[show abstract][hide abstract] ABSTRACT: The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/"metabolizing acquired dioxin-induced skin hamartomas" (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis.
EMBO Molecular Medicine 02/2014; · 7.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal childhood-onset neurodegenerative disorder caused by mutations in ceroid lipofuscinosis neuronal-3 (CLN3), a hydrophobic transmembrane protein of unresolved function. Previous studies indicate blood-brain barrier (BBB) defects in JNCL, and our earlier report showed prominent Cln3 expression in mouse brain endothelium. Here we find that CLN3 is necessary for normal trafficking of the microdomain-associated proteins caveolin-1, syntaxin-6, and multidrug resistance protein 1 (MDR1) in brain endothelial cells. Correspondingly, CLN3-null cells have reduced caveolae, and impaired caveolae- and MDR1-related functions including endocytosis, drug efflux, and cell volume regulation. We also detected an abnormal blood-brain barrier response to osmotic stress in vivo. Evaluation of the plasma membrane with fluorescent sphingolipid probes suggests microdomain destabilization and enhanced fluidity in CLN3-null cells. In further work we found that application of the glycosphingolipid lactosylceramide to CLN3-deficient cells rescues protein transport and caveolar endocytosis. Last, we show that CLN3 localizes to the trans-Golgi network (TGN) and partitions with buoyant microdomain fractions. We propose that CLN3 facilitates TGN-to-plasma membrane transport of microdomain-associated proteins. Insult to this pathway may underlie BBB dysfunction and contribute to JNCL pathogenesis.
Journal of Neuroscience 11/2013; 33(46):18065-79. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Endocytosed (glyco)sphingolipids are degraded, together with other membrane lipids in a stepwise fashion by endolysosomal enzymes with the help of small lipid binding proteins, the sphingolipid activator proteins (SAPs), at the surface of intraluminal lysosomal vesicles. Inherited defects in a sphingolipid-degrading enzyme or SAP cause the accumulation of the corresponding lipid substrates, including cytotoxic lysosphingolipids, such as galactosylsphingosine and glucosylsphingosine, and lead to a sphingolipidosis. Analysis of patients with prosaposin deficiency revealed the accumulation of intra-endolysosmal vesicles and membrane structures (IM). Feeding of prosaposin reverses the storage, suggesting inner membrane structures as platforms of sphingolipid degradation. Water soluble enzymes can hardly attack sphingolipids embedded in the membrane of inner endolysosomal vesicles. The degradation of sphingolipids with few sugar residues therefore requires the help of the SAPs, and is strongly stimulated by anionic membrane lipids. IMs are rich in anionic bis(monoacylglycero)phosphate (BMP).
Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gaucher disease (GD) is the most common inherited lysosomal storage disorder in humans, caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1). GD is clinically heterogeneous and although the type of GBA1 mutation plays a role in determining the type of GD, it does not explain the clinical variability seen among patients. Cumulative evidence from recent studies suggests that GBA2 could play a role in the pathogenesis of GD and potentially interacts with GBA1.
We used a framework of functional and genetic approaches in order to further characterize a potential role of GBA2 in GD. Glucosylceramide (GlcCer) levels in spleen, liver and brain of GBA2-deficient mice and mRNA and protein expression of GBA2 in GBA1-deficient murine fibroblasts were analyzed. Furthermore we crossed GBA2-deficient mice with conditional Gba1 knockout mice in order to quantify the interaction between GBA1 and GBA2. Finally, a genetic approach was used to test whether genetic variation in GBA2 is associated with GD and/ or acts as a modifier in Gaucher patients. We tested 22 SNPs in the GBA2 and GBA1 genes in 98 type 1 and 60 type 2/3 Gaucher patients for single- and multi-marker association with GD.
We found a significant accumulation of GlcCer compared to wild-type controls in all three organs studied. In addition, a significant increase of Gba2-protein and Gba2-mRNA levels in GBA1-deficient murine fibroblasts was observed. GlcCer levels in the spleen from Gba1/Gba2 knockout mice were much higher than the sum of the single knockouts, indicating a cross-talk between the two glucosylceramidases and suggesting a partially compensation of the loss of one enzyme by the other. In the genetic approach, no significant association with severity of GD was found for SNPs at the GBA2 locus. However, in the multi-marker analyses a significant result was detected for p.L444P (GBA1) and rs4878628 (GBA2), using a model that does not take marginal effects into account.
All together our observations make GBA2 a likely candidate to be involved in GD etiology. Furthermore, they point to GBA2 as a plausible modifier for GBA1 in patients with GD.
[show abstract][hide abstract] ABSTRACT: The epidermal permeability barrier of mammalian skin is localized in the stratum corneum. Corneocytes are embedded in an extracellular, highly ordered lipid matrix of hydrophobic lipids consisting of about 50% ceramides, 25% cholesterol and 15% long and very long chain fatty acids. The most important lipids for the epidermal barrier are ceramides. The scaffold of the lipid matrix is built of acylceramides, containing ω-hydroxylated very long chain fatty acids, acylated at the ω-position with linoleic acid. After glucosylation of the acylceramides at Golgi membranes and secretion, the linoleic acid residues are replaced by glutamate residues originating from proteins exposed on the surface of corneocytes. Removal of their glucosyl residues generates a hydrophobic surface on the corneocytes used as a template for the formation of extracellular lipid layers of the water permeability barrier. Misregulation or defects in the formation of extracellular ceramide structures disturbs barrier function. Important anabolic steps are the synthesis of very long chain fatty acids, their ω-hydroxylation, formation of very long chain ceramides and glucosylceramides. The main probarrier precursor lipids, glucosylceramides and sphingomyelins, are packed in lamellar bodies together with hydrolytic enzymes such as glucosylceramide-β-glucosidase and acid sphingomyelinase and secreted into the intercelullar space between the stratum corneum and stratum granulosum. Inherited defects in the extracellular hydrolytic processing of the probarrier acylglucosylceramides impair epidermal barrier formation and cause fatal diseases: such as prosaposin deficiency resulting in lack of lysosomal lipid binding and transfer proteins, or the symptomatic clinical picture of the "collodion baby" in the absence of glucocerebrosidase. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier.
Biochimica et Biophysica Acta 08/2013; · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Early onset of age-related changes in the brain of cannabinoid 1 receptor knockout (Cnr1(-/-)) mice suggests that cannabinoid 1 (CB1) receptor activity significantly influences the progression of brain aging. In the present study we show that lack of CB1 receptors leads to a significant increase in lipofuscin accumulation and a reduced expression and activity of cathepsin D, lysosomal protease implicated in the degradation of damaged macromolecules, in the hippocampus of 12-month-old mice. The impaired clearance of damaged macromolecules due to the low cathepsin D levels and not enhanced oxidative stress may be responsible for the lipofuscin accumulation because macromolecule oxidation levels were comparable between the genotypes within the same age group. The altered levels of autophagy markers p62 and LC3-II suggest that autophagy is upregulated in CB1 knockout mice. Increased autophagic flux in the absence of CB1 receptors is probably a compensatory mechanism to partially counteract decreased lysosomal degradation capacity. Together, these results suggest that CB1 receptor activity affects lysosomal activity, degradation of damaged macromolecules and thus it may influence the course and onset of brain aging.
Mechanisms of ageing and development 08/2013; · 4.18 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gangliosides are the main glycolipids of neuronal plasma membranes. Their surface patterns are generated by coordinated processes, involving biosynthetic pathways of the secretory compartments, catabolic steps of the endolysosomal system, and intracellular trafficking. Inherited defects in ganglioside biosynthesis causing fatal neurodegenerative diseases have been described so far almost exclusively in mouse models, whereas inherited defects in ganglioside catabolism causing various clinical forms of GM1- and GM2-gangliosidoses have long been known. For digestion, gangliosides are endocytosed and reach intra-endosomal vesicles. At the level of late endosomes, they are depleted of membrane-stabilizing lipids like cholesterol and enriched with bis(monoacylglycero)phosphate (BMP). Lysosomal catabolism is catalyzed at acidic pH values by cationic sphingolipid activator proteins (SAPs), presenting lipids to their respective hydrolases, electrostatically attracted to the negatively charged surface of the luminal BMP-rich vesicles. Various inherited defects of ganglioside hydrolases, e.g., of β-galactosidase and β-hexosaminidases, and of GM2-activator protein, cause infantile (with tetraparesis, dementia, blindness) and different protracted clinical forms of GM1- and GM2-gangliosidoses. Mutations yielding proteins with small residual catabolic activities in the lysosome give rise to juvenile and adult clinical forms with a wide range of clinical symptomatology. Apart from patients' differences in their genetic background, clinical heterogeneity may be caused by rather diverse substrate specificities and functions of lysosomal hydrolases, multifunctional properties of SAPs, and the strong regulation of ganglioside catabolism by membrane lipids. Currently, there is no treatment available for neuronal ganglioside storage diseases. Therapeutic approaches in mouse models and patients with juvenile forms of gangliosidoses are discussed.
Journal of Neuroscience 06/2013; 33(25):10195-208. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Unesterified Cholesterol, together with glycolipids, accumulates in cells leading to Niemann-Pick type C disease, an autosomal recessive neurodegenerative disease . Two types of genes, NPC1 and NPC2 responsible for this neurodegenerative disorder have been identified but their respective precise functions are yet to be fully elucidated. NPC1-NPC2 proteins interplay has been elucidated in such a way that NPC2 binds and transfers cholesterol to a cholesterol binding site of NPC1 for export out of the endosomal system. Until now, the study of Cholesterol has been hampered because the lipid lacks a functional group that can be readily tagged without altering its physical and chemical properties. Transfer studies of fluorescent cholesterol esters or cholesterol analogs are biased because of changes in the physicochemical nature of the lipids and might not correctly reflect the properties of unmodified cholesterol. A simple liposomal assay system which allows probing of the intermembrane transfer of radioactive cholesterol from donor to acceptor vesicles is therefore developed as well as a model to measure the vesicle fusion induced by some proteins. Abilities of the lysosomal lipid binding proteins (such as NPC2 protein) to transfer cholesterol in model membranes which mimic the endosomal/lysosomal compartments and to mediate vesicle fusion as well as the influence of lipid compositions were investigated. Other lysosomal lipid binding proteins investigated are saposins A-D and GM2AP while non-lysosomal proteins such as cytochrome C and bovine serum albumin were used as controls.
[show abstract][hide abstract] ABSTRACT: The unraveling of sphingolipid metabolism and function in the last 40 years relied on the extensive study of inherited human disease and specifically-tailored mouse models. However, only few of the achievements made so far would have been possible without chemical biology tools, such as fluorescent and/or radio-labeled and other artificial substrates, (mechanism-based) enzyme inhibitors, cross-linking probes or artificial membrane models. In this review we provide an overview over chemical biology tools that have been used to gain more insight into the molecular basis of sphingolipid-related biology. Many of these tools are still of high relevance for the investigation of current sphingolipid-related questions, others may stimulate the tailoring of novel probes suitable to address recent and future issues in the field. This article is part of a Special Issue entitled Tools to study lipid functions.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 01/2013; · 4.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ceramide synthase 1 (CerS1) catalyzes the synthesis of C18 ceramide and is mainly expressed in the brain. Custom-made antibodies to a peptide from the C-terminal region of the mouse CerS1 protein yielded specific immuno-signals in neurons and no other cell types of wild type brain, but did not detect the CerS1 protein in CerS1 deficient mouse brains. To elucidate the biological function of CerS1-derived sphingolipids in the brain, we generated CerS1 deficient mice by introducing a targeted mutation into the coding region of the cers1 gene. General deficiency of CerS1 in mice causes a foliation defect, progressive shrinkage and neuronal apoptosis in the cerebellum. Mass spectrometric analyses reveal up to 60% decreased levels of gangliosides in cerebellum and forebrain. Expression of Myelin Associated Glycoprotein (MAG) is also decreased by about 60% in cerebellum and forebrain, suggesting that interaction and stabilization of oligodendrocytic MAG protein by neuronal gangliosides is due to the C18-acyl membrane anchor of CerS1 derived precursor ceramides. A behavioral analysis of CerS1 deficient mice yielded functional deficits including impaired exploration of novel objects, locomotion and motor coordination. Our results reveal an essential function of CerS1-derived ceramide in regulation of cerebellar development and neurodevelopmentally-regulated behavior.
Journal of Biological Chemistry 10/2012; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The specificity and the strong binding capacity of the biotin-streptavidin complex makes the system to be one of the most widely used affinity pairs in molecular, immunological and cellular (in-vitro) assays. Previously reported regeneration process of the active streptavidin solid support in DNA sequencing using water at temperatures above 70oC, without denaturing the streptavidin tetramer was shown here to be ineffective when the streptavidin-coated magnetic beads was previously used in the lipid transfer assay. Regeneration of the used beads in chloroform inactivated the beads while the use of 10M NaOH destroyed the solid support of the bead. However, regenerating the paramagnetic beads used in lipid transfer assay with 25% aqueous ammonia only and with 25% aqueous ammonia in methanol (9:1) at 25oC are the most effective methods, with the reuse efficiency of about 90% after recovery. This represents about 50 – 67 % research cost savings, especially in this era of economic depression.
2nd General Assembly/ National Conference of the Nigerian Young Academy; 06/2012
[show abstract][hide abstract] ABSTRACT: The skin provides an efficient permeability barrier and protects from microbial invasion and oxidative stress. Here, we show that these essential functions are linked through the Nrf2 transcription factor. To test the hypothesis that activation of Nrf2 provides skin protection under stress conditions, we determined the consequences of pharmacological or genetic activation of Nrf2 in keratinocytes. Surprisingly, mice with enhanced Nrf2 activity in keratinocytes developed epidermal thickening, hyperkeratosis and inflammation resembling lamellar ichthyosis. This resulted from upregulation of the cornified envelope proteins small proline-rich proteins (Sprr) 2d and 2h and of secretory leukocyte peptidase inhibitor (Slpi), which we identified as novel Nrf2 targets in keratinocytes. Since Sprrs are potent scavengers of reactive oxygen species and since Slpi has antimicrobial activities, their upregulation contributes to Nrf2's protective function. However, it also caused corneocyte fragility and impaired desquamation, followed by alterations in the epidermal lipid barrier, inflammation and overexpression of mitogens that induced keratinocyte hyperproliferation. These results identify an unexpected role of Nrf2 in epidermal barrier function, which needs to be considered for pharmacological use of Nrf2 activators.
EMBO Molecular Medicine 03/2012; 4(5):364-79. · 7.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: Analysis of lipid storage in postmortem brains of patients with amaurotic idiocy led to the recognition of five lysosomal ganglioside storage diseases and identification of their inherited metabolic blocks. Purification of lysosomal acid sphingomyelinase and ceramidase and analysis of their gene structures were the prerequisites for the clarification of Niemann-Pick and Farber disease. For lipid catabolism, intraendosomal vesicles are formed during the endocytotic pathway. They are subjected to lipid sorting processes and were identified as luminal platforms for cellular lipid and membrane degradation. Lipid binding glycoproteins solubilize lipids from these cholesterol poor membranes and present them to water-soluble hydrolases for digestion. Biosynthesis and intracellular trafficking of lysosomal hydrolases (hexosaminidases, acid sphingomyelinase and ceramidase) and lipid binding and transfer proteins (GM2 activator, saposins) were analyzed to identify the molecular and metabolic basis of several sphingolipidoses. Studies on the biosynthesis of glycosphingolipids yielded the scheme of Combinatorial Ganglioside Biosynthesis involving promiscuous glycosyltransferases. Their defects in mutagenized mice impair brain development and function.(Communicated by Kunihiko SUZUKI, M.J.A.).
Proceedings of the Japan Academy Ser B Physical and Biological Sciences 01/2012; 88(10):554-82. · 2.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Defects in the function of the skin barrier are associated with a wide variety of skin diseases, many of which are not well characterized at the molecular level. Using Lef1 (lymphoid enhancer-binding factor 1) dominant-negative mutant mice, we demonstrate here that altered epidermal TCF (T cell factor)/Lef1 signaling results in severe impairment of the stratum corneum skin barrier and early postnatal death. Barrier defects were accompanied by major changes in lipid composition and ultrastructural abnormalities in assembly and extrusion of lipid lamellae of the interfollicular epidermis, as well as abnormal processing of profilaggrin. In contrast, tight-junction formation and stratified organization of the interfollicular epidermis was not obviously disturbed in Lef1 mutant mice. Molecular analysis revealed that TCF/Lef1 signaling regulates expression of lipid-modifying enzymes, such as Elovl3 and stearoyl coenzyme A desaturase 1 (SCD1), which are key regulators of barrier function. Promoter analysis and chromatin immunoprecipitation experiments indeed showed that SCD1 is a direct target of Lef1. Together, our data demonstrate that functional TCF/Lef1 signaling governs important aspects of epidermal differentiation and lipid metabolism, thereby regulating skin barrier function.
Journal of Investigative Dermatology 09/2011; 132(2):337-45. · 6.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sphingolipids represent an important class among lipids, especially when considering their vital roles in lipid metabolism. Thus, a variety of methods have been created to accomplish their analysis and the term "sphingolipidomics" has recently been coined to underline the motivation to enable a comprehensive analysis of all sphingolipid species including the acidic and the neutral ones. In this review, we summarize selected mainly biomedical based mass spectrometric approaches for the analysis of neutral sphingolipids regarding their advantages, applications and limitations. To underline some practical aspects of method development, we focus on a new method recently developed in our laboratory, which enables separation, detection, and mass spectrometric profiling of ceramide, hexosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, sphingomyelin species, and cholesterol in one run. This method can be applied to investigate impairments of neutral sphingolipid metabolism in a variety of disorders such as sphingolipidoses and be employed to screen for sphingolipid profile changes as induced by knockout experiments or related studies.
Biochimica et Biophysica Acta 06/2011; 1811(11):854-60. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recycling and turnover of cell membranes play a critical role in cell metabolism. The internalization of membranes through the different processes of endocytosis, phagocytosis, and autophagy deliver a considerable amount of membranes and lipids to the endosomal and lysosomal system which is tasked with its degradation. Its failure to do so leads to severe fatal neurodegenerative diseases. In order to better understand how membranes are degraded, we have to investigate the complex interactions that take place in this compartment between complex membrane lipids, enzymes and lipid binding and transfer proteins involved. To this end, we developed lipid transfer and fusion assays which allow us to quantify these interactions and assess their specificity. The published results of these investigations are summarized in this article. One of our main conclusions is that we have provided evidence for the hypothesis that acid sphingomyelinase stimulates Niemann pick disease protein type 2-mediated cholesterol export substantially by converting sphingomyelin to ceramide in the inner membranes of late endosomes.
Journal of Neurochemistry 03/2011; 116(5):702-7. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Deposition of amyloid β peptides (Aβs) in extracellular amyloid plaques within the human brain is a hallmark of Alzheimer's disease (AD). Aβ derives from proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretases. The initial cleavage by β-secretase results in shedding of the APP ectodomain and generation of APP C-terminal fragments (APP-CTFs), which can then be further processed within the transmembrane domain by γ-secretase, resulting in release of Aβ. Here, we demonstrate that accumulation of sphingolipids (SLs), as occurs in lysosomal lipid storage disorders (LSDs), decreases the lysosome-dependent degradation of APP-CTFs and stimulates γ-secretase activity. Together, this results in increased generation of both intracellular and secreted Aβ. Notably, primary fibroblasts from patients with different SL storage diseases show strong accumulation of potentially amyloidogenic APP-CTFs. By using biochemical, cell biological, and genetic approaches, we demonstrate that SL accumulation affects autophagic flux and impairs the clearance of APP-CTFs. Thus, accumulation of SLs might not only underlie the pathogenesis of LSDs, but also trigger increased generation of Aβ and contribute to neurodegeneration in sporadic AD.
Journal of Neuroscience 02/2011; 31(5):1837-49. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lysosomal lipid storage diseases, or lipidoses, are inherited metabolic disorders in which typically lipids accumulate in cells and tissues. Complex lipids, such as glycosphingolipids, are constitutively degraded within the endolysosomal system by soluble hydrolytic enzymes with the help of lipid binding proteins in a sequential manner. Because of a functionally impaired hydrolase or auxiliary protein, their lipid substrates cannot be degraded, accumulate in the lysosome, and slowly spread to other intracellular membranes. In Niemann-Pick type C disease, cholesterol transport is impaired and unesterified cholesterol accumulates in the late endosome. In most lysosomal lipid storage diseases, the accumulation of one or few lipids leads to the coprecipitation of other hydrophobic substances in the endolysosomal system, such as lipids and proteins, causing a "traffic jam." This can impair lysosomal function, such as delivery of nutrients through the endolysosomal system, leading to a state of cellular starvation. Therapeutic approaches are currently restricted to mild forms of diseases with significant residual catabolic activities and without brain involvement.
Cold Spring Harbor perspectives in biology 01/2011; 3(6). · 9.63 Impact Factor