Microarrays, Enzymatic Assays, and MALDI-MS for Determining Specific Alterations to Mitochondrial Electron Transport Chain Activity, ROS Formation, and Lipid Composition in a Monkey Model of Parkinson’s Disease

Multiple evidences suggest that mitochondrial dysfunction is implicated in the pathogenesis of Parkinson’s disease via the selective cell death of dopaminergic neurons, such as that which occurs after prolonged exposure to the mitochondrial electron transport chain (ETC) complex I inhibitor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP). However, the effects of chronic MPTP on the ETC complexes and on enzymes of lipid metabolism have not yet been thoroughly determined. To face these questions, the enzymatic activities of ETC complexes and the lipidomic profile of MPTP-treated non-human primate samples were determined using cell membrane microarrays from different brain areas and tissues. MPTP treatment induced an increase in complex II activity in the olfactory bulb, putamen, caudate, and substantia nigra, where a decrease in complex IV activity was observed. The lipidomic profile was also altered in these areas, with a reduction in the phosphatidylserine (38:1) content being especially relevant. Thus, MPTP treatment not only modulates ETC enzymes, but also seems to alter other mitochondrial enzymes that regulate the lipid metabolism. Moreover, these results show that a combination of cell membrane microarrays, enzymatic assays, and MALDI-MS provides a powerful tool for identifying and validating new therapeutic targets that might accelerate the drug discovery process.This research has been supported by grants from the regional Basque Government ITI1454-22 awarded to the “Neurochemistry and Neurodegeneration” consolidated research group and ISCIII Spanish Ministry for Health PI20/00153 and co-funded by the European Union (ERDF “A way to make Europe”), a grant from the Ministry of Economy and Competitiveness (IPT-2011-1205) and Scholarship Program for the Transition from Educational to Occupational Word (UPV-Basque Government)

Structures and proton-pumping strategies of mitochondrial respiratory enzymes

Enzymes of the mitochondrial respiratory chain serve as proton pumps, using the energy made available from electron transfer reactions to transport protons across the inner mitochondrial membrane and create an electrochemical gradient used for the production of ATP. The ATP synthase enzyme is reversible and can also serve as a proton pump by coupling ATP hydrolysis to proton translocation. Each of the respiratory enzymes uses a different strategy for performing proton pumping. In this work, the strategies are described and the structural bases for the action of these proteins are discussed in light of recent crystal structures of several respiratory enzymes. The mechanisms and efficiency of proton translocation are also analyzed in terms of the thermodynamics of the substrate transformations catalyzed by these enzymes

Studies on the Electron Impact Induced Dissociation of Some Organic Compounds

The work discussed in the thesis can be considered In three separate sections: Part I: The Mass Spectra of some Diaryl Ethers. Part II: The Mass Spectra of some Naturally Occurring Phenolic Compounds. Part III: The Mass Spectra of some Carbohydrate Derivatives. Part I: The ethers used in this section are compounds containing fused aromatic rings and homologues of diphenyl ether with methyl substituents on the aromatic nuclei. The spectra of these compounds have been compared with that of diphenyl ether. The degradation of diphenyl ether by loss of carbon monoxide has been noted previously and the extent to which the corresponding reaction takes place in other ethers has been correlated with the structures of these compounds. Other correlations have been made in an attempt to explain the presence of rearranged ions which appear in the ortho-methyl ether spectra. Part II: Some derivatives of tubaic acid were investigated. It was found that mass spectrometric methods can Identify or help to identify the isoprenoid groups present In such molecules. This was confirmed by an examination of the rotenoids. The 2:3-dihydroxyrotenoids can be easily Identified by their mass spectra. Two examples are given of the partial determination of the structure of compounds of this class. An analysis has been mode of the spectra of some flavonoid compounds. Part III: A preliminary examination has been made of the mass spectra of some glycosides and sugar alcohols. Correlations have been made between spectra and structures in each case. Differences in the spectra of two methyl glycosides have been related to differences in the stereochemistry of the compounds. The spectra of all the isomeric inositols are recorded. Proposals have been made for the mechanism of degradation of these compounds

PROTECTIVE ROLE OF BACOPA MONNIERI ON INDUCED PARKINSON'S DISEASE WITH PARTICULAR REFERENCE TO CATECHOLAMINE SYSTEM

Objective: Parkinson's disease (PD) is the second most neurodegenerative disorder. Bacopa monnieri (BM), an Indian herb extensively used in Ayurveda, used in treatments related to neurological complications was used now in our study. Methods: In the present experiment, rats were divided into four groups of six in each group: group 1 received saline water, group 2 received rotenone (RT) through i.p. for 60 days to induce PD, group 3 received for 20 days orally before induction of PD and group 4 received Levodopa (Reference drug; LD) orally. Results: The levels Dopamine (DA), Serotonin (5-HT), Epineprine (EP), Nor-epineprine (NEP) were decreased and Monoamine oxidase (MAO) activity was increased in different brain regions such as Cerebral cortex (CC), Cerebellum (CB), Mid brain (MB) and Pons medulla (PM) during induced PD compared with controls. These results were reversed after treatment with ethanolic extract of BM on par with reference drug (LD). Conclusion: Our results suggest the ability of BM extract to modulate catecholamine system in different brain regions of RT induced rodent model of PD and thus offers protection. When compared overall the BM is better than the LD drug. The BM may provide a platform for future drug discoveries and novel treatment strategies in PD and can act as antiparkinsonian agent

Standardized Centella Asiatica Increased Brain- Derived Neurotrophic Factor and Decreased Apoptosis of Dopaminergic Neuron in Rotenone-Induced Zebrafish

Rotenone is a pesticide that is widely used to kill insects and nuisance fish in lakes. Its used as Parkinson’s Disease (PD) model inducer. The mechanism of toxicity of rotenone is primarily mediated by its potential as mitochondrial complex I inhibition. Centella asiatica (CA) is known as neurotonic, but how its potential protection in Parkinsonism is still unclear. In this study, we examined the effect of CA to Brain-derived Neurotrophic Factor (BDNF) as a neuroprotectant and apoptosis as hallmark of PD in rotenone-induced zebrafish (Danio rerio). Besides, we also measured the zebrafish motility and dopamine (DA) level in the brain. We used adult zebrafish (8 months). Its exposed to 5 g/L rotenone and co-incubated with methanolic extract of CA by several concentrations which are 2.5, 5 and 10 μg/mL for 28 days. Motility observed for 5 minutes at 0, 7, 14, 21 and 28 days. Measurement DA by ELISA, BDNF and apoptosis by immunohistochemistry. The results showed that CA significantly (p<0.05) increased motility and dopamine level in all concentration of extract. Interestingly, BDNF expression in 5 and 10 μg/mL groups had no significantly difference to the control group. Concentration 10 μg/mL could protect dopaminergic neuron from rotenone toxicity due to significantly (p<0.05) decreased compare to rotenone group. Together, these data suggest that methanolic extract of CA could protect Parkinsonian syndrome conserved dopaminergic neuron through increasing BDNF as neurotrophic factor

Isolation and seasonal variation of fruticin in fruits of false indigo-bush (Amorpha fruticosa L. Fabaceae) from Serbia

Fruticin (amorphin) is a constituent of the fruit of the false indigo-bush (Amorpha fruticosa L.), which belongs to the class of rotenoid glycosides, and shows several interesting pharmacological activities. The aim of this study was to isolate and chemically characterize this natural product, as well as to determine the optimal period of the year for A. fruticosa fruits collection. Fruticin was obtained by re-crystallization of the precipitate that formed after partial evaporation of the extract, prepared by 3-fold extraction of powdered plant material by chloroform - ethanol (1:1, v/v). The structure of the final product was determined by various techniques of instrumental analysis (NMR, UV and MS), and confirmed by comparing the obtained spectra with corresponding data in available literature. The content of fruticin in A. fruticosa fruit was determined by LC-DAD-MS, using the external standard method based on the constructed calibration curve. Limits of detection (LOD) and quantification (LOQ) were also determined. A substantial increase in fruticin content was observed during the ripening period (>50%). It has also been established that the optimal time for fruit collection is mid-December. Obtained results indicate that the content of fruticin in the A. fruticosa fruit is highly dependable on the time of collection. Since the biosynthesis of secondary plant metabolites is influenced not only by the time of collection, but by numerous other factors as well, additional studies are needed to define, with greater certainty, the conditions that are necessary for design of prospective efficient and sustainable production process

The Relation of Mitochondrial DNA Mutation with Mitochondrial Diseaseas in Coding Region

AbstractMitochondrial disorders are recognized in several metabolic and degenerative diseases, aging and cancer. Mitochondrial functional deficiency maybe caused by a decrease in the function of complex respiratory enzymes that can inhibit the oxidative phosphorylation chain (OXPHOS) for synthesis of ATP. Based on data MITOMAP in 2013, there are 211 of 466 mutations that have been reported in coding region. The largest number of mutation located on MT-COI region that have 34mutations. In particular, mutations in a subunit of COX have been described associate with various clinical phenotypes. Deficiency of COX is one of disorder that often leads to mitochondrial disease. Based on the database, the dominant disease on subunit of COI is dominated by prostate cancer. Moreover, the highest probability of mutation to the size of gene located on MT-NDI with 2.83%. OXPHOS deficiency which is caused by DNA mitochondrial mutation, mostly appears on subunit ND1. The dominant phenotype on subunit of NDI is Leber's hereditary optic neuropathy (LHON) diseases. This study has revealed that mutations in mitochondrial DNA were not only associated with predisposition to neuromuscular diseases but also to cancer and optical interference

The ND2 subunit is labeled by a photoaffinity analogue of asimicin, a potent complex I inhibitor

AbstractNADH:ubiquinone oxidoreductase (complex I) is the entry enzyme of mitochondrial oxidative phosphorylation. To obtain the structural information on inhibitor/quinone binding sites, we synthesized [3H]benzophenone-asimicin ([3H]BPA), a photoaffinity analogue of asimicin, which belongs to the acetogenin family known as the most potent complex I inhibitor. We found that [3H]BPA was photo-crosslinked to ND2, ND1 and ND5 subunits, by the three dimensional separation (blue-native/doubled SDS–PAGE) of [3H]BPA-treated bovine heart submitochondrial particles. The cross-linking was blocked by rotenone. This is the first finding that ND2 was photo-crosslinked with a potent complex I inhibitor, suggesting its involvement in the inhibitor/quinone-binding

Hypothyroidism decreases the biogenesis in free mitochondria and neuronal oxygen consumption in the cerebral cortex of developing rats

Thyroid hormone plays a critical role in mitochondrial biogenesis in two areas of the developing brain, the cerebral cortex and the striatum. Here we analyzed, in the cerebral cortex of neonatal rats, the effect of hypothyroidism on the biogenesis in free and synaptosomal mitochondria by analyzing, in isolated mitochondria, the activity of respiratory complex I, oxidative phosphorylation, oxygen consumption, and the expression of mitochondrial genome. In addition, we studied the effect of thyroid hormone in oxygen consumption in vivo by determining metabolic flow through C-13 nuclear magnetic resonance spectroscopy. Our results clearly show that in vivo, hypothyroidism markedly reduces oxygen consumption in the neural population of the cerebral cortex. This effect correlates with decreased free mitochondria biogenesis. In contrast, no effect was observed in the biogenesis in synaptosomal mitochondria. The parameters analyzed were markedly improved after T-3 administration. These results suggest that a reduced biogenesis and the subsequent reduction of respiratory capacity in free mitochondria could be the underlying cause of decreased oxygen consumption in the neurons of the cerebral cortex of hypothyroid neonates.This work was supported by Ministerio de Educaciín y Ciencia Grants SAF2004-06263-CO2-02 (to A.S.), SAF2004-06263-CO2-01, and SAF2007-62811 and Comunidad de Madrid Grant GR/SAL/0033/2004 (to A.P.-C.). Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas is funded by the Instituto de Salud Carlos III. T.B.R. is a recipient of a fellowship from the Fundaçâo para a Ciência e Tecnologia, Portugal (SFRH/BPD/26881/2006).Peer reviewe