Mikhail Y. Golovko

Mitochondrial Lipid Abnormality and Electron Transport Chain Impairment in Mice Lacking α-Synuclein

ABSTRACT The presynaptic protein α-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking α-synuclein (Snca−/−), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca −/− brain suggest a relationship between α-synucleins role in brain lipid metabolism, mitochondrial function, and PD.

Deposition of iron and β‐amyloid plaques is associated with cortical cellular damage in rabbits fed with long‐term cholesterol‐enriched diets

Hypercholesterolemia is a potential trigger of Alzheimers disease, and is thought to increase brain levels of β‐amyloid (Aβ) and iron. However, animal models to address the mechanisms by which Aβ and iron accumulation may cause neuronal damage are poorly defined. To address this question, we fed adult rabbits a 1% cholesterol‐enriched diet for 7 months. This diet was associated with increased regional deposition of both iron and Aβ peptide in the brain. Iron preferentially accumulated around Aβ plaques in the adjacent cortex, but was not found in the hippocampus. Co‐localization of iron and Aβ was accompanied by apoptosis, DNA damage, blood–brain barrier (BBB) disruption, as well as dysregulation in the level of the iron‐regulatory proteins, ferritin and heme‐oxygenase‐1. We further demonstrate that the cholesterol diet‐induced apoptosis is mediated by the activation of the endoplasmic reticulum stress pathway, involving the down‐regulation of the endoplasmic reticulum chaperones, calreticulin, grp78 and grp94, and the activation of the growth and arrest DNA damage protein, gadd153. Our results suggest that BBB damage and disturbances in iron metabolism may render the cortex more vulnerable than the hippocampus to the cholesterol‐induced cellular stress.

Fatty acid incorporation is decreased in astrocytes cultured from α-synuclein gene-ablated mice

Because α‐synuclein may function as a fatty acid binding protein, we measured fatty acid incorporation into astrocytes isolated from wild‐type and α‐synuclein gene‐ablated mice. α‐Synuclein deficiency decreased palmitic acid (16:0) incorporation 31% and arachidonic acid [20:4 (n‐6)] incorporation 39%, whereas 22:6 (n‐3) incorporation was unaffected. In neutral lipids, fatty acid targeting of 20:4 (n‐6) and 22:6 (n‐3) (docosahexaenoic acid) to the neutral lipid fraction was increased 1.7‐fold and 1.6‐fold, respectively, with an increase in each of the major neutral lipids. This was consistent with a 3.4‐ to 3.8‐fold increase in cholesteryl ester and triacylglycerol mass. In the phospholipid fraction, α‐synuclein deficiency decreased 16:0 esterification 39% and 20:4 (n‐6) esterification 43% and decreased the distribution of these fatty acids, including 22:6 (n‐3), into this lipid pool. α‐Synuclein gene‐ablation significantly decreased the trafficking of these fatty acids to phosphatidylinositol. This observation is consistent with changes in phospholipid fatty acid composition in the α‐synuclein‐deficient astrocytes, including decreased 22:6 (n‐3) content in the four major phospholipid classes. In summary, these studies demonstrate that α‐synuclein deficiency significantly disrupted astrocyte fatty acid uptake and trafficking, with a marked increase in fatty acid trafficking to cholesteryl esters and triacylglycerols and decreased trafficking to phospholipids, including phosphatidylinositol.

Amyloid precursor protein and proinflammatory changes are regulated in brain and adipose tissue in a murine model of high fat diet-induced obesity.

Background Middle age obesity is recognized as a risk factor for Alzheimers disease (AD) although a mechanistic linkage remains unclear. Based upon the fact that obese adipose tissue and AD brains are both areas of proinflammatory change, a possible common event is chronic inflammation. Since an autosomal dominant form of AD is associated with mutations in the gene coding for the ubiquitously expressed transmembrane protein, amyloid precursor protein (APP) and recent evidence demonstrates increased APP levels in adipose tissue during obesity it is feasible that APP serves some function in both disease conditions. Methodology/Principal Findings To determine whether diet-induced obesity produced proinflammatory changes and altered APP expression in brain versus adipose tissue, 6 week old C57BL6/J mice were maintained on a control or high fat diet for 22 weeks. Protein levels and cell-specific APP expression along with markers of inflammation and immune cell activation were compared between hippocampus, abdominal subcutaneous fat and visceral pericardial fat. APP stimulation-dependent changes in macrophage and adipocyte culture phenotype were examined for comparison to the in vivo changes. Conclusions/Significance Adipose tissue and brain from high fat diet fed animals demonstrated increased TNF-α and microglial and macrophage activation. Both brains and adipose tissue also had elevated APP levels localizing to neurons and macrophage/adipocytes, respectively. APP agonist antibody stimulation of macrophage cultures increased specific cytokine secretion with no obvious effects on adipocyte culture phenotype. These data support the hypothesis that high fat diet-dependent obesity results in concomitant pro-inflammatory changes in brain and adipose tissue that is characterized, in part, by increased levels of APP that may be contributing specifically to inflammatory changes that occur.

Brain neutral lipids mass is increased in α‐synuclein gene‐ablated mice

Because α‐synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that Snca deletion had on whole brain lipid composition. We analysed masses of individual phospholipid (PL) classes and neutral lipid mass as well as PL acyl chain composition in brains from wild‐type and Snca‐/‐ mice. Although total brain PL mass was not altered, cardiolipin and phosphatidylglycerol mass decreased 16% and 27%, respectively, in Snca‐/‐ mice. In addition, no changes were observed in plasmalogen or polyphosphoinositide mass. In ethanolamine glycerophospholipids and phosphatidylserine, docosahexaenoic acid (22 : 6n‐3) was decreased 7%, while 16 : 0 was increased 1.1‐fold and 1.4‐fold, respectively. Surprisingly, brain cholesterol, cholesteryl ester, and triacylglycerol mass were increased 1.1‐fold, 1.6‐fold, and 1.4‐fold, respectively in Snca‐/‐ mice. In isolated myelin, cholesterol mass was also increased 1.3‐fold, but because there was also a net increase in myelin PL mass, the cholesterol to PL ratio was unaltered. No changes in the expression of cholesterogenic enzymes were observed, suggesting these did not account for the observed changes in cholesterol. These data extend our previous results in astrocytes and kinetic studies in vivo demonstrating a role for Snca in brain lipid metabolism and demonstrate a clear impact on brain neutral lipid metabolism.

An improved LC-MS/MS procedure for brain prostanoid analysis using brain fixation with head-focused microwave irradiation and liquid-liquid extraction

High-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) allows a highly selective, sensitive, simultaneous analysis for prostanoids (PG) without derivatization. However, high chemical background noise reduces LC-MS/MS selectivity and sensitivity for brain PG analysis. Four common methods using different solvent systems for PG extraction were tested. Although these methods had the same recovery of PG, the modified acetone extraction followed by liquid/liquid purification had the greatest sensitivity. This method combined with hexane/2-propanol extraction permits the simultaneous analysis of other lipid molecules and PG in the same extract. We also determined that PG mass in brain powder stored at −80°C was reduced 2- to 4- fold in 4 weeks; however, PG were stable for long periods (>3 months) in hexane/2-propanol extracts. PG mass was increased significantly when mice were euthanized by decapitation and the brains rapidly flash-frozen rather than euthanized using head-focused microwave irradiation. This reduction is not the result of PG trapping or destruction in microwave-irradiated brains, demonstrating its importance in limiting mass artifacts during brain PG analysis. Our improved procedure for brain PG analysis provides a reliable, rapid means to detect changes in brain PG mass under both basal and pathological conditions and demonstrates the importance of sample preparation in this process.

α‐Synuclein gene ablation increases docosahexaenoic acid incorporation and turnover in brain phospholipids

Previously, we demonstrated that ablation of α‐synuclein (Snca) reduces arachidonate (20:4n‐6) turnover in brain phospholipids through modulation of an endoplasmic reticulum‐localized acyl‐CoA synthetase (Acsl). The effect of Snca ablation on docosahexaenoic acid (22:6n‐3) metabolism is unknown. In the present study, we examined the effect of Snca gene ablation on brain 22:6n‐3 metabolism. We determined 22:6n‐3 uptake and incorporation into brain phospholipids by infusing awake, wild‐type and Snca−/− mice with [1‐14C]22:6n‐3 using steady‐state kinetic modeling. In addition, because Snca modulates 20:4n‐6‐CoA formation, we assessed microsomal Acsl activity using 22:6n‐3 as a substrate. Although Snca gene ablation does not affect brain 22:6n‐3 uptake, brain 22:6n‐3‐CoA mass was elevated 1.5‐fold in the absence of Snca. This is consistent with the 1.6‐ to 2.2‐fold increase in the incorporation rate and turnover in ethanolamine glycerophospholipid, phosphatidylserine, and phosphatidylinositol pools. Increased 22:6n‐3‐CoA mass was not the result of altered Acsl activity, which was unaffected by the absence of Snca. While Snca bound 22:6n‐3, Kd = 1.0 ± 0.5 μmol/L, it did not bind 22:6n‐3‐CoA. These effects of Snca gene deletion on 22:6n‐3 brain metabolism are opposite to what we reported previously for brain 20:4n‐6 metabolism and are likely compensatory for the decreased 20:4n‐6 metabolism in brains of Snca−/− mice.

The role of α-synuclein in brain lipid metabolism: a downstream impact on brain inflammatory response

Abstractα-Synuclein (Snca) is an abundant small cytosolic protein (140 amino acids) that is expressed in the brain, although its physiological role is poorly defined. Consistent with its ubiquitous distribution in the brain, we and others have established a role for Snca in brain lipid metabolism and downstream events such as neuroinflammation. In astrocytes, Snca is important for fatty acid uptake and trafficking, where its deletion decreases 16:0 and 20:4n-6 uptake and alters targeting to specific lipid pools. Although Snca has no impact on 22:6n-3 uptake into astrocytes, it is important for its targeting to lipid pools. Similar results for fatty acid uptake from the plasma are seen in studies using whole mice coupled with steady-state kinetic modeling. We demonstrate in gene-ablated mice a significant reduction in the incorporation rate of 20:4n-6 into brain phospholipid pools due to reduced recycling of 20:4n-6 through the ER-localized long-chain acyl-CoA synthetases (Acsl). This reduction results in a compensatory increase in the incorporation rate of 22:6n-3 into brain phospholipids. Snca is also important for brain and astrocyte cholesterol metabolism, where its deletion results in an elevation of cholesterol and cholesteryl esters. This increase may be due to the interaction of Snca with membrane-bound enzymes involved in lipid metabolism such as Acsl. Snca is critical in modulating brain prostanoid formation and microglial activities. In the absence of Snca, microglia are basally activated and demonstrate increased proinflammatory cytokine secretion. Thus, Snca, through its modulation of brain lipid metabolism, has a critical role in brain inflammatory responses.

Uptake and metabolism of plasma-derived erucic acid by rat brain

We examined the ability of erucic acid (22:1n-9) to cross the blood-brain barrier (BBB) by infusing [14–14C]22:1n-9 (170 μCi/kg, iv and icv) into awake, male rats. [1-14C]arachidonic acid (20:4n-6) [intravenous (i.v.)] was the positive control. After i.v. infusion, 0.011% of the plasma [14-14C]22:1n-9 was extracted by the brain, compared with 0.055% of the plasma [1-14C]20:4n-6. The [14-14C]22:1n-9 was extensively β-oxidized (60%), compared with 30% for [1-14C]20:4n-6. Although 20:4n-6 was targeted primarily to phospholipid pools, 22:1n-9 was targeted to cholesteryl esters, triglycerides, and phospholipids. When [14-14C]22:1n-9 was infused directly into the fourth ventricle of the brain [intracerebroventricular (i.c.v.)] for 7 days, 60% of the tracer entered the phospholipid pools, similar to the distribution observed for [1-14C]20:4n-6. This demonstrates plasticity in the ability of the brain to esterify 22:1n-9 in an exposure-dependent manner. In i.v. and i.c.v. infused rats, a significant amount of tracer found in the phospholipid pools underwent sequential rounds of chain shortening and was found as [12-14C]20:1n-9 and [10-14C]oleic acid. These results demonstrate for the first time that intact 22:1n-9 crosses the BBB, is incorporated into specific lipid pools, and is chain-shortened.

LC/MS/MS method for analysis of E2 series prostaglandins and isoprostanes

15-series prostaglandins (PGE2s) and isoprostanes (isoPGE2s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE2/D2 and isoPGE2 without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE2/PGD2/isoPGE2 such as PGE2, entPGE2, 8-isoPGE2, 11β-PGE2, PGD2, and 15(R)-PGD2 to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE2 in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE2 levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE2s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.