Drosophila tweety facilitates autophagy to regulate mitochondrial homeostasis and bioenergetics in Glia.

Mitochondria support the energetic demands of the cells. Autophagic turnover of mitochondria serves as a critical pathway for mitochondrial homeostasis. It is unclear how bioenergetics and autophagy are functionally connected. Here, we identify an endolysosomal membrane protein that facilitates autophagy to regulate ATP production in glia. We determined that Drosophila tweety (tty) is highly expressed in glia and localized to endolysosomes. Diminished fusion between autophagosomes and endolysosomes in tty-deficient glia was rescued by expressing the human Tweety Homolog 1 (TTYH1). Loss of tty in glia attenuated mitochondrial turnover, elevated mitochondrial oxidative stress, and impaired locomotor functions. The cellular and organismal defects were partially reversed by antioxidant treatment. We performed live-cell imaging of genetically encoded metabolite sensors to determine the impact of tty and autophagy deficiencies on glial bioenergetics. We found that tty-deficient glia exhibited reduced mitochondrial pyruvate consumption accompanied by a shift toward glycolysis for ATP production. Likewise, genetic inhibition of autophagy in glia resulted in a similar glycolytic shift in bioenergetics. Furthermore, the survival of mutant flies became more sensitive to starvation, underlining the significance of tty in the crosstalk between autophagy and bioenergetics. Together, our findings uncover the role for tty in mitochondrial homeostasis via facilitating autophagy, which determines bioenergetic balance in glia.

Loss of Activity-Induced Mitochondrial ATP Production Underlies the Synaptic Defects in a Drosophila Model of ALS.

Mutations in the gene encoding vesicle-associated membrane protein B (VAPB) cause a familial form of amyotrophic lateral sclerosis (ALS). Expression of an ALS-related variant of vapb (vapbP58S ) in Drosophila motor neurons results in morphologic changes at the larval neuromuscular junction (NMJ) characterized by the appearance of fewer, but larger, presynaptic boutons. Although diminished microtubule stability is known to underlie these morphologic changes, a mechanism for the loss of presynaptic microtubules has been lacking. By studying flies of both sexes, we demonstrate the suppression of vapbP58S -induced changes in NMJ morphology by either a loss of endoplasmic reticulum (ER) Ca2+ release channels or the inhibition Ca2+/calmodulin (CaM)-activated kinase II (CaMKII). These data suggest that decreased stability of presynaptic microtubules at vapbP58S NMJs results from hyperactivation of CaMKII because of elevated cytosolic [Ca2+]. We attribute the Ca2+ dyshomeostasis to delayed extrusion of cytosolic Ca2+ Suggesting that this defect in Ca2+ extrusion arose from an insufficient response to the bioenergetic demand of neural activity, depolarization-induced mitochondrial ATP production was diminished in vapbP58S neurons. These findings point to bioenergetic dysfunction as a potential cause for the synaptic defects in vapbP58S -expressing motor neurons.SIGNIFICANCE STATEMENT Whether the synchrony between the rates of ATP production and demand is lost in degenerating neurons remains poorly understood. We report that expression of a gene equivalent to an amyotrophic lateral sclerosis (ALS)-causing variant of vesicle-associated membrane protein B (VAPB) in fly neurons decouples mitochondrial ATP production from neuronal activity. Consequently, levels of ATP in mutant neurons are unable to keep up with the bioenergetic burden of neuronal activity. Reduced rate of Ca2+ extrusion, which could result from insufficient energy to power Ca2+ ATPases, results in the accumulation of residual Ca2+ in mutant neurons and leads to alterations in synaptic vesicle (SV) release and synapse development. These findings suggest that synaptic defects in a model of ALS arise from the loss of activity-induced ATP production.

Human Prune Regulates the Metabolism of Mammalian Inorganic Polyphosphate and Bioenergetics.

Inorganic polyphosphate (polyP) is an evolutionarily conserved and ubiquitous polymer that is present in all studied organisms. PolyP consists of orthophosphates (Pi) linked together by phosphoanhydride bonds. The metabolism of polyP still remains poorly understood in higher eukaryotes. Currently, only F0F1-ATP synthase, Nudt3, and Prune have been proposed to be involved in this metabolism, although their exact roles and regulation in the context of polyP biology have not been fully elucidated. In the case of Prune, in vitro studies have shown that it exhibits exopolyphosphatase activity on very short-chain polyP (up to four units of Pi), in addition to its known cAMP phosphodiesterase (PDE) activity. Here, we expand upon studies regarding the effects of human Prune (h-Prune) on polyP metabolism. Our data show that recombinant h-Prune is unable to hydrolyze short (13-33 Pi) and medium (45-160 Pi) chains of polyP, which are the most common chain lengths of the polymer in mammalian cells. Moreover, we found that the knockdown of h-Prune (h-Prune KD) results in significantly decreased levels of polyP in HEK293 cells. Likewise, a reduction in the levels of polyP is also observed in Drosophila melanogaster loss-of-function mutants of the h-Prune ortholog. Furthermore, while the activity of ATP synthase, and the levels of ATP, are decreased in h-Prune KD HEK293 cells, the expression of ATP5A, which is a main component of the catalytic subunit of ATP synthase, is upregulated in the same cells, likely as a compensatory mechanism. Our results also show that the effects of h-Prune on mitochondrial bioenergetics are not a result of a loss of mitochondrial membrane potential or of significant changes in mitochondrial biomass. Overall, our work corroborates the role of polyP in mitochondrial bioenergetics. It also demonstrates a conserved effect of h-Prune on the metabolism of short- and medium-chain polyP (which are the predominant chain lengths found in mammalian cells). The effects of Prune in polyP are most likely exerted via the regulation of the activity of ATP synthase. Our findings pave the way for modifying the levels of polyP in mammalian cells, which could have pharmacological implications in many diseases where dysregulated bioenergetics has been demonstrated.

Combination of Lutein and Zeaxanthin, and DHA Regulated Polyunsaturated Fatty Acid Oxidation in H2O2-Stressed Retinal Cells.

Photochemical and oxidative damages in retinal pigment epithelial (RPE) cells are key events in the pathogenesis of age-related macular degeneration. Polyunsaturated fatty acids (PUFA) and carotenoids are rich in retinal cells, and under oxidative stress leads to oxidation and release lipid mediators. We evaluated the impact of carotenoids (lutein, zeaxanthin) and docosahexaenoic acid (DHA) supplementation on RPE cells under oxidative stress. ARPE-19 cells were exposed to H2O2 after pre-treatment with lutein, zeaxanthin, DHA, lutein + zeaxanthin or lutein + zeaxanthin with DHA. The data showed H2O2 reduced cell viability and DHA content, while promoted catalase activity and certain oxidized PUFA products. Treatment with DHA enhanced omega-3 PUFA enzymatic oxidation namely, anti-inflammatory mediators such as hydroxy-DHA, resolvins and neuroprotection compared to control; the effects were not influenced by the carotenoids. Omega-6 PUFA oxidation, namely pro-inflammatory HETE (5-, 9-, 12 and 20-HETE), and isoprostanes (5- and 15-F2t-IsoP and 4-F3t-IsoP) were reduced by lutein + zeaxanthin while the addition of DHA did not further reduce these effects. We observed transcriptional regulation of 5-lipoxygenase by DHA and GPx1 and NEFEL2 by the carotenoids that potentially resulted in decreased HETEs and glutathione respectively. 4-HNE was not affected by the treatments but 4-HHE was reduced by lutein + zeaxanthin with and without DHA. To conclude, carotenoids and DHA appeared to regulate inflammatory lipid mediators while the carotenoids also showed benefits in reducing non-enzymatic oxidation of omega-6 PUFA.

Insight into the contribution of isoprostanoids to the health effects of omega 3 PUFAs.

Omega 3 polyunsaturated fatty acids have been reported to confer beneficial health effects notably in the field of cardiovascular and inflammatory diseases. The current knowledge suggests a significant portion of the effects of omega 3 polyunsaturated fatty acids are mediated by their oxygenated metabolites. This review attempts to cover the current literature about the contribution of specific omega 3 oxygenated metabolites, namely omega 3 isoprostanoids, which are produced through free-radical mediated oxidation. A special emphasis has been given to the most biologically relevant omega 3 polyunsaturated fatty acids namely the α-linolenic, eicosapentaenoic and docosahexaenoic acids. The review includes a comprehensive description of the biosynthetic pathways, a summary of studies related to the biological significance of omega 3 isoprostanoids as well as a critical description of analytical development in the field of omega 3 isoprostanoids profiling in biological samples.

Extra Virgin Olive Oil Reduced Polyunsaturated Fatty Acid and Cholesterol Oxidation in Rodent Liver: Is This Accounted for Hydroxytyrosol-Fatty Acid Conjugation?

The effects of extra virgin olive oil (EVOO) and carbon tetrachloride (CCl4) induced oxidative stress in rats were determined by the generation of isoprostanoids. These are known to be robust biomarkers to evaluate nonenzymatic and free radical related oxidation. Other oxidative stress biomarkers such as hydroxyeicosatetraenoic acid products (HETEs) and cholesterol oxidation products (COPs) were also determined. The rodents received a control diet, high-fat diet (20% w/w) composed of extra virgin olive oil (EVOO), corn oil (CO), or lard, and high-fat diets with CCl4 insult throughout the experimental period. The EVOO diet was found to suppress the formation of isoprostanoids and COPs compared to that of the control. EVOO also had a high total phenolic content and antioxidant activity compared to those of CO and lard and may be contributed to by the hydroxytyrosol component conjugated to fatty acids (HT-FA). This is the first study to identify HT-FA in EVOO, and it was 4-fold higher than that of olive oil, whereas none was found in corn oil. Furthermore, the EVOO diet showed reduced liver lipid vesicles in CCl4 treated rats compared to that of the control. However, liver toxicity measurements of AST (aspartate transaminase) and ALT (alanine transaminase) activities showed augmentation with CCl4 treatment but were not alleviated by the diets given. Our findings suggest that EVOO is a daily functional food capable of enhancing the antioxidant system for liver protection; the effect is potentially attributed to the phenolic and lipophenolic (phenol conjugated by fatty acids) content.

Nonenzymatic oxygenated metabolite of docosahexaenoic acid, 4(RS)-4-F4t -neuroprostane, acts as a bioactive lipid molecule in neuronal cells.

Docosahexaenoic acid (DHA), an abundant fatty acid in the brain, is susceptible to auto-oxidation in situ and releases metabolites such as F4 -neuroprostane (4-F4t -NeuroP). The presence of 4-F4t -NeuroP in the brain is not well explored. In this study, 4-F4t -NeuroP was introduced into neuroblastoma cells (SH-SY5Y) and, by in vivo infusion, into rodents. Targeted lipidomic analysis of liver and brain tissues shows significant elevation of anti-inflammatory hydroxylated DHA metabolites and an isomer of neuroprotectin D1, suggesting potential beneficial bioactivities of 4-F4t -NeuroP. Additionally, 4-F4t -NeuroP treatment in SH-SY5Y cells and primary neuronal culture consistently upregulates the transcriptional level of the antioxidant enzyme heme oxygenase-1, but the effect is reduced when 4-F4t -NeuroP is further oxidized. Our data suggest that 4-F4t -NeuroP could be neuroprotective in the native state but may have disadvantageous bioactivity when oxidized extensively.

Measurement of Enzymatic and Nonenzymatic Polyunsaturated Fatty Acid Oxidation Products in Plasma and Urine of Macular Degeneration Using LC-QTOF-MS/MS.

Oxidized polyunsaturated fatty acids (PUFA) are associated to pathogenesis of diseases including cardiovascular and neurodegeneration. The novel products are not only biomarkers but also lipid mediators in gene regulation and signaling pathways. Herein, simultaneous quantitation of 28 products derived from nonenzymatic and enzymatic oxidation of PUFA i.e. 5-, 15-F2t -isoprostanes, 7-, 17-F2t -dihomo-isoprostanes, 7-, 17-F2t -dihomo-isofurans, 5-, 8-, 18-F3t -isoprostanes, 4-, 10-, 13-, 14-, 20-F4t -neuroprostanes, 5-, 8-, 9-, 11-,12-, 15-, 20-HETE, 4-, 7-, 11-, 14-, 17-HDHA, RvE1, and NPD1 using LC-(ESI)-QTOF-MS/MS was developed. These products were measurable in a single sample and the analytical time was relative short (~15 min). Furthermore, we showed that the use of internal standards is a requisite to normalize matrix effects and preparation loss for the quantitation. Validation assays indicated the method to be robust for plasma and mid-stream urine sample analysis in particular from those of age-related macular degeneration subjects, where the accuracy of quantitation displayed good repeatability.

Lutein Supplementation for Eye Diseases.

Lutein is one of the few xanthophyll carotenoids that is found in high concentration in the macula of human retina. As de novo synthesis of lutein within the human body is impossible, lutein can only be obtained from diet. It is a natural substance abundant in egg yolk and dark green leafy vegetables. Many basic and clinical studies have reported lutein's anti-oxidative and anti-inflammatory properties in the eye, suggesting its beneficial effects on protection and alleviation of ocular diseases such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, myopia, and cataract. Most importantly, lutein is categorized as Generally Regarded as Safe (GRAS), posing minimal side-effects upon long term consumption. In this review, we will discuss the chemical structure and properties of lutein as well as its application and safety as a nutritional supplement. Finally, the effects of lutein consumption on the aforementioned eye diseases will be reviewed.

Limited Antioxidant Effect of Rosemary in Lipid Oxidation of Pan-Fried Salmon.

Consumption of omega-3 polyunsaturated fatty acids (n-3 PUFAs) rich fatty fish is known to provide an array of health benefits. However, high temperature in food preparation, such as pan-frying, potentially degrades eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) of the n-3 PUFAs by heat oxidation. The addition of antioxidant condiments, and herbs in particular, may retard PUFA peroxidation and preserve EPA and DHA during pan-frying. In this study, different types of antioxidant condiments (sage, rosemary, black peppercorn, thyme, basil, and garlic) were tested for antioxidant capacity, and the condiment with the highest capacity was selected for its effect on lipid oxidation of salmon. The changes in fatty acids and lipid peroxidation of salmon, during pan-frying with the selected condiment (olive oil infused with rosemary, RO(infused)), were compared with salmon prepared in extra virgin olive oil, olive oil, or without oil. The total saturated fatty acid was found to be less in pan fried salmon with RO(infused). None of the oil type conserved EPA- and DHA-content in salmon. However, RO(infused) lowered lipid peroxidation by lessening hydroperoxide and 4-HNE formation, but not the other related products (HDHA, HETE, isoprostanes). Our observation indicates that the antioxidant capacity of RO(infused), when it is incorporated with food, becomes limited.

Increase in omega-6 and decrease in omega-3 polyunsaturated fatty acid oxidation elevates the risk of exudative AMD development in adults with Chinese diet.

Appropriate diet is essential for the regulation of age-related macular degeneration (AMD). In particular the type of dietary polyunsaturated fatty acids (PUFA) and poor antioxidant status including carotenoid levels concomitantly contribute to AMD risk. Build-up of oxidative stress in AMD induces PUFA oxidation, and a mix of lipid oxidation products (LOPs) are generated. However, LOPs are not comprehensively evaluated in AMD. LOPs are considered biomarkers of oxidative stress but also contributes to inflammatory response. In this cross-sectional case-control study, plasma omega-6/omega-3 PUFA ratios and antioxidant status (glutathione, superoxide dismutase and catalase), and plasma and urinary LOPs (41 types) were determined to evaluate its odds-ratio in the risk of developing exudative AMD (n = 99) compared to age-gender-matched healthy controls (n = 198) in adults with Chinese diet. The odds ratio of developing exudative AMD increased with LOPs from omega-6 PUFA and decreased from those of omega-3 PUFA. These observations were associated with a high plasma omega-6/omega-3 PUFA ratio and low carotenoid levels. In short, poor PUFA and antioxidant status increased the production of omega-6 PUFA LOPs such as dihomo-isoprostane and dihomo-isofuran, and lowered omega-3 PUFA LOPs such as neuroprostanes due to the high omega-6/omega-3 PUFA ratios; they were also correlated to the risk of AMD development. These findings indicate the generation of specific LOPs is associated with the development of exudative AMD.

Dietary Habits, Fatty Acids and Carotenoid Levels Are Associated with Neovascular Age-Related Macular Degeneration in Chinese.

The role of diet and circulatory carotenoids and docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are implicated in age-related macular degeneration (AMD) but not well studied in Chinese. However, other fatty acids were not comprehensively evaluated if it had additional consequence on AMD. This study investigated the relationship among dietary habits, fatty acids levels, carotenoids and AMD in Hong Kong Chinese adults. In this cross-sectional case-controlled study, plasma fatty acids including, saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA), and carotenoids levels were quantified between patients with neovascular AMD (n = 99) and age-gender-matched controls (n = 198). A food frequency questionnaire was also conducted. Low blood carotenoid levels and omega-3 PUFAs namely DHA, EPA and -linolenic acid increased the odds ratio of developing neovascular AMD. High blood omega-6 PUFAs specifically arachidonic acid and eicosadienoic acid, oleic acid (a MUFA) and SFA levels increased the odds ratio of having neovascular AMD. Neovascular AMD group had significantly less omega-3 PUFA rich food (vegetables, nuts, seafood) intake and higher SFA (meat) intake than controls. In short, neovascular AMD was associated with lower circulatory levels of carotenoids and omega-3 PUFAs, and higher level of omega-6 PUFAs, oleic acid and SFAs in the Hong Kong Chinese population. These findings enhance the understandings of dietary impacts on neovascular AMD and provide a context for future nutritional intervention studies.

Camelina sativa Oil, Fatty Fish, and Lean Fish Do Not Markedly Affect Urinary Prostanoids in Subjects with Impaired Glucose Metabolism.

Dietary fatty acids are suggested to affect oxidative stress; however, results from interventions have been inconclusive. The aim was to examine if fatty fish, lean fish, and Camelina sativa oil (CSO) affect the urinary prostanoid levels in subjects with impaired glucose metabolism. Altogether 79 participants aged 43-72 years completed a randomized controlled study lasting 12 weeks. There were four parallel groups, fatty fish, lean fish (four fish meals/week in both), CSO providing 10 g/day alpha-linolenic acid (ALA), and control diet with limited fish and ALA containing oil consumption. Urinary prostanoids (prostaglandin F2α , 5-F2t -isoprostanes and 15-F2t -isoprostane metabolites, isofuran, 8-F3t -isoprostanes, and 4-(RS)-4-F4t -neuroprostane) of 72 participants (age: mean (±SD) 58.9 ± 6.5 years; body mass index: 29.3 ± 2.5 kg/m2 ) collected over 12-h were measured using liquid chromatography tandem-mass spectrometry. Plasma phospholipid fatty acids were determined using gas chromatography. Our study showed that the proportion of ALA in plasma phospholipids increased in the CSO group (overall difference among the groups p-value <0.001). In the fatty fish group, proportions of eicosapentaenoic and docosahexaenoic acids increased (overall p-value <0.001 for both). Prostaglandin F2α was higher in the CSO group than in the control group (p < 0.05), however, there were no other significant changes in urinary excretion of other prostanoids among the study groups. At baseline, arachidonic acid in plasma phospholipids was positively (r = 0.247, p < 0.05) and ALA negatively (r = -0.326, p < 0.05) associated with urinary total isoprostanes. In conclusion, CSO, fatty fish, and lean fish consumption do not cause major changes in oxidative stress markers in subjects with impaired glucose tolerance.

Short-time UVA exposure to human keratinocytes instigated polyunsaturated fatty acid without inducing lipid peroxidation.

Short-term exposure to ultraviolet A (UVA) radiation can directly injure our skin through inflammatory response and indirectly through oxidative stress, triggering polyunsaturated fatty acid (PUFA) peroxidation in skin cell membrane and formation of DNA adduct, 8-hydroxy-2'-deoxyguanosine (8-OHdG). It is known that UVA exposure leads to photoaging, immunosuppression and skin cancer. However, the changes in PUFA and its oxidized metabolites, and cell cycle after short UVA exposure, are debatable. In this study, human keratinocytes (HaCaT) were exposed to low dose (5 J/cm2) and high dose (20 J/cm2) of UVA and assessed immediately, 8 h, 12 h, and 24 h post-treatment. Both doses showed a transient suppression in S-phase after 8 h of UVA exposure, and G2/M phase arrest after 12-h UVA exposure in the cell cycle but subsequently returned to normal cycle. Also, no observable DNA damage took place, where 8-OHdG levels were below par after 24-h UVA exposure. A dose of 20 J/cm2 UVA stimulated significant amount of arachidonic acid, n-3 docosapentaenoic acid, and docosahexaenoic acid (DHA) but lowered adrenic acid and eicospentaenoic acid after 24-h exposure. Among the 43 oxidized PUFA products determined, enzyme-dependent oxidized PUFAs, namely, 14-hydroxy-DHA (HDoHE) level reduced, and 8- and 13-HDoHE levels elevated significantly in a linear trend with post-treatment time. Out of the nonenzymatic oxidized PUFAs, a significant linear trend with post-treatment time was shown on the reduction of 5-F2t-Isoprostane (IsoP), 15-F2t-IsoP, Isofurans, 5-F3t-IsoP, Neurofurans, and 20-HDoHE. Our observations indicate oxidative stress through short UVA exposure on human keratinocytes did not have detrimental consequences.