Robin B. Chan

Comparative Lipidomic Analysis of Mouse and Human Brain with Alzheimer Disease

Background: Lipid dyshomeostasis has been linked to Alzheimer disease (AD). Results: Lipidomic analyses of brain tissue from AD patients reveal region-specific changes in multiple bioactive lipids, some of which are phenocopied in AD mouse models. Conclusion: Lipid anomalies observed in AD may be linked to pathogenesis, including endolysosomal dysfunction. Significance: This study highlights the hypothesis-generating potential of lipidomics and its applicability to other diseases. Lipids are key regulators of brain function and have been increasingly implicated in neurodegenerative disorders including Alzheimer disease (AD). Here, a systems-based approach was employed to determine the lipidome of brain tissues affected by AD. Specifically, we used liquid chromatography-mass spectrometry to profile extracts from the prefrontal cortex, entorhinal cortex, and cerebellum of late-onset AD (LOAD) patients, as well as the forebrain of three transgenic familial AD (FAD) mouse models. Although the cerebellum lacked major alterations in lipid composition, we found an elevation of a signaling pool of diacylglycerol as well as sphingolipids in the prefrontal cortex of AD patients. Furthermore, the diseased entorhinal cortex showed specific enrichment of lysobisphosphatidic acid, sphingomyelin, the ganglioside GM3, and cholesterol esters, all of which suggest common pathogenic mechanisms associated with endolysosomal storage disorders. Importantly, a significant increase in cholesterol esters and GM3 was recapitulated in the transgenic FAD models, suggesting that these mice are relevant tools to study aberrant lipid metabolism of endolysosomal dysfunction associated with AD. Finally, genetic ablation of phospholipase D2, which rescues the synaptic and behavioral deficits of an FAD mouse model, fully normalizes GM3 levels. These data thus unmask a cross-talk between the metabolism of phosphatidic acid, the product of phospholipase D2, and gangliosides, and point to a central role of ganglioside anomalies in AD pathogenesis. Overall, our study highlights the hypothesis generating potential of lipidomics and identifies novel region-specific lipid anomalies potentially linked to AD pathogenesis.

Retroviruses Human Immunodeficiency Virus and Murine Leukemia Virus Are Enriched in Phosphoinositides

ABSTRACT Retroviruses acquire a lipid envelope during budding from the membrane of their hosts. Therefore, the composition of this envelope can provide important information about the budding process and its location. Here, we present mass spectrometry analysis of the lipid content of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). The results of this comprehensive survey found that the overall lipid content of these viruses mostly matched that of the plasma membrane, which was considerably different from the total lipid content of the cells. However, several lipids are enriched in comparison to the composition of the plasma membrane: (i) cholesterol, ceramide, and GM3; and (ii) phosphoinositides, phosphorylated derivatives of phosphatidylinositol. Interestingly, microvesicles, which are similar in size to viruses and are also released from the cell periphery, lack phosphoinositides, suggesting a different budding mechanism/location for these particles than for retroviruses. One phosphoinositide, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], has been implicated in membrane binding by HIV Gag. Consistent with this observation, we found that PI(4,5)P2 was enriched in HIV-1 and that depleting this molecule in cells reduced HIV-1 budding. Analysis of mutant virions mapped the enrichment of PI(4,5)P2 to the matrix domain of HIV Gag. Overall, these results suggest that HIV-1 and other retroviruses bud from cholesterol-rich regions of the plasma membrane and exploit matrix/PI(4,5)P2 interactions for particle release from cells.

Phospholipase D2 Ablation Ameliorates Alzheimer's Disease-Linked Synaptic Dysfunction and Cognitive Deficits

Growing evidence implicates aberrant lipid signaling in Alzheimers disease (AD). While phospholipases A2 and C have been recently shown to mediate key actions of amyloid β-peptide (Aβ) through a dysregulation of arachidonic acid and phosphatidylinositol-4,5-bisphosphate metabolism, respectively, the role of phospholipase D (PLD) has so far remained elusive. PLD produces phosphatidic acid (PA), a bioactive lipid involved in multiple aspects of cell physiology, including signaling and membrane trafficking processes. Here we show that oligomeric Aβ enhances PLD activity in cultured neurons and that this stimulatory effect does not occur upon ablation of PLD2 via gene targeting. Aβ fails to suppress long-term potentiation in PLD2-deficient hippocampal slices, suggesting that PLD2 is required for the synaptotoxic action of this peptide. In vivo PLD activity, as assessed by detection of phosphatidylethanol levels using mass spectrometry (MS) following ethanol injection, is also increased in the brain of a transgenic mouse model of AD (SwAPP). Furthermore, Pld2 ablation rescues memory deficits and confers synaptic protection in SwAPP mice despite a significant Aβ load. MS-based lipid analysis of Pld2 mutant brains in the presence or absence of the SwAPP transgene unmasks striking crosstalks between different PA species. This lipid analysis shows an exquisite acyl chain specificity and plasticity in the perturbation of PA metabolism. Collectively, our results point to specific molecular species of PA as key modulators of AD pathogenesis and identify PLD2 as a novel potential target for therapeutics.

The phospholipase D1 pathway modulates macroautophagy

Although macroautophagy is known to be an essential degradative process whereby autophagosomes mediate the engulfment and delivery of cytoplasmic components into lysosomes, the lipid changes underlying autophagosomal membrane dynamics are undetermined. Here, we show that phospholipase D1 (PLD1), which is primarily associated with the endosomal system, partially relocalizes to the outer membrane of autophagosome-like structures upon nutrient starvation. The localization of PLD1, as well as the starvation-induced increase in PLD activity, are altered by wortmannin, a phosphatidylinositol 3-kinase inhibitor, suggesting PLD1 may act downstream of Vps34. Pharmacological inhibition of PLD and genetic ablation of PLD1 in mouse cells decreased the starvation-induced expansion of LC3-positive compartments, consistent with a role of PLD1 in the regulation of autophagy. Furthermore, inhibition of PLD results in higher levels of Tau and p62 aggregates in organotypic brain slices. Our in vitro and in vivo findings establish a role for PLD1 in autophagy.

Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system

Defects in endosomal sorting have been implicated in Alzheimers disease. Endosomal traffic is largely controlled by phosphatidylinositol-3-phosphate, a phosphoinositide synthesized primarily by lipid kinase Vps34. Here we show that phosphatidylinositol-3-phosphate is selectively deficient in brain tissue from humans with Alzheimers disease and Alzheimers disease mouse models. Silencing Vps34 causes an enlargement of neuronal endosomes, enhances the amyloidogenic processing of amyloid precursor protein in these organelles and reduces amyloid precursor protein sorting to intraluminal vesicles. This trafficking phenotype is recapitulated by silencing components of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway, including the phosphatidylinositol-3-phosphate effector Hrs and Tsg101. Amyloid precursor protein is ubiquitinated, and interfering with this process by targeted mutagenesis alters sorting of amyloid precursor protein to the intraluminal vesicles of endosomes and enhances amyloid-beta peptide generation. In addition to establishing phosphatidylinositol-3-phosphate deficiency as a contributing factor in Alzheimers disease, these results clarify the mechanisms of amyloid precursor protein trafficking through the endosomal system in normal and pathological states.

The impact of chronic stress on the rat brain lipidome.

Chronic stress is a major risk factor for several human disorders that affect modern societies. The brain is a key target of chronic stress. In fact, there is growing evidence indicating that exposure to stress affects learning and memory, decision making and emotional responses, and may even predispose for pathological processes, such as Alzheimer’s disease and depression. Lipids are a major constituent of the brain and specifically signaling lipids have been shown to regulate brain function. Here, we used a mass spectrometry-based lipidomic approach to evaluate the impact of a chronic unpredictable stress (CUS) paradigm on the rat brain in a region-specific manner. We found that the prefrontal cortex (PFC) was the area with the highest degree of changes induced by chronic stress. Although the hippocampus presented relevant lipidomic changes, the amygdala and, to a greater extent, the cerebellum presented few lipid changes upon chronic stress exposure. The sphingolipid and phospholipid metabolism were profoundly affected, showing an increase in ceramide (Cer) and a decrease in sphingomyelin (SM) and dihydrosphingomyelin (dhSM) levels, and a decrease in phosphatidylethanolamine (PE) and ether phosphatidylcholine (PCe) and increase in lysophosphatidylethanolamine (LPE) levels, respectively. Furthermore, the fatty-acyl profile of phospholipids and diacylglycerol revealed that chronic stressed rats had higher 38 carbon(38C)-lipid levels in the hippocampus and reduced 36C-lipid levels in the PFC. Finally, lysophosphatidylcholine (LPC) levels in the PFC were found to be correlated with blood corticosterone (CORT) levels. In summary, lipidomic profiling of the effect of chronic stress allowed the identification of dysregulated lipid pathways, revealing putative targets for pharmacological intervention that may potentially be used to modulate stress-induced deficits.

Bezafibrate administration improves behavioral deficits and tau pathology in P301S mice

Peroxisome proliferator-activated receptors (PPARs) are ligand-mediated transcription factors, which control both lipid and energy metabolism and inflammation pathways. PPARγ agonists are effective in the treatment of metabolic diseases and, more recently, neurodegenerative diseases, in which they show promising neuroprotective effects. We studied the effects of the pan-PPAR agonist bezafibrate on tau pathology, inflammation, lipid metabolism and behavior in transgenic mice with the P301S human tau mutation, which causes familial frontotemporal lobar degeneration. Bezafibrate treatment significantly decreased tau hyperphosphorylation using AT8 staining and the number of MC1-positive neurons. Bezafibrate treatment also diminished microglial activation and expression of both inducible nitric oxide synthase and cyclooxygenase 2. Additionally, the drug differentially affected the brain and brown fat lipidome of control and P301S mice, preventing lipid vacuoles in brown fat. These effects were associated with behavioral improvement, as evidenced by reduced hyperactivity and disinhibition in the P301S mice. Bezafibrate therefore exerts neuroprotective effects in a mouse model of tauopathy, as shown by decreased tau pathology and behavioral improvement. Since bezafibrate was given to the mice before tau pathology had developed, our data suggest that bezafibrate exerts a preventive effect on both tau pathology and its behavioral consequences. Bezafibrate is therefore a promising agent for the treatment of neurodegenerative diseases associated with tau pathology.

α-Synuclein-Independent Histopathological and Motor Deficits in Mice Lacking the Endolysosomal Parkinsonism Protein Atp13a2

Accumulating evidence from genetic and biochemical studies implicates dysfunction of the autophagic-lysosomal pathway as a key feature in the pathogenesis of Parkinsons disease (PD). Most studies have focused on accumulation of neurotoxic α-synuclein secondary to defects in autophagy as the cause of neurodegeneration, but abnormalities of the autophagic-lysosomal system likely mediate toxicity through multiple mechanisms. To further explore how endolysosomal dysfunction causes PD-related neurodegeneration, we generated a murine model of Kufor–Rakeb syndrome (KRS), characterized by early-onset Parkinsonism with additional neurological features. KRS is caused by recessive loss-of-function mutations in the ATP13A2 gene encoding the endolysosomal ATPase ATP13A2. We show that loss of ATP13A2 causes a specific protein trafficking defect, and that Atp13a2 null mice develop age-related motor dysfunction that is preceded by neuropathological changes, including gliosis, accumulation of ubiquitinated protein aggregates, lipofuscinosis, and endolysosomal abnormalities. Contrary to predictions from in vitro data, in vivo mouse genetic studies demonstrate that these phenotypes are α-synuclein independent. Our findings indicate that endolysosomal dysfunction and abnormalities of α-synuclein homeostasis are not synonymous, even in the context of an endolysosomal genetic defect linked to Parkinsonism, and highlight the presence of α-synuclein-independent neurotoxicity consequent to endolysosomal dysfunction.

Evidence that selective changes in the lipid composition of raft-membranes occur during respiratory syncytial virus infection.

We examined the structure of lipid-raft membranes in respiratory syncytial virus infected cells. Cholesterol depletion studies using methyl-beta-cyclodextrin suggested that membrane cholesterol was required for virus filament formation, but not inclusion bodies. In addition, virus filament formation coincided with elevated 3-hydroxy-3-methylglutaryl-coenzyme A reductase expression, suggesting an increase in requirement for endogenous cholesterol synthesis during virus assembly. Lipid raft membranes were examined by mass spectrometry, which suggested that virus infection induced subtle changes in the lipid composition of these membrane structures. This analysis revealed increased levels of raft-associated phosphatidylinositol (PI) and phosphorylated PI during RSV infection, which correlated with the appearance of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate (PIP(3)) within virus inclusion bodies, and inhibiting the synthesis of PIP(3) impaired the formation of progeny virus. Collectively, our analysis suggests that RSV infection induces specific changes in the composition of raft-associated lipids, and that these changes play an important role in virus maturation.

Role for Lipid Droplet Biogenesis and Microlipophagy in Adaptation to Lipid Imbalance in Yeast

The immediate responses to inhibition of phosphatidylcholine (PC) biosynthesis in yeast are altered phospholipid levels, slow growth, and defects in the morphology and localization of ER and mitochondria. With chronic lipid imbalance, yeast adapt. Lipid droplet (LD) biogenesis and conversion of phospholipids to triacylglycerol are required for restoring some phospholipids to near-wild-type levels. We confirmed that the unfolded protein response is activated by this lipid stress and find that Hsp104p is recruited to ER aggregates. We also find that LDs form at ER aggregates, contain polyubiquitinated proteins and an ER chaperone, and are degraded in the vacuole by a process resembling microautophagy. This process, microlipophagy, is required for restoration of organelle morphology and cell growth during adaptation to lipid stress. Microlipophagy does not require ATG7 but does requires ESCRT components and a newly identified class E VPS protein that localizes to ER and is upregulated by lipid imbalance.