1. Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans

2. Background of Niemann-Pick type C1 (NPC) disease Niemann-Pick type C1 (NPC) is an autosomal recessive, neurodegenerative lipid storage disease NPC1 is a member of the lysosomal storage disease family Lipid storage profile is complex - cholesterol, phospholipids and sphingolipids accumulate in the periphery whereas sphingolipids are the major storage material in the brain NPC patients present with hepatosplenomegaly, progressive loss of motor skills, dementia, and seizures Symptom onset is variable with average life expectancy up to teenage years

3. The NPC1 protein 13 transmembrane domain protein found in the limiting membrane of late endosomes and lysosomes Contains a sterol sensing domain (SSD) of high homology to similar regions found in SCAP, HMG-CoA reductase, and Patched NPC1 is believed to be a cholesterol transporter although no evidence exists for this function, yeast NCR1 functions in sphingolipid sorting NPC1 has highest homology to RND permeases, bacterial transporters

4. (Glyco)Sphingolipids - synthesis/degradation Sphinganine Fatty acid + Ceramide Glucosylceramide UDP glucose + All sphingolipids have a sphingoid backbone More complex sphingolipids have fatty acid side chains and are glycosylated Sphingolipids are synthesized at the ER and the Golgi Are degraded in lysosomes Sphingosine Acid ceramidase Sphingosine-1-P Sphingosine kinase

5. Project aims Very simply - What goes wrong? Can we identify a pathogenic molecule/mechanism that occurs at an early stage of disease progression and then target therapeutically? Hypothetically achieving maximum benefit for the sufferer. Is cholesterol accumulation of any relevance, pathologically to the disease? NPC1 mutation SphingolipidCholesterolNeuronal death

6. GSLs and sphingosine accumulate in NPC brain rafts Genotype GSL (pmol/μg protein) Cholesterol (nmol/μg protein) Sphingosine (pmol/μg protein) NPC1 +/+ 7±27±20.8±0.1 NPC1 -/- 24±51±0.21.6±0.2 te Vruchte et al (2004) J Biol Chem 279:26167-75

7. Which lipid accumulates first? T = 0h + 2  g/ml U18666A T = 2h + 2  g/ml U18666A T = 24h + 2  g/ml U18666A BODIPY-LacCer T = 8h + 2  g/ml U18666A Filipin BODIPY-LacCer Filipin T = 0h + 1  M sphingosine T = 2h + 1  M sphingosine T = 8h + 1  M sphingosine T = 24h + 1  M sphingosine

8. Con BODIPY-LacCer 50pM NPC1 siRNA 12h BODIPY-LacCer Con anti-NPC1 Con Filipin 50pM NPC1 siRNA 12h anti-NPC1 50pM NPC1 siRNA 12h Filipin RAW macrophage NPC1 knock down (12h - partial knock down)

9. Calcium involvement in NPC1 1.U18666a, progesterone, sphingosine, and W7 have all been shown independently to affect calcium homeostasis 2.Previous study suggested a link between abnormal cholesterol transport in NPC1 and inhibited calcium homeostasis 3.Storage diseases with primary sphingolipid accumulation have abnormal calcium homeostasis

10. Fura 2-AM Fura 2 AM Loading at 20°C AM ester hydrolysis 37°C Ca 2+ free buffer 340/380 Time(min) Ca 2+ modulator Ionomycin Saturate Fura 2

11. Calcium involvement in NPC1 AgentTarget ThapsigarginSERCA (ER) RyanodineRyR (ER) CCCPETC (Mitochondria) Wild-type NPC1 2.5min 0.5FU 2  M CCCP 5  M Thaps Wild-type NPC1 2.5min 1.0FU 100  M Ryanodine 2.5min 1.0FU Wild-type NPC1

12. Calcium Release NPC1 LE/Lysosomes Bafilomycin A1 Inhibits vATPase (collapse of proton gradient inducing Ca 2+ release) GPN Peptide inhibitor of Cathepsin A causing osmotic lysis of LE/Lys leading to ion release Wild-type NPC1 2.5min 0.5FU 500nM BafA1 Wild-type NPC1 2.5min 0.5FU 200  M GPN

13. 1  M Sphingosine reduces the late endosome/lysosome calcium pool leading to the induction of an NPC1 phenotype RAW conRAW + 0.1  M Sphingosine 24h RAW + 1  M Sphingosine 24h Sphingoid base 200  M GPN Wild-type Sphingosine 0.1  M Sphinganine 1  M Sphingosine 1  M 1.0FU 2.5min

14. Wild-type NPC1 2.5min 0.5FU Wild-type NPC1 2.5min 0.5FU Wild-type + 10  M NNDMS 2h Sphingosine kinase inhibitors induce an NPC1 phenotype RAW con filipin+ 10  M NNDMS 2h+ 10  M NNDMS 4h+ 10  M NNDMS 8h

15. Does chelation of late endosomal/lysosomal Ca 2+ induce an NPC phenotype? 1.BAPTA-AM: Membrane permeable high affinity Ca 2+ chelator Chelates all accessible stores, modest chelation in LE/Lys 2. BAPTA-Dextran: Only enters endocytic compartments due to high mwt dextran, thus can only chelate Ca 2+ in LE/Lys. Experiment performed in low Ca 2+ buffer. Only in LE when proton gradient is formed does Ca 2+ enter the vesicle and can be chelated by BAPTA-Dex.

16. Lowering endosome calcium induces an NPC1 phenotype in RAW Cells Control 4h 5mg/ml BDex filipin BAPTA-AM 4h 5mg/ml BDex BODIPY-LacCer 4h 50  M BAPTA- AM filipin 4h 50  M BAPTA-AM BODIPY-LacCer RAW BODIPY-LacCer RAW filipin BAPTA-Dex

17. Increasing NPC1 intracellular calcium corrects trafficking Total GSL 0 0.05 0.1 0.15 0.2 0.25 ControlNPC1 pmol GSL/  g protein Control Thaps 1h Thaps 4h Thaps 24h Control Control + 1  M Thapsigargin 1h NPC1 NPC1 + 1  M Thapsigargin 1h BODIPY-LacCer - sphingolipid trafficking

18. Increasing NPC1 [Ca 2+ ] i induces late endosome-lysosome fusion 0 10000 20000 30000 40000 50000 60000 70000 80000 03060 Time (min) Fluorescence emission Control NPC1 Cells loaded biotinylated dextran (10kD, 48 hrs) to label lysosomes Cells pulsed FITC-streptavidin (30 mins) to label late endosomes Thapsigargin added (30, 60 mins), cells fixed. Fusion leads to binding of 2 dyes and increases fluorescence 10x

19. Increasing NPC1 intracellular calcium corrects cholesterol levels 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 ControlNPC1  g cholesterol/  g protein Control Thaps 1h Thaps 4h Thaps 24h Control + 1  M Thapsigargin 1h NPC1 + 1  M Thapsigargin 1h ControlNPC1 Total Cholesterol Filipin staining - cholesterol

20. -0.02 -0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 ControlNPC1  g chol ester/  g protein Control Thaps 1h Increasing NPC1 intracellular calcium increases cholesterol ester levels Nile Red staining - neutral lipids Cholesterol esters Control + 1  M Thapsigargin 1h NPC1 + 1  M Thapsigargin 1h ControlNPC1

21. NPC1 + 1  M Thaps 1h Con + 1  M Thaps 1h ConCon + 25  M W7 NPC1 + 25  M W7 + 1  M Thaps 1h NPC1 Cholesterol transport is Ca 2+ /Calmodulin dependent

22. NPC1 mutation Calcium GSL Trafficking Cholesterol Sphingosine Possible order of events in NPC1 disease leading to pathology Neuronal death **NPC1 is not a cholesterol transporter, cholesterol accumulation is a downstream event in disease pathology**

23. Future Work Measure calcium homeostasis in siRNA treated cells Test other sphingosine analogues Sphingosine levels Molecular target? Mechanism of low Ca 2+ ? (store filling/emptying?) Total ion levels including Ca 2+ (biophysical techniques) Does NPC2 have a Ca 2+ phenotype?

24. Acknowledgements Collaborators Antony Galione (Dept. Pharmacology, Oxford) Anthony Morgan (Dept. Pharmacology, Oxford) Elspeth Garman (LMB, Oxford) Wim van Blitterswijk (Netherlands) Dan Ory (Washington State) Funding Ara Parseghian Medical Research Foundation Oxford Fran Platt Dan Sillence All on 1st floor Raymond Dwek