Estudo da disfunção mitocondrial em modelos de doença de Parkinson in vitro e in vivo e perspectivas terapêuticas

Abstract

Parkinson disease (PD) is a neurodegenerative disease which symptoms are consequence of progressive neuron lost, especially dopaminergic neurons. Other feature related to PD is mitochondrial dysfunction which could target a deficit on the cellular energy maintenance. Experimental models in vitro and in vivo have been used to study PD. The aim of this work is to investigate mitochondrial bioenergetics, as well as, possible interferences with focus on mitochondrial functionality using DP models in vitro and in vivo. In the in vitro study we used slices of cortex, hippocampus and striatum exposed to 6-hydroxydopamine (6-OHDA), for 1 hour at concentration of 100 μM. In cortex brain slices 6-OHDA increased mitochondrial activity which is demonstrated by an increase of oxidative phosphorylation (OXPHOS) related to mitochondrial complex I (CI) and mitochondrial complex II (CII), also there was an increase on lactate dehydrogenase activity (LDH), implying an adaptative response. Striatum brain slices exposed to 6-OHDA presented a mitochondrial impairment demonstrated by decreased OXPHOS and decrease of electron flux related to ATP synthase. There was an increase in peroxide production in slices hippocampus slices exposed to 6-OHDA, but without adaptive response. The simultaneous exposure from brain slices to 6-OHDA and n-acetylcysteine (NAC) was able to counteract 6-OHDA effects on on electron flux in all brain structures analyzed. In the in vivo study we used Drosophila melanogaster with deletion of PTEN-induced putative kinase 1 (pink 1) gene as DP model, evaluating mitochondrial parameters and cellular energy maintenance during aging on the life stages of 3, 15 and 30 days of life. In our study pink1 gene lost decreased survival percentual and impaired the climbing behavioral during aging. Moreover, pink1 flies presented impairment of mitochondrial function evidenced on OXPHOS related to CI&CII and electron transfer (ETS) related to CI&CII at 15 and 30 days of life. Curiously, pink1 flies at 15 days of life presented a decrease of citrate synthase (CS) activity following by an increase of LDH activity and decrease of OXPHOS and ETS related to CII. On the other hand, there was an increase of CS activity and a decrease of LDH activity of pink1 flies at 30 days of life. Furthermore, there was an increase of peroxide levels in pink1 flies at 15 and 30 days of life. These results suggest a distinct bioenergetic profile of pink1 flies during aging, presenting as main result a possible change on energetic metabolism from oxidative to glycolytic pathway at 15 days. Due these results, we used a treatment with caffeine at concentration of 0.5 mg / mL in order to investigate if this xanthine, with antioxidant features, would have potential to reverse or improve parameters related to mitochondrial functionality in pink1 flies. Our results demonstrated that treatment with caffeine in pink 1 flies from the larval stage until 3rd day of adult life was able to increase the levels of OXPHOS CI & CII and ETS CI & CII, moreover increased the coupling efficiencies related to OXPHOS and ETS and improve the mitochondrial network organization. Therefore, this work demonstrates the mitochondrial functionality/ adaptation changes due two different models of PD, in addition, the results confirm mitochondrial function as a possible target for two recognized antioxidant substances, NAC and caffeine.