Synaptic integrity is essential for learning and memory, and its loss is a strong predictor of cognitive decline in Alzheimer’s disease (AD). Although synaptic degeneration has been linked to excitotoxicity, neuroinflammation, and amyloid-β (Aβ) and tau pathology, the underlying mechanisms remain unclear. We investigated whether calcium-dependent cytosolic phospholipase A2 (cPLA2), an enzyme that releases arachidonic acid from membrane phospholipids, contributes to synaptic loss in AD. cPLA2 isoforms were quantified in synaptosomes isolated from postmortem frontal cortex of individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), and AD dementia from the Religious Orders Study, and in human iPSC-derived neurons exposed to Aβ42 oligomers. Both cPLA2α and cPLA2β were elevated in AD synaptosomes and correlated with postsynaptic protein PSD-95, but not presynaptic markers. cPLA2β levels were strongly associated with cognitive dysfunction, particularly in males, and synaptosomal eicosanoids were increased and correlated with cPLA2. In iPSC-derived neurons, Aβ42 oligomers activated cPLA2α, promoted its phosphorylation and translocation to postsynaptic compartments, and induced PSD-95 loss—effects prevented by the selective cPLA2 inhibitor BRI-50460. These findings implicate cPLA2 overactivation in excitatory synaptic pathology and cognitive decline in AD, highlighting cPLA2 as a potential disease-modifying therapeutic target.

Anti-amyloid therapies for Alzheimer's disease (AD) modestly slow cognitive decline but carry significant risk of amyloid-related imaging abnormalities (ARIAs), brain swelling, and hemorrhage, particularly in apolipoprotein E ε4 carriers. Cerebral amyloid angiopathy (CAA) and vascular inflammation drive this vulnerability, highlighting the need for complementary strategies targeting upstream mechanisms of vascular injury. Cytosolic phospholipase A2 (cPLA2) regulates arachidonic acid and lysophosphatidylcholine-derived lipid signaling at the intersection of amyloid burden, oxylipin dysregulation, blood-brain barrier disruption, and neurovascular inflammation. By depleting protective membrane plasmalogens while amplifying inflammatory lipid mediators, cPLA2 creates a state of vascular vulnerability predisposing to ARIAs. This Perspective article synthesizes evidence from human, preclinical, and translational studies positioning cPLA2 as an upstream driver of CAA-related inflammation and vascular vulnerability in AD. We discuss biomarker and imaging approaches to assess cPLA2 activity in vivo and outline how targeting this pathway may enhance anti-amyloid therapy safety by mitigating ARIA risk.

BACKGROUND:Cellular senescence, a hallmark of aging, has been implicated in Alzheimer's disease (AD) pathogenesis. Cholesterol accumulation is known to drive cellular senescence; however, its underlying mechanisms are not fully understood. ATP-binding cassette transporter A1 (ABCA1) plays an important role in cholesterol homeostasis, and its expression and trafficking are altered in APOE4 and AD models. However, the role of ABCA1 trafficking in cellular senescence associated with APOE4 and AD remains unclear. METHODS:We examined the association between cellular senescence and ABCA1 expression in human postmortem brain samples using transcriptomic, histological, and biochemical analyses. Unbiased proteomic screening was performed to identify the proteins that mediate cellular ABCA1 trafficking. We created ABCA1 knock out cell lines and mouse models to validate the role of ABCA1 in cholesterol-induced mTORC1 activation and senescence. Additionally, we used APOE4-TR mice and induced pluripotent stem cell (iPSC) models to explore cholesterol-ABCA1-senescence pathways. RESULTS:Transcriptomic profiling of the human dorsolateral prefrontal cortex from the Religious Order Study/Memory Aging Project (ROSMAP) cohort revealed the upregulation of cellular senescence transcriptome signatures in AD, which correlated with ABCA1 expression and oxysterol levels. Immunofluorescence and immunoblotting analyses confirmed increased lipofuscin-stained lipids and ABCA1 expression in AD brains and an association with mTOR phosphorylation. Discovery proteomics identified caveolin-1, a sensor of cellular cholesterol accumulation, as a key promoter of ABCA1 endolysosomal trafficking. Greater caveolin-1 expression was observed in APOE4-TR mouse models and AD human brains. Oxysterol induced mTORC1 activation and senescence were regulated by ABCA1 lysosomal trapping. Treatment of APOE4-TR mice with cyclodextrin reduced brain oxysterol levels, ABCA1 lysosome trapping, mTORC1 activation, and attenuated senescence and neuroinflammation markers. In human iPSC-derived astrocytes, the reduction of cholesterol by cyclodextrin attenuated inflammatory responses. CONCLUSIONS:Oxysterol accumulation in APOE4 and AD induced ABCA1 and caveolin-1 expression, contributing to lysosomal dysfunction and increased cellular senescence markers. This study provides novel insights into how cholesterol metabolism accelerates features of brain cellular senescence pathway and identifies therapeutic targets to mitigate these processes.

Cellular senescence is a hallmark of aging and has been implicated in several neurodegenerative diseases including Alzheimer’s disease (AD). Senescence cells undergo changes in gene expression and metabolism and can exhibit a so-called “senescence-associated secretory phenotype” (SASP) characterized by increased secretion of pro-inflammatory molecules and factors which can damage nearby cells, contributing to AD pathology progression. In this study, we determined mechanisms of cellular senescence using human postmortem brain samples, cellular models, and APOE4 animal models. Bulk (n = 632) and single-cell nuclei transcriptomic profiling (n = 427) of the human dorsolateral prefrontal cortex (DLPFC) from the Religious Order Study/Memory Aging Project (ROSMAP). Lipidomic profiling was performed on a subset of 200 brains from the midfrontal cortex of ROS. Our findings revealed upregulation of cellular senescence signatures in postmortem AD brain tissues across different cell types in comparison with controls. We identified a strong correlation between SASP and arachidonic acid (AA) metabolism (P<0.001) in bulk RNA. In single cell nuclei transcriptomics, AA Activation was strongly correlated with P2RY12 (homeostatic) microglia (P<0.0001), and was associated with worse performance on all cognitive domains (p<0.001) and AD neuropathology (P<0.001) as shown in the figure. Lipidomic analysis of postmortem brain tissues confirmed activation of AA derived eicosanoids. Pathway analysis implicated the activation of calcium dependent phospholipase A2 (cPLA2). Inhibiting cPLA2 by treatment with ASB14780 reduced senescence-associated eicosanoids in APOE4 mouse models. This work implicates the sustained activation of homeostatic microglia as an underlying mechanism of cellular senescence in the AD brain.

Synaptic loss predicts cognitive decline in Alzheimer’s disease (AD). However, the critical disease modifying molecular mechanisms of synaptic failure remain elusive. Animal studies implicate the increased activation of cytosolic phospholipase (cPLA2) activation in synaptic loss and neuroinflammation. cPLA2 activation in human brain synapses have not yet been demonstrated. cPLA2 preferentially catalyzes the conversion of arachidonic acid (AA) to generate eicosanoids: lipid mediators of inflammation. Synaptosomes represent an excellent ex vivo model to study synaptic function in humans. Both PSD-95 and CamKII are synaptic markers that play essential role in learning and memory. In this study, we sought to determine whether cPLA2β is associated with synaptic markers, AD pathology and cognition in synaptosomes derived from human brain tissues. cPLA2β expression was analyzed in synaptosomes isolated from midfrontal cortex of postmortem brains in participants with no cognitive impairment (NCI, n = 20), mild cognitive impairment (MCI, n = 14) and AD (n = 19) from the clinically and pathologically well-characterized Religious Orders Study (ROS). Experimental methods included Western blotting, immunofluorescence staining, and liquid chromatography-tandem mass spectrometry to measure AA and its metabolites. cPLA2β was increased in synaptosomes/synapses of both MCI (* p < 0.05) and AD (*** p < 0.001) compared to NCI. cPLA2β was inversely corelated with global cognitive function (r = -0.3667, ** p < 0.01), and positively correlated with neurofibrillary tangles (r = 0.2877, * p < 0.05). Interestingly, cPLA2β was highly positively correlated with postsynaptic proteins of PSD-95 (r = 0.7901, **** p < 0.0001) and calcium/calmodulin dependent protein kinase II (CaMKII, r = 0.2825, * p <0.05). Immunofluorescence staining revealed the colocalization of cPLA2β with phospho-tau (AT8), synaptic PSD-95 and CaMKII in both AD and NCI brains. cPLA2β stained degenerative excitatory neurons. On a functional level, cPLA2b was positively correlated with AA (r = 0.5203, * p = 0.01) and its inflammatory metabolites of PGD2 (r = 0.496, * p <0.05) and PGE2 (r = 0.4623, * p <0.05). This is the first report revealing increased expression of cPLA2β in synaptosomes from brains with MCI and AD compared with controls. cPLA2β is associated with tau pathology, AA and its metabolites, synaptic and cognitive performance. Whether reducing cPLA2b reverses AD pathology merits further investigation.

Cellular senescence is a major contributor to aging-related degenerative diseases, including Alzheimer's disease (AD), but much less is known about the key cell types and pathways driving senescence mechanisms in the brain. We hypothesized that dysregulated cholesterol metabolism is central to cellular senescence in AD. We analyzed single-cell RNA-seq data from the ROSMAP and SEA-AD cohorts to uncover cell type-specific senescence pathologies. In ROSMAP snRNA-seq data (982,384 nuclei from postmortem prefrontal cortex), microglia emerged as central contributors to AD-associated senescence phenotypes among non-neuronal cells. Homeostatic, inflammatory, phagocytic, lipid-processing, and neuronal-surveillance microglial states were associated with AD-related senescence in both ROSMAP (152,459 microglia nuclei from six brain regions) and SEA-AD (82,486 microglia nuclei) via integrative analysis. We assessed top senescence-associated bioprocesses and demonstrated that senescent microglia exhibit altered cholesterol-related processes and dysregulated cholesterol metabolism. We identified three gene co-expression modules representing cholesterol-related senescence signatures in postmortem brains. To validate these findings, we applied these signatures to snRNA-seq data from iPSC-derived microglia（iMGs) exposed to myelin, Aβ, apoptotic neurons, and synaptosomes. Treatment with AD-related substrates altered cholesterol-associated senescence signatures in iMGs. This study provides the first human evidence that dysregulated cholesterol metabolism in microglia drives cellular senescence in AD. Targeting cholesterol pathways in senescent microglia is an attractive strategy to attenuate AD progression.

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). ApoE is glycosylated with an O-linked Core-1 sialylated glycan at several sites; however, the impact and function of this glycosylation on AD biomarkers remain unclear. We examined apoE glycosylation (total and secondary) in a cohort of cerebrospinal fluid (CSF, n = 181) and plasma (n = 178) samples from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) stratified into 4 groups: cognitively normal (CN), Mild Cognitive Impairment (MCI), progressors, and non-progressors based on delayed word recall performance over 4 years. We observed decreasing glycosylation (reduced

Human Apolipoprotein (APOE) has three isoforms, ε2 , ε3 , and ε4 among which ε4 ( APOE4 ) confers the highest risk for late-onset Alzheimer’s disease (AD). APOE4 is also the most prone to aggregate among APOE isoforms. Current evidence strongly suggests that APOE aggregation leads to neuronal dysfunction and eventually to AD. APOE4 increases amyloid plaques and neurofibrillary tangles and decreases synapses and neuronal survival. These phenotypes are alleviated by decreasing APOE4 aggregation. We analyzed APOE aggregation using fluorescence lifetime imaging microscopy (FLIM) in combination with Forster resonance energy transfer (FRET). APOE isoforms tagged with E2GFP or mRuby2 were within 10 nm or less, i.e. in an aggregate the fluorescence lifetime of the donor was shortened and signal intensity was diminished which was monitored using FLIM-FRET and analyzed using the Phasor approach. APOE aggregation was also confirmed by using small-angle X-ray scattering (SAXS) of the Sarkosyl extracts of the cells. Secreted APOE-Tdtomato was isolated from BHK cell media and analyzed by ion mobility assay. Human neurons were incubated with APOE-Tdtomato isolated from the media. APOE4 aggregated more than APOE3 in living BHK cells as shown via FLIM-FRET microscopy. APOE4 aggregated less when its lipidation was induced by ABCA1 expression in line with data published by us and others. These results were confirmed via SAXS. APOE was secreted in HDL particles from BHK cells both in the presence or absence of ABCA1 while ABCA1 increased secretion for both APOE3 and APOE4. Finally, tagged-APOE secreted to the media was endocytosed by human neurons. FLIM-FRET is a feasible method to analyze APOE aggregation in living cells. Isolated APOE can be used to study APOE endocytosis in human neurons.

The ATP-binding cassette transporter A1 (ABCA1) is a major regulator of lipid metabolism and plays important roles for neuronal survival and functionality. Rare loss-of-function mutations in ABCA1 are associated with increased Alzheimer's disease (AD) risk. Endosomal dysregulation is associated with AD pathology and affects several AD disease-related mechanisms including synaptic plasticity. However, the impact of ABCA1 on endosomal dysregulation and its effects on synaptic functions are unknown. Here, we show that ABCA1 expression was correlated with Rab family protein expression in bulk RNA-sequencing data from prefrontal cortex tissues of 632 participants in ROSMAP. Endosomal-lysosomal trafficking related genes were enriched by ABCA1 expression in the astrocytes, excitatory neurons, inhibitory neurons, and oligodendrocyte precursor cells in the single nucleus sequencing data from 427 participants in ROSMAP. Lower ABCA1 expression in ABCA1 knockout mice (KO) brain tissues was associated with abnormal trafficking of Rab family proteins, glutamate receptors, and with neuronal and specifically synaptic loss. In addition, protein levels of Rab4, Rab11 and Rab35 were specifically altered in ABCA1 KO mice compared to corresponding wild type mice. Endosomal fractions isolated from brain ABCA1 KO mice identified that ABCA1 reduced GluR2 level and enhanced its internalization and degradation via Rab4 positive endosomes. Our results indicate that ABCA1-associated endosomal dysfunction contributes to the loss of neurons cells and reduces synaptic plasticity by impairing GluR2 membrane recycling. These data further elucidate the critical role of ABCA1 in neuronal synaptic biology.

Purpose of review Most omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation clinical trials report inconsistent or null findings on measures of cognition or Alzheimer's disease (AD) with a relatively large variability in the response to n-3 PUFA supplementation. The purpose of this review is to identify whether the gut microbiome together with the metabolome can provide critical insights to understand this heterogeneity in the response to n-3 PUFA supplementation. Recent findings A Western diet with high saturated fat and omega-6 fatty acid content, obesity, and lack of exercise puts strain on the gut microbiome resulting in imbalance, dysbiosis, reduced bacterial diversity, and increased abundance of the pro-inflammatory taxa. A plant-based diet has beneficial effects on the gut microbiota even when deficient in n-3 PUFAs. Human and animal studies show that increased intake of the n-3 PUFAs correlates with increased beneficial intestinal bacteria when compared to a Western diet. Summary The composition of the gut microbiota can help define the effects of n-3 PUFA supplementation on the brain and lead to more personalized nutritional interventions.

Carrying the apolipoprotein E ε4 (APOE4) allele is the highest known genetic risk factor for late-onset Alzheimer’s disease (LOAD). Although how APOE4 leads to LOAD is not known, dysregulated endo-lysosomal trafficking and lipid metabolism are key defects associated with APOE4. Cholesterol accumulation in glia instigates intracellular trafficking defects, increases cellular stress, and disrupts secretion. Additionally, enlarged endosomes were observed in the frontal lobes of APOE4-carrying individuals, decades before the appearance of Aβ fibrilization. Therefore, we asked if cholesterol and endo-lysosomal trafficking dysregulation in APOE4 carriers were due to differential lipidation of APOE3 and APOE4. Localization of ATP Binding Cassette Subfamily A Member 1 (ABCA1) in early, late, and recycling endosomal compartments were analyzed in human isogenic induced pluripotent stem cell (iPSC)-derived neural cells and in cell lines treated with recombinant APOE in microscopic images. Membrane and cytoplasmic ABCA1 were compared using Western blots. Secreted high-density lipoprotein (HDL) particles containing fluorescently tagged APOE3 or APOE4 were analyzed using ion-mobility spectrometry. CS-6253, an ABCA1 agonist peptide, was used to increase APOE lipidation. The protein levels of ABCA1, which mediates cholesterol and phospholipid efflux to the nascent apolipoprotein particles, were lower in APOE4/4 iPSC-derived astrocytes compared to astrocytes derived from isogenic APOE3/3 iPSCs. Advanced image analysis revealed lower ABCA1 protein levels in Rab11 + recycling endosomes and less colocalization between ABCA1 and Rab7, a late endosome marker, in APOE4/4 astrocytes than APOE3/3 astrocytes. Consequently, less ABCA1 was detected at the cell membrane. Combined with previous data, these findings suggest APOE4 lipidation was reduced due to reduced ABCA1 activity. Additionally, BHK cells treated with recombinant APOE3 or APOE4 showed differences in ABCA1 localization that were altered by increasing APOE lipidation by activating ABCA1 via CS-6253. Finally, fluorescently tagged APOE3 and APOE4 were incorporated in HDL particles and were secreted whose sizes were altered after CS-6253 treatment, suggesting differences in lipidation levels. We demonstrated that diminished APOE4 lipidation is, at least in part, due to dysregulation of endo-lysosomal trafficking of ABCA1 and that increasing APOE4 lipidation could alleviate cellular defects.

Docosahexaenoic acid [22:6(n-3), DHA], a polyunsaturated fatty acid, has an important role in regulating neuronal functions and in normal brain development. Dysregulated brain DHA uptake and metabolism are found in individuals carrying the APOE4 allele, which increases the genetic risk for Alzheimer's disease (AD), and are implicated in the progression of several neurodegenerative disorders. However, there are limited tools to assess brain DHA kinetics in vivo that can be translated to humans. Here, we report the synthesis of an ω-radiofluorinated PET probe of DHA, 22-[18F]fluorodocosahexaenoic acid (22-[18F]FDHA), for imaging the uptake of DHA into the brain. Using the nonradiolabeled 22-FDHA, we confirmed that fluorination of DHA at the ω-position does not significantly alter the anti-inflammatory effect of DHA in microglial cells. Through dynamic PET-MR studies using mice, we observed the accumulation of 22-[18F]FDHA in the brain over time and estimated DHA's incorporation coefficient (K*) using an image-derived input function. Finally, DHA brain K* was validated using intravenous administration of 15 mg/kg arecoline, a natural product known to increase the DHA K* in rodents. 22-[18F]FDHA is a promising PET probe that can reveal altered lipid metabolism in APOE4 carriers, AD, and other neurologic disorders. This new probe, once translated into humans, would enable noninvasive and longitudinal studies of brain DHA dynamics by guiding both pharmacological and nonpharmacological interventions for neurodegenerative diseases.

Automated screening systems in conjunction with machine learning-based methods are becoming an essential part of the healthcare systems for assisting in disease diagnosis. Moreover, manually annotating data and hand-crafting features for training purposes are impractical and time-consuming. We propose a segmentation and classification-based approach for assembling an automated screening system for the analysis of calcium imaging. The method was developed and verified using the effects of disease IgGs (from Amyotrophic Lateral Sclerosis patients) on calcium (Ca2+) homeostasis. From 33 imaging videos we analyzed, 21 belonged to the disease and 12 to the control experimental groups. The method consists of three main steps: projection, segmentation, and classification. The entire Ca2+ time-lapse image recordings (videos) were projected into a single image using different projection methods. Segmentation was performed by using a multi-level thresholding (MLT) step and the Regions of Interest (ROIs) that encompassed cell somas were detected. A mean value of the pixels within these boundaries was collected at each time point to obtain the Ca2+ traces (time-series). Finally, a new matrix called feature image was generated from those traces and used for assessing the classification accuracy of various classifiers (control vs. disease). The mean value of the segmentation F-score for all the data was above 0.80 throughout the tested threshold levels for all projection methods, namely maximum intensity, standard deviation, and standard deviation with linear scaling projection. Although the classification accuracy reached up to 90.14%, interestingly, we observed that achieving better scores in segmentation results did not necessarily correspond to an increase in classification performance. Our method takes the advantage of the multi-level thresholding and of a classification procedure based on the feature images, thus it does not have to rely on hand-crafted training parameters of each event. It thus provides a semi-autonomous tool for assessing segmentation parameters which allows for the best classification accuracy.

Increased brain docosahexaenoic acid (DHA) uptake reflects a compensatory mechanism for maintaining brain DHA homeostasis and can be captured through PET-MRI scan uptake studies using radiolabeled DHA. It has been previously shown that APOE4 status and alcohol intake alter brain DHA uptake in mice. Whether a change in DHA intake affects the brain DHA incorporation coefficient (K*) however is not known. Four-month-old C57BL/6J mice were fed either a diet rich in DHA (15 kcal% Fat, 1.7 g/kg ALA, and 7g/kg DHA) or deficient in DHA (15 kcal% Fat and 0.9 mg/kg ALA) for 4 months. At 8 months, anesthetized mice were subjected to dynamic PET-MRI for 30 min following i.v. injection of 22-[ 18 F]fluorodocosahexaenoic acid ([ 18 F]FDHA), prepared in-house through radiosynthesis. Using an image-derived cardiac input function, brain uptake was assessed by estimating [ 18 F]FDHA K* values using both the Patlak model approach and the irreversible two-tissue compartmental model (Irr2TCM) with and without correction of the brain radioactivity for radioactive spillover from the skull. Mice were then sacrificed, and the plasma and brains were analyzed using liquid chromatography-mass spectrometry (LC-MS) to evaluate differences in the unlabeled concentrations of fatty acids into phospholipids, triglycerides, and other lipids. There was no significant difference in the [ 18 F]FDHA K* estimates (with either Patlak or Irr2TCM) between mice fed a DHA-rich or a DHA-deficient diet. LC-MS based lipidomic analysis showed that the DHA-rich diet significantly increased DHA concentration in triglycerides in plasma and in phosphatidylethanolamine in the brain. These results suggest that under physiological conditions, increased dietary intake of DHA in wild-type mice increases DHA in brain phosphatidylethanolamine and in plasma triglycerides without changing the DHA K*. The net effect likely is increased brain DHA uptake, equal to the product of unesterified DHA plasma concentration and K*. Imaging brain DHA with dynamic [ 18 F]FDHA PET-MRI is a potentially useful tool not only to investigate the APOE4 phenotype, which promotes DHA dysregulation, but also to assess dietary and pharmacological interventions that affect brain DHA homeostasis as well as to guide future treatments.

MOTIVATION:Identifying and prioritizing disease-related proteins is an important scientific problem to develop proper treatments. Network science has become an important discipline to prioritize such proteins. Multiple sclerosis, an autoimmune disease for which there is still no cure, is characterized by a damaging process called demyelination. Demyelination is the destruction of myelin, a structure facilitating fast transmission of neuron impulses, and oligodendrocytes, the cells producing myelin, by immune cells. Identifying the proteins that have special features on the network formed by the proteins of oligodendrocyte and immune cells can reveal useful information about the disease. RESULTS:We investigated the most significant protein pairs that we define as bridges among the proteins providing the interaction between the two cells in demyelination, in the networks formed by the oligodendrocyte and each type of two immune cells (i.e. macrophage and T-cell) using network analysis techniques and integer programming. The reason, we investigated these specialized hubs was that a problem related to these proteins might impose a bigger damage in the system. We showed that 61%-100% of the proteins our model detected, depending on parameterization, have already been associated with multiple sclerosis. We further observed the mRNA expression levels of several proteins we prioritized significantly decreased in human peripheral blood mononuclear cells of multiple sclerosis patients. We therefore present a model, BriFin, which can be used for analyzing processes where interactions of two cell types play an important role. AVAILABILITY AND IMPLEMENTATION:BriFin is available at https://github.com/BilkentCompGen/brifin.

Apolipoprotein E4 (ApoE4) dysregulates cholesterol metabolism in the brain promoting the accumulation of lipid droplets, synaptic dysfunction and associates with a greater Alzheimer’s disease risk. We previously demonstrated that ApoE4 reduces the function of ATP-binding cassette transporters A1 (ABCA1) which has important roles in cholesterol transport. The mechanisms of how ApoE4 induces less ABCA1 activity are poorly understood. ABCA1 binding proteins were analyzed with mass spectrum after immunoprecipitation. Immortalized astrocytes, primary astrocytes, mouse brain and human postmortem brain samples were used to test the association of ABCA1 and caveolin-1 levels. Small interfere RNA was used to knockdown caveolin-1 expression and 3H labelled cholesterol was used to test the cholesterol efflux of ABCA1. Here, we enriched ABCA1 binding protein complexes from ABCA1-overexpressing cells treated with recombinant ApoE3 or ApoE4 proteins for a proteomic screen implicating endocytosis as a major pathway differentiating ApoE3 from ApoE4. Among the protein targets discovered, caveolin-1 expression increased after ApoE4 treatment. Caveolin-1 and ABCA1 interactions were confirmed using immunoprecipitation experiments. Cholesterol accumulation in astrocytes induced caveolin-1 expression and promoted ABCA1 degradation. Greater caveolin-1 and lower ABCA1 expression was observed in brains of 8, 18 and 22 months-old ApoE4-TR mice compared with the corresponding ApoE3-TR mice. Isolation of single cell types from adult mouse ApoE-TR brains confirmed lower ABCA1 and greater caveolin-1 expression was largely driven by astrocytes and not microglia or neurons enriched fractions. In conclusion, cholesterol accumulation induced by ApoE4 promotes ABCA1 degradation in astrocytes partly through a caveolin-1 dependent endocytosis pathway.

Apolipoprotein ε allele 4 (APOE4) influences the metabolism of polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The entorhinal cortex (EC) in the brain is affected early in Alzheimer's disease and is rich in DHA. The purpose of this study is to identify the effect of APOE4 and DHA lipid species on the EC. Plasma and cerebrospinal fluid (CSF) lipidomic measurements were obtained from the DHA Brain Delivery Pilot, a randomized clinical trial of DHA supplementation (n = 10) versus placebo (n = 12) for six months in nondemented older adults stratified by APOE4 status. Wild-type C57B6/J mice were fed a high or low DHA diet for 6 months followed by plasma and brain lipidomic analysis. Levels of phosphatidylcholine DHA (PC 38:6) and cholesterol ester DHA (CE 22:6) had the largest increases in CSF following supplementation (P < 0.001). DHA within triglyceride (TG) lipids in CSF strongly correlated with corresponding plasma TG lipids, and differed by APOE4, with carriers having a lower increase than noncarriers. Changes in plasma PC DHA had the strongest association with changes in EC thickness in millimeters, independent of APOE4 status (P = 0.007). In mice, a high DHA diet increased PUFAs within brain lipids. Our findings demonstrate an exchange of DHA at the CSF-blood barrier and into the brain within all lipid species with APOE having the strongest effect on DHA-containing TGs. The correlation of PC DHA with EC suggests a functional consequence of DHA accretion in high density lipoprotein for the brain.

Myelin is an essential component of the nervous system and myelin damage causes demyelination diseases. Myelin is a sheet of oligodendrocyte membrane wrapped around the neuronal axon. In the fluorescent images, experts manually identify myelin by co-localization of oligodendrocyte and axonal membranes that fit certain shape and size criteria. Because myelin wriggles along x-y-z axes, machine learning is ideal for its segmentation. However, machine-learning methods, especially convolutional neural networks (CNNs), require a high number of annotated images, which necessitate expert labor. To facilitate myelin annotation, we developed a workflow and software for myelin ground truth extraction from multi-spectral fluorescent images. Additionally, to the best of our knowledge, for the first time, a set of annotated myelin ground truths for machine learning applications were shared with the community.