呻吟之王

Dr Dan Sillence

Job: Reader in Cell Biology

Faculty: Health and Life Sciences

School/department: Leicester School of Pharmacy

Address: 呻吟之王, The Gateway, Leicester, LE1 9BH.

T: +44 (0)116 250 6368

E: dsillence@dmu.ac.uk

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Personal profile

The Glycosphingolipid (GSL) storage diseases are a group of diseases where GSLs accumulate in tissues due to a defect in their lysosomal breakdown. GSL storage diseases have varying degrees of central nervous system involvement and in Tay-Sachs and Sandhoff diseases the neuropathology is very severe and frequently lethal in early infancy.

How lysosomal storage of GSL causes such severe neuropathology is not known at present. A strategy that can be applied to treat these diseases is substrate deprivation.

This approach aims to balance the rate of GSL biosynthesis with the impaired rate of catabolism, thus preventing accumulation of GSLs. Two imino sugar inhibitors of Glucosylceramide synthase, which catalyses the first step in GSL synthesis.

How does the storage of lipids lead to disease?

What are the normal functions of the lipids that accumulate?

Research group affiliations

Pharmacology

Publications and outputs

dc.title: Mitochondrial dysfunction in NPC1-deficiency is not rescued by drugs targeting the glucosylceramidase GBA2 and the cholesterol-binding proteins TSPO and StARD1 dc.contributor.author: Sillence, Daniel J.; Wheeler, Simon; Bhardwaj, Meenakshi; Kenyon, Victor; Ferraz, Maria J.; Aerts, Johannes M. F. G. dc.description.abstract: Niemann-Pick type C disease (NPCD) is a rare neurodegenerative disorder most commonly caused by mutations in the lysosomal protein Niemann-Pick C1 (NPC1), which is implicated in cholesterol export. Mitochondrial insufficiency forms a significant feature of the pathology of this disease, yet studies attempting to address this are rare. The working hypothesis is that mitochondria become overloaded with cholesterol which renders them dysfunctional. We examined two potential protein targets-translocator protein (TSPO) and steroidogenic acute regulatory protein D1 (StARD1)-which are implicated in cholesterol transport to mitochondria, in addition to glucocerbrosidase 2 (GBA2), the target of miglustat, which is currently the only approved treatment for NPCD. However, inhibiting these proteins did not correct the mitochondrial defect in NPC1-deficient cells. dc.description: open access article
dc.title: Effect of antidepressant drugs on the brain sphingolipid system dc.contributor.author: Zetterstrom, Tyra; Jaddoa, Estabraq; Masania, Jinit; Masiero, Eva; Sgamma, Tiziana; Arroo, R. R. J.; Sillence, Daniel J. dc.description.abstract: Background: Major depression is a common mood disorder and the central sphingolipid system has been identified as a possible drug target of this condition. Here we investigated the action of antidepressant drugs on sphingolipid levels in rat brain regions, plasma and in cultured mouse macrophages. Methods: Two antidepressant drugs were tested; the serotonin reuptake inhibitor paroxetine and the noradrenaline reuptake inhibitor desipramine, either following acute or chronic treatments. Content of sphingosine and ceramide were analysed using LC-MS or HPLC-UV respectively. This from samples of brain, plasma and cultured mouse macrophages. Antidepressant induced effects on mRNA expression for two key genes of the sphingolipid pathway, SMPD1 and ASAH1 were also measured by using quantitative Real-Time PCR. Results: Chronic but not acute administration of paroxetine or desipramine reduced sphingosine levels in the prefrontal cortex and hippocampus (only paroxetine) but not in the striatum. Ceramide levels were also measured in the hippocampus following chronic paroxetine and likewise to sphingosine this treatment reduced its levels. The corresponding collected plasma samples from chronically treated animals did not show any decrease of sphingosine compared to the corresponding controls. Both drugs failed to reduce sphingosine levels from cultured mouse macrophages. The drug-induced decrease of sphingolipids coincided with reduced mRNA expression of two enzymes of the central sphingolipid pathway, i.e. acid sphingomyelinase (SMPD1) and acid ceramidase (ASAH1). Conclusions: This study supports the involvement of brain sphingolipids in the mechanism of action by antidepressant drugs and for the first time highlights their differential effects on brain versus plasma levels. dc.description: The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.
dc.title: Niemann-Pick type C disease: cellular pathology and pharmacotherapy dc.contributor.author: Wheeler, Simon; Sillence, Daniel J. dc.description.abstract: Niemann-Pick type C disease (NPCD) was first described in 1914 and affects approximately 1 in 150,000 live births. It is characterised clinically by diverse symptoms affecting liver, spleen, motor control and brain; premature death invariably results.Its molecular originswere traced, as late as 1997, to a protein of late endosomes and lysosomes which was named NPC1. Mutation or absence of this protein leads to accumulation of cholesterol in these organelles. In this review we focus on the intracellular events that drive the pathology of this disease. We first introduce endocytosis, a much-studied area of dysfunction in NPCD cells, and survey the various ways in which this process malfunctions. We briefly consider autophagy before attempting to map the more complex pathways by which lysosomal cholesterol storage leads to protein misregulation, mitochondrial dysfunction and cell death. We then briefly introduce the metabolic pathways of sphingolipids (as these emerge as key species for treatment) and critically examine the various treatment approaches that have been attempted to date. dc.description: The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.
dc.title: Cytosolic Glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease dc.contributor.author: Sillence, Daniel J.; Bhardwaj, Meenakshi; Tongue, Paige; Schmid, Ralf; Haberkant, Per; Wheeler, Simon dc.description.abstract: Niemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease. dc.description: A new paradigm for Niemann-Pick C disease is presented where lysosomal storage leads to a deficit in cytoplasmic glucosylceramide (GlcCer) where it performs important functions. Previously it had been reported that Gaucher cells have defective endolysosomal pH. GlcCer also accumulates in Niemann-Pick C disease and also shows this defect. Niemann-Pick C cells were found to have reduced cytoplasmic glucosylceramide (GlcCer) transport. Inhibiting cytoplasmic glucocerebrosidase (GBA2), increased GlcCer, decreased endolysosomal pH in normal cells, reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking and increased expression of vATPase a subunit in Niemann-Pick C fibroblasts. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic GlcCer which are reduced in NPC disease. This work consequently suggests GBA2 and vATPase as new therapeutic targets in Niemann-Pick C and related neurodegenerative diseases. The work was in collaboration with colleagues in the Netherlands and Leicester University. The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.
dc.title: Lipid鈥揚rotein Interactions in Niemann鈥揚ick Type C Disease: Insights from Molecular Modeling dc.contributor.author: Wheeler, Simon; Schmid, R.; Sillence, Daniel J. dc.description.abstract: The accumulation of lipids in the late endosomes and lysosomes of Niemann鈥揚ick type C disease (NPCD) cells is a consequence of the dysfunction of one protein (usually NPC1) but induces dysfunction in many proteins. We used molecular docking to propose (a) that NPC1 exports not just cholesterol, but also sphingosine, (b) that the cholesterol sensitivity of big potassium channel (BK) can be traced to a previously unappreciated site on the channel鈥檚 voltage sensor, (c) that transient receptor potential mucolipin 1 (TRPML1) inhibition by sphingomyelin is likely an indirect effect, and (d) that phosphoinositides are responsible for both the mislocalization of annexin A2 (AnxA2) and a soluble NSF (N-ethylmaleimide Sensitive Fusion) protein attachment receptor (SNARE) recycling defect. These results are set in the context of existing knowledge of NPCD to sketch an account of the endolysosomal pathology key to this disease. dc.description: open access article
dc.title: Mechanisms of Gaucher Disease Pathogenesis dc.contributor.author: Wheeler, Simon; Sillence, Daniel J. dc.description.abstract: Gaucher disease is caused by mutations in the Gba1 gene encoding an acid 尾-glucocerebrosidase (GBA1), the lysosomal hydrolase which breaks down glucosylceramide (GlcCer). In Gaucher type 1 disease the accumulation of this simple glycolipid is mainly restricted to tissue phagocyte lysosomes resulting ultimately in hepatomegaly, splenomegaly and osteopenia. Lower residual GBA1 levels leads to neuronal storage, in types 2 and 3 neurological symptoms are characterised by acute (death at age 2) or sub-acute onset, respectively. The links between cellular changes and clinical manifestations are largely unknown but are the key to the development and monitoring of new therapies. The newcomer to Gaucher disease is likely attracted to the apparent simplicity of an autosomal recessive disorder which promises to unravel the critical GlcCer function in normal cells (GlcCer is widespread, it鈥檚 even present in some bacteria鈥攁lso, mouse and fly GlcCer knockouts die at embryo stage). However, closer acquaintance reveals not a classic Mendelian disorder鈥攕ometimes even monozygotic twins have different symptoms鈥攁nd studies at the cellular level have so far failed to reveal clear GlcCer functions. Now a team led by Ellen Sidransky at the NIH has taken what appears to be a big step forward by producing two in vitro models of Gaucher cells (1).
dc.title: 1H NMR-Linked Urinary Metabolic Profiling of Niemann-Pick Class C1 (NPC1) Disease: Identification of Potential New Biomarkers using Correlated Component Regression (CCR) and Genetic Algorithm (GA) Analysis Strategies dc.contributor.author: Ruiz-Rodado, Victor; Luque-Baena, R. M.; te Vruchte, D. J.; Probert, Fay; Lachmann, R. H.; Hendriksz, Christian J.; Wraith, James E.; Imrie, Jackie; Elizondo, David; Sillence, Daniel J.; Clayton, P.; Platt, Frances M.; Grootveld, Martin dc.description.abstract: Niemann-Pick Class 1 (NPC1) disease is a rare, debilitating neurodegenerative lysosomal storage disease; however, urinary biomarkers available for it and its prognosis are currently limited. In order to identify and establish such biomarkers, we employed high-resolution 1H NMR analysis coupled to a range of multivariate (MV) analysis approaches, i.e. PLS-DA, RFs and uniquely the cross-validated correlated component regression (CCR) strategy in order to discern differences between the urinary metabolic profiles of 13 untreated NPC1 disease and 47 heterozygous (parental) carrier control participants. Novel computational intelligence techniques (CITs) involving genetic algorithms (GAs) were also employed for this purpose
dc.title: Transport of (Glyco)Sphingolipids in and Between Cellular Membranes; Multidrug Transporters and Lateral Domains dc.contributor.author: van Meer, G.; Sillence, Daniel J.; Sprong, H.; K盲lin, N.; Raggers, R. J. dc.description.abstract: Sphingolipids are highly enriched in the outer leaflet of the plasma membrane lipid bilayer. However, the first glycolipid, glucosylceramide, is synthesized in the opposite, cytosolic leaflet of the Golgi membrane. This has led us to experiments which suggest that the level of glucosylceramide in the cytosolic surface is carefully regulated both by the balance between synthesis and hydrolysis and by transport away from this surface through translocators, multidrug transporters, the same molecules that make cancer cells resistant to chemotherapy. Our data suggest a role for newly synthesized glucosylceramide not only in the formation of domains in the luminal leaflet of the Golgi but also on the cytosolic surface of this organelle.
dc.title: Apoptosis and signalling in acid sphingomyelinase deficient cells dc.contributor.author: Sillence, Daniel J. dc.description.abstract: BACKGROUND: Recent evidence suggests that the activation of a non-specific lipid scramblase during apoptosis induces the flipping of sphingomyelin from the cell surface to the cytoplasmic leaftet of the plasma membrane. Inner leaflet sphingomyelin is then cleaved to ceramide by a neutral sphingomyelinase. The production of this non-membrane forming lipid induces blebbing of the plasma membrane to aid rapid engulfment by professional phagocytes. However contrary evidence suggests that cells which are deficient in acid sphingomyelinase are defective in apoptosis signalling. This data has been interpreted as support for the activation of acid sphingomyelinase as an early signal in apoptosis. HYPOTHESIS: An alternative explanation is put forward whereby the accumulation of intracellular sphingomyelin in sphingomyelinase deficient cells leads to the formation of intracellular rafts which lead to the sequestration of important signalling molecules that are normally present on the cell surface where they perform their function. TESTING THE HYPOTHESIS: It is expected that the subcellular distribution of important signalling molecules is altered in acid sphingomyelinase deficient cells, leading to their sequestration in late endosomes/lysosomes. Other sphingolipid storage diseases such as Niemann-Pick type C which have normal acid sphingomyelinase activity would also be expected to show the same phenotype. IMPLICATIONS OF THE HYPOTHESIS: If true the hypothesis would provide a mechanism for the pathology of the sphingolipid storage diseases at the cellular level and also have implications for the role of ceramide in apoptosis.
dc.title: Assays for the transmembrane movement of sphingolipids dc.contributor.author: Sillence, Daniel J.; Raggers, R. J.; van Meer, G.

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Research interests/expertise

Sphingolipid cell biology
Lysosomal storage diseases.

Areas of teaching

Cell Biology
Pharmacology

Qualifications

BSc PhD (Biochemistry, Dundee)

呻吟之王 taught

Cell Biology and Physiology
Pharmacology

Honours and awards

Marie Curie Research Fellow, ‘Transbilayer flipping of sphingolipids’ EU, (2000)

Peter Jones Carlton memorial award (2006) for new therapy for Niemann-Pick C disease with Emyr Lloyd-Evans and Prof Frances Platt, University of Oxford.

Membership of professional associations and societies

American Society for Molecular Biology

Marie Curie Alumni

Consultancy work

Sphingolipid cell biology, Unilever UK.

Externally funded research grants information

Sillence DJ ‘Simple sphingolipids in neurodegeneration’ HEIF Innovation, £15K (Oct 17-18) PI

Grootveld M., F. Platt, D. Sillence ‘Identification of New Biomarkers for the Metabolomics Classification of Batten Disease: A Drug-Targeting Strategy’ Beyond Batten Disease Foundation (Oct 2016-9), £120K Co-PI

Wheeler S and Sillence D ‘Modelling lysosomal storage diseases’ 呻吟之王 High Flier studentship (Sept 2016-19), £45K PI

Silence DJ ‘Niemann Pick C and vATPase’ HEIF5 (sept 15- July 16) £1500

Grootveld M. D. Sillence, F. Platt, A. D’Azzo, C. Tifft, HEIF_5 Collaborative Award (2015-2016), £35K

Establishment and Operation of an Interdisciplinary Global Lysosomal Diseases Metabolomics Research Group (IGLDMRG) Sillence D (with T. Zetterstrom) 呻吟之王 Fees-Only PhD Scholarship Award, 2014-2017, £12.5K

A role for sphingolipids in the action of anti-depressants Rushworth,J, Morris, M. Fretwell, L. Slater, A. Dunford,L. Grootveld, M. Sillence DJ, Ruparelia,K, Bhambra, A, Arroo R. (2014) Cell imaging and analysis facility RCIF2, £77,600

Grootveld, M, Sillence D., Platt, FM (2013) Identification of Novel Biomarker Patterns for the Metabolomic Classification of Niemann-Pick Disease C and Response to Treatment SPARKS, £51,000

Sillence D, Grootveld M (2013) Identification of Novel Biomarker Patterns for the Metabolomic Classification of Niemann-Pick Disease C and its Response to Treatment Small Projects Scheme, £5,000 Co-PI

Assay of sphingomyelinase inhibitors, Off-Label Ltd/Penn Ltd, research project, July 2008 – Nov 2008, PI.

Internally funded research project information

Sphingolipid cell biology, Startup – 1/1/2008 – 2009, PI.

Case studies

Sillence,D J (Nov 2007) New insights into glycosphingolipid functions--storage, lipid rafts, and translocators. International Review of Cytology, 262, pp 151-189.
Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium. Lloyd-Evans,E, Morgan,A J, He,X, Smith,D A, Elliot-Smith,E, Sillence,Daniel J, Churchill,G C, Schuchman,E H, Galione, A, Platt,F M. Nature Med. (2008).

Invited editorial board membership

‘Universal Journal of Medical Science’

‘Conference papers in Cell Biology’

‘International Journal of Clinical Pharmacology and Toxicology’

‘Peertechz Journal of Cytology and Pathology’

Research supervision

Post-Doc

NMR-linked metabolomics in Batten disease, Dr Nikol Sullo, (2016-8)

Post-Doctoral Fellowship in NMR-Linked Metabolomics in Niemann-Pick C disease, Dr Fay Probert, (2013-15)

Research Assistant Danielle TeVruchte (2002-11)

PhD supervision

Simon Wheeler (2016-9) Sphingolipids and Neurodegeneration (1^st)

Victor Ruiz Rodado (2013-6) Metabolomics of Niemann-Pick C disease (2^nd)

Emyr Lloyd-Evans (2003-6) Lysosomal calcium defect in Niemann-Pick C disease (1^st)

Aruna Jeans (2002-5) Immune cell dysfunction in glycolipid storage disease (2^nd)

Renee Raggers (1998-2001) Lipid translocation by multidrug transporters (2^nd)