Microglia sense astrocyte dysfunction and prevent disease progression in an Alexander disease model.

Alexander disease (AxD) is an intractable neurodegenerative disorder caused by GFAP mutations. It is a primary astrocyte disease with a pathological hallmark of Rosenthal fibres within astrocytes. AxD astrocytes show several abnormal phenotypes. Our previous study showed that AxD astrocytes in model mice exhibit aberrant Ca2+ signals that induce AxD aetiology. Here, we show that microglia have unique phenotypes with morphological and functional alterations, which are related to the pathogenesis of AxD. Immunohistochemical studies of 60TM mice (AxD model) showed that AxD microglia exhibited highly ramified morphology. Functional changes in microglia were assessed by Ca2+ imaging using hippocampal brain slices from Iba1-GCaMP6-60TM mice and two-photon microscopy. We found that AxD microglia showed aberrant Ca2+ signals, with high frequency Ca2+ signals in both the processes and cell bodies. These microglial Ca2+ signals were inhibited by pharmacological blockade or genetic knockdown of P2Y12 receptors but not by tetrodotoxin, indicating that these signals are independent of neuronal activity but dependent on extracellular ATP from non-neuronal cells. Our single-cell RNA sequencing data showed that the expression level of Entpd2, an astrocyte-specific gene encoding the ATP-degrading enzyme NTPDase2, was lower in AxD astrocytes than in wild-type astrocytes. In situ ATP imaging using the adeno-associated virus vector GfaABC1D ATP1.0 showed that exogenously applied ATP was present longer in 60TM mice than in wild-type mice. Thus, the increased ATP level caused by the decrease in its metabolizing enzyme in astrocytes could be responsible for the enhancement of microglial Ca2+ signals. To determine whether these P2Y12 receptor-mediated Ca2+ signals in AxD microglia play a significant role in the pathological mechanism, a P2Y12 receptor antagonist, clopidogrel, was administered. Clopidogrel significantly exacerbated pathological markers in AxD model mice and attenuated the morphological features of microglia, suggesting that microglia play a protective role against AxD pathology via P2Y12 receptors. Taken together, we demonstrated that microglia sense AxD astrocyte dysfunction via P2Y12 receptors as an increase in extracellular ATP and alter their morphology and Ca2+ signalling, thereby protecting against AxD pathology. Although AxD is a primary astrocyte disease, our study may facilitate understanding of the role of microglia as a disease modifier, which may contribute to the clinical diversity of AxD.

Severity of Peripheral Infection Differentially Affects Brain Functions in Mice via Microglia-Dependent and -Independent Mechanisms.

Peripheral infection induces inflammation in peripheral tissues and the brain, impacting brain function. Glial cells are key players in this process. However, the effects of peripheral infection on glial activation and brain function remain unknown. Here, we showed that varying degrees of peripheral infection had different effects on the regulation of brain functions by microglia-dependent and -independent mechanisms. Acute mild infection (one-day LPS challenge: 1LPS) exacerbated middle cerebral artery occlusion (MCAO) injury, and severe infection (four-day LPS challenge: 4LPS) for one week suppressed it. MCAO injury was assessed by triphenyltetrazolium chloride staining. We observed early activation of microglia in the 1LPS and 4LPS groups. Depleting microglia with a colony-stimulating factor-1 receptor (CSF1R) antagonist had no effect on 1LPS-induced brain injury exacerbation but abolished 4LPS-induced protection, indicating microglial independence and dependence, respectively. Microglia-independent exacerbation caused by 1LPS involved peripheral immune cells including macrophages. RNA sequencing analysis of 4LPS-treated microglia revealed increased factors related to anti-inflammatory and neuronal tissue repair, suggesting their association with the protective effect. In conclusion, varying degrees of peripheral inflammation had contradictory effects (exacerbation vs. protection) on MCAO, which may be attributed to microglial dependence. Our findings highlight the significant impact of peripheral infection on brain function, particularly in relation to glial cells.

Dynorphinergic Projections from the Central Amygdala to the Parabrachial Nucleus Regulate Itch.

The amygdala plays a key role in the processing of itch and pain signals as well as emotion. A previous study revealed that the central nucleus of the amygdala (CeA)-parabrachial nucleus (PBN) pathway is involved in pain regulation. The same pathway might also control itch. To test this possibility, prodynorphin (Pdyn)-Cre mice were used to optogenetically manipulate Pdyn+ CeA-to-PBN projections. We found that optogenetic stimulation of Pdyn+ amygdala neurons or Pdyn+ CeA-to-PBN projections inhibited histamine-evoked and chloroquine-evoked scratching. The number of Fos-positive neurons in the PBN increased following intradermal injection of chloroquine. Optogenetic stimulation of Pdyn+ CeA-to-PBN projections suppressed the increase in Fos expression in the PBN. Optogenetic stimulation of Pdyn+ CeA-to-PBN projections increased thermal and mechanical thresholds without affecting anxiety-like behavior. These results highlight the importance of dynorphinergic projections from the central amygdala to the parabrachial nucleus in the regulation of itch signaling.SIGNIFICANCE STATEMENT The central nucleus of the amygdala (CeA)-parabrachial nucleus (PBN) pathway regulates pain signaling. Using prodynorphin (Pdyn)-cre mice, we investigated the role of Pdyn+ CeA-to-PBN projections in itch. Optogenetic stimulation of Pdyn+ CeA-to-PBN projections inhibited pruritogen-evoked scratching and neuronal activity (c-Fos expression) in the PBN. Together, dynorphinergic projections from the central amygdala to the parabrachial nucleus are important for regulating itch information.

Extracellular ATP/adenosine dynamics in the brain and its role in health and disease.

Extracellular ATP and adenosine are neuromodulators that regulate numerous neuronal functions in the brain. Neuronal activity and brain insults such as ischemic and traumatic injury upregulate these neuromodulators, which exert their effects by activating purinergic receptors. In addition, extracellular ATP/adenosine signaling plays a pivotal role in the pathogenesis of neurological diseases. Virtually every cell type in the brain contributes to the elevation of ATP/adenosine, and various mechanisms underlying this increase have been proposed. Extracellular adenosine is thought to be mainly produced via the degradation of extracellular ATP. However, adenosine is also released from neurons and glia in the brain. Therefore, the regulation of extracellular ATP/adenosine in physiological and pathophysiological conditions is likely far more complex than previously thought. To elucidate the complex mechanisms that regulate extracellular ATP/adenosine levels, accurate methods of assessing their spatiotemporal dynamics are needed. Several novel techniques for acquiring spatiotemporal information on extracellular ATP/adenosine, including fluorescent sensors, have been developed and have started to reveal the mechanisms underlying the release, uptake and degradation of ATP/adenosine. Here, we review methods for analyzing extracellular ATP/adenosine dynamics as well as the current state of knowledge on the spatiotemporal dynamics of ATP/adenosine in the brain. We focus on the mechanisms used by neurons and glia to cooperatively produce the activity-dependent increase in ATP/adenosine and its physiological and pathophysiological significance in the brain.

IL-23 modulates histamine-evoked itch and responses of pruriceptors in mice.

Accumulating evidence has highlighted the essential roles of cytokines in itch processing. Although IL-23 and Th17 cytokines are elevated in inflammatory skin disorders, their role in itch is unknown. Here, we investigated the role of IL-23 and IL-17A in itch response using an in vitro calcium imaging of mouse dorsal root ganglion (DRG) neurons and an in vivo behaviour test. Calcium imaging studies revealed that a few DRG neurons (~5%) responded to either IL-23 or IL-17A. Pretreatment cells with IL-23 significantly reduced calcium responses to histamine and capsaicin but not chloroquine. Behaviour experiments showed neither IL-23 nor IL-17A evoked scratching. IL-23 significantly decreased histamine-evoked scratching without affecting chloroquine-evoked scratching. There was no difference in scratching between IL-17A- and vehicle-treated groups. These results indicate that IL-23 might play a role in regulating histaminergic itch via modulation of TRPV1 activity.

Low-Threshold Mechanosensitive VGLUT3-Lineage Sensory Neurons Mediate Spinal Inhibition of Itch by Touch.

Innocuous mechanical stimuli, such as rubbing or stroking the skin, relieve itch through the activation of low-threshold mechanoreceptors. However, the mechanisms behind this inhibition remain unknown. We presently investigated whether stroking the skin reduces the responses of superficial dorsal horn neurons to pruritogens in male C57BL/6J mice. Single-unit recordings revealed that neuronal responses to chloroquine were enhanced during skin stroking, and this was followed by suppression of firing below baseline levels after the termination of stroking. Most of these neurons additionally responded to capsaicin. Stroking did not suppress neuronal responses to capsaicin, indicating state-dependent inhibition. Vesicular glutamate transporter 3 (VGLUT3)-lineage sensory nerves compose a subset of low-threshold mechanoreceptors. Stroking-related inhibition of neuronal responses to chloroquine was diminished by optogenetic inhibition of VGLUT3-lineage sensory nerves in male and female Vglut3-cre/NpHR-EYFP mice. Conversely, in male and female Vglut3-cre/ChR2-EYFP mice, optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited firing responses of spinal neurons to pruritogens after the termination of stimulation. This inhibition was nearly abolished by spinal delivery of the κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride, but not the neuropeptide Y receptor Y1 antagonist BMS193885. Optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited pruritogen-evoked scratching without affecting mechanical and thermal pain behaviors. Therefore, VGLUT3-lineage sensory nerves appear to mediate inhibition of itch by tactile stimuli.SIGNIFICANCE STATEMENT Rubbing or stroking the skin is known to relieve itch. We investigated the mechanisms behind touch-evoked inhibition of itch in mice. Stroking the skin reduced the activity of itch-responsive spinal neurons. Optogenetic inhibition of VGLUT3-lineage sensory nerves diminished stroking-evoked inhibition, and optogenetic stimulation of VGLUT3-lineage nerves inhibited pruritogen-evoked firing. Together, our results provide a mechanistic understanding of touch-evoked inhibition of itch.

New insights into the mechanisms behind mechanical itch.

Gentle tactile stimuli, such as insects crawling on the skin, can cause itching sensation called mechanical itch. Recent studies have begun to shed light on the neural mechanisms of mechanical itch. Interestingly, the neural pathway for mechanical itch is apparently different from that for chemical itch triggered by the activation of pruriceptors with various mediators. Mechanical itch dysesthesia is frequently seen in patients with chronic itch. Mechanisms of this dysesthesia are plausibly involved in central sensitization. In this review, we summarize the current knowledge of mechanical itch under normal and pathological conditions.

Peripheral circadian rhythms in the liver and white adipose tissue of mice are attenuated by constant light and restored by time-restricted feeding.

Disturbance of circadian rhythms underlies various metabolic diseases. Constant light exposure (LL) is known to disrupt both central and peripheral circadian rhythms. Here, we attempted to determine whether the effects of LL are different between various peripheral tissues and whether time-restricted feeding restores the circadian rhythms especially in white adipose tissue (WAT). Six-week-old mice were subjected to three feeding regimes: ad libitum feeding under light/dark phase (LD), ad libitum feeding under LL cycle, and restricted feeding at night-time under LL cycle with a normal chow. After 3 weeks, we compared body weight, food intake, plasma levels of lipids and glucose, and the expression patterns of the clock genes and the genes involved in lipid metabolism in the liver and WAT. The mice kept under LL with or without time-restricted feeding were 5.2% heavier (p<0.001, n = 16) than the mice kept under LD even though the food intakes of the two groups were the same. Food intake occurred mostly in the dark phase. LL disrupted this pattern, causing disruptions in circadian rhythms of plasma levels of triglycerides (TG) and glucose. Time-restricted feeding partially restored the rhythms. LL eliminated the circadian rhythms of the expression of the clock genes as well as most of the genes involved in lipid metabolism in both liver and WAT. More notably, LL markedly decreased not only the amplitude but also the average levels of the expression of the genes in the liver, but not in the WAT, suggesting that transcription in the liver is sensitive to constant light exposure. Time-restricted feeding restored the circadian rhythms of most of the genes to various degrees in both liver and WAT. In conclusion, LL disrupted the peripheral circadian rhythms more severely in liver than in WAT. Time-restricted feeding restored the circadian rhythms in both tissues.

A Subpopulation of Amygdala Neurons Mediates the Affective Component of Itch.

Itch consists of both sensory and affective components. For chronic itch patients, the affective component of itch affects both quality of life (leading to psychological comorbidities) and disease prognosis (by promoting scratching of itchy skin). We found that acute itch stimuli, such as histamine, induced anxiety-like behavior and increased activity (c-Fos expression) in the amygdala in adult male C57BL/6 mice. Itch stimuli also increased activity in projection areas to the amygdala, suggesting that these regions form a circuit for affective itch processing. Electrophysiological characterization of histamine-responsive amygdala neurons showed that this population was active on a behaviorally relevant timescale and partially overlapped with pain signaling. Selective optogenetic activation of histamine-responsive amygdala neurons in adult male and female Fos:CreERT2;R26Ai14 mice using the Targeted Recombination in Active Populations system enhanced both scratching and anxiety-like behavior. These results highlight the importance of itch-responsive amygdala neurons in modulating itch-related affect and behavior.SIGNIFICANCE STATEMENT The sensation of itch includes an affective component that leads to stress and anxiety in chronic itch patients. We investigated the neuronal basis of affective itch in mice, with a focus on the amygdala, the key brain region for the generation of anxiety. A subpopulation of amygdala neurons responded to itch stimuli such as histamine. Optogenetic activation of histamine-responsive amygdala neurons affected both scratching and anxiety-like behavior. Therefore, this population appears to be important for mediating the affective component of itch.

Antipruritic Effects of Janus Kinase Inhibitor Tofacitinib in a Mouse Model of Psoriasis.

The Janus kinase 1/3 inhibitor tofacitinib has demonstrated an antipruritic effect in two phase III studies in psoriasis. However, the mechanisms behind this antipruritic effect are still unknown. We presently investigated whether tofacitinib affects spontaneous itch as well as expression of itch-related cytokines and epidermal nerve fiber density (ENFD) in the imiquimod-induced mouse model of psoriasis. Psoriasis-like skin lesions were produced by daily topical application of imiquimod to the back skin. Imiquimod treatment resulted in spontaneous scratching, which was significantly inhibited by tofacitinib treatment. Imiquimod treatment significantly increased mRNA expression of Il22, Il23, and Il31, reduced peptidergic ENFD, and increased nonpeptidergic ENFD compared to naive mice. Tofacitinib significantly decreased the expression of those cytokines and increased peptidergic ENFD without a significant effect on nonpeptidergic ENFD. Tofacitinib may inhibit psoriatic itch through inhibition of cytokine expression as well as modulation of epidermal innervation.

Loss of ACAT1 Attenuates Atherosclerosis Aggravated by Loss of NCEH1 in Bone Marrow-Derived Cells.

AIM: Acyl-CoA cholesterol acyltransferase 1 (ACAT1) esterifies free cholesterol to cholesteryl esters (CE), which are subsequently hydrolyzed by neutral cholesterol ester hydrolase 1 (NCEH1). The elimination of ACAT1 in vitro reduces the amounts of CE accumulated in Nceh1-deficient macrophages. The present study aimed at examining whether the loss of ACAT1 attenuates atherosclerosis which is aggravated by the loss of NCEH1 in vivo. METHODS: Low density lipoprotein receptor (Ldlr)-deficient mice were transplanted with bone marrow from wild-type mice and mice lacking ACAT1, NCEH1, or both. The four types of mice were fed a high-cholesterol diet and, then, were examined for atherosclerosis. RESULTS: The cross-sectional lesion size of the recipients of Nceh1-deficient bone marrow was 1.6-fold larger than that of the wild-type bone marrow. The lesions of the recipients of Nceh1-deficient bone marrow were enriched with MOMA2-positive macrophages compared with the lesions of the recipients of the wild-type bone marrow. The size and the macrophage content of the lesions of the recipients of bone marrow lacking both ACAT1 and NCEH1 were significantly smaller than the recipients of the Nceh1-deficient bone marrow, indicating that the loss of ACAT1 decreases the excess CE in the Nceh1-deficient lesions. The collagen-rich and/or mucin-rich areas and en face lesion size were enlarged in the recipients of the Acat1－/－ bone marrow compared with those of the recipients of the WT bone marrow. CONCLUSION: The loss of ACAT1 in bone marrow-derived cells attenuates atherosclerosis, which is aggravated by the loss of NCEH1, corroborating the in vitro functions of ACAT1 (formation of CE) and NCEH1 (hydrolysis of CE).

Myeloid HMG-CoA (3-Hydroxy-3-Methylglutaryl-Coenzyme A) Reductase Determines Atherosclerosis by Modulating Migration of Macrophages.

Objective- Inhibition of HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is atheroprotective primarily by decreasing plasma LDL (low-density lipoprotein)-cholesterol. However, it is unknown whether inhibition of HMGCR in myeloid cells contributes to this atheroprotection. We sought to determine the role of myeloid HMGCR in the development of atherosclerosis. Approach and Results- We generated mice with genetically reduced Hmgcr in myeloid cells ( Hmgcr m-/m-) using LysM (Cre) and compared various functions of their macrophages to those of Hmgcr fl/fl control mice. We further compared the extent of atherosclerosis in Hmgcr m-/ m- and Hmgcr fl/fl mice in the absence of Ldlr (LDL receptor). Hmgcr m-/ m- macrophages and granulocytes had significantly lower Hmgcr mRNA expression and cholesterol biosynthesis than Hmgcr fl/fl cells. In vitro, Hmgcr m-/ m- monocytes/macrophages had reduced ability to migrate, proliferate, and survive compared with Hmgcr fl/fl monocytes/macrophages. However, there was no difference in ability to adhere, phagocytose, store lipids, or polarize to M1 macrophages between the 2 types of macrophages. The amounts of plasma membrane-associated small GTPase proteins, such as RhoA (RAS homolog family member A), were increased in Hmgcr m-/ m- macrophages. In the setting of Ldlr deficiency, Hmgcr m-/ m- mice developed significantly smaller atherosclerotic lesions than Hmgcr fl/fl mice. However, there were no differences between the 2 types of mice either in plasma lipoprotein profiles or in the numbers of proliferating or apoptotic cells in the lesions in vivo. The in vivo migration of Hmgcr m-/ m- macrophages to the lesions was reduced compared with Hmgcr fl/fl macrophages. Conclusions- Genetic reduction of HMGCR in myeloid cells may exert atheroprotective effects primarily by decreasing the migratory activity of monocytes/macrophages to the lesions.

Modulation of Itch by Localized Skin Warming and Cooling.

Skin thermal changes modulate itch sensitivity. However, the mechanisms of this modulation are still unclear. Using mouse models of acute and chronic itch, we investigated whether local innocuous thermal stimulation of the skin alters itch sensitivity and if blockade of thermosensitive transient receptor potential (TRP) channels can reduce these changes. Localized thermal changes were achieved by placing a thermal probe in contact with the back skin for 30 s. Warming the skin significantly increased serotonin-evoked scratching and spontaneous scratching in the ovalbumin model of atopic dermatitis but decreased histamine-evoked scratching. These changes were blocked by a TRPV4 antagonist. Cooling the skin significantly increased serotonin-evoked scratching but reduced histamine-evoked scratching. The increase in serotonin-evoked scratching, but not the reduction of histamine-evoked scratching, was blocked by TRPM8 antagonism. Chloroquine-evoked scratching was unaffected by either warming or cooling. Our data indicate that different itch signaling pathways are differentially modulated by skin thermal changes.

Role of neurturin in spontaneous itch and increased nonpeptidergic intraepidermal fiber density in a mouse model of psoriasis.

Psoriasis is often accompanied by itch, but the mechanisms behind this symptom remain elusive. Dynamic changes in epidermal innervation have been observed under chronic itch conditions. Therefore, we investigated whether epidermal innervation is altered in the imiquimod-induced psoriasis mouse model, whether blockade of neurotrophic growth factor signaling can reduce these changes, and whether this system can impact psoriatic itch. Over the 7-day time course of imiquimod treatment, the density of epidermal nonpeptidergic nerves significantly increased, whereas the density of peptidergic nerves significantly decreased. The nonpeptidergic epidermal nerves expressed glial cell line-derived neurotrophic factor (GDNF) family receptor GFRα-1 and GFRα-2, the ligand-binding domains for GDNF and neurturin (NRTN). The NRTN mRNA expression was elevated in the skin of imiquimod-treated mice, whereas the GDNF mRNA expression was decreased. Treatment of imiquimod-challenged mice with an NRTN-neutralizing antibody significantly reduced nonpeptidergic nerve density as well as spontaneous scratching. These results indicate that NRTN contributes to an increase in the epidermal density of nonpeptidergic nerves in the imiquimod-induced psoriasis model, and this increase may be a factor in chronic itch for these mice. Therefore, inhibition of NRTN could be a potential treatment for chronic itch in psoriasis.

Mouse model of imiquimod-induced psoriatic itch.

Itch is a major indicator of psoriasis, but the underlying mechanisms behind this symptom are largely unknown. To investigate the neuronal mechanisms of psoriatic itch, we tested whether mice subjected to the imiquimod-induced psoriasis model exhibit itch-associated behaviors. Mice received daily topical applications of imiquimod to the rostral back skin for 7 days. Imiquimod-treated mice exhibited a significant increase in spontaneous scratching behavior directed to the treated area as well as touch-evoked scratching (alloknesis). To characterize this model, we measured the mRNA expression levels of pruritogens and itch-relevant receptors/channels using real-time reverse transcription PCR. The mRNA expression of MrgprA3, MrgprC11, and MrgprD decreased gradually over time in the dorsal root ganglion (DRG) cells. There was no significant change in the mRNA expression of TRPV1 or TRPA1 in DRG cells. TRPV4 mRNA expression was transiently increased in the DRG cells, whereas TRPM8 mRNA was significantly decreased. The mRNA expression levels of histidine decarboxylase and tryptophan hydroxylase 1, as well as the intensity of histamine and serotonin immunoreactivity, were transiently increased in the skin on day 2, returning to baseline by day 7. Histamine H1-receptor antagonists, chlorpheniramine and olopatadine, significantly inhibited spontaneous scratching on day 2, but not day 7. Neither chlorpheniramine nor olopatadine affected alloknesis on day 2 or day 7. These results may reflect the limited antipruritic effects of histamine H1-receptor antagonists on human psoriasis. The imiquimod-induced psoriasis model seems to be useful for the investigation of itch and its sensitization in psoriasis.

Plasma cholesterol-lowering and transient liver dysfunction in mice lacking squalene synthase in the liver.

Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.

Absence of Nceh1 augments 25-hydroxycholesterol-induced ER stress and apoptosis in macrophages.

An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.