Carboxy-terminal blockade of sortilin binding enhances progranulin gene therapy, a potential treatment for frontotemporal dementia.

Frontotemporal dementia is commonly caused by loss-of-function mutations in the progranulin gene. Potential therapies for this disorder have entered clinical trials, including progranulin gene therapy and drugs that reduce progranulin interactions with sortilin. Both approaches ameliorate functional and pathological abnormalities in mouse models of progranulin insufficiency. Here we investigated whether modifying the progranulin carboxy terminus to block sortilin interactions would improve the efficacy of progranulin gene therapy. We compared the effects of treating progranulin-deficient mice with gene therapy vectors expressing progranulin with intact sortilin interactions, progranulin with the carboxy terminus blocked to reduce sortilin interactions, or GFP control. We found that expressing carboxy-terminally blocked progranulin generated higher levels of progranulin both at the injection site and in more distant regions. Carboxy-terminally blocked progranulin was also more effective at ameliorating microgliosis, microglial lipofuscinosis, and lipid abnormalities including ganglioside accumulation and loss of bis(monoacylglycero)phosphate lipids. Finally, only carboxy-terminally blocked progranulin reduced plasma neurofilament light chain, a biomarker of neurodegeneration, in progranulin-deficient mice. These results demonstrate that modifying the progranulin cargo to block sortilin interactions may be important for increasing the effectiveness of progranulin gene therapy. One-sentence Summary: The effectiveness of progranulin gene therapy in models of FTD is improved by blocking the protein's carboxy terminus, which prevents sortilin binding.

PLD3 and PLD4 synthesize S,S-BMP, a key phospholipid enabling lipid degradation in lysosomes.

Bis(monoacylglycero)phosphate (BMP) is an abundant lysosomal phospholipid required for degradation of lipids, in particular gangliosides. Alterations in BMP levels are associated with neurodegenerative diseases. Unlike typical glycerophospholipids, lysosomal BMP has two chiral glycerol carbons in the S (rather than the R) stereo-conformation, protecting it from lysosomal degradation. How this unusual and yet crucial S,S-stereochemistry is achieved is unknown. Here we report that phospholipases D3 and D4 (PLD3 and PLD4) synthesize lysosomal S,S-BMP, with either enzyme catalyzing the critical glycerol stereo-inversion reaction in vitro. Deletion of PLD3 or PLD4 markedly reduced BMP levels in cells or in murine tissues where either enzyme is highly expressed (brain for PLD3; spleen for PLD4), leading to gangliosidosis and lysosomal abnormalities. PLD3 mutants associated with neurodegenerative diseases, including Alzheimer's disease risk, diminished PLD3 catalytic activity. We conclude that PLD3/4 enzymes synthesize lysosomal S,S-BMP, a crucial lipid for maintaining brain health.

Transcriptional determinants of lipid mobilization in human adipocytes.

Defects in adipocyte lipolysis drive multiple aspects of cardiometabolic disease, but the transcriptional framework controlling this process has not been established. To address this, we performed a targeted perturbation screen in primary human adipocytes. Our analyses identified 37 transcriptional regulators of lipid mobilization, which we classified as (i) transcription factors, (ii) histone chaperones, and (iii) mRNA processing proteins. On the basis of its strong relationship with multiple readouts of lipolysis in patient samples, we performed mechanistic studies on one hit, ZNF189, which encodes the zinc finger protein 189. Using mass spectrometry and chromatin profiling techniques, we show that ZNF189 interacts with the tripartite motif family member TRIM28 and represses the transcription of an adipocyte-specific isoform of phosphodiesterase 1B (PDE1B2). The regulation of lipid mobilization by ZNF189 requires PDE1B2, and the overexpression of PDE1B2 is sufficient to attenuate hormone-stimulated lipolysis. Thus, our work identifies the ZNF189-PDE1B2 axis as a determinant of human adipocyte lipolysis and highlights a link between chromatin architecture and lipid mobilization.

Mice lacking triglyceride synthesis enzymes in adipose tissue are resistant to diet-induced obesity.

Triglycerides (TGs) in adipocytes provide the major stores of metabolic energy in the body. Optimal amounts of TG stores are desirable as insufficient capacity to store TG, as in lipodystrophy, or exceeding the capacity for storage, as in obesity, results in metabolic disease. We hypothesized that mice lacking TG storage in adipocytes would result in excess TG storage in cell types other than adipocytes and severe lipotoxicity accompanied by metabolic disease. To test this hypothesis, we selectively deleted both TG synthesis enzymes, DGAT1 and DGAT2, in adipocytes (ADGAT DKO mice). As expected with depleted energy stores, ADGAT DKO mice did not tolerate fasting well and, with prolonged fasting, entered torpor. However, ADGAT DKO mice were unexpectedly otherwise metabolically healthy and did not accumulate TGs ectopically or develop associated metabolic perturbations, even when fed a high-fat diet. The favorable metabolic phenotype resulted from activation of energy expenditure, in part via BAT (brown adipose tissue) activation and beiging of white adipose tissue. Thus, the ADGAT DKO mice provide a fascinating new model to study the coupling of metabolic energy storage to energy expenditure.

Deficiency of the frontotemporal dementia gene GRN results in gangliosidosis.

Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN-mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases.

Profile of tear lipid mediator as a biomarker of inflammation for meibomian gland dysfunction and ocular surface diseases: Standard operating procedures.

Human tear is a biological fluid rich in lipids that is increasingly collected in clinical and biological research. The repertoire of small lipids or lipid mediators (often termed eicosanoids or oxylipins) found in human tear provides insight into metabolism of fatty acids and physiology of the ocular surface and Meibomian glands. Disturbances in the tear lipid mediators profile also occur during inflammation of the ocular surface that is not directly linked to lipid metabolism. The changes in the levels of pro-inflammatory and pro-resolution lipid mediators in the tear help assess the severity and stage of inflammation in ocular surface tissues. Mass spectrometry, used in the evaluation of tear lipid mediators, is an emerging tool in clinical diagnostics and personalized medicine. Here we describe the reproducibility, accuracy, and precision of quantifying lipid mediators in human tears, with a suggested method for tear collection and sample handling. The ranges of lipid mediators concentrations in tear fluid of healthy and diseased individuals with Meibomian gland dysfunction are reported, as well as the impact of age and disease on individual lipid mediators. We would like to recommend a set of guidelines, which can be further discussed in workshops. This will facilitate harmonization of future tear lipid mediators data across different instrument platforms in various laboratories. We hope that other fields requiring lipid mediators assays will also benefit from such an effort.

Host/Malassezia Interaction: A Quantitative, Non-Invasive Method Profiling Oxylipin Production Associates Human Skin Eicosanoids with Malassezia.

Malassezia are common components of human skin, and as the dominant human skin eukaryotic microbe, they take part in complex microbe-host interactions. Other phylogenetically related fungi (including within Ustilagomycotina) communicate with their plant host through bioactive oxygenated polyunsaturated fatty acids, generally known as oxylipins, by regulating the plant immune system to increase their virulence. Oxylipins are similar in structure and function to human eicosanoids, which modulate the human immune system. This study reports the development of a highly sensitive mass-spectrometry-based method to capture and quantify bioactive oxygenated polyunsaturated fatty acids from the human skin surface and in vitro Malassezia cultures. It confirms that Malassezia are capable of synthesizing eicosanoid-like lipid mediators in vitro in a species dependent manner, many of which are found on human skin. This method enables sensitive identification and quantification of bioactive lipid mediators from human skin that may be derived from metabolic pathways shared between skin and its microbial residents. This enables better cross-disciplinary and detailed studies to dissect the interaction between Malassezia and human skin, and to identify potential intervention points to promote or abrogate inflammation and to improve human skin health.

Tailored Polymer-Based Selective Extraction of Lipid Mediators from Biological Samples.

Lipid mediators, small molecules involved in regulating inflammation and its resolution, are a class of lipids of wide interest as their levels in blood and tissues may be used to monitor health and disease states or the effect of new treatments. These molecules are present at low levels in biological samples, and an enrichment step is often needed for their detection. We describe a rapid and selective method that uses new low-cost molecularly imprinted (MIP) and non-imprinted (NIP) polymeric sorbents for the extraction of lipid mediators from plasma and tissue samples. The extraction process was carried out in solid-phase extraction (SPE) cartridges, manually packed with the sorbents. After extraction, lipid mediators were quantified by liquid chromatography-tandem mass spectrometry (LC-MSMS). Various parameters affecting the extraction efficiency were evaluated to achieve optimal recovery and to reduce non-specific interactions. Preliminary tests showed that MIPs, designed using the prostaglandin biosynthetic precursor arachidonic acid, could effectively enrich prostaglandins and structurally related molecules. However, for other lipid mediators, MIP and NIP displayed comparable recoveries. Under optimized conditions, the recoveries of synthetic standards ranged from 62% to 100%. This new extraction method was applied to the determination of the lipid mediators concentration in human plasma and mouse tissues and compared to other methods based on commercially available cartridges. In general, the methods showed comparable performances. In terms of structural specificity, our newly synthesized materials accomplished better retention of prostaglandins (PGs), hydroxydocosahexaenoic acid (HDoHE), HEPE, hydroxyeicosatetraenoic acids (HETE), hydroxyeicosatrienoic acid (HETrE), and polyunsaturated fatty acid (PUFA) compounds, while the commercially available Strata-X showed a higher recovery for dihydroxyeicosatetraenoic acid (diHETrEs). In summary, our results suggest that this new material can be successfully implemented for the extraction of lipid mediators from biological samples.

Changes of tear lipid mediators after eyelid warming or thermopulsation treatment for meibomian gland dysfunction.

Meibomian gland dysfunction (MGD) represents a major cause of dry eye and ocular discomfort. Lipid mediators, often termed oxylipins, can be produced enzymatically or non-enzymatically, and may modulate inflammatory processes in MGD. Here, we aimed to assess the longitudinal changes of lipid mediators after various eyelid treatments (eyelid warming and thermopulsation) over 12 weeks. Secondly, we aimed to assess the chirality of mono-hydroxyl lipid mediators from tears of MGD and healthy participants. Tears lipid mediators were extracted from Schirmer's strips and levels were quantified by liquid chromatography mass spectrometry (LC-MS) techniques. We quantified 33 lipid mediators in the tear, 18 of which (including 11-HETE, 20-OH-LTB4, and 15-oxoETE) were reduced significantly after treatment. Changes in concentrations of 10-HDoHE (r = 0.54) and 15-oxoETE (r = 0.54) were correlated to the number of meibomian gland plugs at baseline, so increased severity of MGD was associated with treatment-induced change in lipid mediators. The chiral analysis demonstrated that 5(S)-HETE, 12(S)-HETE, 15(S)-HETE, 14(S)-HDoHE, 17(S)-HDoHE and 11(R)-HETE were produced with significant enantiomeric excess (ee %) in controls compared to patients, due to enantiomer selective enzymatic action, whereas most lipid mediators were racemates in patients, due to dominance of oxidative effects which have no enantiomeric preference. Treatment of MGD restored the concentrations of 15(S)-HETE, 14(S)-HDoHE and 17(S)-HDoHE with significant ee values, suggesting reduction in oxidative action. Overall, MGD therapy reduced pro-inflammatory molecules generated by lipoxygenase and oxidative stress.

Change of tear lipid mediators in a post-trabeculectomy cohort.

PURPOSE: Trabeculectomy surgery could affect ocular surface disease (OSD) in several ways, through cessation of long term glaucoma eyedrops, exposure to operative mitomycin C and post-operative eyedrops including corticosteroids and aminoglycosides and reduction in eyelid hygiene measures. Previously we showed the relevance of tear lipid mediators (also referred oxylipins) in OSD. Here, we aim to evaluate changes of these lipids in a post-trabeculectomy cohort. METHODS: Patients undergoing trabeculectomy were prospectively evaluated and had tear collected using Schirmer's strips, preoperatively and postoperatively at 0.5, 1.0 and 3.0 years. Lipid mediators were analyzed using liquid chromatography mass spectrometry. RESULTS: The normalized concentrations of 40 lipid mediators were between 0.1 and 8.0 ng/mL, whereas docosahexaenoic acid (DHA), Arachidonic acid (AA) and eicosapentaenoic acid (EPA) ranged up to a few hundred ng/mL. The concentrations of lipid mediators, except DHA, EPA, and thromboxane (TXB1), showed reduction after surgery. At the last visit, these lipids were significantly reduced by 1/3 to ½, compared to pre-operative values: 8-HETE, 15-HETE, 15-oxoETE, 11-HDoHE, 17-HDoHE, and 20-OH-LTB4. To examine collective changes of lipids, clustering analysis revealed 10 groups of lipids consistent with known metabolic pathways. RESULTS: An increase in the level of 2,3-dinor-8-isoPGF2α between 0 and 0.5 year was associated with inferior corneal staining at 0.5 year. In 14 patients who required post-operative needling, six lipid mediators were found to be significantly higher at 1.0 year compared to non-needled patients. CONCLUSIONS: In this 3-years study, trabeculectomy reduced the tear level of pro-inflammatory lipid mediators. Patients who required needling of the bleb to maintain surgical success may have a chronic underlying inflammatory process associated with fibrosis.

Socio-economic determinants of anemia in pregnancy in North Shoa Zone, Ethiopia.

BACKGROUND: Globally, anemia in pregnancy increases maternal, fetal and neonatal mortality and morbidity. According to 2011 Ethiopian Demographic and Health Survey, 22% of pregnant women in Ethiopia were reported to be anemic. However, since the Ethiopian population is diverse with regard to culture, religion and other characteristics, this evidence may not represent the condition in our study area. So, we aimed to determine the prevalence of anemia and its associated factors among women receiving Antenatal Care (ANC) in Debre Berhan Town Hospitals and Clinics. METHODS: We conducted an institution based cross sectional study among women receiving ANC at hospitals and clinics in Debre Berhan Town, Ethiopia from September to November, 2013. Antenatal care providers in the respective health facilities collected the data by interview and observation using closed and open-ended questions. We computed frequencies and percentages to describe the data. We performed bivariate and multivariable binary logistic regression analyses to identify factors associated with anemia in pregnancy. STATA version 12 was used to carry out the analyses. RESULTS: A total of 295 participants completed the study, with a response rate of 89%. This study demonstrated a 10% prevalence of anemia out of which 64.3%, 32% and 4% of the respondents were with mild, moderate and severe anemia respectively. Anemia was statistically significantly associated with education and occupation. CONCLUSION: The prevalence of anemia in our study area is lower than previous studies' findings. Literacy and job status of the women were predictors of anemia in pregnancy. Since this study was conducted on women who had an opportunity to visit health facilities, it is more valuable to conduct community based research to better understand the problem in the study area and thus propose future deliverable.

Tear eicosanoids in healthy people and ocular surface disease.

Meibomian gland (MG) dysfunction is the leading cause of evaporative dry eye and it leads to inflammation of the ocular surface. Eicosanoids may be involved in inflammation of dry eye. This study aimed to profile tear eicosanoid levels in healthy individuals and those with MG dysfunction, and to examine if these levels are associated with clinical factors and expressibility of MG. Forty participants with MG dysfunction and 30 healthy controls were recruited in this study. Clinical signs of MG dysfunction were assessed, and tear lactoferrin concentration was evaluated. Tear eicosanoids were extracted from Schirmer's strips and analyzed using mass spectrometry. We were able to quantify 38 tear eicosanoids and levels were increased in older individuals. In participants with MG dysfunction, higher 5-HETE, LTB4, 18-HEPE, 12-HEPE and 14-HDoHE were associated with poorer MG expressibility. The eicosanoids PGF2α, 18-HEPE, 20-HDoHE and 17-HDoHE were elevated with increased corneal staining; higher 5-HETE, LTB4 were associated with lower tear lactoferrin levels. The receiver-operating-characteristics analysis shows higher levels of 5-HETE, LTB4 and 18-HEPE were able to predict poor expressibility of MGs. In conclusion, tear eicosanoid levels are age-dependent and specific eicosanoids may be indicators of clinical obstruction of MG or the severity of ocular surface damage.