The analysis of drinking pattern in Asm−/− mice showed a higher alcohol preference on the two-bottle free-choice alcohol drinking compared to wt animals. The total fluid intake did not differ between Asm−/− and wt mice reflecting the specificity of the observed pattern of alcohol consumption. Interestingly, the BAC study has observed slightly reduced alcohol availability 3 h after alcohol administration in Asm−/− mice. This might indicate higher metabolic rate of alcohol in these animals compared to wt mice. These results might possibly explain the higher consumption of alcohol in the free-choice alcohol drinking paradigm in Asm−/− mice. In order to reach the same blood alcohol concentration (BAC) over time, Asm−/− mice may have to consume higher amounts of alcohol compared to wt animals. These data are in line with the data obtained in patients with chronic alcoholism. They show greater tolerance to alcohol due to an increase in the alcohol elimination rate and metabolic tolerance (41). Previous animal studies also confirm this hypothesis as alcohol-preferring rats are characterized by a significantly lower blood alcohol concentration compared to non-preferring controls (42), probably due to more rapid alcohol metabolism and higher activity of alcohol dehydrogenase (43).

The analysis of taste preference in Asm−/− mice showed a slightly lower preference of sucrose and lower avoidance to quinine in these animals compared to wt littermates, which reached significance only at high doses of both substances (20 g/dl and 5%). These data might reflect a higher aversion developing in Asm−/− mice and a reduced hedonic perception. Thus, the higher preference of alcohol in these animals compared to wt mice might indicate the specificity of alcohol drinking phenotype, which is unlikely due to the changes in the hedonic and aversion avoidance.

The exposure to stress on the model of forced swimming induced a decrease in alcohol consumption in wt and Asm−/− mice and overwrote the genotype-specific difference in the alcohol drinking pattern. The consumption of alcohol was reduced after stress in both Asm−/− and wt mice. These data are in line with previous studies. It is well-known that stress is a factor triggering alcohol consumption in humans as well as in animal models. However, human studies have observed ambiguous effects of stress on the pattern of drinking in both females and males. Even though the frequency of heavy drinking has positive correlation with the stress level, the frequency of moderate drinking decreased with an increase in stress levels (44, 45). The model of two-bottle free-choice alcohol drinking allows animals to self-titrate with alcohol and to maintain a moderate level of drinking as confirmed by BAC studies (46). Moreover, our data suggested that alcohol treatment enhances depression level in wt and particularly Asm−/− mice. Taking into account that FST may induce a depressive state (47), it may be suggested that Asm−/− mice reduce alcohol consumption after stress to possibly reduce the depression level.

It was proposed that re-balancing of brain sphingolipid composition after stress exposure might play a protective role against a stress-induced increase in alcohol consumption. Previous studies revealed a significant dysregulation of sphingolipid and phospholipid metabolism in rats after chronic unpredictable stress (48). In the prefrontal cortex, chronic unpredictable stress was followed by an increase in ceramide, lysophosphatidylethanolamine, and 38 carbon (38C)-lipid levels, and a reduction in sphingomyelin and dihydrosphingomyelin concentrations, phosphatidylethanolamine, ether phosphatidylcholine, and 36C-lipid levels. Similarly, a decrease in phosphatidylcholine and sphingomyelin, and an increase in phosphatidylserine, ceramide, and dihydrosphingomyelin levels were observed in the hippocampus of rats exposed to chronic unpredictable stress (48). As it was shown, various lipid species contribute to the development of addiction, and particularly alcohol dependence (18), and thus the reestablishment of lipid balance after stress exposure might determine the absence of increase in alcohol consumption in Asm−/− mice.

The analysis of LORR showed higher sedative effects of alcohol in Asm−/− mice compared to wt animals at day 1 and 8 reflecting the higher sensitivity of the CNS to alcohol. These data are in line with previous studies showing that mice with higher alcohol preference display higher initial sensitivity to alcohol, as measured by the static dowel task (49). Similarly, human studies also suggested a higher sensitivity to the subjective effects of alcohol in patients with a family history of alcohol use disorder (50). Interestingly, the latency and duration of LORR significantly decreased in wt animals and tended to decrease in ASM−/− mice at day 8 of the experiment, reflecting the development of alcohol tolerance.

Interestingly, the observed drinking phenotype of Asm−/− mice resembles the phenotype of mice with Asm overexpression, but not heterozygous Asm knockout mice (Asm+/−) as it was shown in our previous study (27). It might be proposed that the complete knockout of the ASM coding gene induces possible alterations in other pathways, particularly other mechanisms of ceramide synthesis, such as de novo pathway, in Asm−/− mice, which may independently contribute to elevated alcohol drinking levels. The de novo pathway was shown to mediate the elevation in ceramide synthesis in the liver during alcohol consumption reflecting that both, sphingomyelinase and de novo pathways, contribute to the ceramide response to alcohol treatment (51–53). Particularly, Tong et al. (54) showed that an inhibitor of the de novo ceramide synthesis, myriocin, inhibits a rise in liver ceramide level and the severity of alcohol-related steatohepatitis in rats exposed to chronic treatment with alcohol. However, single treatment of animals with myriocin does not completely block the observed alcohol-induced changes (51– 53). Thus, it might be speculated that the de novo pathway of ceramide synthesis determines the similarity in the drinking pattern of Asm−/− mice and animals with Asm overexpression. Moreover, recent data showed that pharmacological inhibition of Asm in mice by antidepressants amitriptyline and fluoxetine is followed by the reduction of lysosomal ceramide level, but the accumulation of ceramide in the endoplasmic reticulum (21, 55). It can be proposed that an increase in endoplasmic reticulum ceramide might serve as a compensatory mechanism balancing the ceramide level in ASM−/− mice. However, a variety of additional lipidomic mechanisms determining alcohol consumption, which include several interconnected lipid species besides ceramide (17, 18), might be altered in Asm−/− mice. Thus, further analysis of possible compensatory pathways is needed to clarify the exact pathways.

The analysis of anxiety/depression in Asm−/− mice showed higher anxiety of these animals in the EPM, but not in the OF test. These differences might be related to the specific features of behavior investigated in these tests. Thus, the behavioral pattern of animals investigated in the OF test is mostly based on the exploratory behavior and locomotor activity. In contrast, cue-related behavior is mostly investigated in the EPM (56). It has been proposed that emotionality is a multidimensional parameter, which can be explored from various perspectives. Thus, different kinds of environments, e.g., open spaces, illuminated or elevated areas, might yield different kinds of behavioral responses (57).

Our behavioral data do not replicate the previous findings showing lower anxiety and depression levels in Asm−/− mice manifested in the higher center time in the OF test and time spent in the light compartment of the light-dark box, as well as shorter latency of first eating in the NSF test and shorter immobility time in the FST (21). However, the study by Gulbins et al. (21) was performed on the animals aging up to 7 weeks, which do not develop any behavioral manifestations of Niemann-Pick disease and are not characterized by the excessive sphingolipid accumulation in the tissues. In the present study we used 8–10 weeks old Asm−/− mice, which might be considered as a model of an early stage Niemann-Pick disease. Investigation of these animals showed that they do not possess full symptom expression yet, but have a slight impairment of locomotor activity in the OF test. However, the difference in the age might determine sphingolipid accumulation in the tissues, which might reverse the low depression and anxiety phenotype observed in younger ASM−/− mice. Moreover, the reason of this dissociation might be also based on the differences in the experimental conditions. In our experiment the animals received alcohol/saline injections 30 min before all tests, which can be considered as a mild stress. However, the present investigation also revealed that stress might affect animal behavior and even overwrite the Asm genotypedriven differences (21).

The data on the anxiety/depression state in Asm−/− mice are in line with the clinical studies on patients with Niemann-Pick disease. Niemann-Pick disease is a rare neurovisceral lysosomal storage disorder with a heterogeneous phenotype. The pathogenesis of types A and B of this disorder are based on Asm deficiency followed by sphingomyelin accumulation in the large, lipid-laden foam cells, which are present in the liver, spleen, lymph nodes, adrenal cortex, lung airways, and bone marrow (58). To our knowledge, no data on the anxiety and depression state of these patients are available. However, enhanced anxiety is observed in the patients with Niemann-Pick diseases, type C (59), which is characterized by sphingolipid accumulation in biological tissues and organs due to the mutations in the NPC1 or NPC2 genes (60). It was shown that ASM activity in the cells of these patients is also reduced (61). Moreover, BALB/cJ Npc1 nih mice with NPC1 haploinsufficiency, an animal model of type C Niemann-Pick disease, are characterized by increased anxietylike behavior in early maturity. However, to our knowledge, the level of ASM activity in these animals was not measured (62). Thus, our data on the anxiety/depression state of Asm−/− mice, which might be considered as a model of Niemann-Pick disease type A and B, might add on to the current knowledge of the pathogenesis of this disorder.

The higher anxiety pattern in Asm−/− mice is in line with higher alcohol preference observed in these animals. Previous clinical data show that higher basal anxiety level might result in greater alcohol intake, which can promote alcohol abuse in humans (63). Moreover, a shared genetic risk for both anxiety and alcohol use disorder was observed in family studies (64, 65). Animal data also confirm these observations. For example, Spanagel et al. (66) showed that a high anxiety level in the EPM is associated with higher voluntary drinking in rats.

Interestingly, forced treatment with alcohol in a moderate dose (2 g/kg) had anxiolytic and depressogenic properties in Asm−/− mice. No such effects were observed in wt littermates. Anxiolytic effects of alcohol were manifested in an increase in the time spent, entries and locomotion in the open arms of the EPM. Depressogenic effects became evident by an increase in the floating time in the FST in Asm−/− mice. These data resemble our previous results on Asm+/- mice, which show only partial reduction of Asm activity in the brain. Similar anxiolytic and depressogenic effects of alcohol were observed in these animals (27). A reduction in anxiety level observed in homo- and heterozygous mice with reduced Asm activity are in line with literature. It was shown that rats preselected for high anxiety in the EPM demonstrate more pronounced anxiolytic effects of an alcohol injection compared to low anxiety animals (67). And vice versa, a rodent line preselected for high alcohol consumption showed stronger anxiolytic effects of alcohol in a battery of emotional tests (68, 69).

Similar depressogenic effects of alcohol, as it was shown in Asm−/− mice line, were also shown before (70, 71). A similar dissociation in the effects of alcohol on the emotional behavior was observed in our previous studies on mice with Asm overexpression (27). It should be noted that despite of the high comorbidity of anxiety and depression, certain pathways and neurological mechanisms of these disorders differ in the brain (72). Thus, our findings may suggest that Asmmediated mechanisms might at least partly contribute to the pathways determining this difference, particularly after alcohol treatment. Moreover, the schedule of alcohol administration might mediate this dissociation. The EPM test was performed after two injections of alcohol, and, thus, the anxiolytic effects of alcohol might refer to its acute action. However, the FST was performed after a series of five alcohol injections referring to a rather subchronic alcohol treatment that caused depressogenic effects. Thus, it might be proposed that the duration of treatment might also affect alcohol action in Asm−/− mice.

Our previous data showed that a depressed state in animals with Asm overexpression can be partially reversed by voluntary alcohol consumption (27). Naïve Asm−/− mice are also characterized by higher alcohol preference and anxiety level. As alcohol treatment in these animals has anxiolytic effects, it might be speculated that Asm−/− mice might consume more alcohol compared to wt littermates in order to reduce their anxiety level. It is well-known that individuals use alcohol to enhance coping with stress and to reduce manifestations of mood disorders, especially depression, and anxiety. Chronic moderate alcohol drinking might reduce physiological and behavioral signs of anxiety and depression in humans (73). On another hand, anxiety and depression might enhance alcohol consumption and higher alcohol intake is associated with lower levels of nervousness (74). Thus, our data show that alcohol might be voluntarily used by ASM−/− mice to control their anxiety level. We believe that our study provide an additional confirmation of the hypothesis on the contribution of Asm to alcohol instrumentalization of emotional state, showing that alcohol might be used voluntarily to control possible emotional effects of the Niemann-Pick disorder.

Previously we observed that the reversion of a depressed state in mice with Asm overexpression with alcohol was mediated by partial normalization of Asm activity and downstream monoaminergic deficiency in the nucleus accumbens of these mice (27). Thus, it was proposed that alterations in sphingomyelin pathway of ceramide synthesis might mediate the effects of alcohol on emotional state (28). In the present study we checked if other enzymes of the sphingomyelinase pathway of ceramide metabolism contribute to the mechanisms of alcoholinduced decrease in anxiety and an increase in depression level in Asm−/− animals. The activities of Nsm, a sister enzyme of Asm involved in ceramide production, and Ac and Nc, which catalyze ceramide degradation to sphingosine, were analyzed. It was observed that voluntary alcohol consumption does not affect the activities of these enzymes neither in Asm−/− animals, nor in wt littermates. Thus, it can be suggested that no additional compensatory changes in the sphingomyelinase pathway of ceramide synthesis develop in these animals, confirming the specific contribution of Asm in the observed phenotypes.

Altogether, the present findings might add on to our hypothesis of an involvement of Asm in the mechanisms of comorbidity between alcoholism and anxiety and depression. We suggest that the lack of Asm might facilitate the anxiolytic and depressogenic effects of alcohol as well as an enhanced drinking pattern in mice. Further analysis of specific mechanisms of this comorbidity in Asm−/− mice is needed as the understanding of particular pathways might allow developing a new therapeutic strategy for comorbid psychiatric disorders as well as pathogenetic mechanisms of type A and B Niemann-Pick disease.

#### DATA AVAILABILITY

The datasets generated for this study are available on request to the corresponding author.

#### ETHICS STATEMENT

All experiments were carried out in accordance with the National Institutes of Health guidelines for the humane treatment of

#### REFERENCES

animals and the European Communities Council Directive (86/609/EEC) and were approved by the local governmental commission for animal health (Regierung von Mittelfranken).

#### AUTHOR CONTRIBUTIONS

LK, JK, EG, MR, and CPM initiated the study. LK, CM, VE, and MP performed the experiments. LK, VE, CPM, and CM analyzed the data. MR provided the animals. LK and CPM wrote the manuscript. All authors discussed the manuscript.

### FUNDING

This research was funded by the Deutsche Forschungsgemeinschaft (DFG) grants MU 2789/8-2, KO 947/15-2, and GU 335/32-2 and by funding from the Interdisciplinary Center for Clinical Research (IZKF) Erlangen, Project E13. CM was an associated fellow of the research training group 2162 Neurodevelopment and Vulnerability of the Central Nervous System of the DFG (GRK2162/270949263).

alcohol-dependent patients. Alcohol Clin Exp Res. (2011) 35:1852–9. doi: 10.1111/j.1530-0277.2011.01529.x

niemann-pick C1 carrier status. Neurotherapeutics. (2016) 13:614–22. doi: 10.1007/s13311-016-0427-5

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Kalinichenko, Mühle, Eulenburg, Praetner, Reichel, Gulbins, Kornhuber and Müller. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Comparison of Single Phase and Biphasic Extraction Protocols for Lipidomic Studies Using Human Plasma

Matthew Wai Kin Wong1 , Nady Braidy 1 \*, Russell Pickford2 , Perminder Singh Sachdev 1,3 and Anne Poljak 1,2,4

1 Centre for Healthy Brain Ageing, School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia, 2 Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW, Australia, 3 Euroa Centre, Prince of Wales Hospital, Neuropsychiatric Institute, Sydney, NSW, Australia, 4 School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia

#### Edited by:

Maria Dolores Ledesma, Severo Ochoa Molecular Biology Center (CSIC-UAM), Spain

#### Reviewed by:

Margaret Ellen Flanagan, University of Minnesota Twin Cities, United States Elisabetta Albi, Università Degli Studi Di Perugia, Italy

> \*Correspondence: Nady Braidy<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Dementia, a section of the journal Frontiers in Neurology

Received: 05 March 2019 Accepted: 29 July 2019 Published: 21 August 2019

#### Citation:

Wong MWK, Braidy N, Pickford R, Sachdev PS and Poljak A (2019) Comparison of Single Phase and Biphasic Extraction Protocols for Lipidomic Studies Using Human Plasma. Front. Neurol. 10:879. doi: 10.3389/fneur.2019.00879 Lipidomic profiling of plasma is an emerging field, given the importance of lipids in major cellular pathways, and is dependent on efficient lipid extraction protocols. Recent attention has turned to plasma lipidomics as a means to identify potential diagnostic and prognostic biomarkers related to dementia, neuropsychiatric health and disease. Although several solvent-based lipid extraction protocols have been developed and are currently in use, novel and more efficient methods could greatly simplify lipid analysis in plasma and warrant investigation. Human plasma from normolipidemic adult volunteers was collected to evaluate three different solvent extraction protocols, including the classical Folch method, the methanol/tert-butyl methyl ether (MTBE) (Matyash) method, and a recent single-phase methanol/1-butanol (Alshehry) method. Extracted lipids were analyzed using liquid chromatography mass spectrometry (LC-MS) in positive and negative ion mode. Overall, more than 500 different lipids were identified in positive and negative ion mode combined. Our data show that the single phase Alshehry method was as effective as the Folch and Matyash methods in extracting most lipid classes and was more effective in extraction of polar lipids. Normalized peak areas of the Alshehry method were highly and positively correlated with both the Folch and Matyash methods (r 2 = 0.99 and 0.97, respectively). Within- and between- subject correlations were r = 0.99 and 0.96, respectively. Median intra-assay coefficient of variation (CV%) in positive mode was 14.1, 15.1, and 21.8 for the Alshehry, Folch and Matyash methods, respectively. Median Alshehry inter-assay CV (collected over 5 separate days) was 14.4%. In conclusion, the novel Alshehry method was at least as good as, if not better than the established biphasic extraction methods in detecting a wide range of lipid classes, using as little as 10 µL of plasma, and was highly reproducible, safer and more environmentally-friendly as it doesn't require chloroform.

Keywords: lipidomics, lipid extraction, mass spectrometry, plasma lipids, chromatography

### INTRODUCTION

It is well-documented that lipids have multiple structural and functional roles, including signaling (1, 2), maintenance of membrane structure (3, 4), myelin sheath formation (5, 6), neurotransmission (7), and protein interactions in both plasma and organs including the brain (8, 9). Physiological processes such as synaptic and mitochondrial function, and lipid raft formation are critically dependent on lipid composition (10– 12). By contrast, lipid by-products, particularly metabolites of arachidonic acid and lipid products of oxidative stress are drivers of inflammation (13). Consequently, the plasma lipidome has a considerable impact on the cellular lipid environment, vasculature function, and inflammatory and oxidative processes. The plasma lipid profile therefore also represents a lifestyle modifiable factor which can play a decisive role in the health state and maintenance of cognitive function during aging (14).

The field of lipidomics is constantly expanding, enabling high throughput analysis of lipid analytes in crude extracts for the study of health and disease. Recent advances in mass spectrometry have resulted in greater sensitivity, increased mass accuracy and faster scan speeds (15, 16). This has enabled greater sensitivity and better characterization of lipid changes in bodily fluids, cells, and tissue extracts, leading to renewed understanding of the role of different lipid classes in the pathobiology of diseases. Lipidomics has recently been applied to the study of dementia, where brain, CSF and plasma lipids have been identified as potential diagnostic and prognostic biomarkers for Alzheimer's disease (17, 18), frontotemporal dementia (19), as well as other common neuropsychiatric disorders, such as schizophrenia and bipolar disorder (20, 21).

The future of lipidomics lies in clinical application. As such, blood-based biomarkers are ideal since venepuncture is already routinely used in the clinic and is less invasive compared to CSF extraction via lumbar puncture. Additionally, it is much simpler to take repeat measurements of blood which may be useful for longitudinal analysis. We will discuss further the relationship of centrally derived biological signals with the periphery and vice versa in this paper. Although interest in lipidomics especially in the study of health and disease is expanding, there is still much room for improvement in lipidomic methodologies for research and clinical use. Since lipidomics often involves the study of 100 to 1,000 of individual biological samples within a single experiment, any improvement to the cost or efficiency of sample processing could have a considerable impact on streamlining plasma lipidomics research.

One key area in which improvement is possible is in the extraction process, whereby total lipids within plasma, tissue, and other biological extracts are isolated prior to further analysis. Solvent-based extraction systems must efficiently extract lipids present in biological samples while minimizing bias, lipid degradation/oxidation, or contamination from nonlipid components, such as sugars, peptides and amino acids. Therefore, effective and reliable identification and profiling of lipids in plasma is dependent on the efficiency of the lipid extraction protocol (22). The traditional Folch method (2:1 chloroform/methanol) (23), developed in 1957 is still used as a benchmark extraction process in many laboratories for a wide range of biological fluids, including, blood, tears, urine, saliva, cerebrospinal fluid, human milk, bronchoalveolar lavage fluid and sperm (22). The Folch method is based on the partitioning of lipids in a biphasic mixture of chloroform and methanol. Methanol disrupts hydrogen bonds between lipids and protein following addition of an organic solvent such as chloroform.

Alternative methods, such as the Matyash (tert-butyl methyl ether (MTBE)/methanol) (24), have gained popularity, in particular for the extraction of sphingolipids. Replacement of chloroform with MTBE demonstrated similar extraction of major lipid classes (22, 24). Recently, a single phase (1:1 1 butanol/methanol) extraction developed by Alshehry et al. (25, 26) has also been published. Both newer methods eliminate the need to draw lipids from the lower phase, thereby saving time compared to the Folch method, and are safer and more environmentally friendly as they do not use toxic chloroform. Moreover, the Alshehry method does not involve any biphasic solvent separation and could therefore be even more convenient, and potentially yield better lipid recoveries, than the Matyash method. The Matyash method has already been shown by independent laboratories to be as good, if not better, at extracting lipids as the Folch method (22, 24, 27). A recent comparative study of multiphasic methods has been published (28), however the single phase Alshehry method was only recently published and has not been compared with established biphasic methods.

In this paper, we examine the performance of the singlephase Alshehry extraction system and compare the results against the two biphasic extraction methods, the Matyash method and the traditional Folch method (**Figure 1**) in terms of coverage of lipids extracted and reproducibility from pooled plasma of healthy volunteers. Liquid chromatography mass spectrometry (LC-MS/MS) was used to identify the lipids extracted.

### METHODS

### Reagents and Chemicals

HPLC grade methanol was purchased from Thermo Fisher Scientific (Sydney, Australia). Analytical reagent grade 1-butanol was purchased from Asia Pacific Specialty Chemicals (APS) (Sydney, Australia). Other high grade solvents and reagents were used, including MTBE (Sigma Aldrich, Israel), chloroform (Honeywell, USA), acetonitrile (Honeywell, Korea), formic acid (Chem-supply, Australia), ammonium formate (Honeywell, Germany). Isopropanol was of LC-MS grade (Honeywell, Michigan, USA). SPLASH Lipidomix stable isotope labeled internal standards were purchased from Avanti (Alabaster, Alabama, USA). All other reagents were analytical reagent grade. MilliQ water was used for buffer preparation and had a minimum resistivity of 18m.

#### Internal Standards

Internal standards were purchased from Avanti (Splash Lipidomix, Alabaster, United States) and included phosphatidylcholine 15:0–18:1(d7) PC, phosphatidylethanolamine 15:0–18:1(d7) PE, phosphatidylserine 15:0–18:1(d7) PS, phosphatidylglycerol 15:0–18:1(d7) PG,

phosphatidylinositol 15:0–18:1(d7), PI, phosphatidic acid 15:0–18:1(d7) PA, lysophosphatidylcholine 18:1(d7) LPC, lysophosphatidylethanolamine 18:1(d7) LPE, cholesteryl ester 18:1(d7) CE, monoacylglycerol 18:1(d7) MG, diacylglycerol 15:0–18:1(d7) DG, triacylglycerol 15:0–18:1(d7)-15:0 TG, sphingomyelin 18:1(d9) SM and Cholesterol (d7). The Splash Lipidomix standards are deuterium labeled and not present endogenously, but with concentrations similar to physiological plasma concentrations of lipids for each class and thus are ideal for use as internal standards. A constant amount of internal standards was added to all samples and controls prior to lipid extraction (10 µL of a 1:10 dilution of SPLASH Lipidomix to 10µL neat plasma) for normalization of raw peak areas and to correct for differences in extraction and ionization efficiencies and matrix effects (29).

### Blood Collection

Our study utilized fasting EDTA plasma collected from 10 cognitively "healthy" subjects aged between 65 and 85 years with informed consent, as part of a larger population-based study known as Sydney Memory and Aging Study (MAS) (30, 31). Subjects did not have any forms of mild cognitive impairment or dementia, as assessed by a panel of neuropsychiatrists according to consensus diagnosis criteria (30, 32), All subjects had minimental state examination (MMSE) scores >24 (33) and no history of cardiovascular complications or diabetes mellitus. Plasma taken from the 10 subjects (five males and five females) were then pooled and aliquoted (10 µL). Extractions from 10 pooled plasma aliquots were used to assess the intraassay coefficient of variation (CV%) of each extraction method detailed below, while extractions taken on 5 separate days were used to assess inter-assay CV% of the Alshehry method. Blood collection, processing and storage were performed under strict conditions to minimize preanalytical variability (18, 34), which included restricting plasma collection and aliquot to freezer time to a maximum of 2 h, using EDTA plasma, and minimizing the number of freeze-thaw cycles. In particular, freezer storage of plasma and extracted lipids at −80◦C prior to analysis and limiting the number of freeze thaw cycles, is now part of international standardization guidelines in order to maximize stability of lipids over prolonged periods of time (18, 35, 36).

The study was approved by the Ethics Committees of the University of New South Wales and the South Eastern Sydney and Illawarra Area Health Service (ethics approval HC12313 and HC14327, respectively). All work involving human subjects conformed to the principles of the Declaration of Helsinki of the World Medical Association.

### Lipid Extraction Protocols

#### Single Phase 1-Butanol/Methanol 1:1 (v/v) Alshehry Extraction Method

Lipids were extracted as previously described (25, 26). Briefly, we added 10 µL of internal lipid standards (Avanti SPLASH Lipidomix) that had been first diluted 1:10 in 1 butanol/methanol, to 10 µL aliquots of plasma in 0.5 mL polypropylene tubes (Eppendorf). 1-Butanol/methanol (100 mL, 1:1 v/v) containing 5 mM ammonium formate was then added to the sample, vortexed (10 s), then sonicated (1 h). Tubes were centrifuged (13,000 ×g, 10 min) and the supernatant removed into a clean polypropylene tube. A further 100 µl of 1-butanol/methanol (1:1 v/v) was added to the pellet to re-extract any remaining lipids. The combined supernatant was evaporated by vacuum centrifugation and stored at −80◦ C prior to analysis by LC-ESI MS/MS.

#### Traditional Biphasic 2:1 Chloroform/Methanol (v/v) Folch Extraction Method

Lipids were extracted as previously described (23), but using a 20 fold scale down of volumes used in order to produce comparable results against the same volume of plasma as used in the Alshehry method. Briefly, 10 µL aliquots of plasma and 10 µL of internal lipid standards (Avanti SPLASH Lipidomix) that had been first diluted 1:10 in 1-butanol/methanol were added to 160 µl of ice-cold methanol followed by addition of 320 µl of ice-cold chloroform and vortexed (10 s), then sonicated (1 h). Tubes were centrifuged at 10,000 ×g (10 min). The upper aqueous phase was transferred into a fresh polypropylene tube and re-extracted by addition of 250 µl ice-cold chloroform/methanol as described above. The upper phase was discarded, and the organic phase combined from both extractions, which was then evaporated by vacuum centrifugation and stored at −80◦C prior to analysis by LC-ESI MS/MS.

#### Methanol-Tert-Butyl Methyl Ether Matyash Extraction Method

Lipids were extracted as previously described (24) with a 20 fold scale down of volumes; 10 µL aliquots of plasma and 10 µL of internal lipid standards (Avanti SPLASH Lipidomix) that had been first diluted 1:10 in 1-butanol/methanol were added to 400 µl of ice-cold methanol followed by addition of 500 µl of MTBE, vortexed (10 s), then sonicated (1 h). Afterwards, 500 µl of MilliQ water was added to induce phase-separation. Tubes were centrifuged (10,000 ×g, 10 min). The upper aqueous phase was transferred into a clean polypropylene tube and re-extracted by addition of 200 µl MTBE as described above. The upper phase was discarded, and the organic phase was evaporated by vacuum centrifugation and stored at −80◦ C prior to analysis.

#### LC-MS Analysis

Extracted lipid samples and lipid internal standards (Avanti SPLASH Lipidomix) were removed from the −80◦ C freezer and resuspended in 100 µl of 1-butanol/methanol (1:1 v/v) containing 5 mM ammonium formate and transferred into Chromacol autosampler vials containing a 300 µl glass insert. Lipid analysis was performed by LC ESI-MS/MS (Dionex LC system in-line to a Thermo QExactive Plus Orbitrap mass spectrometer; ThermoFisher Scientific; Waltham, Massachusetts). A Waters ACQUITY UPLC CSHTM C18 1.7µm, 2.1 × 100 mm column was used at a flow rate of 260 µL/min, using the following gradient: 32 to 100% solvent B over 25 min, a return to 32% B and finally isocratic 32% B (5 min) prior to the next injection. Solvents A and B consisted of acetonitrile: MilliQ water (6:4 v/v) and isopropanol: acetonitrile (9:1 v/v) respectively, both containing 10 mM ammonium formate and 0.1% formic acid. The first 3 min of eluent, containing sample salts, was diverted to waste. Product ion scans in positive and negative ion mode were performed on each sample, to maximize numbers of lipid species identified. Sampling order was randomized prior to analysis. LC-MS grade isopropanol was used to minimize intensity of the background signal.

### Alignment and Peak Detection Using LipidSearch 4.2.2

The raw data was aligned, and chromatographic peaks selected using LipidSearch software version 4.2.2 (Thermo Scientific, Tokyo, Japan), the database which now includes masses of the stable isotope labeled SPLASH Lipidomix internal standards. We performed search on raw files using the databases "General" and "labeled standards." For peak detection, recalc isotope was set to "ON," RT interval = 0.0 min. We used product search for LC-MS method and the precursor and product tolerances were set at 5.0 and 8.0 ppm, respectively. The intensity threshold was 1% parent ion, and the m-score threshold was set to 2.0. For quantitation, m/z tolerance was set at −5.0 to 5.0 ppm, and the retention time range was set at −0.5 to 0.5 min. The m-score threshold was 5.0, and all lipid classes were selected for inclusion. Ion adducts included +H, +NH4 for positive ion mode and –H, +HCOO for negative ion mode.

#### Data Analysis

The results from LipidSearch 4.2.2 were exported as a.csv file and opened in Excel software for further data processing and analysis. Peak areas of lipids in samples were normalized by dividing with peak area of the corresponding internal standard for that lipid class (i.e., peak area ratios). Correlations between normalized peak areas from the three extraction methods were calculated using the SPSS statistics software. The intra-assay coefficient of variation (CV) was calculated by dividing the standard deviation of the normalized abundances of each lipid (for each method) by the mean across lipid species. Lipid ion identifications were filtered using the LipidSearch parameters rej=0 and average peak quality>0.75. Recovery was calculated as the average raw intensity of internal standards spiked before extraction and divided by the average raw intensity of internal standards spiked post extraction. The inter-assay CV% for the Alshehry method was also determined by comparing measurements from extractions taken across 5 separate days within a month.

### RESULTS

#### Concordance Between Methods

When we compared the coverage of the different lipid classes based on extracted lipid internal standard peak areas, normalized peak areas in both positive and negative modes were highly and significantly correlated with the biphasic Matyash and Folch methods (r = 0.97 and 0.99 respectively, p < 0.0001, **Figures 2A,B**). This concordance was also reflected when comparing lipid measurements between duplicate extractions of plasma lipids prepared using the Alshehry protocol (r 2 = 0.995), and in two different subjects, which is indicative of high reproducibility (**Figures 2C,D**). Additionally, Bland-Altman analyses revealed that the Alshehry method largely agreed with the Folch and Matyash methods (bias ratio=1.09 and 1.247, respectively), except more for some lowly abundant peak areas, which were higher using the Alshehry method. All three methods produced similar number of LipidSearch molecular ion IDs prior to filtering (381, 400, and 390 IDs in positive ion mode for the Matyash, Folch and Alshehry methods respectively, and 122, 127, and 119 IDs in negative ion mode, similarly).

#### Comparison of Lipid Abundances

One-way ANOVA was used to test whether the raw peak areas for internal standards using the Alshehry method were significantly different from the Matyash or Folch methods. There were no significant differences in peak areas between extraction methods for DG, PE and SM species (p > 0.05). Peak areas of LPC, LPE, PI, PC and PG were higher in the Alshehry method than the Matyash method (p < 0.05, Tukey's post-hoc test), while the peak area of PG and PI were also greater in the Alshehry method relative to the Folch extraction. The Alshehry method was more efficient in extracting the highly polar lipids, such as LPC and LPE internal standard relative to the Matyash method (p < 0.05, one-way ANOVA, Tukey's post-hoc test) (**Figure 3A**).

Interestingly, TG was significantly higher in the Folch method compared to Matyash (p < 0.05, Tukey's post-hoc test), but yielded comparable, or slightly higher peak areas compared to Alshehry method, which did not reach significance (p = 0.08). Similarly, Alshehry peak areas of internal standards were larger than or equal to the Folch, and larger than the Matyash method in negative ion mode (**Figure 3B**, all p < 0.05, Tukey's post-hoc test). Overall, the Alshehry method produces similar, if not greater yields than either the Folch or Matyash method.

### Recovery of Internal Standards

Recoveries for each method were assessed by comparing the ratio of peak areas of internal standards spiked prior to and after extraction (**Figure 4**). The average recoveries were 99, 86, and 73% for the Alshehry, Folch and Matyash extractions, respectively. The highest recoveries for the Alshehry method were achieved with the phospholipids (>95%) and lowest for the less polar TG, DG lipids (<80%).

FIGURE 2 | Correlations of normalized peak areas between methods (A,B), subjects (C) and runs (D). Normalized peak areas of the Alshehry method were highly and positively correlated with both the Folch and Matyash methods. Within-subject correlation across separate runs was also high, though between- subject correlation was lower.

PI, phosphatidylinositol, PS, phosphatidylserine, SM, sphingomyelin; TG, triacylglycerol.

#### Reproducibility of Methods

We also compared intra-assay coefficients of variation (**Figure 5**), defined as the standard deviation of the peak areas across several samples in a single experimental batch divided by the mean of the peak area, expressed as a percentage. A lower CV% then corresponds to consistency of values obtained from extraction of technical replicates. The median intra-assay (repeat injections within a single experiment) and inter-assay (injections from samples processed across separate days) coefficients of variation (CV%) for all lipids in positive ion mode originally reported by Alshehry were 12 and 14% respectively (37). Here, we report median intra-assay and inter-assay CV of 14.2 and 14.4%, respectively for the Alshehry extraction, which is consistent with this previous report. The median intra-assay CV% we obtained using the Alshehry method was lower compared to that obtained for the Matyash and Folch methods (21.8 and 15.1% respectively, p < 0.0001 and p = 0.014 respectively, Mann-Whitney U test). Seventy-five percent of lipids in the Alshehry method had a CV of 21% or lower, and the median CV for internal standards was 8.5%. Median CV for lipids in negative mode were 12.7, 17.5, and 12.2% for the Alshehry, Matyash and Folch recipes respectively (p < 0.0001 for Folch and Alshehry relative to Matyash,

FIGURE 4 | Recoveries of internal standards for extraction methods. The values represent ratios of peak areas ± SD (error bars) relative to unextracted internal standards (i.e., spiked after solvents were added).

Mann-Whitney U-test). The comparative data collected in this experiment is summarized in **Table 1**. Typical representative total ion chromatograms of internal standards (**Figure 6A**), alongside extracted ion chromatograms for each standard (**Figure 6B**), as well as the total ion chromatogram for plasma lipids (**Figure 6C**) are shown.

#### DISCUSSION

In the original study by Alshehry et al. (25) the single phase 1-butanol/methanol 1:1 (v/v) method was reported to be as good an extraction as similar single phase extraction protocols with lipid measurements (based on peak areas normalized against internal standards). In particular, results between the Alshehry method and a single phase 2:1 methanol/chloroform

TABLE 1 | Results summary.

extraction method were strongly correlated (r 2 = 0.976). In the present study, both the Matyash and Folch methods were also strongly correlated with Alshehry method (r = 0.97 and 0.99, respectively) which suggests strong agreeance between the methods, also supported by Bland-Altman bias ratios. Overall, this suggests that methods correspond well, though the Alshehry method may be superior in detection of more lowly abundant lipid species. Number of extracted lipids were also similar between the methods indicating similar coverage of lipids for all three extractions.

Although most lipids classes were extracted similarly between methods, certain lipid classes were more abundantly extracted using the Alshehry method as opposed to the Matyash or Folch methods. This included the lysophospholipids LPC and LPE, as well as some glycerophospholipids containing a polar head group, such as PC, PI and PG. This is consistent with reports that the Alshehry method is more amenable to extraction of highly polar lipids compared to other methods (25), since the hydrophilic phases are discarded in these biphasic methods. A previous pilot analysis also showed markedly greater extraction of the highly polar lipid standards, phosphatidylserine, PS(17:0/17:0) and sphingosine phosphate, SoP(d17:1) in the Alshehry method compared to either the Matyash or Folch methods. The ability of the Alshehry method to capture more polar lipids may be particularly useful in analysis of

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time, and (C) corresponding plasma sample in positive ion mode. Typical retention time ranges of elution (minutes) are provided for various lipid classes.

neuropsychiatric disorders and dementia where these lipids have been implicated in these conditions (17, 38, 39).

Although the Alshehry recipe is ideal for extracting more polar lipids, the method is adaptable for extraction of other lipid classes. For example, a similar study of lipid extraction in cow's milk applied a modified version of the Alshehry single phase method in order to extract higher levels of non-polar lipids (40). This modified version used a single phase mixture of 1 butanol/methanol/chloroform (3:5:4 v:v:v) in order to broaden the coverage of highly lipophilic species, especially triglycerides. Other similar single-phase extraction procedures using different solvent ratios have been described for lipid extraction, such as a 3:1 butanol/methanol extraction for plasma (41) and animal tissue (42), as well as a methanol/MTBE/chloroform (1.33/1/1 v:v:v) extraction (43, 44). All these cases, report comparable, if not greater yields against the Matyash and Folch methods suggesting good feasibility of single-phase extraction methods against traditional biphasic methods, as well as adaptability to capture a wide range of lipids of interest.

Overall, there was strong recovery for a large majority of lipids with the single-phase method, at 99%, especially for polar lipids. The high recovery of the Alshehry method near 100% is consistent with the fact that this method does not experience the risk of dripping losses that occurs with the other two biphasic methods as the bulk of lipids are present in a single phase to handle and readily removable for lipid analysis.

The less than optimal recovery (73%) for the Matyash method compared to previously reported is likely related to the relatively low volume of solvent used during the 20-fold volume scale down procedure, as well as the tendency of MTBE to evaporate quickly, affecting extraction reproducibility (41), which is also reflected in the higher CV% (see below).

Analysis of reproducibility based on CV% revealed the Alshehry method had a median CV% of 14.2%, lower than or equal to the Folch method, and even better than the Matyash method (at 15.1 and 21.8% respectively, in positive ion mode). Thus, the Alshehry protocol produces reliable lipid measurements over technical replicates within experimental batches as well as, or even better than the established Folch and Matyash approaches, which is expected considering reduced dripping losses in a monophasic extraction. Additionally, the inter-assay CV% is very similar to that of the intra-assay CV% for the Alshehry method which suggests that the method produces reliable measurements across experimental batches and any additional variation to this method due to batch differences would be minimal.

Furthermore, the single phase Alshehry method is advantageous in that lipids are drawn into a single phase and can be easily removed from the sample as supernatant without picking up non-extractable matrix. This makes the method easier and potentially faster compared to conventional two-phase solvent partition systems where there is also an increased risk of contamination from unwanted analytes, and the risk of losses during transfer between phases. Additionally, the method can be applied to as little as 10 µl of plasma, and using just 100 µl of solvent per sample, and therefore greatly minimizes the amount of sample and solvent required for extraction. By contrast, the original Matyash extraction reports extractions using 200 µl of plasma, and requires 900 µl of MTBE and methanol combined pre-scale down, which translates to 18 ml of solvent required per plasma sample in the original Matyash method. Considering the broad range of lipids and reduced losses reflected in the high degree of recovery and reproducibility obtainable using a relatively small volume of sample and solvent in the Alshehry method, this represents a strong advantage of the method over existing biphasic methods. The volume savings can be particularly important for high value plasma samples from expensive clinical and population-based studies, since such samples are a finite resource and conservative usage is necessary, to maximize potential experiments and makes extraction of many samples much simpler to handle.

This new method has already been applied for lipid extraction of plasma from large populations for the study of health and disease (37, 45). Nevertheless, we highlight one important limitation of the Alshehry method as reported by the original authors (25): due to the monophasic nature of extraction, ionic contaminants are not readily removed during extraction, leading to an increased risk of ion suppression effects. The Alshehry method is therefore unsuitable for shotgun/direct infusion lipidomics techniques, but is suitable for use with reverse phase LC-ESI MS/MS.

It needs acknowledging that in context of neuropsychiatric disorders and dementia, it is important to establish the connection between putative changes in peripheral signals with that of signals generated by the central nervous system (CNS). Despite all the advantages associated with peripheral biomarkers, a limitation of plasma-based lipidomics research is that the blood brain barrier (BBB) restricts passage of metabolites between the CSF and blood compartments. Therefore, there is not always a strong correspondence between blood and centrally derived metabolites. Nevertheless, many CSF metabolites are selectively reabsorbed back into the vascular system, and essential lipids such as polyunsaturated fatty acids are transported to the brain from blood as free fatty acids (46) or as part of phospholipids, such as lysophosphatidylcholine (47), a lipid class well-detected using the novel method. The BBB may have increased permeability in response to inflammation or disease states (48, 49). Moreover, there is substantial overlap in metabolic changes identified between matched CSF and plasma (50, 51), and some blood lipids may be correlated with CSF tau and amyloid load (52), as well as other imaging measures such as brain atrophy (53).

Other studies have also identified relationships between plasma/serum lipids, apolipoproteins and cognition (54, 55). A recent study identified differential impacts of apolipoprotein E in the CNS and periphery, finding deficits in apolipoprotein E in the CNS induced synaptic loss, whereas a deficit in the periphery lead to impaired cognition (56), though synaptic loss could partially be rescued by increasing plasma apolipoprotein E levels. The potential of plasma lipids to predict memory performance and cognitive decline (or protection) several years in advance (57–59) presents evidence that these lipids may have a substantial influence on the CNS, or that plasma lipids may reflect some underlying pathological process occurring in the CNS. Many of the metabolites reported in the above studies are robustly extracted using the novel Alshehry method, such as sphingolipids, PC, PE and their corresponding lysophospholipids, and have also similarly been found to be altered in post-mortem brain tissue or CSF in patients with Alzheimer's disease (38, 50, 60). We have previously reviewed plasma lipids thought to be altered in Alzheimer's disease (18), which includes plasmalogen phospholipids, sphingomyelins and ceramides. Further, a recent untargeted lipidomics study in plasma found substantial differences in the lipidomic profile between healthy controls, Alzheimer's disease and behavioral variant frontotemporal dementia (bvFTD) (19). Of note, the authors were able to confirm hypertriglyceridemia in bvFTD at the lipid species level, and also detected altered levels of dietary lipids thought to characterize binge eating behaviors among these patients. This study, among others demonstrates the utility of exploring plasma lipids as potential biomarkers for neuropsychiatric and neurological disorders. Even with such a wealth of studies, independent validation and replication remains a high priority concern in the lipid biomarker research community (18). Further, the natural variation in lipids within and between individuals independent of disease is still being characterized (45, 61, 62), which is an important factor to consider before applying comparative lipidomics in disease. Although this pilot study does not focus on inter-individual differences in lipids, we have recently applied this particular method in a larger study of 100 individuals to identify the natural variation of the plasma lipidome in cognitively healthy individuals by age, sex and BMI (63). For future studies, we intend to investigate the influence of APOE genotype on the plasma lipidome, as well as identify lipidomic changes associated with vascular dementia. We believe that a consistent and simple method for plasma lipid extraction, such as explored in this paper, will go a long way toward expanding blood lipidomic studies, give due insight into neurological processes and minimize some of the variance in literature of reported biomarkers inherent in extraction methodology.

### CONCLUSION

Overall, we confirm the findings of the originally published Alshehry single-phase protocol, but also show that it is good as, if not better than the commonly used biphasic Matyash and Folch methods in the general extraction of plasma lipids, based on its ability to cover a broad range of lipid classes, including more polar lipids that may not be extracted readily using the Folch recipe. The Alshehry method also provides reliable and reproducible measurements when lipids are analyzed by LC-MS/MS above that of the Folch and Matyash methods. The single-phase method offers a safe, environmentally friendly (chloroform-free) and economical method for extraction of plasma lipids with high (nearly 100%) recovery that is a good substitute for the traditional approaches. Notably, all methods produce highly correlated results, indicating that reliable data can be obtained with all these protocols. However, the simplicity involved in having most lipids extractable in a single phase and low plasma and solvent volumes required by this Alshehry method may be particularly useful where sample volumes are limited, for projects where high sample numbers need to be assayed for lipidomic analysis, or where plasma needs to be conserved to maximize experiments.

### DATA AVAILABILITY

The datasets generated for this study are available on request to the corresponding author.

### ETHICS STATEMENT

The study was approved by the Ethics Committees of the University of New South Wales and the South Eastern Sydney and Illawarra Area Health Service (ethics approval HC12313 and HC14327, respectively). All work involving human subjects conformed to the principles of the Declaration of Helsinki of the World Medical Association and both informed and written consent was obtained.

### AUTHOR CONTRIBUTIONS

MW, NB, and AP wrote and drafted the manuscript. NB, RP, AP, and PS conceived of the project. MW performed the experiments and analyzed the data. RP provided technical expertise and training for use of the QExactive Plus mass spectrometer and LipidSearch software.

### FUNDING

This work was supported by a National Health & Medical Research Council of Australia Program Grant (APP1054544). MW was the recipient of an Australian Postgraduate Award (APA) from the Australian Commonwealth Government. NB was the recipient of the Australian Research Council Postdoctoral Research Fellowship. The authors thank the Rebecca Cooper Medical Research Foundation for their ongoing financial support.

### ACKNOWLEDGMENTS

The authors would like to acknowledge the contributions of Dr. Kristan Kang and Mahboobeh Hosseini, who provided data collection and biobanking for plasma samples, as well as Ms. Angela Russell and Dr. Sophia Dean, who provided administrative support. The authors also thank the Mark Wainwright Analytical Center in conjunction with the Bioanalytical Mass Spectrometry Facility for providing use of the QExactive mass spectrometer, analytical grade reagents and data processing software. The authors would also like to thank CHeBA and SEALs Pathology (Prince of Wales Hospital) for handling and preparing of the MAS samples, as well as the MAS research teams and participants for making this project possible.

in a community cohort of older individuals. PLoS ONE. (2012) 7:e34078. doi: 10.1371/journal.pone.0034078

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Wong, Braidy, Pickford, Sachdev and Poljak. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Plasma Lipid Profiling Identifies Biomarkers of Cerebral Microvascular Disease

Ida Azizkhanian1 , Sunil A. Sheth2 , Anthony T. Iavarone3 , Songmi Lee2 , Visesha Kakarla4 and Jason D. Hinman4 \*

*1 New York Medical College School of Medicine, Valhalla, NY, United States, 2 Department of Neurology, UT Health McGovern School of Medicine, Houston, TX, United States, 3 QB3/Chemistry Mass Spectrometry Facility, University of California, Berkeley, Berkeley, CA, United States, 4 Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States*

Brain-specific sphingolipids (SLs) may serve as effective biomarkers of white matter hyperintensities (WMH). Here, we investigate the efficacy of SLs as a novel fluid-based biomarker to identify WMH reflective of chronic ischemia. Patients presenting to our stroke center for evaluation of acute neurological deficits were enrolled in the Advanced Serum Profiling in Recent Stroke (ASPIRE) study. From this cohort of 202 individuals, 58 patients who underwent an MRI and did not have a clinical stroke event were included in this study. Plasma samples were collected at the time of MRI, and targeted SL profiling was performed by HPLC/tandem mass spectrometry. T2 FLAIR imaging was evaluated for WMH and scored according to the Fazekas scoring (FS) method and manually quantified. Twenty two SLs were definitively identified, consisting of ceramide (Cer) and sphingomyelin (SM) species. Of these, two sphingolipids, SM 38:1 and Cer 34:1, significantly correlated with high FS (*r* = 0.287, *p* = 0.029, and *r* = 0.356, *p* = 0.006, respectively) and were used in subsequent analysis. SM 38:1 (OR 1.129, 95% CI 1.032, 1.236, *p* = 0.008) and Cer 34:1 (OR 1.118, 95% CI 1.031, 1.212, *p* = 0.007), accurately differentiated between FS 0–2 vs. 2.5–6 in regression analysis. A combined lipid score demonstrated fair discrimination in ROC analysis (AUC = 0.729, *p* = 0.003) and was cross-validated using leave-one-out analysis. Plasma levels of brain-specific SLs may serve as effective biomarkers of subacute white matter disease.

#### Reviewed by: *Michael Alosco,*

Edited by: *Fanny M. Elahi,*

*United States*

*Boston University, United States Magda Tsolaki, Aristotle University of Thessaloniki, Greece*

*University of California, San Francisco,*

\*Correspondence: *Jason D. Hinman<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Dementia, a section of the journal Frontiers in Neurology*

Received: *26 May 2019* Accepted: *16 August 2019* Published: *29 August 2019*

#### Citation:

*Azizkhanian I, Sheth SA, Iavarone AT, Lee S, Kakarla V and Hinman JD (2019) Plasma Lipid Profiling Identifies Biomarkers of Cerebral Microvascular Disease. Front. Neurol. 10:950. doi: 10.3389/fneur.2019.00950* Keywords: sphingolipids, stroke, white matter (WM), biomarker, ceramide (CER), sphingomyelin (SM)

### INTRODUCTION

White matter hyperintensities (WMH) detected on T2 weighted images are associated with white matter damage and can be identified in 11% of adults over 60 and in 94% of adults over 82 (1). While age is the most consistent risk factor for WMH; diabetes, smoking, and hypertension are also independent risk factors, suggesting that WMHs are pathological rather than a part of the normal aging process (2). A meta-analysis of 22 imaging studies evaluating WMH shows that these lesions are associated with increased risk of stroke (hazard ratio 3.3) and dementia (hazard ratio 1.9) (3). Increased WMH load is also associated with increased severity of cognitive dysfunction (4). WMH are associated with poor recovery after stroke and hemorrhagic transformation after thrombolysis (3, 5–9). Other evidence supports an association with gait disturbances and impaired balance, putting those with significant WMH load at risk for falls and hospitalizations (2). Populations with limited access to healthcare and MRI studies may have underreported WMH and unrecognized cognitive impairment mandating alternative detection methods for WMH in these populations to promote screening, early intervention and appropriate management to prevent complications and hospitalizations.

Existing blood biomarkers for cerebral vascular disease are not specific to WMH and are not reliably detected in subclinical, or early stages of disease (10). While BNP and CRP are elevated in acute stroke and have shown modest ability to predict risk of future stroke, these biomarkers fail to reflect current extent of cerebral injury (11, 12). Non-specific inflammatory markers such as CRP, TNF, and IL-6, have not been shown to relate to WMH load (13). While some markers of endothelial damage, such as von Willebrand factor and intercellular adhesion molecule 1 are not associated with increasing WMH volume, others, including circulating vascular adhesion molecule-1, are (13, 14). Therefore, the role of different inflammatory pathways in the progression of WMH remains unclear. Serum neurofilament light chain (sNfL) has been recently shown to be a brain-specific biomarker for acute ischemic stroke (15, 16). While sNfL levels are reported to increase with acute ischemic damage and WMH, levels also increase in other neurological disorders including motor neuron disease (MND), multiple sclerosis (MS), and Alzheimer's disease (AD) (17–20). Because AD, MND, and WMH are all more prevalent in the older patient population, sNfL can only be used as a non-specific biomarker for WMH.

Tissue pathology from regions of WMH reveals demyelination, axonal damage, and mild gliosis (21), arising from chronic microvascular dysfunction. Oligodendrocyte vacuolization around venules in perivascular spaces contributes to loss of myelination, axonal damage and white matter fiber disruption in regions of WMH. Both histological and diffusion tensor imaging evidence suggest that white matter changes extend into a penumbra surrounding the region of WMH detected by routine MRI (22–24). Therefore, MRI may underestimate extent of brain injury and can fail to detect early changes. Plasma biomarkers that reflect direct damage to white matter may serve a critical role in identifying early white matter injury.

We previously demonstrated that sphingolipids (SLs) are highly enriched in the brain and that plasma SLs are sensitive markers of acute ischemic cerebral injury. SLs, and in particular one subset of SLs known as sphingomyelins, are known to be key components of cerebral white matter, as major constituents of the cerebral myelin sheaths that contribute to membrane fluidity and structural integrity (25). In this study, we determine the ability of circulating SLs to serve as accurate biomarkers of WMH.

#### SUBJECT/MATERIALS AND METHODS

#### Ethics Statement

Research involving human subjects was approved by the Institutional Review Board of the University of California, Los Angeles (IRB # 14-001798) and was conducted in compliance with the Health Information Portability and Accountability Act. A board-certified Neurologist interviewed all patients to determine the capacity of each individual participant to provide informed consent. If in the opinion of the physician the patient suffered from a compromised ability to provide consent, a surrogate consent procedure was instituted whereby the next of kin or legally authorized representative was given the opportunity to consent on the patient's behalf. Formal written consent was obtained for all participants prior to the collection of blood samples.

### Study Design

This cohort study was prospectively designed to build from the results of our previous discovery-phase study, to validate findings on lipid biomarkers of acute ischemic stroke in a larger, real-world clinical cohort. Consecutive participants were patients presenting to the Ronald Reagan UCLA Emergency Department with symptoms concerning for stroke between December 2014 and June 2016 and who were offered the opportunity to participate in the study. Patients were included in this study if they had onset of stroke symptoms within 8 h of presentation (or within 2 h of presentation if symptoms were present upon awakening from sleep and the time of symptom onset was unknown); were >18 years of age and were able to offer informed consent or had a suitable surrogate individual who could consent on their behalf. The final clinical diagnosis was based on the results of a detailed evaluation of each patient by the Neurovascular Neurology service. Blood samples were collected by peripheral vein venipuncture into heparin-containing tubes. Samples were kept on ice and then centrifuged immediately at 13,000x g for 5 min at 4◦C. The plasma was collected and aliquoted into freezer vials for storage at −80◦C. Subjects with evidence of CNS infection, known CNS malignancy, or recent head trauma as a potential cause of neurologic symptoms were excluded. There were no other neurological conditions that met criteria for exclusion. In this pre-specified post hoc subset analysis, patients were included if they underwent MRI at the time of blood collection, and were not ultimately diagnosed as having an acute ischemic stroke or TIA. Descriptive statistics are presented on the cohort as a whole, and then for the purposes of model derivation to identify potential biomarkers of subacute cerebral microvascular disease, only stroke mimic cases were used and a leave-one-out validation was performed. Patients with stroke mimics had a range of pathologies including migraines, brain tumors, and syncope (**Table 1**).

#### Sphingolipid Extraction

Lipid extraction and detection were performed in an identical manner as previously reported (26). Briefly, 20 µL of plasma was placed in 13 × 100 mm screw-capped borosilicate glass test tubes with Teflon caps (Fisher Scientific Catalog Number 14- 933A, New Jersey, USA). 0.5 mL of methanol followed by 0.25 mL of chloroform (Fisher Scientific, New Jersey, USA) and 10 µL of internal standards (Sph/Cer Mixture I, Catalog Number LM-6002, Avanti Polar Lipids, Alabaster, AL) were added. Samples were then sonicated in an ultrasonic bath until they appeared evenly dispersed and then incubated for 2 h at 48◦C in a heating block. Tubes were then cooled to room temperature and 75 µL of

TABLE 1 | Stroke mimic pathologies.

1M KOH was added. This mixture was sonicated briefly and then incubated for 30 min at 37◦C. Samples were then cooled to room temperature and neutralized by addition of 16 µL of glacial acetic acid (Fisher Scientific, New Jersey, USA). pH was checked with test strips to verify near return to neutral pH 7.0. One milliliter of chloroform and 2 mL of water were added to each tube. The solution was mixed gently and centrifuged at 300 x g for 5 min to separate the phases. A Pasteur pipette was rinsed with chloroform and then used to remove the lower layer into another glass test tube, and the solvent was removed using vacuum centrifugation. The lipid residue was then re-dissolved in 75 µL methanol. The upper phase was re-extracted by adding 1 mL chloroform, mixing gently, and centrifuging as above. The lower layer was again transferred to another glass test tube using a Pasteur pipette that had been rinsed with chloroform, and the solvent was removed using vacuum centrifugation. The lipid residue was re-dissolved in 75 µL methanol. These two extracts were then pooled for a total of 150 µL, vortexed, and then centrifuged at 13,000 x g to clarify. The supernatant was then transferred to an autosampler vial (Catalog number 225180, Wheaton, New Jersey, USA). Vials were stored at −80◦C until ready for LC-MS analysis.

### High Performance Liquid Chromatography-Mass Spectrometry

Methanol (Optima grade, Fisher Scientific, New Jersey, USA), formic acid (99+%, Thermo Fisher Scientific, Waltham, MA), ammonium formate (99%, Alfa Aesar, Ward Hill, MA), and water purified to a resistivity of 18.2 M·cm (at 25◦C) using a Milli-Q Gradient ultrapure water purification system (Millipore, Billerica, MA), were used to prepare mobile phase solvents for liquid chromatography-mass spectrometry. Lipid extracts were analyzed using an Agilent 1200 liquid chromatography system (Agilent, Santa Clara, CA) that was connected in-line with an LTQ Orbitrap XL mass spectrometer equipped with an electrospray ionization source (ESI; Thermo Fisher Scientific, Waltham, MA). The LC was equipped with a C4 analytical column (Viva C4, length: 150 mm, inner diameter: 1.0 mm, particle size: 5µm, pore size: 300 Å, Restek, Bellefonte, PA), and a 100-µL sample loop. Solvent A was 99.8% water/0.2% formic acid and solvent B was 99.8% methanol/0.2% formic acid (volume/volume). Solvents A and B both contained 5 mM ammonium formate. Lipid extract samples in autosampler vials sealed with septa caps were loaded into the autosampler compartment prior to analysis. The sample injection volume was 100 µL. The elution program consisted of isocratic flow at 30% B for 3 min, a linear gradient to 50% B over 0.5 min, a linear gradient to 100% B over 11.5 min, isocratic flow at 100% B for 5 min, a linear gradient to 30% B over 0.5 min, and isocratic flow at 30% B for 19.5 min, at a flow rate of 150 µL per min. The column and sample compartments were maintained at 40 and 4◦C, respectively. The injection needle was rinsed with a 1:1 methanol:water (volume/volume) solution after each injection to avoid cross-contamination between samples.

The column exit was connected to the ESI probe of the mass spectrometer using PEEK tubing (0.005′′ inner diameter × 1/16′′ outer diameter, Agilent, Santa Clara, CA). Mass spectra were acquired in the positive ion mode over the range m/z = 270– 1,150 using the Orbitrap mass analyzer, in profile format, with a mass resolution setting of 100,000 (at m/z = 400, measured at full width at half-maximum peak height). In the data-dependent mode, the five most intense ions exceeding an intensity threshold of 30,000 counts were selected from each full-scan mass spectrum for tandem mass spectrometry (MS/MS) analysis using collisioninduced dissociation (CID) or pulsed-Q dissociation (PQD). MS/MS spectra were acquired in the positive ion mode using the linear ion trap, in centroid format, with the following parameters: isolation width 3 m/z units, normalized collision energy 30%, activation time 30 ms, activation Q 0.25 for CID, activation Q 0.50 for PQD, and default charge state 1+. A parent mass list was used to preferentially select ions of interest for MS/MS analysis. To avoid the occurrence of redundant MS/MS measurements, real-time dynamic exclusion was enabled to preclude re-selection of previously analyzed precursor ions, using the following parameters: repeat count 2, repeat duration 30 s, exclusion list size 500, exclusion duration 60 s, and exclusion width 0.1 m/z unit.

#### Sphingolipid Identification

Raw Thermo MS data files were converted into centroided data and into the mzXML format using the MS Convert tool in the ProteoWizard 3.0 package (27). Retention time correction and automated peak picking were then performed using XCMS online (28). Feature detection was performed using the centWave method at 2 parts per million (ppm) m/z deviation, minimum peak width of 20 s and maximum peak width of 80 s. A 20 s retention time deviation (bw) was used with a width of 0.01 s for overlapping m/z slices (mzwid). Peaks were annotated by searching for isotopes plus selected adducts (H+ and Na+) with an m/z absolute error of 0.015 and relative error of 5 ppm. Parameters were iteratively optimized by manual review of the selective peaks.

From the automatically selected peaks, target lipids were identified from the METLIN database with the annotation parameters described above (Scripps Research Institute, La Jolla, CA). Peaks were then filtered to only include SLs, and then manually reviewed to ensure accuracy. Peaks that were felt to be inappropriately selected were excluded. Thus, the final list of included SLs consisted of automatically selected peaks that were manually curated.

#### Imaging Analysis

MRI was performed on either Siemens Avanto 1.5T or Siemens Trio 3T machines. Axial T2-weighted images were obtained continuously in 5-mm-thick sections with repetition time of 3,800 ms and time to echo of 116 ms. The field of view was 220 cm, and the matrix was 384 × 384. Axial FLAIR images were obtained continuously in 5-mm-thick sections with repetition time of 9,000 ms and time to echo of 89 ms. The field of view was 220 cm, and the matrix was 320 × 216. Axial diffusion-weighted images were obtained continuously in 5-mm-thick sections with repetition time of 5,600 ms and time to echo of 106 ms. The field of view was 255 cm, and the matrix was 192 × 192.

Two authors (I.A. and V.K.), who were blinded to clinical data, evaluated WMH on axial T2-weighted FLAIR images. The raters used the Fazekas subjective scoring method to assign a score from 0 to 3 in periventricular white matter (PVWM) and deep white mater (DWM) regions of the brain (29, 30). PVWM hyperintensities were scored 0, no hyperintensities; 1, caps or pencil thin lining; 2, smooth halo; 3, irregular periventricular signal extending into the DWM. The DWM was scored 0, no hyperintensities; 1, punctate foci; 2, beginning confluence; 3, large confluent areas. The total Fazekas score (FS) was obtained by summing the scores from periventricular and deep white matter regions. Interrater reliability as measured by quadratic weighted Cohen's Kappa was excellent with κ = 0.81. The average of the scores assigned by the two authors was used in subsequent analysis. This endpoint was dichotomized to separate low WMH load (FS 0–2) from high WMH load (FS > 2). Other features of cerebral microvascular disease including microbleeds and perivascular spaces were also evaluated using axial gradient-echo sequences and axial T2-weighted sequences.

### White Matter Hyperintensity Segmentation

WMH area was quantified using manual ROI segmentation in OsiriX Lite (Pixmeo SARL). WMH areas on individual T2- Weighted FLAIR MRI slices were carefully outlined using the wand tool and the total area of each lesion measured. Lesion areas were summed across slices, and classified as deep or periventricular. Deep and periventricular areas were calculated and then correlated with Fazekas scores and lipid species.

### Statistical Analysis

Statistical analysis was performed using SPSS, Graphpad v 7.0, and Matlab v2018a software. Continuous variables with a normal distribution were described as mean ± SD, and nonnormally distributed variables were described as median and interquartile range. For univariate analysis, categorical variables were compared by χ 2 statistics. A Spearman's correlation was used to measure the association between lipid species, FS, and measured WMH areas. Logistic regression analyses were performed to distinguish independent predictors of heavy white matter damage defined as previously described FS cut-offs. Leaveone-out cross-validation was performed in Matlab using defined code. Statistical significance was defined as p < 0.05.

### RESULTS

### Subjects

Demographic, clinical, radiographic, and plasma specimens were collected as part of the Advanced Serum Profiling in Recent (ASPIRE) Stroke study, a previously conducted UCLA IRB approved, single-center observational study that aimed to identify lipid biomarkers of acute stroke. Data for the ASPIRE trial were collected from all consenting patients over the age of 18 years old presenting to the emergency department (ED) between December 2014 and June 2015 with acute neurological symptoms within the prior 24 h. Subjects with evidence of CNS infection, known CNS malignancy, or recent head trauma as a potential cause of neurologic symptoms were excluded. Demographic data and neurodiagnostic studies are available for all enrolled patients.

To identify only those subjects with subacute white matter disease, the total study population was selectively reduced to exclude those presenting with imaging or clinically confirmed TIA/stroke and those without MRI imaging. Of the 202 patients enrolled in the ASPIRE Stroke study, 34 were excluded as they had not undergone MRI, and an additional 110 were excluded for being diagnosed ultimately with acute ischemic stroke or TIA (**Figure 1**). Among the 58 patients who presented with a stroke mimic syndrome and were included in this analysis, median age was 69.5 and 51.7% were female (**Table 2**). Higher FS was associated with advanced age (29 vs. 70% over 70 yo, FS 0–2 vs. 2.5–6, p = 0.002). While sex did not have an effect on FS in χ 2 analysis (p = 0.32), age did (p = 0.0041). Fazekas positive status was given to 46.5% of the cohort for having FS > 2. Rates of other stigmata of cerebral microvascular disease in the stroke mimic cohort were low with only 5.5% having cerebral microbleeds and 17.8% having prominent perivascular spaces, the majority (>70%) of which also have FS > 2. **Figure 2A** demonstrates the distribution of Fazekas scores in the total ASPIRE cohort and the smaller stroke mimic cohort. Stroke mimics represented a variety of non-ischemic diagnoses ranging from migraine to syncope (**Table 1**).

### Sphingolipid Analysis

Two species, SM 38:1 and Cer 34:1, significantly correlated with FS in a Spearman's correlation model in both the overall ASPIRE cohort and the stroke mimic subgroup (**Tables 3**, **4**). In multivariate logistic regression, SM 38:1 (OR 1.129, 95% CI 1.032, 1.236, p = 0.008) and Cer 34:1 (OR 1.118, 95% CI 1.031, 1.212, p = 0.007) also accurately differentiated between FS 0– 2 vs. 2.5–6. When controlling for age, the two species were still independently predictive of WMH load: SM 38:1 (OR 1.125, 95% CI 1.010, 1.253, p = 0.032) and Cer 34:1 (OR 1.096, 95% CI, 1.002, 1.200, p = 0.046).

A composite lipid score (LS) was computed from the sum of these two lipid species and demonstrated fair discrimination in ROC analysis (**Figures 2B,C**). Leave-one-out validation

demonstrated a cross-validated R 2 of 12.54%, indicating reproducibility of the LS to predict WMHs resulting from cerebral microvascular disease. To further validate the specificity of the LS, we measured WMH area using FLAIR sequences within the stroke mimic cohort of ASPIRE. Of the 58 stroke mimic subjects, 44 subjects had scans allowing precise measurement of WMH. WMH burden correlated with FS (r = 0.828; p = 4.1 × 10−12) and significantly correlated with LS (r = 0.459; p = 0.0017). **Figure 3** demonstrates the relationship between FS, LS, and WMH in this cohort. In linear regression analysis Cer 34:1 significantly associated with objective white matter measurements (p = 0.003), even when controlling for age as a covariate (p = 0.003). To evaluate other potential variables to include in the model, mean LS was compared in patients with and without hypertension, diabetes, history of statin use, sex, and low HDL levels. No significant differences in mean LS exist between these groups. Furthermore, these variables were not predictive of FS+ status in univariate binary logistic regression and were therefore excluded from the model.

To demonstrate the specificity of LS for clinically subacute WMH and not the presence of acute ischemic damage, SM 38:1, Cer 34:1, and LS levels were compared between the stroke mimic cohort (n = 58) and DWI positive stroke group (n = 110). While LS correlated with FS in acute stroke patients (r = 0.440, p < 0.001), overall SM 38:1 and LS levels were significantly lower in the acute stroke patients compared to stroke mimics (**Table 5**). There was a trend showing lower Cer 34:1 in the acute

*n (%), number (percent); p, level of significance; MetS, Metabolic Syndrome;* χ *2 , chi squared statistic; Hx Statin use, History of statin use; Hx Stroke, History of Stroke; Fazekas –, Fazekas score* ≤ *2; Fazekas* +*, Fazekas score* >*2; x Age (SD), mean age (standard deviation); MSBP (IQR), median systolic blood pressure (interquartile range); M DBP (IQR), median diastolic blood pressure (interquartile range).* \*\**Correlation is significant at the 0.01 level (2-tailed).* \**Correlation is significant at the 0.05 level (2-tailed).*

stroke patients as well, however, this difference did not reach statistical significance.

#### DISCUSSION

Biomarkers play an important role in timely detection and management of neurological diseases. The availability of a fluidbased biomarker for subacute ischemic white matter injury may aid in early detection of subacute ischemic brain damage and prevention of its progression to symptomatic stroke, global cognitive decline, and functional loss. Currently, no brainspecific biomarkers are in clinical use for the evaluation of WMH. WMH are a neuroimaging finding that may be indicative of ischemic brain damage and are often found incidentally yet convey considerable cerebrovascular risk. The high cost and relative inaccessibility of MRI studies for subacute neurological disease necessitate an alternative to neuroimaging for evaluation of chronic white matter disease.

A number of studies have suggested protein-based circulating biomarkers for ischemic white matter injury but these associations are highly variable among populations (31). Recent work suggests that serum detection of neurofilament light chain is transiently increased within 3 months of a recent small subcortical infarct, adding sensitivity to MRI confirmation of acute stroke but not necessarily reliable in the detection of subacute ischemic white matter injury (32). Here, we demonstrate that serum levels of two brain-specific lipid species, SM 38:1 and Cer 34:1, correlate with WMH load as measured by FS specifically in subjects with imaging confirmation of no recent brain ischemia. Summing the relative intensities in serum of these lipids yields a LS that reliably distinguishes between low and high WMH load with a cut off of FS >2. Quantitative assessments of WMH area further validated the role of LS as a blood-based biomarker for WMH in a clinically relevant study population.

An ideal biomarker for cerebrovascular disease is specific to the brain and can cross the blood-brain barrier (BBB) to be measured in serum or plasma from peripheral circulation. Leakage across the BBB is increasingly recognized as central to the pathogenesis of cerebral microvascular disease with evidence of fibrinogen and other plasma proteins leaking into the brain and white matter in individuals with cerebral microvascular disease (32, 33). While acute stroke can cause adequate disruption to the BBB for lipid species to cross over into systemic circulation, other conditions such as acute infection, migraines, or seizures may cause similar leaks and lead to false positive signals. However, subclinical ischemia is generally confined to the cerebral white matter and using a biomarker specific to myelin components may help tune specificity to white matter damage. Indeed, characterization of specific sphingolipid species indicates that those species increased in acute stroke (Cer 42:1 and SM 36:0) are independent and distinguishable from those relevant to WMHs (Cer 34:1 and SM 38:1) (26).

Brain white matter is enriched for the lipid species investigated in this study.  Human myelin within white matter tracts comprises many different sphingomyelin species (26, 35). Both ceramide and sphingomyelin are key components of lipid rafts that play a key role in myelination and the maintenance of myelin (36, 37). CSF measurement of sphingomyelin species has been proposed as a biomarker for demyelination in peripheral neuropathies (38). Given the relative

*p, level of significance; r, Spearman's rho; Fazekas* +*, Fazekas score* >*2; M (IQR), median (interquartile range).* \*\**Correlation is significant at the 0.01 level (2-tailed).* \**Correlation is significant at the 0.05 level (2-tailed).*

FIGURE 2 | Combined lipid score predicts WMH. The distribution of Fazekas scoring for the total ASPIRE study cohort (black) and the stroke mimic cohort (gray) (A). Correlation of the combined Cer 34:1 + SM 38:1 lipid score with Fazekas scoring in the stroke mimic cohort (B). ROC curve for the combined lipid score as a predictor of WMH (C).

*p, level of significance; r, Spearman's rho; 95% CI, 95% confidence interval.* \*\**Correlation is significant at the 0.01 level (2-tailed).*

abundance of myelin lipids to axonal protein components such as NF-L, leakage of these lipids into the periphery after injury may be a more sensitive indicator of subacute white matter injury. The design of this study enriches for chronic WMH but limits the ability to judge the specificity of LS in other demyelinating injuries.

The original ASPIRE Stroke study cohort of over 200 patients was initially reduced to only 58 to stringently select for those without acute stroke or TIA yet with evidence of WMH TABLE 5 | Lipid biomarker levels in stroke mimic and tissue-defined stroke cohorts.

*Medians were compared with a one tailed Mann Whitney U-test.* \*\**, significance at the 0.01 level. LS, combined lipid score from sum of SM 38:1 and Cer 34:1; M, median; p, p-value.*

reflective of chronic ischemic damage. Despite excluding patients with acute stroke, this cohort is enriched for systemic disease and advanced age due to the original recruitment criteria of the ASPIRE study. Moreover, our approach of using routine clinical imaging as opposed to research grade MRI sequencing approaches that are often used in evaluation of white matter hyperintensity burden, necessarily reduces the accuracy of white matter hyperintensity load measurements but increases the potential applicability of the LS biomarker in clinical practice. Additional studies using precise volumetric measurements of WMH should be pursued to further validate this biomarker. Details on the microstructural integrity of white matter available using DTI images may also enhance the biological significance of sphingolipids through indirect measurement of myelin sheath breakdown. In ASPIRE, rates of other imaging metrics of cerebral microvascular disease including cerebral microbleeds and perivascular spaces were low or potentially confounded by recent ischemia in the stroke cohort and therefore not included in this analysis. However, since SLs are a major constituent of the myelin sheath, WMH are the logical imaging variable to utilize in determining the value of a fluid-based biomarker for cerebral microvascular disease. Further investigation of this biomarker in healthier and more diverse populations and those with other neurologic and white matter injury phenotypes will help to establish baseline values in both healthy and diseased patients. Furthermore, following patients over months and years will establish the longitudinal value of plasma sphingolipid levels to track longitudinal progression of WMH and clinical outcomes associated with WMH such as stroke risk, cognitive decline, gait changes, and other neuropsychiatric variables.

A fluid-based biomarker for cerebral microvascular disease can serve as a valuable tool for detecting subacute brain ischemia. Exposing subclinical ischemia before its progression to symptomatic stroke or clinically apparent cognitive impairment can reduce disease burden in elderly and frail individuals already at elevated risk for acute stroke. Future studies will likely expand the role of this and other fluid-based biomarkers for WMH and provide a critical role for risk stratification in the study of stroke and vascular cognitive impairment and dementia.

#### DATA AVAILABILITY

The datasets generated for this study are available on request to the corresponding author.

### AUTHOR CONTRIBUTIONS

IA, JH, and SS: conception and design of the study and drafting a significant portion of the manuscript or figures. IA, JH, SS, AI, SL, and VK: acquisition and analysis of data.

### FUNDING

This study was supported by funding from the American Heart Association Grants #15CRP22900006 (SS) and Grant-in-Aid #16GRNT31080021 (JH) as well as the Medical Student

### REFERENCES

Training in Aging Research Program sponsored by the National Institute on Aging (T35AG026736) and the Lillian R. Gleitsman Foundation. The QB3/Chemistry Mass Spectrometry Facility at UC Berkeley received support from the National Institutes of Health (#1S10OD020062-01).

### ACKNOWLEDGMENTS

The authors are grateful to current and past members of the UCLA Student Stroke Force and UCLA Stroke Center Members who assisted in ASPIRE Trial enrollment and data collection.

(sVCAM-1) is associated with concurrent depressive symptoms and cerebral white matter Hyperintensities in older adults. BMC Geriatr. (2015) 15:62. doi: 10.1186/s12877-015-0063-7

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor declared a past co–authorship with author JH.

Copyright © 2019 Azizkhanian, Sheth, Iavarone, Lee, Kakarla and Hinman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Novel Results and Concepts Emerging From Lipid Cell Biology Relevant to Degenerative Brain Aging and Disease

Ole Isacson\*, Oeystein R. Brekk and Penelope J. Hallett

*McLean Hospital and Harvard Medical School, Neuroregeneration Research Institute, Belmont, MA, United States*

While very rare familial forms of proteinopathy can cause Parkinson's disease (PD), Lewy body dementia (LBD) and age-related dementias, recent in-depth studies of lipid disturbances in the majority of the common forms of these diseases instead suggest a primary pathogenesis in lipid pathways. This review synthesizes a perspective from new data that point to an interdependence of lipids and proteinopathy. This article describes disturbed relationships in lipid homeostasis that causes neuropathology to develop over time and with age, which includes altered mechanisms of glia-neuron exchange of lipids and inflammatory signals.

Edited by:

#### *Elisabetta Albi, University of Perugia, Italy*

Reviewed by:

#### *Magda Tsolaki, Aristotle University of Thessaloniki, Greece Matthew Betts, German Center for Neurodegenerative Diseases (DZNE), Germany*

\*Correspondence: *Ole Isacson<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Dementia, a section of the journal Frontiers in Neurology*

Received: *17 June 2019* Accepted: *17 September 2019* Published: *09 October 2019*

#### Citation:

*Isacson O, Brekk OR and Hallett PJ (2019) Novel Results and Concepts Emerging From Lipid Cell Biology Relevant to Degenerative Brain Aging and Disease. Front. Neurol. 10:1053. doi: 10.3389/fneur.2019.01053* Keywords: neurons, lipids, astrocytes, microglia, inflammation, α-synuclein, apolipoprotein, Parkinson's disease

### LIPID CHANGES IN PARKINSON'S DISEASE AND RELATED NEURODEGENERATION

The relationship between lysosomal storage disorders (LSDs) and Lewy body disorders became apparent through evidence of increased risk for developing Lewy body dementia (LBD) and Parkinson's disease (PD) in carriers of LSD gene mutations, and through glycosphingolipid dysregulation and lysosomal dysfunction implicated in the normal pathophysiology of PD (1, 2) (**Table 1**). Homozygous mutations in the lysosomal hydrolase glucocerebrosidase, GBA1, are associated with the LSD, Gaucher disease. Haploinsufficiency of GBA1, which causes reduced activity of glucocerebrosidase (GCase), is associated clinically with a significantly increased risk of PD and LBD, and with faster rate of cognitive decline in α-synucleinopathies, including LBD and PD (3, 4). Brain GCase activity is also decreased and corresponding glycosphingolipid substrate levels are elevated in the brain in PD without GBA1 mutations (1, 5–7) pointing to a much broader age-related decline, and to more complex mechanisms (8). Similar dysfunction of lysosomal hydrolases and disturbances in glycosphingolipid levels to those found clinically in PD are observed in normal aging of both mouse and human brain (1, 9).

Furthermore, PD gene expression and genetic analyses of large cohorts also point to an early involvement of biological processes upstream of accumulating alpha-synuclein (αSyn), including involvement of lipids and lipoproteins, oxidative stress, endosomallysosomal functioning, endoplasmic reticulum stress, and immune response activation (8, 10). In addition, with age, lysosomal enzyme function, chaperones and transporters present in the endoplasmic reticulum-Golgi complex, may become compromised at an early stage of pathogenesis. Critically, biochemical evidence shows that lysosomal enzyme loss of function and lipid disturbances creates PD-like pathology (see **Table 1**). TABLE 1 | Biochemical and clinical evidence for lysosomal enzyme loss of function and lipid disturbances creating Parkinson's disease-like pathology.

With age, many mechanisms can compromise lysosomal enzyme function, including loss of chaperones and transporters present in the endoplasmic reticulum-Golgi complex. Insufficiency of such lysosomal enzymes puts certain cells and brain regions at risk over the longer time frame associated with a relative increase of longevity in humans, creating risk for age-related neurodegenerative diseases (8).

Over the last decades there has been a large emphasis placed on the idea of "proteinopathy", conceptualized primarily in two versions. First, as a primary mechanism for cell dysfunction and degeneration in PD and other diseases with cellular protein aggregates, which however may only be true in very rare genetic cases with gene copy number variations (CNVs) or rare mutations (26). For example, whilst familial PD cases with CNVs of αSyn (duplication, triplication) cause genetic PD and LBD and protein elevations, such cases are very rare; there are currently ∼80 individuals worldwide carrying these CNVs diagnosed with PD/LBD (26), compared to an estimated 6 million cases of PD worldwide (27). Second, the theory of causative proteinopathy has been extended to encompass extra-neural spread of toxic proteins in order to explain regional patterns of chronic cellular pathology seen in many neurodegenerative disorders. Evidence of physical αSyn spread between cells in human PD and related diseases remains to be established, and so far is demonstrated only in artificial experimental model systems.

Instead, more obvious causes for cell dysfunction and pathologies in PD and other related disorders, are primary disturbances from lipids and other metabolic stressors, which in turn can produce protein elevation and aggregation. Lewy body inclusions, widely believed to be predominantly composed of proteinaceous filaments, are in fact more co-labeled with lipids (28, 29). Importantly, recent ultrastructural findings demonstrate that Lewy bodies and neurites in PD post-mortem brain are composed of abundant membranous structures, abnormal vesicles and autophagosome-like structures, in addition to disrupted cytoskeletal elements and dysmorphic mitochondria (30). In summary, the previous almost exclusive focus on aggregating proteins in familial and sporadic cases of PD and LBD, may be replaced by a critical analysis of intracellular lipids and dysfunctional lipid transport as primary mechanisms of disease; in concert with inflammatory processes for PD and LBD. Such analyses may be very useful in the future for selecting candidates, biomarkers and modalities for treatments.

How can perturbation of glycolipid metabolism and lysosomal homeostasis in the aging brain precede or be upstream of protein handling? Glycolipids are abundant in plasma and intracellular membranes and are particularly enriched in the brain where they act as cell surface recognition molecules, as well as having essential roles in the regulation of membrane fluidity and lipid raft formation, modulation of membraneprotein function and signal transduction (31). When GCase in inhibited in vivo, there is large accumulation of high molecular weight aggregates of αSyn (32) and in vivo genetic models of primary GBA mutations also produce significant αsynucleinopathy over time (33, 34). These α-synucleinopathies can be reversed or prevented by agents that increase GCase, or otherwise reduce the accumulation of glycolipids (33, 35). In conclusion, the physiological burden of elevated neuronal glycolipid load in aging and in PD/LBD would affect multiple organelles and biological pathways, and may lower the threshold for developing PD and related neurodegenerative disorders, or accelerate pathophysiological processes in vulnerable neurons.

### NEURON-GLIA INTERACTIONS DURING LIPID DYSHOMEOSTASIS

Abnormal glycolipid, neutral lipid and protein homeostasis within PD and LBD susceptible neurons are likely to signal to surrounding cells, including microglia and astrocytes, accelerating neurodegeneration (36, 37) **(Figure 1A)**. Understanding how glycolipid changes can drive the inflammatory and neurodegenerative mechanisms will be crucial in enabling the development of novel therapeutics. Elevation of reactive oxygen species, mitochondrial dysfunction and loss of autophagy in neurons leads to elevated lipid particle formation (and peroxidated fatty acids). Such lipid dyshomeostasis in neurons may lead to subsequent accumulation of lipid droplets and eventually larger undigested lipid particles, in neurons, and potentially neighboring microglia (39–41) and Isacson, Brekk, Hallett unpublished observation. Appropriate lipid transfer between these cells via lipid transporters such as apolipoproteins, is essential for this process, and for maintaining metabolic integrity of the neuron (40). Peroxidated fatty acids released by neurons are bound to lipoproteins, which are endocytosed by glia (41). Disrupting the transport of lipids from neurons to glia for lipid droplet formation under conditions of neuronal stress, leads to neurodegeneration (39).

FIGURE 1 | Interdependence of lipid processing and proteinopathy. Lipids, and glia-neuron exchange of lipids and inflammatory signals (A) Proposed model for pathogenic lipid perturbations in human PD. Overall accumulation of glycolipids in the substantia nigra with differentially altered lipid droplet deposition in neurons and glial cells could facilitate aberrant protein-lipid interactions (e.g. with αSyn and pTau), in turn perturbing the neuron-glia lipid exchange and activating GPNMB inflammatory protein signals. Cellular dysfunction caused by elevated glycolipids could converge on downstream cytokine-signaling, and other immune responses, in neurons, microglia and astrocytes, causing excessive and aberrant neurite and synaptic damage ultimately leading to neurodegeneration. (B) αSyn has a role in lipid binding through its 11-amino acid repeat amphipathic helical region (shaded in yellow). Mutations in αSyn associated with familial PD, A30P, E46K, H50Q, G51D, and A53T, all occur within this region. αSyn shares conserved tandem repeat regions with the apolipoproteins, ApoA4, ApoE, and ApoA1 (yellow shading). Under lipid stress or aging, αSyn can become significantly lipidated, which may also create dysfunction leading to Lewy bodies. Schematic in (A) is original by the authors. Schematic in (B), showing apolipoprotein binding motifs, is adapted and modified from Emamzadeh (38), under terms of the Creative Commons CC BY license.

Glycoprotein non-metastatic protein B (GPNMB) is a type-I transmembrane glycoprotein that seems to be mechanistically related to altered glycolipid levels (42). In the brain, GPNMB is expressed primarily in glial cells and is thought to play a role as an inflammatory regulator to prevent chronic inflammation (43). Polymorphisms in GPNMB are found to associate with idiopathic PD (44), and GPNMB protein levels are selectively increased in the substantia nigra of PD patients (42). In Gaucher disease, increased protein levels of GPNMB correlate with disease severity and progression (45, 46). Upon systemic pharmacological induction of lipidopathy in mice, which also causes α-synucleinopathy, GPNMB is elevated in a brain-region specific manner including the hippocampus, substantia nigra and cerebral cortex (42). This regional specific upregulation of GPNMB may reflect a differential response of brain regions to lysosomal dysfunction and subsequent differential vulnerability of neuronal populations to degeneration (42, 47). In this lipidopathy model, GPNMB elevation is also associated with robust glial activation and GPNMB is localized in astrocytes and microglia (42). Of note, GPNMB is not elevated by increased αSyn alone in transgenic mice overexpressing human αSyn, indicating a selective response to lipid perturbations (42).

### αSYN AS A LIPID-CARRYING PROTEIN

αSyn is abundant in neurons and is highly enriched at presynaptic terminals, and it is also associated with some organelles. αSyn has important functional roles in the regulation of vesicles, such as synaptic vesicles, neurotransmitter release, dopamine metabolism, synaptic activity, and plasticity (48). αSyn can interact with various lipid species through its amphipathic N-terminus (amino acid) domain. The N-terminal region of αSyn contains six hexameric repeats of 11 amino acids which are characteristic of the 11 amino acid repeats that mediate lipid interactions of apolipoproteins (38, 49) (**Figure 1B**). Significantly, all mutations in αSyn which are associated with familial PD, are located in this lipid-binding region. In the brain, apolipoprotein E (apoE) is the most abundant lipoprotein, forming lipoprotein particles and binding to ApoE cell surface receptors for the delivery of cholesterol and other lipids to neurons. The APOE4 isoform allele is associated with increased risk for dementias in Lewy body diseases, including LBD and PD (50). Interestingly, plasma protein levels of another apolipoprotein, apolipoprotein A1, are associated with age of onset and motor severity in early PD (51). αSyn has several apolipoprotein-like characteristics, including regulation of cholesterol efflux in neuronal cells and formation of lipoprotein nanoparticles (52, 53) providing a clear premise for αSyn's function as a lipid-carrying molecule. Statin treatment in a rodent α-synucleinopathy model reduces α-synuclein aggregates and neuronal pathology (54). The role of αSyn as a functional apolipoprotein is also highlighted by the finding that there is a worsening of ApoE4-mediated pathology in mice that carry human apoE4 when mouse αSyn is ablated (55). Levels of neutral lipids are elevated in the brain in mice that lack endogenous αSyn (56). In perspective, given these data it appears that αSyn normally is not participating in lipid transport in roles that involve synaptic transmission and vesicular functions at the synapse, but can become lipidated under cellular stressful conditions that involve glycolipid, sphingolipid, neutral lipid, lipid peroxidation and age-related disturbances (7). In such circumstances, αSyn becomes part of a pathological adaptation to resolve the lipid problems (55), which likely leads to vesicular binding and transport changes that precede Lewy body formation.

Apolipoprotein function is also linked with inflammation, and ApoE is a modulator of immune responses (57) (**Figure 1A**). Mice lacking ApoE show similar immune activation to mice expressing human ApoE4 in response to lipopolysaccharide (58), and expression of complement pathway genes are upregulated in ApoE knockout mice (59). ApoE has also recently been shown to form a complex with C1q within lipid compartments where it is a regulator of the classical complement cascade (59). C1q is implicated as an early mediator of neuronal dysfunction in preclinical models of AD, whereby reduced expression or blockade of C1q rescues synaptic loss and dysfunction upon exposure to toxic amyloid-beta (60); similarly, C1qa knockdown mitigates neurotoxicity in an in vivo model of frontotemporal dementia (61). Furthermore, C1qa deficiency delays functional cognitive decline associated with normal aging in mice (62). Activation of the complement system is induced in lysosomal storage disorders, including models of neuronopathic Gaucher disease by inhibiting GCase (using CBE), where protein expression of C1q is robustly elevated in several brain regions (32). Inhibition of the complement pathway, through genetic deficiency of C5R1a completely prevents glycolipid accumulation and inflammation in the brain following similar paradigms of systemic CBE (63).

### SUMMARY AND FUTURE PERSPECTIVE

In summary, much evidence points to disruption of lipid cell biology; as glycosphingolipids, gangliosides and possibly several other lipids with metabolic influence can be early initiating factors for age-related neurodegenerative disorders such as PD and LBD (**Table 1** and **Figure 2**). Lipid disturbances in cell types of the brain and/or in specific compartments of such cells, including neurons, astrocytes, microglia and oligodendrocytes, are involved in a large number of neurological diseases. In particular, it is now clear that PD can be triggered by lipid disturbances that are caused by lysosomal genetic or similar age-induced enzymatic loss of function. Relevant to such lipid changes, we find that lipid transport may be compromised by pathological accommodation of αSyn to lipid binding and altered transport roles which are not optimal for normal neuronal function (**Figure 1**). In particular, it is important in future research to identify lipid binding and abnormal lipid droplet or other cellular lipid formations under specific cell

biological conditions. Lipid storage diseases with excessive lipid handling demand can lead to astrocytic and microglial disturbances. There are several contexts in which such lipid and associated lipid-protein interactions could eventually become pathological. The fact that the lipid-carrying APOE4 variant is associated with increased risk for AD and dementias may be the most explicit biological situation where apolipoprotein functions are a major driver of brain dementias. Under some conditions, αSyn may even have a cooperative role with apolipoproteins and lipid transport. In addition, basic research demonstrates that several proteins including αSyn (**Figure 1B**) can accommodate pathological lipid disturbances in astrocytic, neuronal and microglial compartments. In such situations, for a time affected cells will handle genetic and age-acquired lipid and metabolic disturbances, and clearly, such cells may even return to a healthier condition when the pathological stimuli are removed, or a treatment is devised that addresses the cause or initiating factor. Regardless, in chronic neurodegenerative diseases when neurons and glia are unable to maintain such cellular component functions, the pathogenic mechanisms will lead to cellular functional failures that are irreversible (see **Figure 2**). Inflammatory responses can be present at any of these pathogenic steps but are potentially most damaging in the later stages of degeneration, as such processes can remove cellular structures,

including synapses, permanently at a structural level **(Figure 2)**. The continuous expression of elevated amounts or aberrant lipids inside and outside neurons and glia can activate the immune system. In our opinion, novel research needs to focus on the interactions between neurons and glia as an interdependent system that attempts to regulate lipid and protein changes. When such lipid disturbances are significant they can lead to inflammatory reactions and eventually synaptic pathobiology (**Figure 1A**).

Lipid dyshomeostasis, transport and clearance are emerging as central causative factors in neurodegenerative diseases and should help in selecting molecular targets for medical treatments, as well as diagnostic insights to both corrective and antiinflammatory action to prevent structural degeneration in the brain. This new perspective of pathogenesis relevant, upstream causative mechanisms in several neurodegenerative diseases in PD, LBD and potentially also age-related dementias, provides optimism in developing new therapies for these devastating diseases.

The implication for the new understanding presented here; that lipid and inflammatory mechanisms can precede proteinopathies (**Figure 2**), provides clinical opportunities for identification of relevant and specific lipid and inflammatory biomarkers. It is already possible to measure specific abnormalities from GCase in the blood and brain (1, 64) of patients with PD. Such patient stratification in other lipid species and specific metabolic disturbances may help to better define effective treatments of neurodegenerative diseases in clinical trials. It is also important that systemic and peripheral biomarkers can be coincident with brain pathology observed in neurodegenerative diseases. There is evidence of such peripheral biomarkers in patients with LSDs, PD, and AD for pathways involved in lipids and inflammation (51, 64–68). In conclusion, determining altered cellular lipid accumulation, transfer and clearance mechanisms in PD and related disorders can be of significant value to helping patients and at-risk individuals.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

OI drafted the article with PH. OB contributed to the discussion and analysis of the results mentioned in the article.

#### FUNDING

This research was supported by NIH/NINDS 1R01NS092667, NIH/NIA R01AG060195, the Consolidated Anti Aging Foundation, and the Orchard Foundation. OB was supported by the Harold and Ronna Cooper Post Doctoral Fellowship for Parkinson's Disease Research.

B in type 1 Gaucher disease patients and mouse models. FEBS Open Bio. (2016) 6:902–13. doi: 10.1002/2211-5463.12078

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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# Mitochondrial Cholesterol in Alzheimer's Disease and Niemann–Pick Type C Disease

Sandra Torres 1,2, Carmen M. García-Ruiz 1,2,3 and Jose C. Fernandez-Checa1,2,3 \*

1 Department of Cell Death and Proliferation, Instituto de Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, Spain, 2 Liver Unit and Hospital Clinc I Provincial, Centro de Investigación Biomédica en Red (CIBEREHD), Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain, 3 Southern California Research Center for ALDP and Cirrhosis, Los Angeles, CA, United States

Mitochondrial dysfunction has been recognized as a key player in neurodegenerative diseases, including Alzheimer's disease (AD) and Niemann–Pick type C (NPC) disease. While the pathogenesis of both diseases is different, disruption of intracellular cholesterol trafficking has emerged as a common feature of both AD and NPC disease. Nutritional or genetic mitochondrial cholesterol accumulation sensitizes neurons to Aβ-mediated neurotoxicity in vitro and promotes cognitive decline in AD models. In addition to the primary accumulation of cholesterol and sphingolipids in lysosomes, NPC disease is also characterized by an increase in mitochondrial cholesterol levels in affected organs, predominantly in brain and liver. In both diseases, mitochondrial cholesterol accumulation disrupts membrane physical properties and restricts the transport of glutathione into mitochondrial matrix, thus impairing the mitochondrial antioxidant defense strategy. The underlying mechanisms leading to mitochondrial cholesterol accumulation in AD and NPC diseases are not fully understood. In the present manuscript, we discuss evidence for the potential role of StARD1 in promoting the trafficking of cholesterol to mitochondria in AD and NPC, whose upregulation involves an endoplasmic reticulum stress and a decrease in acid ceramidase expression, respectively. These findings imply that targeting StARD1 or boosting the mitochondrial antioxidant defense may emerge as a promising approach for both AD and NPC disease.

#### Keywords: cholesterol, mitochondria, lysosomal disorders, sphingolipids, acid ceramidase

### INTRODUCTION

Neurodegenerative diseases encompass a wide range of neurological disorders caused by different causes, most notably genetic mutations in specific genes. Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases in which the progressive loss of neurons is associated with the upregulation of peptides and activation of proteins, such as amyloid beta (Aβ) or tau phosphorylation, that trigger specific signaling pathways that ultimately contribute to the progression of the disease. In addition, accumulation of other cellular components, such as specific types of lipids, can cause neuronal death and mitochondrial dysfunction in the brain and in peripheral organs. The role of mitochondrial dysfunction in neurodegenerative diseases remains to be fully elucidated. Recent studies have provided evidence that alterations in lipid metabolism can have a deleterious impact on mitochondrial function, which can contribute to the progression

#### Edited by:

Maria Dolores Ledesma, Severo Ochoa Molecular Biology Center (CSIC-UAM), Spain

#### Reviewed by:

Alejandra Rojas Alvarez, Pontifical Catholic University of Chile, Chile Andrew P. Lieberman, University of Michigan, United States

> \*Correspondence: Jose C. Fernandez-Checa<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Dementia, a section of the journal Frontiers in Neurology

Received: 30 July 2019 Accepted: 18 October 2019 Published: 07 November 2019

#### Citation:

Torres S, García-Ruiz CM and Fernandez-Checa JC (2019) Mitochondrial Cholesterol in Alzheimer's Disease and Niemann–Pick Type C Disease. Front. Neurol. 10:1168. doi: 10.3389/fneur.2019.01168 not only of AD but also of the lysosomal storage disorder Niemann–Pick type C (NPC) disease, a neurovisceral disorder primarily characterized by the accumulation of lipids in intracellular organelles, most predominantly in lysosomes. Here, we briefly summarize evidence indicating that increased cholesterol trafficking to mitochondria has emerged as a putative key player in AD and NPC disease through the disruption of mitochondrial routine performance, leading to oxidative stress and cell death.

### MITOCHONDRIAL CHOLESTEROL TRAFFICKING IN NEURODEGENERATIVE DISEASES

Cholesterol is an essential component of membrane bilayers, which determines their physico-chemical and functional properties. Cholesterol is particularly enriched in the brain, where it regulates key biological functions, such as signal transduction pathways, myelin formation, and synaptogenesis (1). In the central nervous system (CNS), cholesterol is synthesized de novo from acetyl-CoA in the mevalonate pathway in the endoplasmic reticulum (ER) (2). Since cholesterol does not cross the blood–brain barrier (BBB), to ensure steady-state turnover, cholesterol synthesis is matched by its metabolism to 24S-hydroxycholesterol (24-OHC), which crosses the BBB and is delivered to peripheral organs and hence constitutes an intrinsic mechanism to prevent cholesterol accumulation in the brain. During the perinatal period, cholesterol accumulation in the brain is mainly determined by oligodendrocytes due to their key role in myelination, while neurons synthesize their own cholesterol de novo needed for neuronal plasticity and function (3). In the adult life, however, the rate of de novo cholesterol synthesis declines, forcing neurons to acquire cholesterol from a cross-talk between neurons and astrocytes (1, 4). Although the regulation of cholesterol homeostasis in brain diseases has been reviewed elsewhere (2), here we discuss the specific role of mitochondrial cholesterol in neurodegenerative diseases.

While much of what it is known about the trafficking of cholesterol into mitochondria has been elucidated in the context of steroidogenesis, the relevance of mitochondrial cholesterol in neurodegeneration has been less recognized. Emerging evidence indicates that mitochondrial cholesterol loading can influence mitochondrial function independently of its conversion to pregnenolone or oxysterols, arising as a key factor in the pathology of several neurological diseases associated with mitochondrial dysfunction, such as AD and NPC disease (5). Although the physiological levels of cholesterol in mitochondrial membranes are low compared to other membrane bilayers (e.g., plasma membrane), the limited content of mitochondrial cholesterol is essential for the maintenance of mitochondrial membrane physical properties and synthesis of neurosteroids. The key regulatory enzymes responsible for steroid synthesis in the CNS include the cytochrome P450 sidechain cleavage (P450scc), and the steroidogenic acute regulatory protein StARD1, the founder member of a family of lipid transporting proteins that contain StAR-related lipid transfer (StART) domains. The rate-limiting step in the synthesis of neurosteroids is the availability of mitochondrial cholesterol in the mitochondrial inner membrane (MIM) for metabolism by P450scc. Cholesterol is imported to mitochondrial membranes by the action of lipid transfer multiprotein complex acting at membrane contact sites. StARD1 plays an essential role in the transfer of cholesterol to the MIM, as inferred from the outcome of mice with global StARD1 deletion, which undergo a lethal adrenal lipoid hyperplasia, indicating that other members of the StAR family cannot replace its function in intramitochondrial cholesterol trafficking (6, 7). StARD1 contains a mitochondrial localization sequence and a steroid-binding domain. The exact location of StARD1 in mitochondria has been much discussed, but among StARD1 forms, the 30 kDa phosphorylated form has been described to be localized on the MIM (8). MLN64 (also known as StARD3) provides cholesterol to the mitochondrial outer membrane (MOM) from endosomes (5, 9) and together with StARD1 work in tandem in the net import of cholesterol to the MIM for metabolism.

In the last decade, significant progress has been made on the impact of cholesterol accumulation in mitochondrial function and routine performance in AD and NPC disease using genetic mouse models, such as the APP/PSEN1 transgenic mice overexpressing SREPB-2 (APP/PSEN1/SREBP2) and the Npc1−/− knockout mice (10–14). While much of the deleterious effects of mitochondrial cholesterol accumulation in both diseases is accounted for by the depletion of mitochondrial GSH (mGSH), unfortunately the underlying mechanisms whereby cholesterol accumulates in mitochondria in AD and NPC are not fully understood. Our hypothesis posits that StARD1 is a critical player in mitochondrial cholesterol loading and hence emerges as a putative novel target for intervention in both diseases (**Figure 1**).

### INTRACELLULAR CHOLESTEROL HOMEOSTASIS AND MITOCHONDRIAL FUNCTION IN AD

AD is one of the most common neurodegenerative disorders in older adults. The pathological hallmark of AD is the cognitive impairment and associated dementia due to neuronal death caused in part by the accumulation of amyloid plaques in the cortex and hippocampus (15–18). Currently, there is no cure for AD, which reflects our incomplete understanding of AD pathogenesis. AD is a multifactorial disease and several players contribute to its progression, including the disruption of cholesterol homeostasis. In this regard, epidemiological findings showed that hypercholesterolemia is a major risk factor for AD development (19). However, in spite of the association between hypercholesterolemia and AD, the role of cholesterol in AD is controversial and not fully understood. In this regard, a body of literature supports a link between increased cholesterol levels in the brain with the progression of AD. For instance, the specific presence of the enzymes involved in the generation of toxic Aβ peptides in lipid rafts, specific domains of membrane bilayers

properties and impairs the transport of GSH into mitochondria, via the 2-oxoglutarate carrier (OGC), disrupting the mitochondrial antioxidant defense and subsequent

highly enriched in cholesterol, provides a strong association between high cholesterol levels with Aβ generation and AD development (20, 21). Moreover, in the CNS, cholesterol is transported between different cell types by a multicellular trafficking process largely regulated by ApoE (22–24). Consistent with its function in cholesterol trafficking within the brain, ApoE polymorphisms, particularly APOEε4 allele, have emerged as a risk for AD development and correlate with higher levels of Aβ in the serum (25, 26). Moreover, experimental models fed diets enriched in cholesterol have been shown to develop AD-like pathology [(27); reviewed in (28)]. Quite interestingly, besides the association between the increase in cholesterol levels with AD, there is also evidence indicating that low cholesterol levels in the brain can contribute to the AD progression (2). This inverse relationship is of particular significance during aging, as low levels of cholesterol in hippocampus are characteristic of the aged human brain (29). Furthermore, as cholesterol metabolism in the brain to 24-OHC by the action of CYP46A1 represents

oxidative stress. Chol, cholesterol; mGSH, mitochondrial glutathione; ROS, reactive oxygen species.

a unique mechanism to control brain cholesterol homeostasis, CYP46A1 polymorphisms correlate with lower brain cholesterol levels and increased risk of AD (30, 31). In addition, hippocampal cholesterol loss has been shown to contribute to the poor cognition in old rodents, and hence, cholesterol replenishment in aging animals improves hippocampal-dependent learning and memory (32).

In the early stage of AD development, mitochondria undergo significant functional deficits, which correlate with accumulation of neurotoxic Aβ (33–36). Interestingly, it has been shown that Aβ can target mitochondria to stimulate ROS generation, thus contributing to Aβ toxicity in neurons (37, 38). Immunoelectron microscopy analysis indicated the association of APP with mitochondrial protein translocation components, TOM20 and TIM23, which correlated with decreased import of respiratory chain subunits, lower cytochrome oxidase activity, and increased ROS generation (39). Moreover, functional complexes with γ-secretase activity have been found in mitochondria while insulin-degrading enzyme (IDE), which is known to contribute to Aβ removal, can be targeted to mitochondria (40, 41). Thus, mitochondrial dysfunction is associated with increased Aβ generation and AD progression.

In line with the potential relevance linking intracellular cholesterol to mitochondrial dysfunction in AD, mitochondrial cholesterol enrichment has been shown to sensitize to Aβmediated neurotoxicity through depletion of mGSH levels (10). Moreover, APP/PS1/SREBP-2 mice, which exhibit an early mitochondrial cholesterol loading and mGSH depletion, exhibited an accelerated β-secretase activation, Aβ accumulation, and cognitive decline compared to APP/PS1 mice (11), further supporting the correlation between mitochondrial cholesterol accumulation and the subsequent mGSH depletion in AD. Consequently, in vivo treatment of APP/PS1/SREBP-2 mice with the cell-permeable GSH ethyl ester, which restored mGSH levels, attenuated synaptic degeneration and improved cognition, suggesting that therapeutic strategy to prevent mitochondrial cholesterol accumulation or the restoration of mGSH levels may represent a relevant approach in the treatment of AD. Further evidence indicated that ER stress acts as a link between Aβ generation and cholesterol upregulation and subsequent mitochondrial cholesterol trafficking due to increased expression of StARD1 (12). Furthermore, administration of chemical chaperones that prevent ER stress ameliorates StARD1 upregulation and cognitive decline in APP/PS1/SREBP-2 mice.