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. 2021 May;10(7):e12089.
doi: 10.1002/jev2.12089. Epub 2021 May 11.

Characterization of brain-derived extracellular vesicle lipids in Alzheimer's disease

Huaqi Su  1   2 , Yepy H Rustam  2 , Colin L Masters  1 , Enes Makalic  3 , Catriona A McLean  1 , Andrew F Hill  4 , Kevin J Barnham  1 , Gavin E Reid  2   5 , Laura J Vella  1   6
Affiliations

Characterization of brain-derived extracellular vesicle lipids in Alzheimer's disease

Huaqi Su et al. J Extracell Vesicles. 2021 May.

Abstract

Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimer's disease (AD). Substantial progress has been made in identifying positron emission tomography and cerebrospinal fluid biomarkers for AD, but they have limited use as front-line diagnostic tools. Extracellular vesicles (EVs) are released by all cells and contain a subset of their parental cell composition, including lipids. EVs are released from the brain into the periphery, providing a potential source of tissue and disease specific lipid biomarkers. However, the EV lipidome of the central nervous system is currently unknown and the potential of brain-derived EVs (BDEVs) to inform on lipid dyshomeostasis in AD remains unclear. The aim of this study was to reveal the lipid composition of BDEVs in human frontal cortex, and to determine whether BDEVs have an altered lipid profile in AD. Using semi-quantitative mass spectrometry, we describe the BDEV lipidome, covering four lipid categories, 17 lipid classes and 692 lipid molecules. BDEVs were enriched in glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we further report that BDEVs are enriched in ether-containing PS lipids, a finding that further establishes ether lipids as a feature of EVs. BDEVs in the AD frontal cortex offered improved detection of dysregulated lipids in AD over global lipid profiling of this brain region. AD BDEVs had significantly altered glycerophospholipid and sphingolipid levels, specifically increased plasmalogen glycerophosphoethanolamine and decreased polyunsaturated fatty acyl containing lipids, and altered amide-linked acyl chain content in sphingomyelin and ceramide lipids relative to CTL. The most prominent alteration was a two-fold decrease in lipid species containing anti-inflammatory/pro-resolving docosahexaenoic acid. The in-depth lipidome analysis provided in this study highlights the advantage of EVs over more complex tissues for improved detection of dysregulated lipids that may serve as potential biomarkers in the periphery.

Keywords: Alzheimer's disease; brain; exosomes; extracellular vesicles; frontal cortex; glycerophospholipids; lipid biomarkers; lipidome; polyunsaturated fatty acids; sphingolipids; tissue.

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Figures

FIGURE 1
FIGURE 1
Characterization of BDEVs from human frontal cortex. [(A) Western blot analysis. Equivalent amount of protein from human frontal cortex brain homogenates (Brain Total), brain homogenates after collagenase treatment (Brain+C) and BDEV suspensions (F1, F2, and F3) were subjected to SDS‐PAGE. Total proteins were visualized using stain‐free technology to ensure similar loading. Frontal cortex brain tissue homogenates, ‘Brain Total’ and ‘Brain+C’, were enriched in calnexin, while calnexin was not detectable in an equivalent amount of BDEV protein. Proteins typical of endosome derived exosomes, TSG101 and syntenin, were observed in F2, illustrating that F2 is enriched in exosome‐like vesicles. The densities in F1, F2 and F3 were approximately 1.02 g/ml, 1.08 g/ml and 1.17 g/ml respectively. Immunoblots images are representative of 8 independent CTL and 8 AD human tissue samples. (B) Transmission electron microscopy (TEM) of BDEV. All BDEVs from F2 were fixed with 1% (w/v) glutaraldehyde, negatively stained with 2% (w/v) uranyl acetate and visualized by a FEI Tecnai F30 transmission electron microscope. The zoomed‐out image (left) provides an overview of the BDEV suspensions with scale bar representing 500 nm. The close‐up image (right) shows clearer small, cup‐shaped BDEVs which is consistent with the morphology of vesicles with scale bar representing 200 nm. The TEM images of BDEVs in F2 are representative of the BDEVs from all samples (AD and CTL). AD = Alzheimer's disease, CTL = control, BDEV = brain derived extracellular vesicles]
FIGURE 2
FIGURE 2
Comparison of total lipid abundance between frontal cortex and the BDEVs in this tissue from control subjects. [(A) Mol% total lipid abundance distributions at the lipid category level. Four lipid categories, covering glycerophospholipids (GPs), sphingolipids (SPs), glycerolipids (GLs) and sterol lipids (STs) were included in this study. The inset shows the low abundant GL and ST categories for clarity. BDEVs contained significantly higher levels of GPs and corresponding lower levels of SPs in comparison to tissue. (B) Mol% total lipid abundance distributions at the lipid class level. A total of 17 lipid classes were identified in this study. The inset shows the low abundant PA, PI, PG, CL, Cer, Hex1Cer, Hex2Cer, sulfatide, MG, DG, TG and CE classes for clarity. BDEVs were found to be significantly enriched in PS lipids, making up approx. 30% of the total lipid abundance, compared to tissue (approx. 17%). Ganglioside lipids were significantly downregulated in BDEV compared to tissue. Data represent the average mol% total lipid abundances ± standard error of the mean. Statistical significance was determined by ANOVA followed by Sidak's multiple comparison test, with multiplicity adjusted p value < 0.01. ### Adjusted P value < 0.0001. CTL = control, BDEV = brain derived extracellular vesicles. N = 8 CTL subjects]
FIGURE 3
FIGURE 3
Comparison of PC, PE and PS lipid subclasses abundance and individual PS lipid molecules between tissue and BDEVs. [(A) Mol% total PC lipid subclass abundance distributions. A significant increase in diacyl‐PC was observed in BDEV vs. tissue, accompanied with a decrease in PC‐O. The inset shows the low abundant PC‐O, PC‐P, acyl‐LPC and LPC‐O for clarity. (B) Mol% total PE lipid subclass abundance distributions. Significant increase in diacyl‐PE was observed in BDEV, accompanied with decreased in acyl‐LPE. (C) Mol% total PS lipid subclass abundance distributions. A significant decrease was observed in diacyl‐PS and LPS‐O in BDEV relative to tissue, accompanied with an enrichment of PS‐O and PS‐P. (D) Mol% total PS lipid abundance distributions of individual PS molecules. Diacyl PS species, PS(36:2), PS(36:1) and PS(40:6) were significantly decreased in BDEV relative to tissue. An overall increase in ether PS species, including PS(O‐37:6), PS(O‐37:5), PS(O‐37:4), PS(O‐39:4), PS(P‐36:4), PS(P‐38:6) and PS(P‐38:5), was observed in BDEV. Only the most abundant lipid molecules in PS lipid class are shown for clarity. Data represent the average mol% total lipid class abundance ± standard error of the mean. Statistical significance was determined by ANOVA followed by Sidak's multiple comparison test, with multiplicity adjusted P value < 0.01.#Adjusted P value < 0.01, ## adjusted P value < 0.001, and ### adjusted P value < 0.0001. CTL = control, BDEV = brain derived extracellular vesicles. N = 8 CTL subjects]
FIGURE 4
FIGURE 4
Comparison of mol% total lipid abundance differences between BDEV from control versus Alzheimer's disease tissue. [(A) Mol% total lipid abundance distributions at the lipid category level. Four lipid categories, covering glycerophospholipids (GPs), sphingolipids (SPs), glycerolipids (GLs) and sterol lipids (STs) were included in this study. The inset shows the low abundant GL and ST categories for clarity. GPs were significantly decreased in AD vs. CTL BDEV, with a corresponding significant increase in SPs. (B) Mol% total PE lipid subclass abundance distributions. Diacyl‐PE was decreased and PE‐P was increased in AD BDEV. The inset shows the ratio of PE‐O and PE‐P to diacyl PE and a significant shift from diacyl‐PE towards PE‐P in AD. (C) Mol% total PE lipid abundance distributions of individual PE molecules. LPE(18:1) was increased in AD relative to CTL. A group of polyunsaturated fatty acid (PUFA) containing PE molecules, including LPE(22:6), LPE(22:4), PE(38:4), PE(40:6) and PE(40:4), were significantly decreased in AD. A group of the most abundant PE‐P lipids, including PE(P‐36:2) and PE(P‐38:4), was significantly increased in AD. Only the most abundant lipid molecules in each lipid class are shown for clarity. Data represent the average mol% total lipid class abundance ± standard error of the mean. Statistical significance was determined by ANOVA followed by Sidak's multiple comparison test, with multiplicity adjusted P value < 0.01. * Adjusted P value < 0.01, ** adjusted P value < 0.001, and *** adjusted P value < 0.0001. CTL, control; AD, Alzheimer's disease; BDEV, brain derived extracellular vesicles. N = 8 AD subjects and N = 8 CTL subjects]
FIGURE 5
FIGURE 5
Comparison of SM and Cer individual lipid molecules (mol% class) of BDEV from control versus Alzheimer's disease tissue. [(A) Mol% total SM lipid class abundance distributions. Significant decreases in SM(d36:1) and SM(d38:1), accompanied with an increase in SM(d42:2), predominantly the SM(d18:1_24:1) species, were observed in AD vs. CTL BDEV. (B) Mol% total Cer lipid class abundance distributions. Cer(d36:1) was found significantly lower in AD vs. CTL BDEV. Only the most abundant lipid molecules in each lipid class are shown for clarity. Data represent the average mol% total lipid class abundance ± standard error of the mean. Statistical significance was determined by ANOVA followed by Sidak's multiple comparison test, with multiplicity adjusted P value < 0.01. * Adjusted P value < 0.01, ** adjusted P value < 0.001, and *** adjusted P value < 0.0001. CTL, control; AD, Alzheimer's disease; BDEV, brain derived extracellular vesicles. N = 8 AD subjects and N = 8 CTL subjects]
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