Mechanisms of the neurodegenerative effect produced by ochratoxin A

The hypothesis of the key role of environmental factors on the development of Parkinson’s Disease (PD) has gained importance in recent years. Thus, some lines of research have set their focus on environmental neurotoxins, such as mycotoxins. Low doses of the mycotoxin ochratoxin A (OTA) have been reported to cause neurotoxicity. Indeed, previous in vivo studies with Balb/c mice demonstrated that OTA treatment p.o. (0.21 and 0.5 mg/kg of body weight) induced motor and dopaminergic (DA) alterations, associated with the phosphorylation of a-syn, at both intestinal and brain levels. These alterations were reported 6 months after the end of the OTA treatment. In vitro, it was observed that subtoxic doses of OTA caused a decrease in the levels of LAMP-2A, a protein involved in a-syn degradation, as well as an increase of a-syn levels, and its half-life, when treating intestinal and neuronal cell models with 100 and 200 nM OTA for 72 h. With this in mind, the present work is first focused on a description of the current available strategies for the assessment of the neurotoxic potential of a substance, as well as analysing if said strategies covers the endpoints of PD. To achieve this, the Organisation for Economic Cooperation and Development (OECD) test guidelines (TG) for the testing of chemicals and the European Food Safety Authority (EFSA) publications concerning neurotoxicity were reviewed. Among the different OECD TGs, only TG 424 collects methods for the specific evaluation of adult neurotoxicity (ANT). Most available strategies for the evaluation of neurotoxicity are centred on effects observable immediately after the exposure, yet there are not described strategies for the evaluation of long-term neurotoxic potential of a compound. Therefore, both EFSA and OECD organisms recognise the need for the development of on an in vitro test battery for the evaluation of neurodegeneration. A systematic search of scientific articles studying OTA neurotoxic effects was then carried out, analysing those articles according to the Adverse Outcome Pathway (AOP) of PD. Authors agree that in vitro and in vivo exposure to OTA causes mitochondrial dysfunction, impaired proteostasis, degeneration of DA neurons and neuroinflammation. However, further research might be needed to provide more information about OTA neurodegenerative potential and deepening in the elaboration of its neurotoxic profile. On another hand, this project aimed, through in vitro and in vivo studies, to unravel the timeline of the dysregulation of cellular processes, caused by OTA, that could be leading to mechanistical features observed in PD, as well as studying if these alterations are occurring at an intestinal level. To achieve this, proliferating and differentiated neuroblastoma cells, SH-SY5Y overexpressing wild type human a-syn (WT a-syn SH-SY5Y), were exposed to subtoxic concentrations of OTA (100 and 200 nM), for 24 h, 48 h and 72 h, and Balb/c mice were orally treated with 0.21 mg OTA/kg b.w. daily for 28 days, and sacrificed immediately after the end of the treatment, and 3 and 6 months after the end of the treatment (AET). In vitro, the effects of OTA treatment over a-SYN mRNA and protein levels were studied, as well as over the levels and/or activities of some molecules (LAMP-2A, LC3, p62, GBA, HEX, ß-GALACT, TFEB) involved in different a-syn degradation routes (chaperon-mediated autophagy (CMA) and macroautophagy). In vivo, the influence of OTA over a-syn and LAMP-2A expression was studied at both brain and intestinal level, as well as the presence of phosphorylated a-syn (p-syn) aggregates in midbrain. Effects of OTA over the nigrostriatal DA pathway were also assessed. Our results showed that OTA induced a time-dependent CMA dysfunction by decreasing the levels of LAMP-2A, in both proliferating and differentiated SH-SY5Y cells, starting at 24 h, and being worsen after longer OTA exposures (48 h and 72 h). A similar outcome was also observed in vivo, at brain and intestinal levels, being detected a reduction in LAMP-2A protein levels immediately after the OTA treatment, remaining downregulated over time (3- and 6-months AET timepoint). No alterations were detected in a-syn mRNA and protein levels both, in vitro and in vivo, but progressive apparition over time of p-syn aggregates in midbrain was observed in animals exposed to the mycotoxin. Also, a significant decrease in the total DA cell number was observed 3 months AET. Therefore, putting all this evidence together, we could state that exposure to subtoxic doses of OTA first causes malfunction of CMA at brain and intestinal levels (observable immediately AET and maintained throughout time), which promotes a-syn aggregation and this, in turn, leads to DA cell death (both detectable from 3 months AET). We also demonstrated, in vitro, that, when exposing SH-SY5Y cells to subtoxic concentrations of OTA, in presence and absence of bafilomycin A1 (BAF), an inhibitor of the autophagosomelysosome fusion, LC3 and p62 protein levels were increased, being this effect more pronounced in the presence of BAF. This suggested that exposure to OTA causes an increase in the formation of autophagosomes, indicating an enhancement of the macroautophagy route, possibly as a compensatory mechanism to face the CMA impairment.

Esta tesis se ha llevado a cabo gracias a la ayuda para la formación de personal investigador de la Asociación de Amigos de la Universidad de Navarra y al impulso recibido por la Obra Social “La Caixa.” Asimismo, esta tesis se encuentra enmarcada dentro del proyecto “Micotoxinas y Parkinson: ¿el eslabón perdido?”, financiado por el Gobierno de Navarra (Proyecto-43, modalidad A) y el Fondo de Desarrollo Regional Europeo (ERDF under Operational Program for Navarra 2014-2020).