Cholesterol secosterol aldehyde adduction and aggregation of Cu,Zn-superoxide dismutase: Potential implications in ALS

Lucas S Dantas,Adriano B Chaves-Filho,Fernando R Coelho,Thiago C Genaro-Mattos,Keri A Tallman,Ned A Porter,Ohara Augusto,Sayuri Miyamoto

doi:10.1016/j.redox.2018.08.007

Lucas S Dantas, Adriano B Chaves-Filho + Show 6 more

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https://doi.org/10.1016/j.redox.2018.08.007

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Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by degeneration of upper and lower motor neurons. While the fundamental causes of the disease are still unclear, the accumulation of Cu,Zn-superoxide dismutase (SOD1) immunoreactive aggregates is associated with familial ALS cases. Cholesterol 5,6-secosterol aldehydes (Seco A and Seco B) are reported to contribute to neurodegenerative disease pathology by inducing protein modification and aggregation. Here we have investigated the presence of secosterol aldehydes in ALS SOD1-G93A rats and their capacity to induce SOD1 aggregation. Secosterol aldehydes were analyzed in blood plasma, spinal cord and motor cortex of ALS rats at the pre-symptomatic and symptomatic stages. Seco B was significantly increased in plasma of symptomatic ALS rats compared to pre-symptomatic animals, suggesting an association with disease progression. In vitro experiments showed that both Seco A and Seco B induce the formation of high molecular weight (HMW) SOD1 aggregates with amorphous morphology. SOD1 adduction to ω-alkynyl-secosterols analyzed by click assay showed that modified proteins are only detected in the HMW region, indicating that secosterol adduction generates species highly prone to aggregate. Of note, SOD1-secosterol adducts containing up to five secosterol molecules were confirmed by MALDI-TOF analysis. Interestingly, mass spectrometry sequencing of SOD1 aggregates revealed preferential secosterol adduction to Lys residues located at the electrostatic loop (Lys 122, 128 and 136) and nearby the dimer interface (Lys 3 and 9). Altogether, our results show that secosterol aldehydes are increased in plasma of symptomatic ALS rats and represent a class of aldehydes that can potentially modify SOD1 enhancing its propensity to aggregate.

Highlights

Cholesterol is a neutral lipid found in the membranes of all mammalian cells
Secosterol aldehydes were significantly higher in blood plasma of symptomatic compared to pre-symptomatic Amyotrophic lateral sclerosis (ALS) animals (Fig. 2A)
The MS spectrum showed the presence of up to five modifications in the protein, differing from the intact protein by the addition of 402 Da (Fig. 6C and D). Such mass difference is consistent with the formation of Schiff base adducts between the secosterol aldehyde (M=418 Da) and basic amino acid residues, such as lysine, after a reduction step with sodium borohydride. These results suggest that secosterol aldehydes (Seco A and Seco B) produce covalently modified SOD1-adducts that are much more prone to undergo olygomerization into high molecular weight aggregates when compared to 4-HNE

Summary

Introduction

Cholesterol is a neutral lipid found in the membranes of all mammalian cells. The central nervous system is rich in cholesterol, presenting a concentration near to 20 mg/g in the brain and 40 mg/g in spinal cord, which represents about 23% of the total sterol present in the body [1]. Cholesterol, like all other unsaturated lipids, is susceptible to oxidation, giving rise to a variety of oxidized derivatives, collectively known as oxysterols [2,3,4]. This lipid can be oxidized by enzymatic and non-enzymatic mechanisms. Attention has been given to cholesterol 5,6-secosterol aldehydes (Seco A and Seco B) (Fig. 1), two electrophilic oxysterols known to be formed from cholesterol oxidation intermediates formed by O3 [9,10] and 1O2 [8,11].

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