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Traumatic brain injury (TBI) leads to major membrane lipid breakdown. We investigated plasma lipids over 3 days post-TBI, to identify a signature of acute human TBI and assess its correlation with neuronal injury and inflammation. Plasma from patients with TBI (Abbreviated Injury Scale (AIS)3 - serious injury, n = 5; AIS4 - severe injury, n = 8), and controls (n = 13) was analysed for lipidomic profile, neurofilament light (NFL) and cytokines, and the omega-3 index was measured in red blood cells. A lipid signature separated TBI from controls, at 24 and 72 h. Major species driving the separation were: lysophosphatidylcholine (LPC), phosphatidylcholine (PC) and hexosylceramide (HexCer). Docosahexaenoic acid (DHA, 22:6) and LPC (0:0/22:6) decreased post-injury. NFL levels were increased at 24 and 72 h post-injury in AIS4 TBI vs. controls. Interleukin (IL-)6, IL-2 and IL-13 were elevated at 24 h in AIS4 patients vs. controls. NFL and IL-6 were negatively correlated with several lipids. The omega-3 index at admission was low in all patients (controls: 4.3 ± 1.1% and TBI: 4.0 ± 1.1%) and did not change significantly over 3 days post-injury. We have identified specific lipid changes, correlated with markers of injury and inflammation in acute TBI. These observations could inform future lipid-based therapeutic approaches. [ABSTRACT FROM AUTHOR]

The review presents an analysis of published works concerning the various aspects of studies of antioxidants in heterogeneous systems, in particular, in oil-in-water emulsions. It is shown that the discussed methodological approach is an important and integral part of a comprehensive study and evaluation of the antioxidant activity of a wide range of natural and synthetic compounds, their mixtures and extracts obtained from various sources of natural origin. This approach makes it possible to objectively evaluate the properties of these compounds and their potential using models simulating native biological disperse systems. Examples of a convenient, accessible, and biologically relevant model are emulsions obtained based on animal tissue homogenates, which contain polyunsaturated fatty acids within their lipid composition. Lipid peroxidation in these emulsions that is initiated by various methods is used for screening new substances, studying the molecular and cellular mechanisms of action of antioxidants of various origins, as well as investigating the structure—activity dependence. The results obtained in the test system using emulsions based on animal tissue homogenates open up new promising opportunities and can contribute to the development of new effective antioxidants possessing specified properties and meeting the stringent requirements imposed on them in the biomedical and food industries. The methodology for assessing the antioxidant activity of various substances using emulsions based on animal tissue homogenates that is described in this review may be useful for applications in other interdisciplinary studies, including those of an ecological nature. [ABSTRACT FROM AUTHOR]

Aging induces complex changes in the lipid profiles across different areas of the brain. These changes can affect the function of brain cells and may contribute to neurodegenerative diseases such as Alzheimer's disease. Research shows that while the overall lipid profile in the human brain remains quite steady throughout adulthood, specific changes occur with age, especially after the age of 50. These changes include a slow decline in total lipid content and shifts in the composition of fatty acids, particularly in glycerophospholipids and cholesterol levels, which can vary depending on the brain region. Lipid rafts play a crucial role in maintaining membrane integrity and facilitating cellular signaling. In the context of Alzheimer's disease, changes in the composition of lipid rafts have been associated with the development of the disease. For example, alterations in lipid raft composition can lead to increased accumulation of amyloid β (Aβ) peptides, contributing to neurotoxic effects. Lipid droplets store neutral lipids and are key for cellular energy metabolism. As organisms age, the dynamics of lipid droplets in the brain change, with evidence suggesting a decline in metabolic activity over time. This reduced activity may lead to an imbalance in lipid synthesis and mobilization, contributing to neurodegenerative processes. In model organisms like Drosophila, studies have shown that lipid metabolism in the brain can be influenced by diet and insulin signaling pathways, crucial for maintaining metabolic balance. The interplay between lipid metabolism, oxidative stress, and inflammation is critical in the context of aging and Alzheimer's disease. Lipid peroxidation, a consequence of oxidative stress, can lead to the formation of reactive aldehydes that further damage neurons. Inflammatory processes can also disrupt lipid metabolism, contributing to the pathology of AD. Consequently, the accumulation of oxidized lipids can affect lipid raft integrity, influencing signaling pathways involved in neuronal survival and function. [ABSTRACT FROM AUTHOR]

Apolipoprotein B (APOB), a receptor-binding protein present in cholesterol-rich lipoproteins, has been implicated in Alzheimer's disease (AD). High levels of APOB-containing low-density lipoproteins (LDL) are linked to the pathogenesis of both early-onset familial and late-onset sporadic AD. Rare coding mutations in the APOB gene are associated with familial AD, suggesting a role for APOB-bound lipoproteins in the central nervous system. This research explores APOB gene regulation across the AD spectrum using four cohorts: BRAINEAC (elderly control brains), DBCBB (controls, AD brains), ROSMAP (controls, MCI, AD brains), and ADNI (control, MCI, AD clinical subjects). APOB protein levels, measured via mass spectrometry and ELISA, positively correlated with AD pathology indices and cognition, while APOB mRNA levels showed negative correlations. Brain APOB protein levels are also correlated with cortical Aβ levels. A common coding variant in the APOB gene locus affected its expression but didn't impact AD risk or brain cholesterol concentrations, except for 24-S-hydroxycholesterol. Polymorphisms in the CYP27A1 gene, notably rs4674344, were associated with APOB protein levels. A negative correlation was observed between brain APOB gene expression and AD biomarker levels. CSF APOB correlated with Tau pathology in presymptomatic subjects, while cortical APOB was strongly associated with cortical Aβ deposition in late-stage AD. The study discusses the potential link between blood-brain barrier dysfunction and AD symptoms in relation to APOB neurobiology. Overall, APOB's involvement in lipoprotein metabolism appears to influence AD pathology across different stages of the disease.

Ischemic stroke remains a leading cause of mortality and long-term disability worldwide, necessitating efforts to identify biomarkers for diagnosis, prognosis, and treatment monitoring. The present study aimed to identify novel plasma biomarkers of neurodegeneration and inflammation in a mouse model of stroke induced by distal middle cerebral artery occlusion. Using targeted lipidomic and global untargeted metabolomic profiling of plasma collected from aged male mice 24 h after stroke and weekly thereafter for 7 weeks, we discovered distinct acute and chronic signatures. In the acute phase, we observed elevations in myelin-associated lipids, including sphingomyelin (SM) and hexosylceramide (HCER) lipid species, indicating brain lipid catabolism. In the chronic phase, we identified 12-hydroxyeicosatetraenoic acid (12-HETE) as a putative biomarker of prolonged inflammation, consistent with our previous observation of a biphasic pro-inflammatory response to ischemia in the mouse brain. These results provide insight into the metabolic alterations detectable in the plasma after stroke and highlight the potential of myelin degradation products and arachidonic acid derivatives as biomarkers of neurodegeneration and inflammation, respectively. These discoveries lay the groundwork for further validation in human studies and may improve stroke management strategies.

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Cytochrome P450 46A1 (CYP46A1) is the CNS-specific cholesterol 24-hydroxylase that controls cholesterol elimination and turnover in the brain. In mouse models, pharmacologic CYP46A1 activation with low-dose efavirenz or by gene therapy mitigates the manifestations of various brain disorders, neurologic, and nonneurologic, by affecting numerous, apparently unlinked biological processes. Accordingly, CYP46A1 is emerging as a promising therapeutic target; however, the mechanisms underlying the multiplicity of the brain CYP46A1 activity effects are currently not understood. We proposed the chain reaction hypothesis, according to which CYP46A1 is important for the three primary (unifying) processes in the brain (sterol flux through the plasma membranes, acetyl-CoA, and isoprenoid production), which in turn affect a variety of secondary processes. We already identified several processes secondary to changes in sterol flux and herein undertook a multiomics approach to compare the brain proteome, acetylproteome, and metabolome of 5XFAD mice (an Alzheimer’s disease model), control and treated with low-dose efavirenz. We found that the latter had increased production of phospholipids from the corresponding lysophospholipids and a globally increased protein acetylation (including histone acetylation). Apparently, these effects were secondary to increased acetyl-CoA production. Signaling of small GTPases due to their altered abundance or abundance of their regulators could be affected as well, potentially via isoprenoid biosynthesis. In addition, the omics data related differentially abundant molecules to other biological processes either reported previously or new. Thus, we obtained unbiased mechanistic insights and identified potential players mediating the multiplicity of the CYP46A1 brain effects and further detailed our chain reaction hypothesis.

The lanthanide metal gadolinium has been used in the healthcare industry as a paramagnetic contrast agent for years. Gadolinium deposition in brain tissue and kidneys has been reported following gadolinium-based contrast agent administration to patients undergoing MRI. This study demonstrates the detrimental effects of gadolinium exposure at the level of the cell membrane. Biophysical analysis using fluorescence spectroscopy and dynamic light scattering illustrates differential interactions of gadolinium ions with key classes of brain membrane lipids, including phosphatidylcholines and sphingomyelins, as well as brain polar extracts and biomimetic brain model membranes. Electrostatic attraction to negatively charged lipids like phosphatidylserine facilitates metal complexation but zwitterionic phosphatidylcholine and sphingomyelin interaction was also significant, leading to membrane rigidification and increases in liposome size. Effects were stronger for fully saturated over monounsaturated acyl chains. The metal targets key lipid classes of brain membranes and these biophysical changes could be very detrimental in biological membranes, suggesting that the potential negative impact of gadolinium contrast agents will require more scientific attention. [ABSTRACT FROM AUTHOR]

The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%–80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis.

GM1 gangliosidosis is a neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes lysosomal β-galactosidase. The enzyme deficiency blocks GM1 ganglioside catabolism, leading to accumulation of GM1 ganglioside and asialo-GM1 ganglioside (GA1 glycolipid) in brain. This disease can present in varying degrees of severity, with the level of residual β-galactosidase activity primarily determining the clinical course. Glb1 null mouse models, which completely lack β-galactosidase expression, exhibit a less severe form of the disease than expected from the comparable deficiency in humans, suggesting a potential species difference in the GM1 ganglioside degradation pathway. We hypothesized this difference may involve the sialidase NEU3, which acts on GM1 ganglioside to produce GA1 glycolipid. To test this hypothesis, we generated Glb1/Neu3 double KO (DKO) mice. These mice had a significantly shorter lifespan, increased neurodegeneration, and more severe ataxia than Glb1 KO mice. Glb1/Neu3 DKO mouse brains exhibited an increased GM1 ganglioside to GA1 glycolipid ratio compared with Glb1 KO mice, indicating that NEU3 mediated GM1 ganglioside to GA1 glycolipid conversion in Glb1 KO mice. The expression of genes associated with neuroinflammation and glial responses were enhanced in Glb1/Neu3 DKO mice compared with Glb1 KO mice. Mouse NEU3 more efficiently converted GM1 ganglioside to GA1 glycolipid than human NEU3 did. Our findings highlight NEU3’s role in ameliorating the consequences of Glb1 deletion in mice, provide insights into NEU3’s differential effects between mice and humans in GM1 gangliosidosis, and offer a potential therapeutic approach for reducing toxic GM1 ganglioside accumulation in GM1 gangliosidosis patients.

Previously, we and others reported a rapid and dramatic increase in brain prostanoids (PG), including prostaglandins, prostacyclins, and thromboxanes, under ischemia that is traditionally explained through the activation of esterified arachidonic acid (20:4n6) release by phospholipases as a substrate for cyclooxygenases (COX). However, the availability of another required COX substrate, oxygen, has not been considered in this mechanism. To address this mechanism for PG upregulation through oxygen availability, we analyzed mouse brain PG, free 20:4n6, and oxygen levels at different time points after ischemic onset using head-focused microwave irradiation (MW) to inactivate enzymes in situ before craniotomy. The oxygen half-life in the ischemic brain was 5.32 ± 0.45 s and dropped to undetectable levels within 12 s of ischemia onset, while there were no significant free 20:4n6 or PG changes at 30 s of ischemia. Furthermore, there was no significant PG increase at 2 and 10 min after ischemia onset compared to basal levels, while free 20:4n6 was increased ∼50 and ∼100 fold, respectively. However, PG increased ∼30-fold when ischemia was followed by craniotomy of nonMW tissue that provided oxygen for active enzymes. Moreover, craniotomy performed under anoxic conditions without MW did not result in PG induction, while exposure of these brains to atmospheric oxygen significantly induced PG. Our results indicate, for the first time, that oxygen availability is another important regulatory factor for PG production under ischemia. Further studies are required to investigate the physiological role of COX/PG regulation through tissue oxygen concentration.

Intracellular lipolysis—the enzymatic breakdown of lipid droplet-associated triacylglycerol (TAG)—depends on the cooperative action of several hydrolytic enzymes and regulatory proteins, together designated as lipolysome. Adipose triglyceride lipase (ATGL) acts as a major cellular TAG hydrolase and core effector of the lipolysome in many peripheral tissues. Neurons initiate lipolysis independently of ATGL via DDHD domain-containing 2 (DDHD2), a multifunctional lipid hydrolase whose dysfunction causes neuronal TAG deposition and hereditary spastic paraplegia. Whether and how DDHD2 cooperates with other lipolytic enzymes is currently unknown. In this study, we further investigated the enzymatic properties and functions of DDHD2 in neuroblastoma cells and primary neurons. We found that DDHD2 hydrolyzes multiple acylglycerols in vitro and substantially contributes to neutral lipid hydrolase activities of neuroblastoma cells and brain tissue. Substrate promiscuity of DDHD2 allowed its engagement at different steps of the lipolytic cascade: In neuroblastoma cells, DDHD2 functioned exclusively downstream of ATGL in the hydrolysis of sn-1,3-diacylglycerol (DAG) isomers but was dispensable for TAG hydrolysis and lipid droplet homeostasis. In primary cortical neurons, DDHD2 exhibited lipolytic control over both, DAG and TAG, and complemented ATGL-dependent TAG hydrolysis. We conclude that neuronal cells use noncanonical configurations of the lipolysome and engage DDHD2 as dual TAG/DAG hydrolase in cooperation with ATGL.

Although pregnant women’s fish consumption is beneficial for the brain development of the fetus due to the DHA in fish, seafood also contains methylmercury (MeHg), which adversely affects fetal brain development. Epidemiological studies suggest that high DHA levels in pregnant women’s sera may protect the fetal brain from MeHg-induced neurotoxicity, but the underlying mechanism is unknown. Our earlier study revealed that DHA and its metabolite 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP) produced by cytochrome P450s (P450s) and soluble epoxide hydrolase (sEH) can suppress MeHg-induced cytotoxicity in mouse primary neuronal cells. In the present study, DHA supplementation to pregnant mice suppressed MeHg-induced impairments of pups’ body weight, grip strength, motor function, and short-term memory. DHA supplementation also suppressed MeHg-induced oxidative stress and the decrease in the number of subplate neurons in the cerebral cortex of the pups. DHA supplementation to dams significantly increased the DHA metabolites 19,20-epoxydocosapentaenoic acid (19,20-EDP) and 19,20-DHDP as well as DHA itself in the fetal and infant brains, although the expression levels of P450s and sEH were low in the fetal brain and liver. DHA metabolites were detected in the mouse breast milk and in human umbilical cord blood, indicating the active transfer of DHA metabolites from dams to pups. These results demonstrate that DHA supplementation increased DHA and its metabolites in the mouse pup brain and alleviated the effects of MeHg on fetal brain development. Pregnant women’s intake of fish containing high levels of DHA (or DHA supplementation) may help prevent MeHg-induced neurotoxicity in the fetus.

Introduction: Peroxisomes are essential organelles in lipid metabolism. They contain enzymes for β-oxidation of very long-chain fatty acids (VLCFA) that cannot be broken down in mitochondria. Reduced expression in hepatic acyl-CoA oxidase 1 (ACOX1), a peroxisome β-oxidation enzyme, followed by modification of the brain fatty acid profile has been observed in aged rodents. These studies have suggested a potential role for peroxisome β-oxidation in brain aging. This study was designed to examine the effect of hepatic ACOX1 inhibition on brain fatty acid composition and neuronal cell activities of young rats (200-250 g). Methods: A specific ACOX1 inhibitor, 10, 12- tricosadiynoic acid (TDYA), 100 μg/kg (in olive oil) was administered by daily gavage for 25 days in male Wistar rats. The brain fatty acid composition and electrophysiological properties of dentate gyrus granule cells were determined using gas chromatography and whole-cell patch-clamp, respectively. Results: A significant increase in C20, C22, C18:1, C20:1, and a decrease of C18, C24, C20:3n6, and C22:6n3 were found in 10, 12- tricosadiynoic acid (TDYA) treated rats compared to the control group. The results showed that ACOX1 inhibition changes fatty acid composition similar to old rats. ACOX1 inhibition caused hyperpolarization of resting membrane potential, and also reduction of input resistance, action potential duration, and spike firing. Moreover, ACOX1 inhibition increased rheobase current and afterhyperpolarization amplitude in granule cells. Conclusion: The results indicated that systemic inhibition of ACOX1 causes hypo-excitability of neuronal cells. These results provide new evidence on the involvement of peroxisome function and hepatic ACOX1 activity in brain fatty acid profile and the electrophysiological properties of dentate gyrus cells. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of soybean lecithin and plasmalogens concentrating on a variety of physiological tests and biochemical analyses in healthy Wistar rats. For six weeks, male Wistar rats were given a standard diet that included plasmalogens or soybean lecithin. We measured anxiety levels, overall exploratory activity, short- and long-term memory, cognitive abilities, and grip strength. Lecithin increased significantly anxiety and enhanced memory and cognitive functions. Plasmalogens significantly improved appetite and increased grip strength. When compared to plasmalogens, lecithin significantly raised HDL levels while lowering LDL levels. The plasmalogens group showed a significant increase in the C16:0DMA/C16:0 ratio, which led us to assume that plasmalogen consumption could increase their synthesis in neural tissue. The study's findings imply that, despite their various modes of action, soy lecithin and plasmalogens may both be significant nutritional components for enhancing cognitive functions. [ABSTRACT FROM AUTHOR]

PUFAs are precursors to bioactive oxylipin metabolites that increase in the brain following CO2-induced hypercapnia/ischemia. It is not known whether the brain-dissection process and its duration also alter these metabolites. We applied CO2 with or without head-focused microwave fixation for 2 min to evaluate the effects of CO2-induced asphyxiation, dissection, and dissection time on brain oxylipin concentrations. Compared with head-focused microwave fixation (control), CO2 followed by microwave fixation prior to dissection increased oxylipins derived from lipoxygenase (LOX), 15-hydroxyprostaglandin dehydrogenase (PGDH), cytochrome P450 (CYP), and soluble epoxide hydrolase (sEH) enzymatic pathways. This effect was enhanced when the duration of postmortem ischemia was prolonged by 6.4 min prior to microwave fixation. Brains dissected from rats subjected to CO2 without microwave fixation showed greater increases in LOX, PGDH, CYP and sEH metabolites compared with all other groups, as well as increased cyclooxygenase metabolites. In nonmicrowave-irradiated brains, sEH metabolites and one CYP metabolite correlated positively and negatively with dissection time, respectively. This study presents new evidence that the dissection process and its duration increase brain oxylipin concentrations, and that this is preventable by microwave fixation. When microwave fixation is not available, lipidomic studies should account for dissection time to reduce these artifacts.

PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo. Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function. [ABSTRACT FROM AUTHOR]

PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo. Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function.

Abstract—This article presents the main stages in the development of brain biochemistry studies at St. Petersburg (Leningrad) State University, starting from the foundation of the Biochemistry Department in 1928. The contribution to neurochemistry of professors E.S. London, G.E. Vladimirov, I.P. Ashmarin, and M.I. Prokhorova is discussed, and brief information about the training of young neurochemists at St. Petersburg State University at the present time is given. [ABSTRACT FROM AUTHOR]

Phosphatidic acid is a key signaling molecule heavily implicated in exocytosis due to its protein-binding partners and propensity to induce negative membrane curvature. One phosphatidic acid-producing enzyme, phospholipase D (PLD), has also been implicated in neurotransmission. Unfortunately, due to the unreliability of reagents, there has been confusion in the literature regarding the expression of PLD isoforms in the mammalian brain which has hampered our understanding of their functional roles in neurons. To address this, we generated epitope-tagged PLD1 and PLD2 knockin mice using CRISPR/Cas9. Using these mice, we show that PLD1 and PLD2 are both localized at synapses by adulthood, with PLD2 expression being considerably higher in glial cells and PLD1 expression predominating in neurons. Interestingly, we observed that only PLD1 is expressed in the mouse retina, where it is found in the synaptic plexiform layers. These data provide critical information regarding the localization and potential role of PLDs in the central nervous system.

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

N-acylethanolamines (NAEs) are endogenous lipid-signaling molecules derived from fatty acids that regulate numerous biological functions, including in the brain. Interestingly, NAEs are elevated in the absence of fatty acid amide hydrolase (FAAH) and following CO2-induced ischemia/hypercapnia, suggesting a neuroprotective response. Tetracosahexaenoic acid (THA) is a product and precursor to DHA; however, the NAE product, tetracosahexaenoylethanolamide (THEA), has never been reported. Presently, THEA was chemically synthesized as an authentic standard to confirm THEA presence in biological tissues. Whole brains were collected and analyzed for unesterified THA, total THA, and THEA in wild-type and FAAH-KO mice that were euthanized by either head-focused microwave fixation, CO2 + microwave, or CO2 only. PPAR activity by transient transfection assay and ex vivo neuronal output in medium spiny neurons (MSNs) of the nucleus accumbens by patch clamp electrophysiology were determined following THEA exposure. THEA in the wild-type mice was nearly doubled (P < 0.05) following ischemia/hypercapnia (CO2 euthanization) and up to 12 times higher (P < 0.001) in the FAAH-KO compared with wild-type. THEA did not increase (P > 0.05) transcriptional activity of PPARs relative to control, but 100 nM of THEA increased (P < 0.001) neuronal output in MSNs of the nucleus accumbens. Here were identify a novel NAE, THEA, in the brain that is elevated upon ischemia/hypercapnia and by KO of the FAAH enzyme. While THEA did not activate PPAR, it augmented the excitability of MSNs in the nucleus accumbens. Overall, our results suggest that THEA is a novel NAE that is produced in the brain upon ischemia/hypercapnia and regulates neuronal excitation.

Myelin is a unique lipid-rich membrane structure that accelerates neurotransmission and supports neuronal function. Sphingolipids are critical myelin components. Yet sphingolipid content and synthesis have not been well characterized in oligodendrocytes, the myelin-producing cells of the CNS. Here, using quantitative real-time PCR, LC-MS/MS-based lipid analysis, and biochemical assays, we examined sphingolipid synthesis during the peak period of myelination in the postnatal rat brain. Importantly, we characterized sphingolipid production in isolated oligodendrocytes. We analyzed sphingolipid distribution and levels of critical enzymes and regulators in the sphingolipid biosynthetic pathway, with focus on the serine palmitoyltransferase (SPT) complex, the rate-limiting step in this pathway. During myelination, levels of the major SPT subunits increased and oligodendrocyte maturation was accompanied by extensive alterations in the composition of the SPT complex. These included changes in the relative levels of two alternative catalytic subunits, SPTLC2 and -3, in the relative levels of isoforms of the small subunits, ssSPTa and -b, and in the isoform distribution of the SPT regulators, the ORMDLs. Myelination progression was accompanied by distinct changes in both the nature of the sphingoid backbone and the N-acyl chains incorporated into sphingolipids. We conclude that the distribution of these changes among sphingolipid family members is indicative of a selective channeling of the ceramide backbone toward specific downstream metabolic pathways during myelination. Our findings provide insights into myelin production in oligodendrocytes and suggest how dysregulation of the biosynthesis of this highly specialized membrane could contribute to demyelinating diseases.

Steroids that contain a 3-hydroxyl group (3-OH steroids) are widely distributed in nature. During analysis with ESI-MS, they easily become dehydrated while in the protonated form, resulting in the production of several precursor ions and leading to low sensitivity of detection. To address this analytical challenge, here, we developed a method for the quantitation of 3-OH steroids by LC-MS/MS coupled with post-column addition of lithium (Li) ions to the mobile phase. The Li ion has a high affinity for the keto group of steroids, stabilizing their structures during ionization and permitting detection of analytes exclusively as the lithiated form. This not only improved the intensities of the precursor ions, but also promoted the formation of typical lithiated fragment ions. This improvement made the quantitation by multiple reaction monitoring more sensitive and reliable, as evidenced by 1.53–188 times enhanced detection sensitivity of 13 steroids that contained at least one keto and two hydroxyl groups or one keto and one 5-olefinic double bond, among 16 different 3-OH steroids. We deployed our newly developed method for profiling steroids in mouse brain tissue and identified six steroids in one tissue sample. Among these, 16-hydroxyestrone, tetrahydrocorticosterone, and 17α-hydroxypregnenolone were detected for the first time in the mouse brain. In summary, the method described here enables the detection of lithiated steroids by LC-MS/MS, including three 3-OH steroids not previously reported in the mouse brain. We anticipate that this new method may allow the determination of 3-OH steroids in different brain regions.

The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b−/− mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b−/− mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.

Background: Vascular cognitive impairment (VCI) is a major public health problem. The current diagnosis of VCI is made based on the assessment of clinical symptoms and neuropsychological measurements, and is supported by neuroimaging. These methods are both time-consuming and expensive, which leads to needs for alternative biomarkers for VCI. Metabolomics is an emerging and powerful tool to discover of new biomarkers of disease, which can investigate variations in different metabolic processes such as lipid, since the brain is highly enriched in lipids and that lipid changes may lead to pathology in the brain. Vascular cognitive impairment is vulnerable to the disturbance of lipid metabolism. Furthermore, blood samples, which could be identified as reliable clinical biomarkers are relatively convenient to obtain and provide a non-invasive assessment. Therefore, our study aims to understand whether peripheral lipid biomarkers can be used as diagnostic biomarkers and monitor the progression of VCI.Methods: We systematically searched the PubMed, Embase, CNKI, and VIP databases to find VCI and lipid metabolism in reports from inception through February 2021. Studies meeting the following criteria were eligible: (1) original studies in humans; (2) lipid metabolites in blood; (3) reports of VCI.Results: Through our review, nine original articles were eligible. Blood-based metabolites that might be potential biomarkers were identified. Most of them including PC, PE, Cers, and ChEs were significantly lower, while elevation of FAs and DGs were associated with VCI. Most importantly, these blood-based metabolites might be proposed as potential biomarkers for VCI, which provides direction for further validation.Discussion and Conclusion: To the best of our knowledge, this is the first systemic review concerning the relationship of lipid metabolism and VCI. It identifies potential biomarkers and provides insights into the disease pathobiology. However, more advanced studies and researches on a lipidomic platform must be done to understand the exact pathology behind and identify potential lipid biomarkers, which might help achieve the goal of discovering novel therapeutics.

Background: Vascular cognitive impairment (VCI) is a major public health problem. The current diagnosis of VCI is made based on the assessment of clinical symptoms and neuropsychological measurements, and is supported by neuroimaging. These methods are both time-consuming and expensive, which leads to needs for alternative biomarkers for VCI. Metabolomics is an emerging and powerful tool to discover of new biomarkers of disease, which can investigate variations in different metabolic processes such as lipid, since the brain is highly enriched in lipids and that lipid changes may lead to pathology in the brain. Vascular cognitive impairment is vulnerable to the disturbance of lipid metabolism. Furthermore, blood samples, which could be identified as reliable clinical biomarkers are relatively convenient to obtain and provide a non-invasive assessment. Therefore, our study aims to understand whether peripheral lipid biomarkers can be used as diagnostic biomarkers and monitor the progression of VCI. Methods: We systematically searched the PubMed, Embase, CNKI, and VIP databases to find VCI and lipid metabolism in reports from inception through February 2021. Studies meeting the following criteria were eligible: (1) original studies in humans; (2) lipid metabolites in blood; (3) reports of VCI. Results: Through our review, nine original articles were eligible. Blood-based metabolites that might be potential biomarkers were identified. Most of them including PC, PE, Cers, and ChEs were significantly lower, while elevation of FAs and DGs were associated with VCI. Most importantly, these blood-based metabolites might be proposed as potential biomarkers for VCI, which provides direction for further validation. Discussion and Conclusion: To the best of our knowledge, this is the first systemic review concerning the relationship of lipid metabolism and VCI. It identifies potential biomarkers and provides insights into the disease pathobiology. However, more advanced studies and researches on a lipidomic platform must be done to understand the exact pathology behind and identify potential lipid biomarkers, which might help achieve the goal of discovering novel therapeutics. [ABSTRACT FROM AUTHOR]

This study has extended previous metabolic measures in postmortem tissues (frontal and parietal lobes, pons, cerebellum, hippocampus, and cerebral cortex) obtained from a 37-year-old male patient with succinic semialdehyde dehydrogenase deficiency (SSADHD) who expired from SUDEP (sudden unexplained death in epilepsy). Histopathologic characterization of fixed cortex and hippocampus revealed mild to moderate astrogliosis, especially in white matter. Analysis of total phospholipid mass in all sections of the patient revealed a 61% increase in cortex and 51% decrease in hippocampus as compared to (n = 2-4) approximately age-matched controls. Examination of mass and molar composition of major phospholipid classes showed decreases in phospholipids enriched in myelin, such as phosphatidylserine, sphingomyelin, and ethanolamine plasmalogen. Evaluation of gene expression (RT2 Profiler PCR Arrays, GABA, glutamate; Qiagen) revealed dysregulation in 14/15 GABAA receptor subunits in cerebellum, parietal, and frontal lobes with the most significant downregulation in ∊, θ, ρ1, and ρ2 subunits (7.7-9.9-fold). GABAB receptor subunits were largely unaffected, as were ionotropic glutamate receptors. The metabotropic glutamate receptor 6 was consistently downregulated (maximum 5.9-fold) as was the neurotransmitter transporter (GABA), member 13 (maximum 7.3-fold). For other genes, consistent dysregulation was seen for interleukin 1β (maximum downregulation 9.9-fold) and synuclein α (maximal upregulation 6.5-fold). Our data provide unique insight into SSADHD brain function, confirming astrogliosis and lipid abnormalities previously observed in the null mouse model while highlighting long-term effects on GABAergic/glutamatergic gene expression in this disorder. [ABSTRACT FROM AUTHOR]

PUFAs are precursors to bioactive oxylipin metabolites that increase in the brain following CO2-induced hypercapnia/ischemia. It is not known whether the brain-dissection process and its duration also alter these metabolites. We applied CO2 with or without head-focused microwave fixation for 2 min to evaluate the effects of CO2-induced asphyxiation, dissection, and dissection time on brain oxylipin concentrations. Compared with head-focused microwave fixation (control), CO2 followed by microwave fixation prior to dissection increased oxylipins derived from lipoxygenase (LOX), 15-hydroxyprostaglandin dehydrogenase (PGDH), cytochrome P450 (CYP), and soluble epoxide hydrolase (sEH) enzymatic pathways. This effect was enhanced when the duration of postmortem ischemia was prolonged by 6.4 min prior to microwave fixation. Brains dissected from rats subjected to CO2 without microwave fixation showed greater increases in LOX, PGDH, CYP and sEH metabolites compared with all other groups, as well as increased cyclooxygenase metabolites. In nonmicrowave-irradiated brains, sEH metabolites and one CYP metabolite correlated positively and negatively with dissection time, respectively. This study presents new evidence that the dissection process and its duration increase brain oxylipin concentrations, and that this is preventable by microwave fixation. When microwave fixation is not available, lipidomic studies should account for dissection time to reduce these artifacts.

EPA and DHA protect against multiple metabolic and neurologic disorders. Although DHA appears more effective for neuroinflammatory conditions, EPA is more beneficial for depression. However, the brain contains negligible amounts of EPA, and dietary supplements fail to increase it appreciably. We tested the hypothesis that this failure is due to absorption of EPA as triacylglycerol, whereas the transporter at the blood-brain barrier requires EPA as lysophosphatidylcholine (LPC). We compared tissue uptake in normal mice gavaged with equal amounts (3.3 μmol/day) of either LPC-EPA or free EPA (surrogate for current supplements) for 15 days and also measured target gene expression. Compared with the no-EPA control, LPC-EPA increased brain EPA >100-fold (from 0.03 to 4 μmol/g); free EPA had little effect. Furthermore, LPC-EPA, but not free EPA, increased brain DHA 2-fold. Free EPA increased EPA in adipose tissue, and both supplements increased EPA and DHA in the liver and heart. Only LPC-EPA increased EPA and DHA in the retina, and expression of brain-derived neurotrophic factor, cyclic AMP response element binding protein, and 5-hydroxy tryptamine (serotonin) receptor 1A in the brain. These novel results show that brain EPA can be increased through diet. Because LPC-EPA increased both EPA and DHA in the brain, it may help in the treatment of depression as well as neuroinflammatory diseases, such as Alzheimer's disease.

Lysophospholipids (LysoPLs) are bioactive lipid species involved in cellular signaling processes and the regulation of cell membrane structure. LysoPLs are metabolized through the action of lysophospholipases, including lysophospholipase A1 (LYPLA1) and lysophospholipase A2 (LYPLA2). A new X-ray crystal structure of LYPLA2 compared with a previously published structure of LYPLA1 demonstrated near-identical folding of the two enzymes; however, LYPLA1 and LYPLA2 have displayed distinct substrate specificities in recombinant enzyme assays. To determine how these in vitro substrate preferences translate into a relevant cellular setting and better understand the enzymes' role in LysoPL metabolism, CRISPR-Cas9 technology was utilized to generate stable KOs of Lypla1 and/or Lypla2 in Neuro2a cells. Using these cellular models in combination with a targeted lipidomics approach, LysoPL levels were quantified and compared between cell lines to determine the effect of losing lysophospholipase activity on lipid metabolism. This work suggests that LYPLA1 and LYPLA2 are each able to account for the loss of the other to maintain lipid homeostasis in cells; however, when both are deleted, LysoPL levels are dramatically increased, causing phenotypic and morphological changes to the cells.

Cholesterol, a critical component of the cellular plasma membrane, is essential for normal neuronal function. Cholesterol content is highest in the brain, where most cholesterol is synthesized de novo; HMG-CoA reductase controls the synthesis rate. Despite strict control, elevated blood cholesterol levels are common and are associated with various neurological disorders. G protein-gated inwardly rectifying potassium (GIRK) channels mediate the actions of inhibitory brain neurotransmitters. Loss of GIRK function enhances neuron excitability; gain of function reduces neuronal activity. However, the effect of dietary cholesterol or HMG-CoA reductase inhibition (i.e., statin therapy) on GIRK function remains unknown. Using a rat model, we compared the effects of a high-cholesterol versus normal diet both with and without atorvastatin, a widely prescribed HMG-CoA reductase inhibitor, on neuronal GIRK currents. The high-cholesterol diet increased hippocampal CA1 region cholesterol levels and correspondingly increased neuronal GIRK currents. Both phenomena were reversed by cholesterol depletion in vitro. Atorvastatin countered the high-cholesterol diet effects on neuronal cholesterol content and GIRK currents; these effects were reversed by cholesterol enrichment in vitro. Our findings suggest that high-cholesterol diet and atorvastatin therapy affect ion channel function in the brain by modulating neuronal cholesterol levels.

Brain contains the highest lipid content involved in various structural and physiological activities such as structural development, neurogenesis, synaptogenesis, signal transduction and myelin sheath formation. Lipids bilayer is essential to maintain the structural integrity for the physiological functions of protein. Impairments in lipid metabolism and its composition can lead to the progression of various brain ailments such as neurodegenerative and neuropsychiatric disorders. Aluminium (Al), the potent neurotoxin has been linked to Alzheimer's disease (AD) like pathology. Al can bind to biomembrane and influence oligomerization and conformational changes of proteins by acting as cross-linkers. The present study evaluated the influence of Ginkgo biloba (GBE) on the lipid profile alterations induced by Al lactate in hippocampal and cortical regions using FTIR spectroscopy. Rats were exposed with 10 mg/kg b.w. (intraperitoneal) of Al lactate for 6 weeks. This was followed by a treatment protocol of GBE (100 mg/kg b.w.) both preexposure (2 weeks) and conjunctive (6 weeks) exposure. A self recovery group was also included, where Al withdrawal was done for 2 weeks post Al exposure. A significant decrease in peak areas of cholesterol, sphingolipids and phospholipids was observed in Al treated groups. Further, polyunsaturated fatty acids and membrane fluidity has also decreased, as revealed by olefinic and methyl asymmetric stretching bands. Al treatment significantly increased the fluorescence polarization, anisotropy and order parameter, which however were normalized following GBE supplementation. Results also showed that pretreatment with GBE provided more beneficial effects on the adverse changes following Al in membrane composition and behavioral outcome. [ABSTRACT FROM AUTHOR]

Polyunsaturated fatty acids (PUFAs) and eicosanoids are important mediators of inflammation. The functional role of eicosanoids in metabolic-syndrome-related diseases has been extensively studied. However, their role in neuroinflammation and the development of neurodegenerative diseases is still unclear. The aim of this study was the development of a sample pretreatment protocol for the simultaneous analysis of PUFAs and eicosanoids in mouse liver and brain. Liver and brain samples of male wild-type C57BL/6J mice (11–122 mg) were used to investigate conditions for tissue rinsing, homogenization, extraction, and storage. A targeted liquid chromatography–negative electrospray ionization tandem mass spectrometry method was applied to quantify 7 PUFAs and 94 eicosanoids. The final pretreatment protocol consisted of a 5-min homogenization step by sonication in 650 μL n-hexane/2-propanol (60:40 v/v) containing 2,6-di-tert-butyl-4-methylphenol at 50 μg/mL. Homogenates representing 1 mg tissue were extracted in a single step with n-hexane/2-propanol (60:40 v/v) containing 0.1% formic acid. Autoxidation was prevented by addition of 2,6-di-tert-butyl-4-methylphenol at 50 μg/mL and keeping the samples at 4 °C during sample preparation. Extracts were dried under nitrogen and reconstituted in liquid chromatography eluent before analysis. Recovery was determined to range from 45% to 149% for both liver and brain tissue. Within-run and between-run variability ranged between 7% and 18% for PUFAs and between 1% and 24% for eicosanoids. In liver, 7 PUFAs and 15 eicosanoids were quantified; in brain, 6 PUFAs and 21 eicosanoids had significant differences within the brain substructures. In conclusion, a robust and reproducible sample preparation protocol for the multiplexed analysis of PUFAs and eicosanoids by liquid chromatography–tandem mass spectrometry in liver and discrete brain substructures was developed. [ABSTRACT FROM AUTHOR]

Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of β-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB-corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB-corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB-corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses.

Inhibiting the glutamate/cystine antiporter system xc−, a key antioxidant defense machinery in the CNS, could trigger a novel form of regulated necrotic cell death, ferroptosis. The underlying mechanisms of system xc−-dependent cell demise were elucidated using primary oligodendrocytes (OLs) treated with glutamate to block system xc− function. Pharmacological analysis revealed ferroptosis as a major contributing factor to glutamate-initiated OL death. A sphingolipid profile showed elevations of ceramide species and sphingosine that were preventable by inhibiting of an acid sphingomyelinase (ASM) activity. OL survival was enhanced by both downregulating ASM expression and blocking ASM activity. Glutamate-induced ASM activation seems to involve posttranscriptional mechanisms and was associated with a decreased GSH level. Further investigation of the mechanisms of OL response to glutamate revealed enhanced reactive oxygen species production, augmented lipid peroxidation, and opening of the mitochondrial permeability transition pore that were attenuated by hindering ASM. Of note, knocking down sirtuin 3, a deacetylase governing the mitochondrial antioxidant system, reduced OL survival. The data highlight the importance of the mitochondrial compartment in regulated necrotic cell death and accentuate the novel role of ASM in disturbing mitochondrial functions during OL response to glutamate toxicity, which is essential for pathobiology in stroke and traumatic brain injury.

Alzheimer's disease (AD) is the most common form of dementia in older adults. Currently, there is no cure for AD. The hallmark of AD is the accumulation of extracellular amyloid plaques composed of amyloid-β (Aβ) peptides (especially Aβ1-42) and neurofibrillary tangles, composed of hyperphosphorylated tau and accompanied by chronic neuroinflammation. Aβ peptides are derived from the amyloid precursor protein (APP). The oligomeric form of Aβ peptides is probably the most neurotoxic species; its accumulation eventually forms the insoluble and aggregated amyloid plaques. ApoE is the major apolipoprotein of the lipoprotein(s) present in the CNS. ApoE has three alleles, of which the Apoe4 allele constitutes the major risk factor for late-onset AD. Here we describe the complex relationship between ApoE4, oligomeric Aβ peptides, and cholesterol homeostasis. The review consists of four parts: 1) key elements involved in cellular cholesterol metabolism and regulation; 2) key elements involved in intracellular cholesterol trafficking; 3) links between ApoE4, Aβ peptides, and disturbance of cholesterol homeostasis in the CNS; 4) potential lipid-based therapeutic targets to treat AD. At the end, we recommend several research topics that we believe would help in better understanding the connection between cholesterol and AD for further investigations.

Compartmentalization of metabolism into specific regions of the cell, tissue, and organ is critical to life for all organisms. Mass spectrometric imaging techniques have been valuable in identifying and quantifying concentrations of metabolites in specific locations of cells and tissues, but a true understanding of metabolism requires measurement of metabolite flux on a spatially resolved basis. Here, we utilize desorption ESI-MS (DESI-MS) to measure lipid turnover in the brains of mice. We show that anatomically distinct regions of the brain have distinct lipid turnover rates. These turnover measurements, in conjunction with relative concentration, will enable calculation of regiospecific synthesis rates for individual lipid species in vivo. Monitoring spatially dependent changes in metabolism has the potential to significantly facilitate research in many areas, such as brain development, cancer, and neurodegeneration.

Alzheimer's disease (AD) is one of the fastest-growing causes of death and disability in persons 65 years of age or older, affecting more than 5 million Americans alone. Clinical manifestations of AD include progressive decline in memory, executive function, language, and other cognitive domains. Research efforts within the last three decades have identified APOE as the most significant genetic risk factor for late-onset AD, which accounts for >99% of cases. The apoE protein is hypothesized to affect AD pathogenesis through a variety of mechanisms, from its effects on the blood-brain barrier, the innate immune system, and synaptic function to the accumulation of amyloid-β (Aβ). Here, we discuss the role of apoE on the biophysical properties and metabolism of the Aβ peptide, the principal component of amyloid plaques and cerebral amyloid angiopathy (CAA). CAA is characterized by the deposition of amyloid proteins (including Aβ) in the leptomeningeal medium and small arteries, which is found in most AD cases but sometimes occurs as an independent entity. Accumulation of these pathologies in the brain is one of the pathological hallmarks of AD. Beyond Aβ, we will extend the discussion to the potential role of apoE on other amyloidogenic proteins found in AD, and also a number of diverse neurodegenerative diseases.

We present here a quantitative molecular blueprint of the three major glycerophospholipid (GPL) classes, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), in retina and six regions of the brain in C57Bl6 mice at 2, 10, and 26 months of age. We found an age-related increase in molecular species containing saturated and monoenoic FAs and an overall decrease in the longer-chain PUFA molecular species across brain regions, with loss of DHA-containing molecular species as the most consistent and dramatic finding. Although we found very-long-chain PUFAs (VLC-PUFAs) (C28) in PC in the retina, no detectable levels were found in any brain region at any of the ages examined. All brain regions (except hippocampus and retina) showed a significant increase with age in PE plasmalogens. All three retina GPLs had di-PUFA molecular species (predominantly 44:12), which were most abundant in PS (∼30%). In contrast, low levels of di-PUFA GPL (1–2%) were found in all regions of the brain. This study provides a regional and age-related assessment of the brain's lipidome with a level of detail, inclusion, and quantification that has not heretofore been published.

Perturbations of ganglioside homeostasis have been observed following stroke whereby toxic simple gangliosides GM2 and GM3 accumulate, while protective complex species GM1 and GD1 are reduced. Thus, there is a need for therapeutic interventions which can prevent ganglioside dysregulation after stroke. A pharmacological intervention using chloroquine was selected for its transient lysosomotropic properties which disrupt the activity of catabolic ganglioside enzymes. Chloroquine was administered both in vitro (0.1 μM), to primary cortical neurons exposed to GM3 toxicity, and in vivo (45 mg/kg i.p.), to 3-month-old male Wistar rats that underwent a severe stroke injury. Chloroquine was administered for seven consecutive days beginning 3 days prior to the stroke injury. Gangliosides were examined using MALDI imaging mass spectrometry at 3 and 21 days after the injury, and motor deficits were examined using the ladder task. Chloroquine treatment prevented ganglioside dysregulation 3 days post-stroke and partially prevented complex ganglioside depletion 21 days post-stroke. Exogenous GM3 was found to be toxic to primary cortical neurons which was protected by chloroquine treatment. Motor deficits were prevented in the forelimbs of stroke-injured rats with chloroquine treatment and was associated with decreased inflammation, neurodegeneration, and an increase in cell survival at the site of injury. Chloroquine administration prevents ganglioside dysregulation acutely, protects against GM3 toxicity in neurons, and is associated with long-term functional and pathological improvements after stroke in the rat. Therefore, targeting lipid dysregulation using lysosomotropic agents such as chloroquine may represent a novel therapeutic avenue for stroke injuries. [ABSTRACT FROM AUTHOR]

Endocannabinoids, a class of lipid messengers, have emerged as crucial regulators of synaptic communication in the CNS. Dysregulation of these compounds has been implicated in many brain disorders. Although some studies have identified and quantified a limited number of target compounds, a method that provides comprehensive quantitative information on endocannabinoids and related N-acylethanolamines (NAEs) in cerebrospinal fluid (CSF) is currently lacking, as measurements are challenging due to low concentrations under normal physiological conditions. Here we developed and validated a high-throughput nano LC-ESI-MS/MS platform for the simultaneous quantification of endocannabinoids (anandamide and 2-arachidonoylglycerol), ten related NAEs, and eight additional putatively annotated NAEs in human CSF. Requiring only 200 μl of CSF, our method has limits of detection from 0.28 to 61.2 pM with precisions of relative SD

As neurons die, cholesterol is released in the central nervous system (CNS); hence, this sterol and its metabolites may represent a biomarker of neurodegeneration, including in amyotrophic lateral sclerosis (ALS), in which altered cholesterol levels have been linked to prognosis. More than 40 different sterols were quantified in serum and cerebrospinal fluid (CSF) from ALS patients and healthy controls. In CSF, the concentration of cholesterol was found to be elevated in ALS samples. When CSF metabolite levels were normalized to cholesterol, the cholesterol metabolite 3β,7α-dihydroxycholest-5-en-26-oic acid, along with its precursor 3β-hydroxycholest-5-en-26-oic acid and product 7α-hydroxy-3-oxocholest-4-en-26-oic acid, were reduced in concentration, whereas metabolites known to be imported from the circulation into the CNS were not found to differ in concentration between groups. Analysis of serum revealed that (25R)26-hydroxycholesterol, the immediate precursor of 3β-hydroxycholest-5-en-26-oic acid, was reduced in concentration in ALS patients compared with controls. We conclude that the acidic branch of bile acid biosynthesis, known to be operative in-part in the brain, is defective in ALS, leading to a failure of the CNS to remove excess cholesterol, which may be toxic to neuronal cells, compounded by a reduction in neuroprotective 3β,7α-dihydroxycholest-5-en-26-oic acid.

In mammals, because they share a single synthetic pathway, n-6/n-3 ratios of dietary PUFAs impact tissue arachidonic acid (ARA) and DHA content. Likewise, SNPs in the human fatty acid desaturase (FADS) gene cluster impact tissue ARA and DHA. Here we tested the feasibility of using heterozygous Fads2-null-mice (HET) as an animal model of human FADS polymorphisms. WT and HET mice were fed diets with linoleate/α-linolenate ratios of 1:1, 7:1, and 44:1 at 7% of diet. In WT liver, ARA and DHA in phospholipids varied >2× among dietary groups, reflecting precursor ratios. Unexpectedly, ARA content was only

To date, sterylglucosides have been reported to be present in various fungi, plants, and animals. In bacteria, such as Helicobacter pylori, proton NMR spectral analysis of isolated 1-O-cholesteryl-β-d-glucopyranoside (GlcChol) demonstrated the presence of an α-glucosidic linkage. By contrast, in animals, no detailed structural analysis of GlcChol has been reported, in part because animal-derived samples contain a high abundance of glucosylceramides (GlcCers)/galactosylceramides, which exhibit highly similar chromatographic behavior to GlcChol. A key step in vertebrate GlcChol biosynthesis is the transglucosylation reaction catalyzed by glucocerebrosidase (GBA)1 or GBA2, utilizing GlcCer as a glucose donor. These steps are expected to produce a β-glucosidic linkage. Impaired GBA1 and GBA2 function is associated with neurological disorders, such as cerebellar ataxia, spastic paraplegia, and Parkinson's disease. Utilizing a novel three-step chromatographic procedure, we prepared highly enriched GlcChol from embryonic chicken brain, allowing complete structural confirmation of the β-glucosidic linkage by 1H-NMR analysis. Unexpectedly, during purification, two additional sterylglucoside fractions were isolated. NMR and GC/MS analyses confirmed that the plant-type sitosterylglucoside in vertebrate brain is present throughout embryonic development. The aglycon structure of the remaining sterylglucoside (GSX-2) remains elusive due to its low abundance. Together, our results uncovered unexpected aglycon heterogeneity of sterylglucosides in vertebrate brain.

Long-chain fatty acid amides are signaling lipids found in mammals and other organisms; however, details of the metabolic pathways for the N-acylglycines and primary fatty acid amides (PFAMs) have remained elusive. Heavy-labeled precursor and subtraction lipidomic experiments in mouse neuroblastoma N18TG2 cells, a model cell line for the study of fatty acid amide metabolism, establish the biosynthetic pathways for the N-acylglycines and the PFAMs. We provide evidence that the N-acylglycines are formed by a long-chain specific glycine-conjugating enzyme, glycine N-acyltransferase-like 3 (GLYATL3). siRNA knockdown of GLYATL3 in the N18TG2 cells resulted in a decrease in the levels of the N-acylglycines and the PFAMs. This is the first report of an enzyme responsible for long-chain N-acylglycine production in cellula. The production of the PFAMs in N18TG2 cells was reported to occur by the oxidative cleavage of the N-acylglycines, as catalyzed by peptidylglycine α-amidating monooxygenase (PAM). siRNA knockdown of PAM resulted in an accumulation of [13C18]N-oleoylglycine and decreased levels of [13C18]oleamide when the N18TG2 cells were grown in the presence of [13C18]oleic acid. The addition of [1-13C]palmitate to the N18TG2 cell growth media led to the production of a family of [1-13C]palmitoylated fatty acid amides, consistent with the biosynthetic pathways detailed herein.

The number, position, and configuration of double bonds in lipids affect membrane fluidity and the recruitment of signaling proteins. Studies on mammalian sphingolipids have focused on those with a saturated sphinganine or mono-unsaturated sphingosine long chain base. Using high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), we observed a marked accumulation of lipids containing a di-unsaturated sphingadiene base in the hippocampus of mice lacking the metabolic enzyme sphingosine kinase 2 (SphK2). The double bonds were localized to positions C4–C5 and C14–C15 of sphingadiene using ultraviolet photodissociation-tandem mass spectrometry (UVPD-MS/MS). Phosphorylation of sphingoid bases by sphingosine kinase 1 (SphK1) or SphK2 forms the penultimate step in the lysosomal catabolism of all sphingolipids. Both SphK1 and SphK2 phosphorylated sphinga-4,14-diene as efficiently as sphingosine, however deuterated tracer experiments in an oligodendrocyte cell line demonstrated that ceramides with a sphingosine base are more rapidly metabolized than those with a sphingadiene base. Since SphK2 is the dominant sphingosine kinase in brain, we propose that the accumulation of sphingadiene-based lipids in SphK2-deficient brains results from the slower catabolism of these lipids, combined with a bottleneck in the catabolic pathway created by the absence of SphK2. We have therefore uncovered a previously unappreciated role for SphK2 in lipid quality control.

Flaviviruses, such as the dengue virus and the West Nile virus (WNV), are arthropod-borne viruses that represent a global health problem. The flavivirus lifecycle is intimately connected to cellular lipids. Among the lipids co-opted by flaviviruses, we have focused on SM, an important component of cellular membranes particularly enriched in the nervous system. After infection with the neurotropic WNV, mice deficient in acid sphingomyelinase (ASM), which accumulate high levels of SM in their tissues, displayed exacerbated infection. In addition, WNV multiplication was enhanced in cells from human patients with Niemann-Pick type A, a disease caused by a deficiency of ASM activity resulting in SM accumulation. Furthermore, the addition of SM to cultured cells also increased WNV infection, whereas treatment with pharmacological inhibitors of SM synthesis reduced WNV infection. Confocal microscopy analyses confirmed the association of SM with viral replication sites within infected cells. Our results unveil that SM metabolism regulates flavivirus infection in vivo and propose SM as a suitable target for antiviral design against WNV.