Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination.

Regeneration of myelin is mediated by oligodendrocyte progenitor cells-an abundant stem cell population in the central nervous system (CNS) and the principal source of new myelinating oligodendrocytes. Loss of myelin-producing oligodendrocytes in the CNS underlies a number of neurological diseases, including multiple sclerosis and diverse genetic diseases1-3. High-throughput chemical screening approaches have been used to identify small molecules that stimulate the formation of oligodendrocytes from oligodendrocyte progenitor cells and functionally enhance remyelination in vivo4-10. Here we show that a wide range of these pro-myelinating small molecules function not through their canonical targets but by directly inhibiting CYP51, TM7SF2, or EBP, a narrow range of enzymes within the cholesterol biosynthesis pathway. Subsequent accumulation of the 8,9-unsaturated sterol substrates of these enzymes is a key mechanistic node that promotes oligodendrocyte formation, as 8,9-unsaturated sterols are effective when supplied to oligodendrocyte progenitor cells in purified form whereas analogous sterols that lack this structural feature have no effect. Collectively, our results define a unifying sterol-based mechanism of action for most known small-molecule enhancers of oligodendrocyte formation and highlight specific targets to propel the development of optimal remyelinating therapeutics.

Studies of ApoD-/- and ApoD-/-ApoE-/- mice uncover the APOD significance for retinal metabolism, function, and status of chorioretinal blood vessels.

Apolipoprotein D (APOD) is an atypical apolipoprotein with unknown significance for retinal structure and function. Conversely, apolipoprotein E (APOE) is a typical apolipoprotein with established roles in retinal cholesterol transport. Herein, we immunolocalized APOD to the photoreceptor inner segments and conducted ophthalmic characterizations of ApoD-/- and ApoD-/-ApoE-/- mice. ApoD-/- mice had normal levels of retinal sterols but changes in the chorioretinal blood vessels and impaired retinal function. The whole-body glucose disposal was impaired in this genotype but the retinal glucose metabolism was unchanged. ApoD-/-ApoE-/- mice had altered sterol profile in the retina but apparently normal chorioretinal vasculature and function. The whole-body glucose disposal and retinal glucose utilization were enhanced in this genotype. OB-Rb, both leptin and APOD receptor, was found to be expressed in the photoreceptor inner segments and was at increased abundance in the ApoD-/- and ApoD-/-ApoE-/- retinas. Retinal levels of Glut4 and Cd36, the glucose transporter and scavenger receptor, respectively, were increased as well, thus linking APOD to retinal glucose and fatty acid metabolism and suggesting the APOD-OB-Rb-GLUT4/CD36 axis. In vivo isotopic labeling, transmission electron microscopy, and retinal proteomics provided additional insights into the mechanism underlying the retinal phenotypes of ApoD-/- and ApoD-/-ApoE-/- mice. Collectively, our data suggest that the APOD roles in the retina are context specific and could determine retinal glucose fluxes into different pathways. APOD and APOE do not play redundant, complementary or opposing roles in the retina, rather their interplay is more complex and reflects retinal responses elicited by lack of these apolipoproteins.

Discovery of a Redox Thiol Switch: Implications for Cellular Energy Metabolism.

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H2S has been shown to persulfidate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H2S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTSGS). We propose that the RTSGS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

Deficiency in Acyl-CoA:Wax Alcohol Acyltransferase 2 causes evaporative dry eye disease by abolishing biosynthesis of wax esters.

Lipids secreted by the meibomian glands (MGs) of the eyelids are essential to the protection of the eye's surface. An altered meibum composition represents the primary cause of evaporative dry eye disease (DED). Despite the critical importance of the meibum, its biosynthetic pathways and the roles of individual lipid components remain understudied. Here, we report that the genetic deletion of Acyl-CoA:wax alcohol acyltransferase 2 (AWAT2) causes the obstruction of MGs and symptoms of evaporative DED in mice. The lipid composition of the meibum isolated from Awat2-/- mice revealed the absence of wax esters, which was accompanied by a compensatory overproduction of cholesteryl esters. The resulting increased viscosity of meibum led to the dilation of the meibomian ducts, and the progressive degeneration of the MGs. Overall, we provide evidence for the main physiological role of AWAT2 and establish Awat2-/- mice as a model for DED syndrome that can be used in studies on tear film-oriented therapies.

Effect of Bubble Concentration on the in Vitro and in Vivo Performance of Highly Stable Lipid Shell-Stabilized Micro- and Nanoscale Ultrasound Contrast Agents.

Ultrasound (US) is a widely used diagnostic imaging tool because it is inexpensive, safe, portable, and broadly accessible. Ultrasound contrast agents (UCAs) are employed to enhance backscatter echo and improve imaging contrast. The most frequently utilized UCAs are echogenic bubbles made with a phospholipid or protein-stabilized hydrophobic gas core. While clinically utilized, applications of UCAs are often limited by rapid signal decay (<5 min) in vivo under typical ultrasound imaging protocols. Here, we report on a formulation of lipid shell-stabilized perfluoropropane (C3F8) microbubbles and nanobubbles with a significantly prolonged in vivo stability. Microbubbles (875 ± 280 nm) of the target size were prepared by utilizing a multiple-step centrifugation cycle, while nanobubbles (299 ± 189 nm) were isolated from the activated vial using a single centrifugation step. To provide in-depth acoustic characterization of the new construct we evaluated the effect of size and concentration on their in vitro and in vivo performance. In vitro and in vivo characterization were carried out for a range of bubble concentrations normalized by total gas volume quantified via headspace gas chromatography/mass spectrometry (GC/MS). In vitro characterization revealed that nanobubbles at different concentrations are more consistently stable over time with the highest and lowest dilutions (50-fold decrease) only differing in US signal after 8 min exposure by 10.34%, while for microbubbles the difference was 86.46%. As expected, due to the difference in hydrodynamic diameter and scattering cross section difference, nanobubbles showed lower overall initial signal intensity. In vivo experiments showed that both microbubbles and nanobubbles with similar initial peak signal intensity are comparably stable over time with 66.8% and 60.6% remaining signal after 30 min, respectively. This study demonstrates that bubble concentration has significant effects on the persistence of both microbubbles and nanobubbles in vitro and in vivo, but the effects are more pronounced in larger bubbles. These effects should be taken into account when selecting the appropriate bubble parameters for future imaging applications.

Small adipose stores in cystic fibrosis mice are characterized by reduced cell volume, not cell number.

Cystic fibrosis (CF) is a lethal genetic disorder that affects many organ systems of the body, including various endocrine and exocrine tissues. Health and survival positively associate with body mass, and as a consequence, CF clinical care includes high-fat, high-calorie diets to maintain and increase adipose tissue stores. Such strategies have been implemented without a clear understanding of the cause and effect relationship between body mass and patients' health. Here, we used CF mouse models, which display small adipose stores, to begin examining body fat as a prelude into mechanistic studies of low body growth in CF, so that optimal therapeutic strategies could be developed. We reasoned that low adiposity must result from reduced number and/or volume of adipocytes. To determine relative contribution of either mechanism, we quantified volume of intraperitoneal and subcutaneous adipocytes. We found smaller, but not fewer, adipocytes in CF compared with wild-type (WT) animals. Specifically, intraperitoneal CF adipocytes were one-half the volume of WT cells, whereas subcutaneous cells were less affected by the Cftr genotype. No differences were found in cell types between CF and WT adipose tissues. Adipose tissue CFTR mRNA was detected, and we found greater CFTR expression in intraperitoneal depots as compared with subcutaneous samples. RNA sequencing revealed that CF adipose tissue exhibited lower expression of several key genes of adipocyte function ( Lep, Pck1, Fas, Jun), consistent with low triglyceride storage. The data indicate that CF adipocytes contain fewer triglycerides than WT cells, and a role for CFTR in these cells is proposed. NEW & NOTEWORTHY Adipocytes in cystic fibrosis mice exhibit smaller size due to low triglyceride storage. Adipocyte cell number per fat pad is similar, implying triglyceride storage problem. The absence of CFTR function in adipose tissue has been proposed as a direct link to low triglyceride storage in cystic fibrosis.

Absence of leptin signaling allows fat accretion in cystic fibrosis mice.

Negative energy balance is a prevalent feature of cystic fibrosis (CF). Pancreatic insufficiency, elevated energy expenditure, lung disease, and malnutrition, all characteristic of CF, contribute to the negative energy balance causing low body-growth phenotype. As low body weight and body mass index strongly correlate with poor lung health and survival of patients with CF, improving energy balance is an important clinical goal (e.g., high-fat diet). CF mouse models also exhibit negative energy balance (growth retardation and high energy expenditure), independent from exocrine pancreatic insufficiency, lung disease, and malnutrition. To improve energy balance through increased caloric intake and reduced energy expenditure, we disrupted leptin signaling by crossing the db/db leptin receptor allele with mice carrying the R117H Cftr mutation. Compared with db/db mice, absence of leptin signaling in CF mice (CF db/db) resulted in delayed and moderate hyperphagia with lower de novo lipogenesis and lipid deposition, producing only moderately obese CF mice. Greater body length was found in db/db mice but not in CF db/db, suggesting CF-dependent effect on bone growth. The db/db genotype resulted in lower energy expenditure regardless of Cftr genotype leading to obesity. Despite the db/db genotype, the CF genotype exhibited high respiratory quotient indicating elevated carbohydrate oxidation, thus limiting carbohydrates for lipogenesis. In summary, db/db-linked hyperphagia, elevated lipogenesis, and morbid obesity were partially suppressed by reduced CFTR activity. CF mice still accrued large amounts of adipose tissue in contrast to mice fed a high-fat diet, thus highlighting the importance of dietary carbohydrates and not simply fat for energy balance in CF. NEW & NOTEWORTHY We show that cystic fibrosis (CF) mice are able to accrue fat under conditions of carbohydrate overfeeding, increased lipogenesis, and decreased energy expenditure, although length was unaffected. High-fat diet feeding failed to improve growth in CF mice. Morbid db/db-like obesity was reduced in CF double-mutant mice by reduced CFTR activity.

Retinal Cholesterol Content Is Reduced in Simvastatin-Treated Mice Due to Inhibited Local Biosynthesis Albeit Increased Uptake of Serum Cholesterol.

Statins, a class of cholesterol-lowering drugs, are currently being investigated for treatment of age-related macular degeneration, a retinal disease. Herein, retinal and serum concentrations of four statins (atorvastatin, simvastatin, pravastatin, and rosuvastatin) were evaluated after mice were given a single drug dose of 60 mg/kg body weight. All statins, except rosuvastatin, were detected in the retina: atorvastatin and pravastatin at 1.6 pmol and simvastatin at 4.1 pmol. Serum statin concentrations (pmol/ml) were 223 (simvastatin), 1401 (atorvastatin), 2792 (pravastatin), and 9050 (rosuvastatin). Simvastatin was then administered to mice daily for 6 weeks at 60 mg/kg body weight. Simvastatin treatment reduced serum cholesterol levels by 18% and retinal content of cholesterol and lathosterol (but not desmosterol) by 24% and 21%, respectively. The relative contributions of retinal cholesterol biosynthesis and retinal uptake of serum cholesterol to total retinal cholesterol input were changed as well. These contributions were 79% and 21%, respectively, in vehicle-treated mice and 69% and 31%, respectively, in simvastatin-treated mice. Thus, simvastatin treatment lowered retinal cholesterol because a compensatory upregulation of retinal uptake of serum cholesterol was not sufficient to overcome the effect of inhibited retinal biosynthesis. Simultaneously, simvastatin-treated mice had a 2.9-fold increase in retinal expression of Cd36, the major receptor clearing oxidized low-density lipoproteins from Bruch's membrane. Notably, simvastatin treatment essentially did not affect brain cholesterol homeostasis. Our results reveal the statin effect on the retinal and brain cholesterol input and are of value for future clinical investigations of statins as potential therapeutics for age-related macular degeneration.

S-Nitrosoglutathione formation at gastric pH is augmented by ascorbic acid and by the antioxidant vitamin complex, Resiston.

CONTEXT: Exogenous nitrogen oxides must be made bioavailable to sustain normal physiology because nitric oxide synthase (NOS) deficient mice are viable. In the stomach, S-nitrosoglutathione (GSNO) is formed from ingested nitrite and high levels of airway glutathione (GSH) that are cleared and swallowed. However, gastric GSNO may be broken down by nutrients like ascorbic acid (AA) before it is absorbed. OBJECTIVE: To study the effect of AA on GSNO formation and stability. MATERIALS AND METHODS: GSH and nitrite were reacted with or without 5 mM AA or Resiston (5 mM AA with retinoic acid and α-tocopherol). GSNO was measured by reduction/chemiluminescence and HPLC. AA and reduced thiols were measured colorimetrically. O-Nitrosoascorbate and AA were measured by gas chromatography-mass spectrometry (GC-MS). RESULTS: GSNO was formed in saline and gastric samples (pH ∼4.5) from physiological levels of GSH and nitrite. Neither AA nor Resiston decreased [GSNO] at pH >3; rather, they increased [GSNO] (0.12 ± 0.19 µM without AA; 0.42 ± 0.35 µM with AA; and 0.43 ± 0.23 µM with Resiston; n = 4 each; p ≤ 0.05). However, AA compounds decreased [GSNO] at lower pH and with incubation >1 h. Mechanistically, AA, but not dehydroascorbate, increased GSNO formation; and the O-nitrosoascorbate intermediate was formed. CONCLUSIONS: AA, with or without other antioxidants, did not deplete GSNO formed from physiological levels of GSH and nitrite at pH >3. In fact, it favoured GSNO formation, likely through O-nitrosoascorbate. Gastric GSNO could be a NOS-independent source of bioavailable nitrogen oxides.

Cholesterol in mouse retina originates primarily from in situ de novo biosynthesis.

The retina, a thin tissue in the back of the eye, has two apparent sources of cholesterol: in situ biosynthesis and cholesterol available from the systemic circulation. The quantitative contributions of these two cholesterol sources to the retinal cholesterol pool are unknown and have been determined in the present work. A new methodology was used. Mice were given separately deuterium-labeled drinking water and chow containing 0.3% deuterium-labeled cholesterol. In the retina, the rate of total cholesterol input was 21 µg of cholesterol/g retina • day, of which 15 µg of cholesterol/g retina • day was provided by local biosynthesis and 6 µg of cholesterol/g retina • day was uptaken from the systemic circulation. Thus, local cholesterol biosynthesis accounts for the majority (72%) of retinal cholesterol input. We also quantified cholesterol input to mouse brain, the organ sharing important similarities with the retina. The rate of total cerebral cholesterol input was 121 µg of cholesterol/g brain • day with local biosynthesis providing 97% of total cholesterol input. Our work addresses a long-standing question in eye research and adds new knowledge to the potential use of statins (drugs that inhibit cholesterol biosynthesis) as therapeutics for age-related macular degeneration, a common blinding disease.

ADEMA: an algorithm to determine expected metabolite level alterations using mutual information.

Metabolomics is a relatively new "omics" platform, which analyzes a discrete set of metabolites detected in bio-fluids or tissue samples of organisms. It has been used in a diverse array of studies to detect biomarkers and to determine activity rates for pathways based on changes due to disease or drugs. Recent improvements in analytical methodology and large sample throughput allow for creation of large datasets of metabolites that reflect changes in metabolic dynamics due to disease or a perturbation in the metabolic network. However, current methods of comprehensive analyses of large metabolic datasets (metabolomics) are limited, unlike other "omics" approaches where complex techniques for analyzing coexpression/coregulation of multiple variables are applied. This paper discusses the shortcomings of current metabolomics data analysis techniques, and proposes a new multivariate technique (ADEMA) based on mutual information to identify expected metabolite level changes with respect to a specific condition. We show that ADEMA better predicts De Novo Lipogenesis pathway metabolite level changes in samples with Cystic Fibrosis (CF) than prediction based on the significance of individual metabolite level changes. We also applied ADEMA's classification scheme on three different cohorts of CF and wildtype mice. ADEMA was able to predict whether an unknown mouse has a CF or a wildtype genotype with 1.0, 0.84, and 0.9 accuracy for each respective dataset. ADEMA results had up to 31% higher accuracy as compared to other classification algorithms. In conclusion, ADEMA advances the state-of-the-art in metabolomics analysis, by providing accurate and interpretable classification results.

Transcriptional programming of translation by BCL6 controls skeletal muscle proteostasis.

Skeletal muscle is dynamically controlled by the balance of protein synthesis and degradation. Here we discover an unexpected function for the transcriptional repressor B cell lymphoma 6 (BCL6) in muscle proteostasis and strength in mice. Skeletal muscle-specific Bcl6 ablation in utero or in adult mice results in over 30% decreased muscle mass and force production due to reduced protein synthesis and increased autophagy, while it promotes a shift to a slower myosin heavy chain fibre profile. Ribosome profiling reveals reduced overall translation efficiency in Bcl6-ablated muscles. Mechanistically, tandem chromatin immunoprecipitation, transcriptomic and translational analyses identify direct BCL6 repression of eukaryotic translation initiation factor 4E-binding protein 1 (Eif4ebp1) and activation of insulin-like growth factor 1 (Igf1) and androgen receptor (Ar). Together, these results uncover a bifunctional role for BCL6 in the transcriptional and translational control of muscle proteostasis.

Clonal Parabacteroides from Gut Microfistulous Tracts as Transmissible Cytotoxic Succinate-Commensal Model of Crohn's Disease Complications.

Crohn's disease (CD) has been traditionally viewed as a chronic inflammatory disease that cause gut wall thickening and complications, including fistulas, by mechanisms not understood. By focusing on Parabacteroides distasonis (presumed modern succinate-producing commensal probiotic), recovered from intestinal microfistulous tracts (cavernous fistulous micropathologies CavFT proposed as intermediate between 'mucosal fissures' and 'fistulas') in two patients that required surgery to remove CD-damaged ilea, we demonstrate that such isolates exert pathogenic/pathobiont roles in mouse models of CD. Our isolates are clonally-related; potentially emerging as transmissible in the community and mice; proinflammatory and adapted to the ileum of germ-free mice prone to CD-like ileitis (SAMP1/YitFc) but not healthy mice (C57BL/6J), and cytotoxic/ATP-depleting to HoxB8-immortalized bone marrow derived myeloid cells from SAMP1/YitFc mice when concurrently exposed to succinate and extracts from CavFT-derived E. coli , but not to cells from healthy mice. With unique genomic features supporting recent genetic exchange with Bacteroides fragilis -BGF539, evidence of international presence in primarily human metagenome databases, these CavFT Pdis isolates could represent to a new opportunistic Parabacteroides species, or subspecies (' cavitamuralis' ) adapted to microfistulous niches in CD.

Time course of hepatic gluconeogenesis during hindlimb suspension unloading.

The goal of this work was to determine the time-dependent changes in fractional hepatic gluconeogenesis (GNG) during conditions of hindlimb suspension unloading (HSU), a 'ground-based' method for inducing muscular atrophy to simulate space flight. We hypothesized that GNG would increase in HSU conditions as a result of metabolic shifts in the liver and skeletal muscle. A significant and progressive atrophy was observed in the soleus (30, 47 and 55%) and gastrocnemius muscles (0, 15 and 17%) after 3, 7 and 14 days of HSU, respectively. Fractional hepatic GNG was determined following the incorporation of deuterium from deuterated water ((2)H(2)O) into C-H bonds of newly synthesized glucose after an 8 h fast. Enrichment of plasma glucose with (2)H was measured using the classic method of Landau et al. (the 'hexamethylenetetramine (HMT) method'), based on specific (2)H labelling of glucose carbons, and the novel method of Chacko et al. ('average method'), based on the assumption of equal (2)H enrichment on all glucose carbons (except C2). After 3 days of HSU, fractional GNG was significantly elevated in the HSU group, as determined by either method (∼13%, P < 0.05). After 7 and 14 days of HSU, gluconeogenesis was not significantly different. Both analytical methods yielded similar time-dependent trends in gluconeogenic rates, but GNG values determined using the average method were consistently lower (∼30%) than those found by the HMT method. To compare and validate the average method against the HMT method further, we starved animals for 13 h to allow for hepatic GNG to contribute 100% to endogenous glucose production. The HMT method yielded 100% GNG, while the average method yielded GNG of ∼70%. As both methods used the same values of precursor enrichment, we postulated that the underestimation of gluconeogenic rate was as a result of differences in the measurements of product enrichment ((2)H labelling of plasma glucose). This could be explained by the following factors: (i) loss of deuterium via exchange between acetate and glucose; (ii) interference caused by fragment m/z 169, representing multiple isobaric species; and (iii) interference from other sugars at m/z 169. In conclusion, HSU caused a time-dependent increase in hepatic gluconeogenesis, irrespective of the analytical methods used.

Increased glucose availability sensitizes pancreatic cancer to chemotherapy.

Pancreatic Ductal Adenocarcinoma (PDAC) is highly resistant to chemotherapy. Effective alternative therapies have yet to emerge, as chemotherapy remains the best available systemic treatment. However, the discovery of safe and available adjuncts to enhance chemotherapeutic efficacy can still improve survival outcomes. We show that a hyperglycemic state substantially enhances the efficacy of conventional single- and multi-agent chemotherapy regimens against PDAC. Molecular analyses of tumors exposed to high glucose levels reveal that the expression of GCLC (glutamate-cysteine ligase catalytic subunit), a key component of glutathione biosynthesis, is diminished, which in turn augments oxidative anti-tumor damage by chemotherapy. Inhibition of GCLC phenocopies the suppressive effect of forced hyperglycemia in mouse models of PDAC, while rescuing this pathway mitigates anti-tumor effects observed with chemotherapy and high glucose.

Regulatory role of ß-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells.

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of ß-arrestin-2 (ßarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that ßarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged ßarr2 (ßarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The ßarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This ßarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting ßarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/ßarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for ßarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of ßarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of ßarr2-mediated alterations on cholesterol processing. It is concluded that ßarr2 expression contributes to altered cholesterol trafficking observed in CF cells.

Altered de novo lipogenesis contributes to low adipose stores in cystic fibrosis mice.

Cystic fibrosis (CF) mouse models exhibit exocrine pancreatic function, yet they do not develop adipose stores to the levels of non-CF mice. CF mice homozygous for the Cftr mutation (F508del) at 3 wk (postweaning) and 6 wk (young adult) of age had markedly less adipose tissue than non-CF mice. Food intake was markedly lower in 3-wk-old CF mice but normalized by 6 wk of age. Both 3- and 6-wk-old mice had dietary lipid absorption and fecal lipid excretion comparable to non-CF mice. Hepatic de novo lipogenesis (DNL), determined by (2)H incorporation, was reduced in CF mice. At 3 wk, F508del mice had significantly decreased DNL of palmitate and stearate, by 83% and 80%, respectively. By 6 wk, DNL rates in non-CF mice remained unchanged compared with 3-wk-old mice, while DNL rates of F508del mice were still reduced, by 33% and 40%, respectively. Adipose tissue fatty acid (FA) profiles were comparable in CF and non-CF mice, indicating that adipose differences are quantitative, not qualitative. A correspondingly lower content of (2)H-labeled FA was found in CF adipose tissue, consistent with reduced deposition of newly made hepatic triglycerides and/or decreased adipose tissue lipogenesis. Hepatic transcriptome analysis revealed lower mRNA expression from several genes involved in FA biosynthesis, suggesting downregulation of this pathway as a mechanism for the reduced lipogenesis. These novel data provide a model for altered lipid metabolism in CF, independent of malabsorption, and may partly explain the inability of pancreatic enzyme replacement therapy to completely restore normal body mass to CF patients.

Intracellular vesicle entrapment of nanobubble ultrasound contrast agents targeted to PSMA promotes prolonged enhancement and stability in vivo and in vitro.

Gas-core nanoscale bubbles (or nanobubbles) have gained significant recent attention as promising contrast agents for cancer molecular imaging using medical ultrasound. Previous work has shown that active targeting of nanobubbles to tumor biomarkers such as the prostate-specific membrane antigen (PSMA) significantly prolongs ultrasound signal enhancement, which is a critical feature for successful tumor diagnosis. However, the specific mechanism behind this effect is not well understood, and has not been previously studied in detail. Thus, in the current work, we investigated the process of PMSA- targeted nanobubble transport in tumors across different scales from in vivo whole tumor imaging using high-frequency dynamic contrast-enhanced ultrasound to intracellular confocal imaging and, molecularly using headspace gas chromatography/mass spectrometry. Data demonstrated that, indeed, molecular targeting of nanobubbles to the PSMA biomarker prolongs their tumor uptake and retention across the entire tumor volume, but with variability due to the expected tumor heterogeneity. Importantly, in vitro, the active targeting of NBs results in internalization via receptor-mediated endocytosis into the target cells, and the co-localization with intracellular vesicles (late-stage endosomes/lysosomes) significantly prolongs perfluorocarbon gas retention within the cells. This has not been directly observed previously. These results support the potential for nanobubbles to enable highly specific, background-free diagnostic imaging of the target cells/tissues using ultrasound.

Enhancers of Human and Rodent Oligodendrocyte Formation Predominantly Induce Cholesterol Precursor Accumulation.

Regeneration of myelin in the central nervous system is being pursued as a potential therapeutic approach for multiple sclerosis. Several labs have reported small molecules that promote oligodendrocyte formation and remyelination in vivo. Recently, we reported that many such molecules function by inhibiting a narrow window of enzymes in the cholesterol biosynthesis pathway. Here we describe a new high-throughput screen of 1,836 bioactive molecules and a thorough re-analysis of more than 60 molecules previously identified as promoting oligodendrocyte formation from human, rat, or mouse oligodendrocyte progenitor cells. These studies highlight that an overwhelming fraction of validated screening hits, including several molecules being evaluated clinically for remyelination, inhibit cholesterol pathway enzymes like emopamil-binding protein (EBP). To rationalize these findings, we suggest a model that relies on the high druggability of sterol-metabolizing enzymes and the ability of cationic amphiphiles to mimic the transition state of EBP. These studies further establish cholesterol pathway inhibition as a dominant mechanism among screening hits that enhance human, rat, or mouse oligodendrocyte formation.

Inhibition of SC4MOL and HSD17B7 shifts cellular sterol composition and promotes oligodendrocyte formation.

While the cholesterol biosynthesis pathway has been extensively studied, recent work has forged new links between inhibition of specific sterol pathway enzymes, accumulation of their unique sterol substrates, and biological areas as diverse as cancer, immunology, and neurodegenerative disease. We recently reported that dozens of small molecules enhance formation of oligodendrocytes, a glial cell type lost in multiple sclerosis, by inhibiting CYP51, Sterol 14-reductase, or EBP and inducing cellular accumulation of their 8,9-unsaturated sterol substrates. Several adjacent pathway enzymes also have 8,9-unsaturated sterol substrates but have not yet been evaluated as potential targets for oligodendrocyte formation or in many other biological contexts, in part due to a lack of available small-molecule probes. Here, we show that genetic suppression of SC4MOL or HSD17B7 increases the formation of oligodendrocytes. Additionally, we have identified and optimized multiple potent new series of SC4MOL and HSD17B7 inhibitors and shown that these small molecules enhance oligodendrocyte formation. SC4MOL inhibitor CW4142 induced accumulation of SC4MOL's sterol substrates in mouse brain and represents an in vivo probe of SC4MOL activity. Mechanistically, the cellular accumulation of these 8,9-unsaturated sterols represents a central driver of enhanced oligodendrocyte formation, as exogenous addition of purified SC4MOL and HSD17B7 substrates but not their 8,9-saturated analogs promotes OPC differentiation. Our work validates SC4MOL and HSD17B7 as novel targets for promoting oligodendrocyte formation, underlines a broad role for 8,9-unsaturated sterols as enhancers of oligodendrocyte formation, and establishes the first high-quality small molecules targeting SC4MOL and HSD17B7 as novel tools for probing diverse areas of biology.