) activity. Because the dependence on and the stimulation by calcium of base-exchange reactions is a well known mechanism and alterations in calcium levels in rat brain have been reported, we decided to investigate PS synthesis in the presence of endogenous and exogenous calcium (2.5mM). PS synthesis increased in cerebral cortex (CC) and cerebellum (CRBL) of aged rats with respect to adult rats in basal conditions (without the addition of exogenous calcium), but more significant changes were observed in serine base exchange activity during aging when exogenous calcium was added. PEMT activity in aged CC increased by 100%, the principal modification being observed in the first methylated product of the sequential reaction. Furthermore, the transphosphatidyl reaction was higher in aged brain as indicated by the increased PG synthesis. Our findings allow us to conclude that age affects some alternative pathways for phospholipid synthesis in the central nervous system, and indicate the presence of a compensatory mechanism to provide a pool of phospholipid classes for the maintenance of cellular membrane lipid composition during aging.

Effect of light on phosphatidate phosphohydrolase activity of retina rod outer segments: the role of transducin.

The aim of the present paper is to evaluate the modulation of phosphatidate phosphohydrolase (PAPase) and diacylglyceride lipase (DGL) activities in bovine rod outer segment (ROS) under dark and light conditions and to evaluate the role of transducin (T) in this phenomenon. In dark-adapted ROS membranes exposed to light, PAPase activity is inhibited by 20% with respect to the activity found under dark conditions. To determine whether the retinal G protein, T, participates in the regulation of PAPase activity in these membranes, the effects of GTPgammaS and GDPbetaS on enzyme activity were examined. Under dark conditions in the presence of GTPgammaS, which stabilizes T in its active form (Talpha + Tbetagamma), enzyme activity was inhibited and approached control values under light conditions. GDPbetaS, on the other hand, which stabilizes the inactive state of T (Talphabetagamma), stimulated PAPase activity by 36% with respect to control light conditions. ADP-ribosylation by cholera and pertussis toxin was also studied. In ADP-rybosilated ROS membranes with pertussis toxin under dark conditions, PAPase activity was 36% higher than the activity found under control light conditions. ADP-ribosylation by CTx, on the other hand, inhibited PAPase activity by 22%, with respect to dark control conditions, mimicking light effect. The effects of GTPgammaS and GDPbetaS and conditions of ADP-ribosylation by PTx and CTx on DGL activity were similar to those of PAPase activities. Based on NEM sensitivity we have also demonstrated that the PAPase present in ROS is the PAP 2 isoform. Our findings therefore suggest that light inhibition of PAP 2 in ROS is a transducin-mediated mechanism.

Light activation of phosphatidylethanolamine N-methyltransferase in rod outer segments and its modulation by association states of transducin.

Phosphatidylethanolamine N-Methyltransferase (PE N-MTase) is the enzyme responsible for the synthesis of phosphatidylcholine from phosphatidylethanolamine by successive transfer of methyl groups. This enzyme is present in bovine rod outer segments (ROS) and it is the only pathway for the synthesis of phosphatidylcholine in the outer segment of rod photoreceptor cells. In dark-adapted ROS membranes PE N-MTase activity is stimulated by 100% when ROS membranes are incubated under light condition. To determine whether the retinal G protein, transducin (Gt), intervenes in the regulation of PE N-MTase in these membranes, the effects of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and guanosine 5'-O-(2-thiodiphosphate (GDPbetaS) on the enzyme activity were examined. In dark, GTPgammaS which induces dissociation of Gt, stimulates the enzyme activity mimicking the stimulation by light. On the contrary, GDPbetaS stabilizes the inactive state of Gt, inhibiting the stimulation by light of PE N-MTase without affecting basal activities. In addition, adenosine 5'-diphosphate (ADP)-ribosylation by cholera and pertussis toxin was studied. ADP-ribosylation of ROS membrane with pertussis toxin, which stabilizes transducin in its inactive state, prevents the light-induced increase in PE N-MTase activity. On the contrary ADP-ribosylation with cholera toxin stimulates the enzyme activity. Our findings therefore suggest that light-stimulated effect of PE N-MTase activity is transducin-mediated.

Characterization of phospholipase D activity in bovine photoreceptor membranes.

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2(3)H]glycerol-3-phosphocholine (PC), or [14C]ethanol. Using [3H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [3H]phosphatidic acid and phosphatidylethanol [3H]PtdEt; whereas with [14C]ethanol or [3H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [14C]PtdEt or [3H]phosphatidylglycerol, respectively). The use of varying concentrations of [3H]PC and 400 mM of ethanol gave an apparent Km value for PC of 0.51 mM and a Vmax value of 111 nmol x h(-1) x (mg protein)(-1). The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent Km of 202 mM and a Vmax value for ethanol of 125 nmol x h(-1) x (mg protein)(-1). A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca2+ or Mg2+ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.