Our body produces the vast majority of our cholesterol and only a small amount comes directly from the diet. Cholesterol is a type of lipid molecule and is a primary component of the membrane that surrounds each cell. It serves as a precursor for the biosynthesis of steroid hormones, bile acids and vitamin D. Cholesterol is present in high concentrations in the Central Nervous System. In the brain and nerve tissue, it is required for the formation of the myelin sheath, a fatty material that surrounds long portions of nerve fibers. Myelin insulates nerve cells and enhances the transmission of electrical signals throughout the nervous system’s circuitry.

However, like all unsaturated lipids, cholesterol is susceptible to oxidation, giving rise to a variety of oxidized derivatives, known as oxysterols. Singlet oxygen, ozone and free radicals oxidize cholesterol, generating hydroperoxides, epoxides and aldehydes, including cholesterol 5,6-secosterol aldehydes (Seco A and Seco B). These aldehydes are extremely reactive molecules capable of modifying proteins and inducing the formation of aggregates.

Studying an animal model of amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease, the team of Sayuri Miyamoto, professor at the Instituto de Química of Universidade de São Paulo (USP) and a member of the Center for Research of Redox Processes in Biomedicine (RIDC Redoxoma), showed that cholesterol aldehydes are ubiquitously present in the motor cortex and spinal cord of the animals, and they modify Cu,Zn-superoxide dismutase (SOD1) protein in vitro, leading to the production of high molecular weight aggregates. SOD1 is associated with familial cases of ALS. The results of the study were published in the journal Redox Biology.

"Our results are important because they show the formation in vivo of this reactive cholesterol derivatives in the brain. The presence of cholesterol in the brain is poorly explored and we have growing evidence in the literature and in the work done in our laboratory, indicating that oxidative changes and cholesterol metabolism in the Central Nervous System may be implicated in neurodegenerative diseases and aging”, said Miyamoto.

The Central Nervous System is particularly rich in cholesterol, with a concentration of about 20 mg/g in the brain and 40 mg/g in the spinal cord, which represents about 23% of the total sterol present in the body.

Amyotrophic lateral sclerosis

Considering the high abundance of cholesterol in the brain and spinal cord and the characteristics of its oxidation products, the researchers investigated both the presence of cholesterol aldehydes in neural tissues and plasma of ALS rats, and their potential to induce SOD1 aggregation.

First, the researchers sought to detect cholesterol aldehydes in the motor cortex, spinal cord and plasma of transgenic rats overexpressing the SOD1-G93A mutant, which leads them to develop amyotrophic lateral sclerosis (ALS). Using chromatographic techniques, the researchers investigated the presence of aldehydes in animals with and without the diseased and also in the symptomatic and pre-symptomatic stages.

The results showed that seco B (the most abundant form of cholesterol aldehyde) was significantly increased in plasma of symptomatic ALS rats compared to pre-symptomatic animals, suggesting an association with disease progression.

Regardless of the disease progression, however, researchers found cholesterol aldehydes, mostly detected in the form of seco B, in neural tissues and plasma of all animals analyzed. "It's a tiny amount, but we can detect it. It seems that there is a basal cholesterol oxidation in the Central Nervous System and this may be involved in other neurodegenerative diseases besides ALS", said Miyamoto.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects mainly adults aged 50-60 years, characterized by progressive dysfunction and death of motor neurons in the motor cortex, brainstem and spinal cord. The causes of the disease are still unclear. Whereas 90-95% of diagnosed cases have been characterized of sporadic origin, it is estimated that 5-10% of the cases are genetic, classified as familiar ALS. Among familial cases, 20 to 25% are caused by mutations in the gene encoding the enzyme copper / zinc superoxide dismutase (Cu / Zn-SOD, or SOD1).

SOD1 is one of the most important antioxidant defenses of the body, and it is present in the cytosol, nucleus, peroxisomes, and mitochondrial intermembrane space of eukaryotic cells. It catalyzes the dismutation of the free radical superoxide to hydrogen peroxide and molecular oxygen.

The mutation in SOD1 results in a gain of toxic function, which has been suggested to cause pro-oxidant effects and/or formation of cytotoxic aggregates. As in other neurodegenerative diseases, protein unfolding, a process in which they lose their three-dimensional structure, and protein aggregation are hallmarks of ALS.
According to Miyamoto, the aldehydes seco A and seco B can modify specific proteins in the brain, leading to the formation of protein aggregates. They are considered important intermediates in the pathogenesis of cardiovascular and neurodegenerative diseases and have already been detected in atherosclerotic tissue and in the brain of patients with Parkinson's disease (in this case, involved in alpha-synuclein aggregation).

To evaluate the ability of seco A and B to promote SOD1 aggregation, the researchers performed in vitro experiments, incubating the recombinant SOD1 with the aldehydes. They found that protein aggregation was greatly accelerated in the presence of the aldehydes, reaching a plateau at 12-24 h. This can be due to the hydrophobic nature of the secosterols that increases the hydrophobicity of protein regions where they are attached. Aggregation of unfolded proteins occurs through exposed hydrophobic surfaces.

"We compared cholesterol aldehydes with other aldehydes also derived from lipid oxidation and found that the latter also modify the proteins, but do not lead to extensive aggregation. The main difference between them is the hydrophobicity", said the researcher.

Using mass spectrometry to sequence the SOD1 aggregates, the researchers found that cholesterol aldehydes bind to the lysine amino acids at the electrostatic loop and nearby the dimer interface, corroborating studies in the literature that point out the involvement of these regions in aggregation. SOD1 is composed of two identical subunits that form a homodimer through hydrogen bonds and hydrophobic interactions.

Miyamoto said that more research is needed to detect in vivo the presence of SOD1-secosterol aldehyde adducts, as well as to identify other protein targets, since cholesterol aldehydes can modify any protein located nearby its site of formation.

The article Cholesterol Secosterol Aldehyde Adduction and Aggregation of Cu,Zn-Superoxide Dismutase: Potential Implications in ALS, by Lucas S. Dantas, Adriano B. Chaves Filho, Fernando R. Coelho, Thiago C. Genaro-Mattos, Keri A. Tallman, Ned A. Porter, Ohara Augusto and Sayuri Miyamoto, can be read in https://www.sciencedirect.com/science/article/pii/S2213231718303707