Parkinson’s disease (PD) is a neurodegenerative disorder characterized by fibrillar cytoplasmic aggregates of α-synuclein (i.e., Lewy bodies [LB]) and the associated loss of dopaminergic cells in the substantia nigra. But, mutations in genes such as α-synuclein (SNCA) account for only 10% of PD occurrences.  The exposure to environmental toxicants including pesticides (e.g. paraquat [PQ]) and manganese (Mn), are also recognized as important PD risk factors. Thus, aging, genetic alterations and environmental factors all contribute to the etiology of PD.  In fact, both genetic and environmental factors are thought to interact in the promotion of idiopathic PD, but the mechanisms involved are still unclear.  In this study, we report a toxic synergistic effect between α-synuclein and either paraquat or Mn treatment. We identified an essential role for central carbon (glucose) metabolism in dopaminergic cell death induced by paraquat or Mn treatment that is enhanced by the overexpression of α-synuclein. PQ “hijacks” the pentose phosphate pathway (PPP) to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. PQ also stimulated an increase in glucose uptake, the translocation of glucose transporters to the plasma membrane, and AMPK activation. The overexpression of α-synuclein further stimulated an increase in glucose uptake and AMPK activity, but impaired glucose metabolism. In effect, α-synuclein activity directs additional carbon to the PPP to supply paraquat redox cycling. Alternatively, Mn induces an upregulation in glycolysis and the malate-aspartate shuttle to compensate for energy depletion due to Mn toxicity. Mn treatment causes a decrease in carbon flow through the TCA cycle and a disruption in pyruvate metabolism, which are consistent with a dysfunctional mitochondria and inhibition of pyruvate dehydrogenase. The overexpression of α-synuclein was shown to potentiate Mn toxicity by glycolysis impairment by inhibiting aldolase activity. In effect, α-synuclein overexpression negates the metabolic response to alleviate Mn toxicity that results in an increase in cell death.

Alzheimer’s disease diagnosis is mainly based in the identification of the cognitive disorder in patients who have already overt the advanced stage of dementia, when is too late for some kind of therapeutic adjustment. Therefore, in order to improve early recognition of Alzheimer’s disease, novel approaches for biomarkers identification, such as metabolomics, are been developed and the potential of Fourier Transform Infrared Spectroscopy (FTIR) in the clinical field is receiving particular attention.

The present work aims to contribute to identification of the main pathological changes that occurred during neurodegeneration, by identifying plasma biochemical alterations that might be related to dementia and discriminate control from cognitive impaired samples, through FTIR analysis. Multivariate analysis was applied to spectra data (n=45). Plasma samples from cognitive impaired subjects presented a higher content of saturated lipids in relation to the unsaturated ones, which translates in high potential brain damage. It was also noticed the presence of carboxylic acids (usually related to lipid hyperoxidation), production of reactive carbonyls, and proteins structural and functional alterations. Differences in protein conformation were also identified between control and disease samples and were mainly related with occurrence of protein aggregates.Some changes were also associated with oxidative cellular damage in disease samples.

In conclusion, FTIR has potential to be applied in future not only for cognitive impairment diagnosis but also for identification of disease stage and prognostic evaluation, besides assessment of disease developing risk for control subjects.

Metabolomics has demonstrated a great potential in numerous biomedical research fields in the last years, such as the study of the underlying pathology of diseases, discovery of diagnostic biomarkers or drug development. Nowadays, the main challenge in metabolomics is to obtain comprehensive and unbiased metabolomic profiles due to the huge complexity, heterogeneity and dynamism of metabolome. For this purpose, mass spectrometry represents a very interesting analytical platform, since complexity of metabolome may be overcome through the use of different orthogonal separation techniques, including liquid chromatography, gas chromatography and capillary electrophoresis. Alternatively, direct mass spectrometry analysis, either by direct infusion or flow injection, has been postulated as an alternative in metabolomics, complementing hyphenated approaches. These techniques exhibit several advantages such as the ability for high-throughput screening, fast analysis and wide metabolomic coverage, since there is not exclusion of compounds due to the separation device.

The present work explores the potential of metabolomic platforms based on direct infusion mass spectrometry for metabolic fingerprinting of serum samples. The most important issues to be considered in this type of approaches were reviewed, including sample handling, comprehensive analysis, data processing, as well as further identification of metabolites and global characterization of metabolomic fingerprints.

Introduction

Spodoptera frugiperda (Lepidoptera: Noctuidae) is one of the main plagues of maize cultivation and it is resistant to the most used insecticides. Plants are important source of biopesticides due to their own mechanisms of interaction and diversified composition. Thus, fractions (hexane, acetate and hydro-ethanol ) of extracts from different parts of Caesalpinia pluviosa var. peltophoroides (Fabaceae) have insecticidal activity investigated. Chemical composition of the fractions were evaluated by UHPLC-HRFTMS, thereafter processed on MZmine 2.2 software  and exported to do multivariate statistical analysis (MSA) - O2PLS-DA (SIMCA-P 13.0). The dereplication of compounds were performed using DNP^© , Scifinder^® and our in house Caesalpinia database.

Results and Discussion

Six fractions were inactive and five displayed pupal or larval mortality. The O2PLS-DA analysis found a clear separation of inactive and insecticidal group (R²= 0,72) and indicated the metabolites highly correlated with the insecticidal property (VIP>1.95): eugenyl vicianoside; gluconic acid lactone and gallic acid.

Conclusions

Thus, through metabolomic strategies was possible to dereplicate the compounds most correlated with insecticidal property and this knowledge can be very useful to  development of new techniques to the crop pest control.

Acknowledgements

FAPEMIG and CNPQ for the financial funding and the AsterBioChem for the UHPLC-HRMS analysis.

It is recognized that altered metabolic states reports on the chronic and acute disease statuses. Decades of research have shown that metabolism is not a self-regulating network operating independently but rather heavily integrated into every cellular process and involved in organ system functions. Therefore global monitoring of metabolic processes is recommended for more comprehensive understanding of the initiation and advancement of disease. Mass spectrometry based metabolomics, in particular, demonstrates tremendous promise in delivering high throughput quantitative information on alterations in metabolism associated with disease onset/progression and response to pharmaceutical intervention. Recent advances in mass spectrometry and informatics tools have facilitated emerging in house OMICS platforms capable of translating biological output into viable therapeutic candidates and assist in stratifying patient populations. At BERG, we have implemented an industrial level high throughput metabolomics platform providing both high quality and depth of information allowing for reliable and broadest capture of the metabolome for the pre-clinical and clinical matrixes analyzed. Global metabolomics platform dedicated for theranostic and clinical studies as well as tracer metabolomics are harvested to facilitate CDx biomarkers discovery in a unique way. Highlights of the BERG’s in-depth patient stratification approaches as well as biology based drugs will be presented.