Polyunsaturated fatty acids in amyotrophic lateral sclerosis: role of DHA, peroxidative modifications and sexual dimorphism

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Cacabelos Barral, Daniel
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Portero Otín, ManuelPortero Otín, Manuel - ORCID ID
Pamplona Gras, ReinaldPamplona Gras, Reinald - ORCID ID
Universitat de Lleida. Departament de Medicina Experimental
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In the present work we focus into the potential relevance of PUFAs in some models and human samples from patients suffering amyotrophic lateral sclerosis (ALS). Due to its pathological implication, oxidative stress was our first goal. We started from simple oxidative methodology screening to search for an antioxidant substance (among 21 different candidates) available in a Mediterranean diet. The results demonstrated high heterogeneity in carbonyl (measured by DNP) accumulation, regarding the oxidative source, substrate suffering it and the antioxidant structure. Further, thanks to GC/MS and LCQTOF, we detailed the protection over specific accrual of protein and lipid peroxidation markers as well as lipid profile modifications (as % of total fatty acids -FA) in oxLDL thanks to those dietary compounds. Moreover, we demonstrated its in vitro relevance, in terms of survival, when two cell lines (HMEC-1, HepG2) were treated with this oxidized (and protected) compounds, and finally address in vivo importance of those findings, demonstrating decreased carbonyl and oxidative accumulation in hamsters under an atherogenic diet supplemented with antioxidants. Once described the protective effect of antioxidants and specific signatures found regarding lipid oxidation markers, we extend the study focusing in different ALS samples. From previous work, we demonstrated an altered docosohexaenoic acid (DHA) composition in different location for patients suffering sporadic ALS. Hence, we though necessary to define whether the enzymatic machinery aimed to synthesize DHA from its precursors, are affected in sALS. Interestingly, we found a tissue specific variation (spinal cord vs cortex), compatible with our previous FA results. Further, thanks to inmunohistochemistry, differential involvement was unveiled for motor neurons (MN) and surrounding glia. Therefore, trying to depict cellular contribution, we switch to a neuronal model (N2A under oxidative stressors and/or aggregation-prone-TDP-43) and a tissular one (OT). There, we showed decreased desaturase (Δ6) and drebrin expression as well as increased DHA synthesis and an unreported inverse correlation of drebrin loss and aberrant p-TDP-43 expression under oxidative conditions in the cell culture. In the OT model, lipidomic analysis showed specific accretion of 8-iso-PGF2α and NPD1 as well as increased DHA (and dramatically decreased precursors) and reduced AA concentrations (GC measured). Analysis of slice O2 consumption showed decreased O2 levels under excitotoxic treatment and alleviation by antioxidant (tocopherol) addition. Treatment of OT slices with Ω-3 precursors (better than final products) and DHA plus tocopherol ameliorated MNs number. Finally, we wanted to disclose PUFA’s implication and phenothype of an animal model (SODG93A) under a dietary intervention with opposed FA unsaturation levels. Not surprisingly, FA profile was difficult to be altered in nervous system, although subtle specific variations were found. More importantly, differences in survival and clinical manifestations, UPR (Ubiquitin inclusions), mt-DNA (8- oxo-dG) and protein oxidative modifications revealed sex as a relevant factor in lipid handling for this model. Hence, whereas male under a low PUFA diet showed increased survival, females lack this beneficial outcome. Last but not least, we wanted to dig deeper regarding this sexual dimorphism. For this purpose, we focused in mitochondria and analyzed spinal cord oxygen consumption, oxidative damage to proteins and lipid profile along disease progression and also in a neuronal model (N2A overexpressing SODG93A, treated with 17β-estradiol).We could demonstrate a clear sexual implication, with females having late onset clinical symptoms concomitant to an upgraded mitochondrial function and lower protein and mitochondrial damaged proteins compared with males. Finally, to further confirmed steroid potential as a protective element in disease progression, in vitro estradiol pretreatmet of N2A showed increased oxygen consumption, with no relation with the mitochondrial complex expression.
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