Cellular and Mitochondrial NAD Homeostasis in Health and Disease.

The mitochondrion has a unique position among other cellular organelles due to its dynamic properties and symbiotic nature, which is reflected in an active exchange of metabolites and cofactors between the rest of the intracellular compartments. The mitochondrial energy metabolism is greatly dependent on nicotinamide adenine dinucleotide (NAD) as a cofactor that is essential for both the activity of respiratory and TCA cycle enzymes. The NAD level is determined by the rate of NAD synthesis, the activity of NAD-consuming enzymes, and the exchange rate between the individual subcellular compartments. In this review, we discuss the NAD synthesis pathways, the NAD degradation enzymes, and NAD subcellular localization, as well as NAD transport mechanisms with a focus on mitochondria. Finally, the effect of the pathologic depletion of mitochondrial NAD pools on mitochondrial proteins' post-translational modifications and its role in neurodegeneration will be reviewed. Understanding the physiological constraints and mechanisms of NAD maintenance and the exchange between subcellular compartments is critical given NAD's broad effects and roles in health and disease.

Melatonin Mitigates Rotenone-Induced Oxidative Stress and Mitochondrial Dysfunction in the Drosophila melanogaster Model of Parkinson's Disease-like Symptoms.

Parkinson's disease (PD) is the second most common neurodegenerative disorder; however, its etiology remains elusive. Antioxidants are considered to be a promising approach for decelerating neurodegenerative disease progression owing to extensive examination of the relationship between oxidative stress and neurodegenerative diseases. In this study, we investigated the therapeutic effect of melatonin against rotenone-induced toxicity in the Drosophila model of PD. The 3-5 day old flies were divided into four groups: control, melatonin alone, melatonin and rotenone, and rotenone alone groups. According to their respective groups, flies were exposed to a diet containing rotenone and melatonin for 7 days. We found that melatonin significantly reduced the mortality and climbing ability of Drosophila because of its antioxidative potency. It alleviated the expression of Bcl 2, tyrosine hydroxylase (TH), NADH dehydrogenase, mitochondrial membrane potential, and mitochondrial bioenergetics and decreased caspase 3 expression in the Drosophila model of rotenone-induced PD-like symptoms. These results indicate the neuromodulatory effect of melatonin, and that it is likely modulated against rotenone-induced neurotoxicity by suppressing oxidative stress and mitochondrial dysfunctions.

Mitochondria-Related Apoptosis Regulation by Minocycline: A Study on a Transgenic Drosophila Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a very complicated and multifactorial neurological disorder having limited therapeutic interventions illustrated by the impairment in memory and cognitive function. Several lines of confirmation are stoutly connected with mitochondrial function perturbation as a significant causative factor in AD, while the molecular mechanisms involved in AD pathogenesis are still poorly understood. Minocycline, a well-known antibiotic, has confirmed efficacy against mitochondrial defects and oxidative stress as a neuroprotective effect. In view of this property, we examined the remedial effect of minocycline on AD. To attain insight into the molecular machinery responsible for AD pathogenesis, we preferred the UAS/GAL4 scheme for the development of AD in flies that overexpress the Aß42 protein in the brain of Drosophila. The warning signs like the declined lifespan, locomotion deficit and memory loss, impaired mitochondrial membrane potential, and increased caspase 3 expression with mitogen-associated protein kinases linked with AD pathogenesis were examined in the existence of minocycline. Minocycline halted the Aß42-induced symptoms including behavioral changes and altered the mitochondrial membrane potential along with apoptotic factors' protein expression (JNK/p-JNK and caspase 3). Thus, the current study could be functional to find out the role of minocycline in human Aß42-overexpressed transgenic AD flies.

Cross Talk between Mitochondria and Other Targets in Alzheimer's Disease.

Among the neurodegenerative diseases, Alzheimer's disease (AD) is a predominant public health issue, affecting 16 million people around the world. It is clinically manifested by the presence of amyloid plaques (Aß) and neurofibrillary tangles (NFT) within the brain. Due to intraneuronal processing, Aß interacts with cellular targets such as mitochondria, ER, and Golgi apparatus and hampers their normal functions. Alteration in the mitochondrial function, closely related to the production of reactive oxygen species (ROS), Ca+2 overload, and apoptosis in the brain, is one of the key pathological events studied in AD pathogenesis. It is also an important pivot for the intracellular interaction with ER and Golgi through signal transduction and membrane contact to regulate cell survival and death mechanism. Alteration in mitochondrial function is intimately connected with abnormal ER or Golgi function. Stimuli that enhance perturbation in the normal ER or Golgi organelles function can involve mitochondria mediated apoptotic cell death. In this review, we address the importance of the mitochondria and their cross talk with ER and Golgi in AD pathogenesis and animal models with a therapeutic strategy to improve the mitochondrial functions.

Effect of melatonin on Aß42 induced changes in the mitochondrial function related to Alzheimer's disease in Drosophila melanogaster.

Alzheimers's disease is one of the alarming neurodegenerative disease and it is of global concern.The hallmarks of the disease are the amyloid beta (Aß) aggregation and presence of neurofibrillary tangles (NFTs). The interaction of Aß with macromolecular targets affects the normal cellular functions. The amyloid peptide interaction with cellular surfaces may trigger the intracellular cascades of signalling. Interaction of Aß with mitochondria leads to the generation of free radicals. Many studies have suggested the involvement of mitochondrial dysfunction in Alzheimer's disease. Melatonin prevents mitochondria stress and by means of activating the antioxidant systems it protects the death of neurons. Although the study have been already conducted on Aß42 infused or injury induced animal models but till date there is no reports of such studies on transgenic model of Drosophila melanogaster. In the present study, we have taken UAS/Gal4 system for the development of transgenic flies that overexpress Aß42 in the brain of Drosophila. With the help of these transgenic flies we have analyse different experiments like behavioural parameters, oxidative stress parameters and protein expression through western blotting in the presence of melatonin. We have found that melatonin significantly ameliorated the toxicity caused by the Aß42 overexpression. Thus the present study could be beneficial to find out the role of melatonin in transgenic flies overexpressing human Aß42.

Dme-miR-314-3p modulation in Cr(VI) exposed Drosophila affects DNA damage repair by targeting mus309.

microRNAs (miRNAs) as one of the major epigenetic modulators negatively regulate mRNAs at post transcriptional level. It was therefore hypothesized that modulation of miRNAs by hexavalent Chromium [Cr(VI)], a priority environmental chemical, can affect DNA damage. In a genetically tractable model, Drosophila melanogaster, role of maximally up-regulated miRNA, dme-miR-314-3p, on DNA damage was examined by exposing the third instar larvae to 5.0-20.0 µg/ml Cr(VI) for 24 and 48 h. mus309, a Drosophila homologue of human Bloom's syndrome and predicted as one of the potential targets of this miRNA, was confirmed as its target by 5'RLM-RACE assay. A significant down-regulation of mus309 was observed in dme-miR-314-3p overexpression strain (myo-gal4>UAS-miR-314-3p) as compared with that in parental strains (myo-gal4 and UAS-miR-314-3p) and in w(1118). A significant increase in DNA damage including double strand breaks generation was observed in exposed myo-gal4>UAS-miR-314 and mus309 mutants as compared with that in parental strain and in unexposed control. A significant down-regulation of cell cycle regulation genes (CycA, CycB and cdc2) was observed in these exposed genotypes. Collectively, the study demonstrates that dme-miR-314-3p can mediate the downregulation of repair deficient gene mus309 leading to increased DNA damage and cell cycle arrest in exposed organism which may affect Cr(VI) mediated carcinogenesis.

Overexpression of hsp27 Rescued Neuronal Cell Death and Reduction in Life- and Health-Span in Drosophila melanogaster Against Prolonged Exposure to Dichlorvos.

Long-term exposure to dichlorvos (O,O-dimethyl-2,2-dichlorovinyl phosphate (DDVP), an organophosphate pesticide) is reported to exert neurotoxicity, i.e., generation of reactive oxygen species (ROS), oxidative damage, and neuronal cell death along with life- and health-span reduction in nontarget organisms including humans. However, studies on genetic modulation towards neuroprotection against prolonged DDVP exposure are elusive. Hsp27 (a small heat shock protein) is involved in various cellular processes and thus has attained emphasis as a therapeutic target. We aimed to examine the protective effect of hsp27 overexpression against prolonged DDVP exposure using an in vivo model Drosophila melanogaster. Flies were exposed to 15.0 ng/ml DDVP for a prolonged period to examine neuronal cell death, locomotor performance, and lifespan. After prolonged exposure, cell death, ROS level, glutathione depletion, nicotinamide adenine dinucleotide phosphate level (NADPH), glucose-6-phosphate dehydrogenase (G6PD), and thioredoxin reductase (TrxR) activities were examined in fly brain tissues at different days of age (days 10, 20, and 30). Flies with ubiquitous overexpression of hsp27 showed better resistance (improved lifespan and locomotor performance) in comparison to that targeted to motor neurons and nervous system. These flies also exhibited lesser intracellular ROS level and glutathione depletion by restoring G6PD activity, NADPH level, and TrxR activity in their brains thereby resisted neuronal cell death. Conversely, hsp27 knockdown flies exhibited reversal of the above endpoints. The study evidenced the neuroprotective efficacy of hsp27 overexpression against prolonged DDVP exposure and favored Hsp27 as a therapeutic target towards achieving better organismal (including human) health against long-term chemical exposure.

Efficacy of methuselah gene mutation toward tolerance of dichlorvos exposure in Drosophila melanogaster.

Adverse reports on the exposure of organisms to dichlorvos (DDVP; an organophosphate insecticide) necessitate studies of organismal resistance/tolerance by way of pharmacological or genetic means. In the context of genetic modulation, a mutation in methuselah (mth; encodes a class II G-protein-coupled receptor (GPCR)) is reported to extend (~35%) the life span of Drosophila melanogaster and enhance their resistance to oxidative stress induced by paraquat exposure (short term, high level). A lack of studies on organismal tolerance of DDVP by genetic modulation prompted us to examine the protective efficacy of mth mutation in exposed Drosophila. Flies were exposed to 1.5 and 15.0 ng/ml DDVP for 12-48 h to examine oxidative stress endpoints and chemical resistance. After prolonged exposure of flies to DDVP, antioxidant enzyme activities, oxidative stress, glutathione content, and locomotor performance were assayed at various days (0, 10, 20, 30, 40, 50) of age. Flies with the mth mutation (mth(1)) showed improved chemical resistance and rescued redox impairment after acute DDVP exposure. Exposed mth(1) flies exhibited improved life span along with enhanced antioxidant enzyme activities and rescued oxidative perturbations and locomotor insufficiency up to middle age (~20 days) over similarly exposed w(1118) flies. However, at late (≥30 days) age, these benefits were undermined. Further, similarly exposed mth-knockdown flies showed effects similar to those observed in mth(1) flies. This study provides evidence of tolerance in organisms carrying a mth mutation against prolonged DDVP exposure and further warrants examination of similar class II GPCR signaling facets toward better organismal health.