Our brain is rich in lipids. Found in large amounts and a wide variety in the central nervous system (CNS), these hydrophobic molecules control the fluidity of cell membranes and form the myelin sheath, improving the transmission of electrical impulses, and also are signaling molecules. CNS lipids are highly polyunsaturated and therefore susceptible to oxidation by enzymes as well as by reactive oxygen species. Alterations of lipid metabolism in neurons and glial cells modulate processes linked to aging and neurodegenerative disease.

To investigate the link between lipidome alterations in the CNS and amyotrophic lateral sclerosis (ALS), a fatal disease characterized by progressive loss of motor neurons, RIDC Redoxoma researchers led by Professor Sayuri Miyamoto at the Instituto de Química of Universidade de São Paulo (USP) performed a comparative lipidomics analysis of motor cortex and spinal cord tissues from an animal model of ALS and showed that lipid metabolism is altered in symptomatic animals, with increased levels of cholesterol esters and decreased levels of cardiolipin in the spinal cord as the main lipid signatures of the disease.

These results suggest dysfunctional mitochondria in motor neurons and lipid droplets accumulation in aberrant astrocytes. Based on this, the researchers hypothesized that cholesterol esters are over synthesized in neurons, later accumulating in esterified form in astrocytes, as a neuroprotective mechanism against oxidative stress. The results of the study were published in the journal Scientific Reports.

“Our work confirms data from the literature showing increased cholesterol esters in ALS, but now with a more powerful tool to characterize the entire lipidome of the motor cortex and spinal cord tissues from an ALS model. We characterized about ten cholesterol esters and all of them are polyunsaturated. Increased cholesterol esters have also been shown in Alzheimer's disease. We are proposing a mechanism based on these findings that may be common to many neurodegenerative diseases,” Miyamoto said.

Cholesterol Esters in the CNS

The SOD1-G93A mice used in this study are transgenic animals that overexpress the human Cu/Zn-superoxide dismutase enzyme gene and develop amyotrophic lateral sclerosis. The researchers analyzed the motor cortex and spinal cord tissues of these animals in two stages: at 70 days, when they are still asymptomatic, and at 120 days when they already have the symptoms of the disease. As a control, they analyzed tissues from respective age-matched wild type animals (WT).

In the motor cortex, 285 lipid species were identified and quantified, which were sorted into 26 subclasses. In the older mice (SOD1-G93A120d and WT 120d), the researchers found an increase in sphingolipids, which are components of the myelin sheath and are related to age. At 120 days, the mice are young adults - their life expectancy is two to three years - and they are still in a neuronal development stage, which is accompanied by increased sphingolipids concentration.

The most interesting results were found in the spinal cord, where they identified and quantified 406 lipid species, sorted into 33 subclasses. Compared to both control and asymptomatic animals, symptomatic rats have six-fold higher levels of storage lipids, the cholesterol esters, and a significant decrease in cardiolipin concentrations.

Cholesterol esters accumulate due to increased cholesterol synthesis induced by a cellular redox state imbalance, involving, among other factors, mitochondrial dysfunction, which leads to increased production of reactive oxygen species. In the study, mitochondrial dysfunction was evidenced by the decreased levels of cardiolipin, which is a lipid found in the inner membrane of mitochondria, playing an important role in the structure and function of the organelles. Impaired energy metabolism has been observed in patients and ALS animal models. Reactive oxygen species generation, protein aggregation and mitochondrial dysfunction in motor neurons are hallmarks of the disease.

Storage lipid accumulation in the spinal cord of the same ALS animal model had already been evidenced in other studies by the isolation of astrocytes with aberrant features containing abundant lipid droplets, the contents of which were not characterized.

“Storage lipid synthesis is associated with cell survival and protection mechanisms. In general, cells subjected to stress conditions accumulate storage lipids. Little is known about the chemical composition of these lipid droplets. The droplets are believed to be predominantly triacylglycerols, popularly known as fat, which is the most common form of energy storage, and are found in abundance in adipose tissue. It is not common for the cell to store fatty acids in the form of cholesterol esters,” explains Adriano de Britto Chaves-Filho, one of the first authors of the paper. The function assigned to circulating cholesterol esters is cholesterol transport. In the central nervous system, the function of these lipids is poorly known.

The researchers now propose that cholesterol esters are the major components of lipid droplets in aberrant astrocytes. According to the model proposed in this paper, with disease progression, elevate reactive oxygen species and mitochondrial defects in neurons trigger lipid droplet formation.

According to the researchers, accurate analysis of lipid species as performed in this study may help to further explore the role of oxidative stress regulating lipid metabolism in neurodegenerative diseases and aging.

Lipidomics

Lipidomics is an emerging area of study when compared to other omics such as proteomics, genomics, and transcriptomics. It means the global analysis of all types of lipids in biological systems. Characterizing and quantifying lipidomes - the set of lipids in a cell - is a complex task, as lipids have a great diversity in chemical structure and have distinct properties. There are currently about 43,000 unique lipid structures annotated in the Lipid Maps Structure Database, which is the world's largest public lipid database.

The advance of the mass spectrometry technique, with the discovery of soft ionization sources, allowed the analysis of biomolecules, and with that, it advanced lipidomics. “With soft ionization, we can convert molecules from the liquid phase to the gas phase without breaking them,” explains the researcher. Mass spectrometry is the process of ionizing a sample, separating the ions by mass and charge, detecting the ions, and recording a spectrum.

In addition to the advancement in ionization, high-resolution mass spectrometers have also emerged, such as that acquired by RIDC Redoxoma in 2016, which, in addition to giving precision in structural determination of the molecules, has a high acquisition speed. “This equipment allows the acquisition of several spectra of intact and fragmented ions, allowing qualitative and quantitative lipidomic analyzes. And in our lab, postdoc Marcos Yukio Yoshinaga brought the know-how to interpret mass spectra and identify lipids,” Miyamoto said.

Software for lipid analysis still produces inaccurate results, with errors in lipid identification, so in this paper, the researchers made the manual identification of the lipids. According to Miyamoto, “All identified lipids have undergone rigorous molecular characterization, in addition we used internal standards to ensure more accurate quantification. So our data is very robust.”

The article Alterations in lipid metabolism of spinal cord linked to amyotrophic lateral sclerosis, by Adriano Britto Chaves-Filho, Isabella Fernanda Dantas Pinto, Lucas Souza Dantas, Andre Machado Xavier, Alex Inague, Marisa HG Medeiros, Isaias Glezer, Marcos Yukio Yoshinaga and Sayuri Miyamoto, can be accessed at https://www.nature.com/articles/s41598-019-48059-7