Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a

killer

Emyr Lloyd-Evans

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

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

(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-PSphingosi

ne kinase

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

SphingolipidCholesterol Neuronal death

GSLs and sphingosine accumulate in NPC brain rafts

GenotypeGSL

(pmol/μg protein)

Cholesterol (nmol/μg protein)

Sphingosine (pmol/μg

protein)

NPC1+/+ 7±2 0.8±0.1 0.8±0.1

NPC1-/- 24±5 1±0.2 1.6±0.2

te Vruchte et al (2004) J Biol Chem 279:26167-75

Which lipid accumulates first?

T = 0h + 2g/ml U18666A

T = 2h + 2g/ml U18666A

T = 24h + 2g/ml U18666A

BODIPY-LacCer

T = 8h + 2g/ml U18666A

Filipin

BODIPY-LacCer

Filipin

T = 0h + 1M sphingosine

T = 2h + 1M sphingosine

T = 8h + 1M sphingosine

T = 24h + 1M sphingosine

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)

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

Fura 2-AM

Fura 2

AM

Loading at 20°C AM ester hydrolysis 37°C

Ca2+ freebuffer

340/380

Time(min)

Ca2+ modulator Ionomycin

Saturate Fura 2

Calcium involvement in NPC1

Agent Target

Thapsigargin

SERCA (ER)

Ryanodine RyR (ER)

CCCP ETC (Mitochondria)

Wild-type

NPC1

2.5min

0.5FU

2M CCCP

5M Thaps

Wild-type

NPC1

2.5min

1.0FU

100M Ryanodine2.5min

1.0FU

Wild-type

NPC1

Calcium Release NPC1 LE/Lysosomes

Bafilomycin A1Inhibits vATPase (collapse of protongradient inducing Ca2+ release)

GPNPeptide inhibitor of Cathepsin Acausing 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

200M GPN

1M Sphingosine reduces the late endosome/lysosome calcium pool leading to the induction of an NPC1 phenotype

RAW con RAW + 0.1M Sphingosine 24h

RAW + 1M Sphingosine 24h

Sphingoid base

200M GPN

Wild-type

Sphingosine 0.1M

Sphinganine 1M

Sphingosine 1M

1.0FU

2.5min

Wild-type

NPC1

2.5min

0.5FU

Wild-type

NPC1

2.5min

0.5FU

Wild-type + 10M NNDMS 2h

Sphingosine kinase inhibitors induce an NPC1 phenotype

RAW con filipin + 10M NNDMS 2h + 10M NNDMS 4h + 10M NNDMS 8h

Does chelation of late endosomal/lysosomal Ca2+ induce an NPC phenotype?

1. BAPTA-AM: Membrane permeable high affinity Ca2+ 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 Ca2+ in LE/Lys. Experiment performed in low Ca2+ buffer.

Only in LE when proton gradient is formed does Ca2+ enter the vesicle and can be chelated by BAPTA-Dex.

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 50M BAPTA-AM filipin

4h 50M BAPTA-AM BODIPY-LacCer

RAW BODIPY-LacCer

RAW filipin

BAPTA-Dex

Increasing NPC1 intracellular calcium corrects trafficking

Total GSL

0

0.05

0.1

0.15

0.2

0.25

Control NPC1

pm

ol

GS

L/

g p

rote

in

ControlThaps 1hThaps 4hThaps 24h

Control

Control + 1M Thapsigargin

1h

NPC1

NPC1 + 1M Thapsigargin

1h

BODIPY-LacCer - sphingolipid trafficking

Increasing NPC1 [Ca2+]i induces late endosome-lysosome fusion

0

10000

20000

30000

40000

50000

60000

70000

80000

0 30 60

Time (min)

Flu

ore

scen

ce e

mis

sio

nControl

NPC1

Cells loaded biotinylated dextran (10kD, 48 hrs) to label lysosomesCells pulsed FITC-streptavidin (30 mins) to label late endosomesThapsigargin added (30, 60 mins), cells fixed.Fusion leads to binding of 2 dyes and increases fluorescence 10x

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

Control NPC1

g

ch

ole

ste

rol/

g p

rote

in

ControlThaps 1hThaps 4hThaps 24h

Control + 1M Thapsigargin

1h

NPC1 + 1M Thapsigargin

1h

Control NPC1

Total Cholesterol

Filipin staining - cholesterol

-0.02

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

Control NPC1

g c

ho

l est

er/

g p

rote

in

ControlThaps 1h

Increasing NPC1 intracellular calcium increases cholesterol ester levels

Nile Red staining - neutral lipids

Cholesterol esters

Control + 1M Thapsigargin

1h

NPC1 + 1M Thapsigargin

1h

Control NPC1

NPC1 + 1M Thaps 1h

Con + 1M Thaps 1hCon Con + 25M W7

NPC1 + 25M W7 + 1M Thaps 1h

NPC1

Cholesterol transport is Ca2+/Calmodulin dependent

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**

Future Work

• Measure calcium homeostasis in siRNA treated cells

• Test other sphingosine analogues

• Sphingosine levels

• Molecular target? Mechanism of low Ca2+?

(store filling/emptying?)

• Total ion levels including Ca2+ (biophysical techniques)

• Does NPC2 have a Ca2+ phenotype?

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 PlattDan Sillence

All on 1st floor

Raymond Dwek