Publications by authors named "Elma Aflaki"

24 Publications

  • Page 1 of 1

GPR110 ligands reduce chronic optic tract gliosis and visual deficit following repetitive mild traumatic brain injury in mice.

J Neuroinflammation 2021 Jul 17;18(1):157. Epub 2021 Jul 17.

Laboratory of Molecular Signaling, NIAAA, NIH, 5625 Fishers Lane, Rockville, MD, 20852, USA.

Background: Repetitive mild traumatic brain injury (mTBI) can result in chronic visual dysfunction. G-protein receptor 110 (GPR110, ADGRF1) is the target receptor of N-docosahexaenoylethanolamine (synaptamide) mediating the anti-neuroinflammatory function of synaptamide. In this study, we evaluated the effect of an endogenous and a synthetic ligand of GPR110, synaptamide and (4Z,7Z,10Z,13Z,16Z,19Z)-N-(2-hydroxy-2-methylpropyl) docosa-4,7,10,13,16,19-hexaenamide (dimethylsynaptamide, A8), on the mTBI-induced long-term optic tract histopathology and visual dysfunction using Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA), a clinically relevant model of mTBI.

Methods: The brain injury in wild-type (WT) and GPR110 knockout (KO) mice was induced by CHIMERA applied daily for 3 days, and GPR110 ligands were intraperitoneally injected immediately following each impact. The expression of GPR110 and proinflammatory mediator tumor necrosis factor (TNF) in the brain was measured by using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in an acute phase. Chronic inflammatory responses in the optic tract and visual dysfunction were assessed by immunostaining for Iba-1 and GFAP and visual evoked potential (VEP), respectively. The effect of GPR110 ligands in vitro was evaluated by the cyclic adenosine monophosphate (cAMP) production in primary microglia isolated from adult WT or KO mouse brains.

Results: CHIMERA injury acutely upregulated the GPR110 and TNF gene level in mouse brain. Repetitive CHIMERA (rCHIMERA) increased the GFAP and Iba-1 immunostaining of glia cells and silver staining of degenerating axons in the optic tract with significant reduction of N1 amplitude of visual evoked potential at up to 3.5 months after injury. Both GPR110 ligands dose- and GPR110-dependently increased cAMP in cultured primary microglia with A8, a ligand with improved stability, being more effective than synaptamide. Intraperitoneal injection of A8 at 1 mg/kg or synaptamide at 5 mg/kg significantly reduced the acute expression of TNF mRNA in the brain and ameliorated chronic optic tract microgliosis, astrogliosis, and axonal degeneration as well as visual deficit caused by injury in WT but not in GPR110 KO mice.

Conclusion: Our data demonstrate that ligand-induced activation of the GPR110/cAMP system upregulated after injury ameliorates the long-term optic tract histopathology and visual impairment caused by rCHIMERA. Based on the anti-inflammatory nature of GPR110 activation, we suggest that GPR110 ligands may have therapeutic potential for chronic visual dysfunction associated with mTBI.
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http://dx.doi.org/10.1186/s12974-021-02195-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8286622PMC
July 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R).

Invest Ophthalmol Vis Sci 2021 02;62(2):30

Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.

Purpose: To examine the contribution of pigment epithelium-derived factor receptor (PEDF-R) to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by the PNPLA2 gene, as a phospholipase A2 in the retinal pigment epithelium (RPE). During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. The role of PEDF-R in RPE phagocytosis is not known.

Methods: Mice in which PNPLA2 was conditionally knocked out (cKO) in the RPE were generated. Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2 silencing duplexes. POSs were isolated from bovine retinas. The phospholipase A2 inhibitor bromoenol lactone was used. Transmission electron microscopy, immunofluorescence, lipid labeling, pulse-chase experiments, western blots, and free fatty acid and β-hydroxybutyrate assays were performed.

Results: The RPE of the cKO mice accumulated lipids, as well as more abundant and larger rhodopsin particles, compared to littermate controls. Upon POS exposure, RPE explants from cKO mice released less β-hydroxybutyrate compared to controls. After POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered β-hydroxybutyrate release from phagocytic RPE cells. PNPLA2 knockdown also resulted in a decline in fatty acids and β-hydroxybutyrate release from phagocytic RPE cells.

Conclusions: PEDF-R downregulation delayed POS digestion during phagocytosis. The findings imply that the efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE.
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http://dx.doi.org/10.1167/iovs.62.2.30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900850PMC
February 2021

A characterization of Gaucher iPS-derived astrocytes: Potential implications for Parkinson's disease.

Neurobiol Dis 2020 02 10;134:104647. Epub 2019 Nov 10.

Section of Molecular Neurogenetics, National Human Genome Research Institute, NIH, Bethesda, MD 20892, United States of America. Electronic address:

While astrocytes, the most abundant cells found in the brain, have many diverse functions, their role in the lysosomal storage disorder Gaucher disease (GD) has not been explored. GD, resulting from the inherited deficiency of the enzyme glucocerebrosidase and subsequent accumulation of glucosylceramide and its acylated derivative glucosylsphingosine, has both non-neuronopathic (GD1) and neuronopathic forms (GD2 and 3). Furthermore, mutations in GBA1, the gene mutated in GD, are an important risk factor for Parkinson's disease (PD). To elucidate the role of astrocytes in the disease pathogenesis, we generated iAstrocytes from induced pluripotent stem cells made from fibroblasts taken from controls and patients with GD1, with and without PD. We also made iAstrocytes from an infant with GD2, the most severe and progressive form, manifesting in infancy. Gaucher iAstrocytes appropriately showed deficient glucocerebrosidase activity and levels and substrate accumulation. These cells exhibited varying degrees of astrogliosis, Glial Fibrillary Acidic Protein (GFAP) up-regulation and cellular proliferation, depending on the level of residual glucocerebrosidase activity. Glutamte uptake assays demonstrated that the cells were functionally active, although the glutamine transporter EEAT2 was upregulated and EEAT1 downregulated in the GD2 samples. GD2 iAstrocytes were morphologically different, with severe cytoskeletal hypertrophy, overlapping of astrocyte processes, pronounced up-regulation of GFAP and S100β, and significant astrocyte proliferation, recapitulating the neuropathology observed in patients with GD2. Although astrocytes do not express α-synuclein, when the iAstrocytes were co-cultured with dopaminergic neurons generated from the same iPSC lines, excessive α-synuclein released from neurons was endocytosed by astrocytes, translocating into lysosomes. Levels of aggregated α-synuclein increased significantly when cells were treated with monomeric or fibrillar α-synuclein. GD1-PD and GD2 iAstrocytes also exhibited impaired Cathepsin D activity, leading to further α-synuclein accumulation. Cytokine and chemokine profiling of the iAstrocytes demonstrated an inflammatory response. Thus, in patients with GBA1-associated parkinsonism, astrocytes appear to play a role in α-synuclein accumulation and processing, contributing to neuroinflammation.
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http://dx.doi.org/10.1016/j.nbd.2019.104647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980699PMC
February 2020

Validation of anti-glucocerebrosidase antibodies for western blot analysis on protein lysates of murine and human cells.

Biochem J 2019 01 25;476(2):261-274. Epub 2019 Jan 25.

Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, U.S.A.

Gaucher disease (GD) is a rare lysosomal storage disorder caused by mutations in the gene, encoding the lysosome-resident glucocerebrosidase enzyme involved in the hydrolysis of glucosylceramide. The discovery of an association between mutations in and the development of synucleinopathies, including Parkinson disease, has directed attention to glucocerebrosidase as a potential therapeutic target for different synucleinopathies. These findings initiated an exponential growth in research and publications regarding the glucocerebrosidase enzyme. The use of various commercial and custom-made glucocerebrosidase antibodies has been reported, but standardized in-depth validation is still not available for many of these antibodies. This work details the evaluation of several previously reported glucocerebrosidase antibodies for western blot analysis, tested on protein lysates of murine and immortalized neurons and primary human wild-type and type 2 GD fibroblasts.
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http://dx.doi.org/10.1042/BCJ20180708DOI Listing
January 2019

Induced pluripotent stem cell models of lysosomal storage disorders.

Dis Model Mech 2017 06;10(6):691-704

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA

Induced pluripotent stem cells (iPSCs) have provided new opportunities to explore the cell biology and pathophysiology of human diseases, and the lysosomal storage disorder research community has been quick to adopt this technology. Patient-derived iPSC models have been generated for a number of lysosomal storage disorders, including Gaucher disease, Pompe disease, Fabry disease, metachromatic leukodystrophy, the neuronal ceroid lipofuscinoses, Niemann-Pick types A and C1, and several of the mucopolysaccharidoses. Here, we review the strategies employed for reprogramming and differentiation, as well as insights into disease etiology gleaned from the currently available models. Examples are provided to illustrate how iPSC-derived models can be employed to develop new therapeutic strategies for these disorders. We also discuss how models of these rare diseases could contribute to an enhanced understanding of more common neurodegenerative disorders such as Parkinson's disease, and discuss key challenges and opportunities in this area of research.
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http://dx.doi.org/10.1242/dmm.029009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483008PMC
June 2017

The Complicated Relationship between Gaucher Disease and Parkinsonism: Insights from a Rare Disease.

Neuron 2017 Feb;93(4):737-746

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3708, USA. Electronic address:

The discovery of a link between mutations in GBA1, encoding the lysosomal enzyme glucocerebrosidase, and the synucleinopathies directly resulted from the clinical recognition of patients with Gaucher disease with parkinsonism. Mutations in GBA1 are now the most common known genetic risk factor for several Lewy body disorders, and an inverse relationship exists between levels of glucocerebrosidase and oligomeric α-synuclein. While the underlying mechanisms are still debated, this complicated association is shedding light on the role of lysosomes in neurodegenerative disorders, demonstrating how insights from a rare disorder can direct research into the pathogenesis and therapy of seemingly unrelated common diseases.
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http://dx.doi.org/10.1016/j.neuron.2017.01.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5327952PMC
February 2017

Chaperoning glucocerebrosidase: a therapeutic strategy for both Gaucher disease and Parkinsonism.

Neural Regen Res 2016 Nov;11(11):1760-1761

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.

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http://dx.doi.org/10.4103/1673-5374.194717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204226PMC
November 2016

Efferocytosis is impaired in Gaucher macrophages.

Haematologica 2017 04 23;102(4):656-665. Epub 2016 Dec 23.

Section of Molecular Neurogenetics, Medical Genetics Branch, National Institutes of Health, Bethesda, MD, USA

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylceramide-laden macrophages resulting from impaired digestion of aged erythrocytes or apoptotic leukocytes. Studies of macrophages from patients with type 1 Gaucher disease with genotypes N370S/N370S, N370S/L444P or N370S/c.84dupG revealed that Gaucher macrophages have impaired efferocytosis resulting from reduced levels of p67 and Rab7. The decreased Rab7 expression leads to impaired fusion of phagosomes with lysosomes. Moreover, there is defective translocation of p67 to phagosomes, resulting in reduced intracellular production of reactive oxygen species. These factors contribute to defective deposition and clearance of apoptotic cells in phagolysosomes, which may have an impact on the inflammatory response and contribute to the organomegaly and inflammation seen in patients with Gaucher disease.
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http://dx.doi.org/10.3324/haematol.2016.155093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395106PMC
April 2017

A new glucocerebrosidase-deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease.

Dis Model Mech 2016 07 19;9(7):769-78. Epub 2016 May 19.

Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA

Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1 Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1 To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba(-/-) mice and the control littermate (gba(+/+)) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba(-/-) neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba(+/+) neurons. This null allele gba(-/-) mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies.
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http://dx.doi.org/10.1242/dmm.024588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958308PMC
July 2016

A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism.

J Neurosci 2016 07;36(28):7441-52

Section of Molecular Neurogenetics, National Human Genome Research Institute,

Unlabelled: Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease.

Significance Statement: Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.
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http://dx.doi.org/10.1523/JNEUROSCI.0636-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945664PMC
July 2016

Applications of iPSC-derived models of Gaucher disease.

Ann Transl Med 2015 Nov;3(19):295

Section of Molecular Neurogenetics, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

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http://dx.doi.org/10.3978/j.issn.2305-5839.2015.10.41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671880PMC
November 2015

Lysosomal storage and impaired autophagy lead to inflammasome activation in Gaucher macrophages.

Aging Cell 2016 Feb 21;15(1):77-88. Epub 2015 Oct 21.

Section of Molecular Neurogenetics, National Human Genome Research Institute, Bethesda, MD, 20892, USA.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylcer-amide macrophages, the accumulation of glucosylceramide in lysosomes and the secretion of inflammatory cytokines. However, the connection between this lysosomal storage and inflammation is not clear. Studying macrophages derived from peripheral monocytes from patients with type 1 Gaucher disease with genotype N370S/N370S, we confirmed an increased secretion of interleukins IL-1β and IL-6. In addition, we found that activation of the inflammasome, a multiprotein complex that activates caspase-1, led to the maturation of IL-1β in Gaucher macrophages. We show that inflammasome activation in these cells is the result of impaired autophagy. Treatment with the small-molecule glucocerebrosidase chaperone NCGC758 reversed these defects, inducing autophagy and reducing IL-1β secretion, confirming the role of the deficiency of lysosomal glucocerebrosidase in these processes. We found that in Gaucher macrophages elevated levels of the autophagic adaptor p62 prevented the delivery of inflammasomes to autophagosomes. This increase in p62 led to activation of p65-NF-kB in the nucleus, promoting the expression of inflammatory cytokines and the secretion of IL-1β. This newly elucidated mechanism ties lysosomal dysfunction to inflammasome activation, and may contribute to the massive organomegaly, bone involvement and increased susceptibility to certain malignancies seen in Gaucher disease. Moreover, this link between lysosomal storage, impaired autophagy, and inflammation may have implications relevant to both Parkinson disease and the aging process. Defects in these basic cellular processes may also provide new therapeutic targets.
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http://dx.doi.org/10.1111/acel.12409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717273PMC
February 2016

Active autophagy but not lipophagy in macrophages with defective lipolysis.

Biochim Biophys Acta 2015 Oct 2;1851(10):1304-1316. Epub 2015 Jul 2.

Institute of Molecular Biology & Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.

During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.
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http://dx.doi.org/10.1016/j.bbalip.2015.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562370PMC
October 2015

New macrophage models of Gaucher disease offer new tools for drug development.

Macrophage (Houst) 2015;2(1):e712

Section of Molecular Neurogenetics, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

Gaucher disease is an inherited enzyme deficiency resulting in the lysosomal accumulation of specific glycolipids in macrophages and, in some cases, neurons. While current treatments are effective at reducing this glycolipid storage in macrophages, they are expensive and ineffective in treating neurological manifestations of the disease, driving the search for novel therapeutics. Moreover, mutations in , the gene implicated in Gaucher disease, are an important risk factor for the development of Parkinson disease and related disorders, an association that has further heightened interest in Gaucher disease research. However, the development of therapeutic strategies has been hampered by a shortage of appropriate cellular models of Gaucher disease. We have generated two novel macrophage models of Gaucher disease, one through the differentiation of peripheral blood monocytes from patients with Gaucher disease and the other through the differentiation of induced pluripotent stem cells derived from patient fibroblasts. Both disease models demonstrate similar cellular phenotypes and exhibit extensive glycolipid storage when exposed to exogenous lipid sources such as erythrocyte membranes. Furthermore, we have used these models to confirm the efficacy of a novel small molecule in clearing glycolipid storage and restoring normal macrophage function. These results demonstrate the usefulness of these models in exploring new therapeutics for Gaucher disease and related disorders.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4469193PMC
January 2015

Macrophage models of Gaucher disease for evaluating disease pathogenesis and candidate drugs.

Sci Transl Med 2014 Jun;6(240):240ra73

Section on Molecular Neurogenetics, Medical Genetics Branch, National Institutes of Health, Bethesda, MD 20892, USA.

Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes, particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore, we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition, we created induced pluripotent stem cell (iPSC)-derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages, reduced glycolipid storage, and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development.
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http://dx.doi.org/10.1126/scitranslmed.3008659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161206PMC
June 2014

Toxicity of oxidized phospholipids in cultured macrophages.

Lipids Health Dis 2012 Sep 7;11:110. Epub 2012 Sep 7.

Institute of Biochemistry, Graz University of Technology, Petersgasse 12/2, A-8010, Graz, Austria.

Background: The interactions of oxidized low-density lipoprotein (LDL) and macrophages are hallmarks in the development of atherosclerosis. The biological activities of the modified particle in these cells are due to the content of lipid oxidation products and apolipoprotein modification by oxidized phospholipids.

Results: It was the aim of this study to determine the role of short-chain oxidized phospholipids as components of modified LDL in cultured macrophages. For this purpose we investigated the effects of the following oxidized phospholipids on cell viability and apoptosis: 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and oxidized alkylacyl phospholipids including 1-O-hexadecyl-2-glutaroyl-sn-glycero-3-phosphocholine (E-PGPC) and 1-O-hexadecyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (E-POVPC). We found that these compounds induced apoptosis in RAW264.7 and bone marrow-derived macrophages. The sn-2 carboxyacyl lipid PGPC was more toxic than POVPC which carries a reactive aldehyde function in position sn-2 of glycerol. The alkylacyl phospholipids (E-PGPC and E-POVPC) and the respective diacyl analogs show similar activities. Apoptosis induced by POVPC and its alkylether derivative could be causally linked to the fast activation of an acid sphingomyelinase, generating the apoptotic second messenger ceramide. In contrast, PGPC and its ether analog only negligibly affected this enzyme pointing to an entirely different mechanism of lipid toxicity. The higher toxicity of PGPC is underscored by more efficient membrane blebbing from apoptotic cells. In addition, the protein pattern of PGPC-induced microparticles is different from the vesicles generated by POPVC.

Conclusions: In summary, our data reveal that oxidized phospholipids induce apoptosis in cultured macrophages. The mechanism of lipid toxicity, however, largely depends on the structural features of the oxidized sn-2 chain.
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http://dx.doi.org/10.1186/1476-511X-11-110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3533736PMC
September 2012

Adipose triglyceride lipase in immune response, inflammation, and atherosclerosis.

Biol Chem 2012 Sep;393(9):1005-11

Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21, A-8010 Graz, Austria.

Consistent with its central importance in lipid and energy homeostasis, lipolysis occurs in essentially all tissues and cell types, including macrophages. The hydrolytic cleavage of triacylglycerol by adipose triglyceride lipase (ATGL) generates non-esterified fatty acids, which are subsequently used as essential precursors for lipid and membrane synthesis, mediators in cell signaling processes or as energy substrate in mitochondria. This review summarizes the current knowledge concerning the consequences of ATGL deficiency in macrophages with particular emphasis on macrophage (dys)-function, apoptosis, and atherosclerosis.
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http://dx.doi.org/10.1515/hsz-2012-0192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3520003PMC
September 2012

Lack of acyl-CoA:diacylglycerol acyltransferase 1 reduces intestinal cholesterol absorption and attenuates atherosclerosis in apolipoprotein E knockout mice.

Biochim Biophys Acta 2011 Dec 1;1811(12):1011-20. Epub 2011 Sep 1.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Harrachgasse 21, Graz, Austria.

Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in several tissues. The final step in TG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Lack of whole body DGAT1 is associated with reduced lipid-induced inflammation. Since one major component of atherosclerosis is chronic inflammation we hypothesized that DGAT1 deficiency might ameliorate atherosclerotic lesion development. We therefore crossbred Apolipoprotein E-deficient (ApoE(-/-)) mice with Dgat1(-/-) mice. ApoE(-/-) and ApoE(-/-)Dgat1(-/-) mice were fed Western-type diet (WTD) for 9weeks and thereafter examined for plaque formation. The mean atherosclerotic lesion area was substantially reduced in ApoE(-/-)Dgat1(-/-) compared with ApoE(-/-) mice in en face and aortic valve section analyses. The reduced lesion size was associated with decreased cholesterol uptake and absorption by the intestine, reduced plasma TG and cholesterol concentrations and increased cholesterol efflux from macrophages. The expression of adhesion molecules was reduced in aortas of ApoE(-/-)Dgat1(-/-) mice, which might be the reason for less migration capacities of monocytes and macrophages and the observed decreased amount of macrophages within the plaques. From our results we conclude that the lack of DGAT1 is atheroprotective, implicating an additional application of DGAT1 inhibitors with regard to maintaining cholesterol homeostasis and attenuating atherosclerosis.
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http://dx.doi.org/10.1016/j.bbalip.2011.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3223411PMC
December 2011

Impaired Rho GTPase activation abrogates cell polarization and migration in macrophages with defective lipolysis.

Cell Mol Life Sci 2011 Dec 2;68(23):3933-47. Epub 2011 May 2.

Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.

Infiltration of monocytes and macrophages into the site of inflammation is critical in the progression of inflammatory diseases such as atherosclerosis. Cell migration is dependent on the continuous organization of the actin cytoskeleton, which is regulated by members of the small Rho GTPase family (RhoA, Cdc42, Rac) that are also important for the regulation of signal transduction pathways. We have recently reported on reduced plaque formation in an atherosclerotic mouse model transplanted with bone marrow from adipose triglyceride lipase-deficient (Atgl-/-) mice. Here we provide evidence that defective lipolysis in macrophages lacking ATGL, the major enzyme responsible for triacylglycerol hydrolysis, favors an anti-inflammatory M2-like macrophage phenotype. Our data implicate an as yet unrecognized principle that insufficient lipolysis influences macrophage polarization and actin polymerization, resulting in impaired macrophage migration. Sustained phosphorylation of focal adhesion kinase [due to inactivation of its phosphatase by elevated levels of reactive oxygen species (ROS)] results in defective Cdc42, Rac1 and RhoA activation and in increased and sustained activation of Rac2. Inhibition of ROS production restores the migratory capacity of Atgl-/- macrophages. Since monocyte and macrophage migration are a prerequisite for infiltrating the arterial wall, our results provide a molecular link between lipolysis and the development of atherosclerosis.
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http://dx.doi.org/10.1007/s00018-011-0688-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3214256PMC
December 2011

GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils.

Cell Res 2011 Oct 5;21(10):1452-69. Epub 2011 Apr 5.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Universitätsplatz 4, Graz A-8010, Austria.

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB(2) receptor (CB(2)R), but additional modulatory sites distinct from CB(2)R have recently been suggested to impact CB(2)R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB(2)R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB(2)R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB(2)R interfere with each other's signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB(2)R, while it synergizes with CB(2)R in recruiting neutrophils to sites of inflammation.
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http://dx.doi.org/10.1038/cr.2011.60DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3132458PMC
October 2011

Triacylglycerol accumulation activates the mitochondrial apoptosis pathway in macrophages.

J Biol Chem 2011 Mar 1;286(9):7418-28. Epub 2011 Jan 1.

Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.

Programmed cell death of lipid-laden macrophages is a prominent feature of atherosclerotic lesions and mostly ascribed to accumulation of excess intracellular cholesterol. The present in vitro study investigated whether intracellular triacylglycerol (TG) accumulation could activate a similar apoptotic response in macrophages. To address this question, we utilized peritoneal macrophages isolated from mice lacking adipose triglyceride lipase (ATGL), the major enzyme responsible for TG hydrolysis in multiple tissues. In Atgl(-/-) macrophages, we observed elevated levels of cytosolic Ca(2+) and reactive oxygen species, stimulated cytochrome c release, and nuclear localization of apoptosis-inducing factor. Fragmented mitochondria prior to cell death were indicative of the mitochondrial apoptosis pathway being triggered as a consequence of defective lipolysis. Other typical markers of apoptosis, such as externalization of phosphatidylserine in the plasma membrane, caspase 3 and poly(ADP-ribose) polymerase cleavage, were increased in Atgl(-/-) macrophages. An artificial increase of cellular TG levels by incubating wild-type macrophages with very low density lipoprotein closely mimicked the apoptotic phenotype observed in Atgl(-/-) macrophages. Results obtained during the present study define a novel pathway linking intracellular TG accumulation to mitochondrial dysfunction and programmed cell death in macrophages.
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http://dx.doi.org/10.1074/jbc.M110.175703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044998PMC
March 2011

Macrophage adipose triglyceride lipase deficiency attenuates atherosclerotic lesion development in low-density lipoprotein receptor knockout mice.

Arterioscler Thromb Vasc Biol 2011 Jan 28;31(1):67-73. Epub 2010 Oct 28.

Division of Biopharmaceutics, Gorlaeus Laboratories, Einsteinweg 55, 2333CC Leiden, the Netherlands.

Objective: The consequences of macrophage triglyceride (TG) accumulation on atherosclerosis have not been studied in detail so far. Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for the initial step in TG hydrolysis. Because ATGL knockout (KO) mice exhibit massive TG accumulation in macrophages, we used ATGL KO mice to study the effects of macrophage TG accumulation on atherogenesis.

Methods And Results: Low-density lipoprotein receptor (LDLr) KO mice were transplanted with bone marrow from ATGL KO (ATGL KO→LDLr KO) or wild-type (WT→LDLr KO) mice and challenged with a Western-type diet for 9 weeks. Despite TG accumulation in ATGL KO macrophages, atherosclerosis in ATGL KO→LDLr KO mice was 43% reduced associated with decreased plasma monocyte chemoattractant protein-1 (MCP-1) and macrophage interleukin-6 concentrations. This coincided with a reduced amount of macrophages, possibly because of a 39% increase in intraplaque apoptosis and a decreased migratory capacity of ATGL KO macrophages. The reduced number of white blood cells might be due to a 36% decreased Lin(-)Sca-1(+)cKit(+) hematopoietic stem cell population.

Conclusions: We conclude that the attenuation of atherogenesis in ATGL KO→LDLr KO mice is due to decreased infiltration of less inflammatory macrophages into the arterial wall and increased macrophage apoptosis.
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http://dx.doi.org/10.1161/ATVBAHA.110.215814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063945PMC
January 2011

Efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase.

J Biol Chem 2010 Jun 27;285(26):20192-201. Epub 2010 Apr 27.

Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.

Macrophage phagocytosis is an essential biological process in host defense and requires large amounts of energy. To date, glucose is believed to represent the prime substrate for ATP production in macrophages. To investigate the relative contribution of free fatty acids (FFAs) in this process, we determined the phagocytosis rates in normal mouse macrophages and macrophages of adipose triglyceride lipase (ATGL)-deficient mice. ATGL was shown to be the rate-limiting enzyme for the hydrolysis of lipid droplet-associated triacylglycerol (TG) in many tissues. Here, we demonstrate that Atgl(-/-) macrophages fail to efficiently hydrolyze cellular TG stores leading to decreased cellular FFA concentrations and concomitant accumulation of lipid droplets, even in the absence of exogenous lipid loading. The reduced availability of FFAs results in decreased cellular ATP concentrations and impaired phagocytosis suggesting that fatty acids must first go through a cycle of esterification and re-hydrolysis before they are available as energy substrate. Exogenously added glucose cannot fully compensate for the phagocytotic defect in Atgl(-/-) macrophages. Hence, phagocytosis was also decreased in vivo when Atgl(-/-) mice were challenged with bacterial particles. These findings imply that phagocytosis in macrophages depends on the availability of FFAs and that ATGL is required for their hydrolytic release from cellular TG stores. This novel mechanism links ATGL-mediated lipolysis to macrophage function in host defense and opens the way to explore possible roles of ATGL in immune response, inflammation, and atherosclerosis.
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http://dx.doi.org/10.1074/jbc.M110.107854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2888432PMC
June 2010
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