Sarah E. Gale

Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death.

Cell dysfunction and death induced by lipid accumulation in nonadipose tissues, or lipotoxicity, may contribute to the pathogenesis of obesity and type 2 diabetes. However, the mechanisms leading to lipotoxic cell death are poorly understood. We recently reported that, in Chinese hamster ovary (CHO) cells and in H9c2 cardiomyoblasts, lipid overload induced by incubation with 500 μM palmitate leads to intracellular accumulation of reactive oxygen species, which subsequently induce endoplasmic reticulum (ER) stress and cell death. Here, we show that palmitate also impairs ER function through a more direct mechanism. Palmitate was rapidly incorporated into saturated phospholipid and triglyceride species in microsomal membranes of CHO cells. The resulting membrane remodeling was associated with dramatic dilatation of the ER and redistribution of protein-folding chaperones to the cytosol within 5 h, indicating compromised ER membrane integrity. Increasing β-oxidation, through the activation of AMP-activated protein kinase, decreased palmitate incorporation into microsomes, decreased the escape of chaperones to the cytosol, and decreased subsequent caspase activation and cell death. Thus, palmitate rapidly increases the saturated lipid content of the ER, leading to compromised ER morphology and integrity, suggesting that impairment of the structure and function of this organelle is involved in the cellular response to fatty acid overload.

Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease

Oxysterols are biomarkers for diagnosis and drug treatment in Niemann-Pick C1 disease. Turning the Tables on Cholesterol A big push in disease research is to identify biochemical markers (biomarkers) in the blood that are early indicators of a disease that is already silently under way. By detecting the disease in its earliest stages, drugs and other therapeutic interventions have the best chance of halting or reversing the course of the disease before major tissue damage has been done. In a new study, Porter and colleagues set out to identify blood biomarkers for Niemann-Pick C1, a childhood neurological disease that is usually fatal. Niemann-Pick C1 disease is caused by mutations in the NPC1 or NPC2 proteins that result in mishandling of cholesterol and lipids in the endolysosomal system of cells. This leads to aberrant deposition of free cholesterol in the central nervous system, the death of neurons, and increasing motor and intellectual impairment, usually resulting in death during adolescence. The early symptoms of the disease are often difficult to distinguish from other childhood diseases, and thus, intervention in the form of a drug such as miglustat often comes too late. This prompted Porter and coworkers to search for possible molecules in the blood that could be used for early diagnosis of the disease and also to monitor the effectiveness of new drugs. On the basis of reports that aberrantly deposited free cholesterol is associated with increased oxidative stress, these investigators reasoned that cholesterol oxidation products (oxysterols) might be the long-sought biomarkers for Niemann-Pick C1 disease. Working in mice lacking the Npc1 gene, the researchers quickly identified two oxysterols that were markedly elevated in the plasma and tissues of the sick mice but not their healthy counterparts. Furthermore, the concentrations of these two oxysterols increased as the disease progressed. Moving into cats carrying an NPC1 mutation, which exhibit similar disease symptoms and progression as human patients, Porter and coworkers were able to decrease elevated concentrations of the two oxysterols and ameliorate disease symptoms by treating the animals with the experimental drug cyclodextrin. But could oxysterols be used as biomarkers in the human disease? The investigators demonstrated that the blood concentrations of two related oxysterol molecules were almost 10 times higher in Niemann-Pick C1 patients than in age-matched healthy controls or those with other diseases such as atherosclerosis or diabetes. Together, these compelling results suggest that the two oxysterol molecules are accurate diagnostic markers of early clinical disease and can be used not only to monitor disease progression but also to demonstrate drug efficacy. Free cholesterol may be at the root of Niemann-Pick C1 disease, but now, there is a way to turn the tables on cholesterol by using its oxidation products to diagnose and treat the disease in its earliest stages. Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1−/− mouse model and found several cholesterol oxidation products that were elevated in Npc1−/− mice, were detectable before the onset of symptoms, and were associated with disease progression. Nonenzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with the age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.

Pregnane X receptor (PXR) activation: A mechanism for neuroprotection in a mouse model of Niemann–Pick C disease

Niemann–Pick type C1 (NPC1) disease is a fatal neurodegenerative disease characterized by neuronal lipid storage and progressive Purkinje cell loss in the cerebellum. We investigated whether therapeutic approaches to bypass the cholesterol trafficking defect in NPC1 disease might delay disease progression in the npc1−/− mouse model. We show that the neurosteroid allopregnanolone (ALLO) and T0901317, a synthetic oxysterol ligand, act in concert to delay onset of neurological symptoms and prolong the lifespan of npc1−/− mice. ALLO and T0901317 therapy preserved Purkinje cells, suppressed cerebellar expression of microglial-associated genes and inflammatory mediators, and reduced infiltration of activated microglia in the cerebellar tissue. To establish whether the mechanism of neuroprotection in npc1−/− mice involves GABAA receptor activation, we compared treatment of natural ALLO and ent-ALLO, a stereoisomer that has identical physical properties of natural ALLO but is not a GABAA receptor agonist. ent-ALLO provided identical functional and survival benefits as natural ALLO in npc1−/− mice, strongly supporting a GABAA receptor-independent mechanism for ALLO action. On the other hand, the efficacy of ALLO, ent-ALLO, and T0901317 therapy correlated with the ability of these compounds to activate pregnane X receptor-dependent pathways in vivo. These findings suggest that treatment with pregnane X receptor ligands may be useful clinically in delaying the progressive neurodegeneration in human NPC disease.

A Regulatory Role for 1-Acylglycerol-3-phosphate-O-acyltransferase 2 in Adipocyte Differentiation

Mutations in the 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) gene have been identified in individuals affected with congenital generalized lipodystrophy (CGL). AGPAT2 catalyzes acylation of lysophosphatidic acid to phosphatidic acid, a precursor for both triacylglycerol (TAG) and phospholipid synthesis. Recent studies suggest that reduced AGPAT2 enzymatic activity may underlie the CGL clinical phenotype. To gain insight into how altered AGPAT2 activity causes lipodystrophy, we examined the effect of knockdown of AGPAT2 expression in preadipocytes on TAG synthesis and storage, and on adipocyte differentiation. We show that AGPAT2 mRNA expression is induced 30-fold during adipocyte differentiation and that AGPAT2 enzymatic activity is required for TAG mass accumulation in mature adipocytes. We demonstrate that small interference RNA-mediated knockdown of AGPAT2 expression prevents appropriate early induction of C/EBPβ and PPARγ, key transcriptional activators of the adipogenic program, and delays expression of multiple adipocyte-related genes. The unexpected finding, that levels of several phospholipid species, including phosphatidic acid (PA), are elevated in TAG-depleted adipocytes with AGPAT2 knockdown, suggests that impaired AGPAT2 activity affects availability of PA for TAG synthesis but not overall PA synthesis nor utilization of PA for phospholipid synthesis. These findings underscore the importance of an AGPAT2-mediated metabolic pathway in adipocyte differentiation.

Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding

Over 200 disease-causing mutations have been identified in the NPC1 gene. The most prevalent mutation, NPC1I1061T, is predicted to lie within the cysteine-rich luminal domain and is associated with the classic juvenile-onset phenotype of Niemann-Pick type C disease. To gain insight into the molecular mechanism by which the NPC1I1061T mutation causes disease, we examined expression of the mutant protein in human fibroblasts homozygous for the NPC1I1061T mutation. Despite similar NPC1 mRNA levels between wild type and NPC1I1061T fibroblasts, NPC1 protein levels are decreased by 85% in NPC1I1061T cells. Metabolic labeling studies demonstrate that unlike wild type protein, which undergoes a glycosylation pattern shift from Endo H-sensitive to Endo H-resistant species, NPC1I1061T protein remains almost exclusively Endo H-sensitive and exhibits a reduced half-life (t½ 6.5 h) versus wild type Endo H-resistant species (t½ 42 h). Treatment with chemical chaperones, growth at permissive temperature, or inhibition of proteasomal degradation increases NPC1I1061T protein levels, indicating that the mutant protein is likely targeted for endoplasmic reticulum-associated degradation (ERAD) due to protein misfolding. Overexpression of NPC1I1061T in NPC1-deficient cells results in late endosomal localization of the mutant protein and complementation of the NPC mutant phenotype, likely due to a small proportion of the nascent NPC1I1061T protein that is able to fold correctly and escape the endoplasmic reticulum quality control checkpoints. Our findings provide the first description of an endoplasmic reticulum trafficking defect as a mechanism for human NPC disease, shedding light on the mechanism by which the NPC1I1061T mutation causes disease and suggesting novel approaches to treat NPC disease caused by the NPC1I1061T mutation.

Side Chain Oxygenated Cholesterol Regulates Cellular Cholesterol Homeostasis through Direct Sterol-Membrane Interactions

Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.

Development of a bile acid–based newborn screen for Niemann-Pick disease type C

A newborn screen for Niemann-Pick disease type C was developed on the basis of discovery of a bile acid marker. Expanding the newborn screen Niemann-Pick disease type C (NPC) is a fatal neurologic disorder caused by the deficiency of an enzyme involved in cholesterol storage. Although this disease was untreatable in the past, new therapeutics are now in clinical trials, but they are most likely to be effective if treatment is started as early as possible, before neurodegeneration has occurred. Jiang et al. identified three bile acids that are greatly increased in the blood of patients with NPC compared to healthy controls. The authors also demonstrated that one of these bile acids can be reliably measured in dried blood spots using mass spectrometry, suggesting that this bile acid test should be evaluated for potential addition to neonatal screening programs. Niemann-Pick disease type C (NPC) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, it is imperative to improve diagnostics and facilitate early intervention. We used metabolomic profiling to identify potential markers and discovered three unknown bile acids that were increased in plasma from NPC but not control subjects. The bile acids most elevated in the NPC subjects were identified as 3β,5α,6β-trihydroxycholanic acid and its glycine conjugate, which were shown to be metabolites of cholestane-3β,5α,6β-triol, an oxysterol elevated in NPC. A high-throughput mass spectrometry–based method was developed and validated to measure the glycine-conjugated bile acid in dried blood spots. Analysis of dried blood spots from 4992 controls, 134 NPC carriers, and 44 NPC subjects provided 100% sensitivity and specificity in the study samples. Quantification of the bile acid in dried blood spots, therefore, provides the basis for a newborn screen for NPC that is ready for piloting in newborn screening programs.

Box C/D Small Nucleolar RNA (snoRNA) U60 Regulates Intracellular Cholesterol Trafficking

Background: The mechanisms regulating internalization of plasma membrane cholesterol in mammalian cells are not well understood. Results: A cell line haploinsufficient for U60 snoRNA expression exhibits impaired plasma membrane to ER cholesterol trafficking and increased de novo cholesterol synthesis. Conclusion: U60 snoRNA expression regulates cholesterol homeostasis by affecting internalization of plasma membrane cholesterol. Significance: This is the first study to implicate a snoRNA in regulation of cholesterol homeostasis. Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.

Side-chain oxysterols modulate cholesterol accessibility through membrane remodeling.

Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), are key regulators of cholesterol homeostasis. New evidence suggests that the alteration of membrane structure by 25-HC contributes to its regulatory effects. We have examined the role of oxysterol membrane effects on cholesterol accessibility within the membrane using perfringolysin O (PFO), a cholesterol-dependent cytolysin that selectively binds accessible cholesterol, as a sensor of membrane cholesterol accessibility. We show that 25-HC increases cholesterol accessibility in a manner dependent on the membrane lipid composition. Structural analysis of molecular dynamics simulations reveals that increased cholesterol accessibility is associated with membrane thinning, and that the effects of 25-HC on cholesterol accessibility are driven by these changes in membrane thickness. Further, we find that the 25-HC antagonist LY295427 (agisterol) abrogates the membrane effects of 25-HC in a nonenantioselective manner, suggesting that agisterol antagonizes the cholesterol-homeostatic effects of 25-HC indirectly through its membrane interactions. These studies demonstrate that oxysterols regulate cholesterol accessibility, and thus the availability of cholesterol to be sensed and transported throughout the cell, by modulating the membrane environment. This work provides new insights into how alterations in membrane structure can be used to relay cholesterol regulatory signals.

A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol

Cholesterol homeostasis is regulated not only by cholesterol, but also by oxygenated cholesterol species, referred to as oxysterols. Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), regulate cholesterol homeostasis through feedback inhibition and feed-forward activation of transcriptional pathways that govern cholesterol synthesis, uptake, and elimination, as well as through direct nongenomic actions that modulate cholesterol accessibility in membranes. Elucidating the cellular distribution of 25-HC is required to understand its biological activity at the molecular level. However, studying oxysterol distribution and behavior within cells has proven difficult due to the lack of fluorescent analogs of 25-HC that retain its chemical and physical properties. To address this, we synthesized a novel intrinsically fluorescent 25-HC mimetic, 25-hydroxycholestatrienol (25-HCTL). We show that 25-HCTL modulates sterol homeostatic responses in a similar manner as 25-HC. 25-HCTL associates with lipoproteins in media and is taken up by cells through LDL-mediated endocytosis. In cultured cells, 25-HCTL redistributes among cellular membranes and, at steady state, has a similar distribution as cholesterol, being enriched in both the endocytic recycling compartment as well as the plasma membrane. Our findings indicate that 25-HCTL is a faithful fluorescent 25-HC mimetic that can be used to investigate the mechanisms through which 25-HC regulates sterol homeostatic pathways.