identifying the key events behind niemann-pick type c1 disease pathology, cholesterol (for once) is...
TRANSCRIPT
![Page 1: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/1.jpg)
Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a
killer
Emyr Lloyd-Evans
![Page 2: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/2.jpg)
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
![Page 3: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/3.jpg)
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
![Page 4: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/4.jpg)
(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
![Page 5: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/5.jpg)
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
![Page 6: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/6.jpg)
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
![Page 7: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/7.jpg)
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
![Page 8: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/8.jpg)
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)
![Page 9: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/9.jpg)
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
![Page 10: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/10.jpg)
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
![Page 11: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/11.jpg)
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
![Page 12: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/12.jpg)
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
![Page 13: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/13.jpg)
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
![Page 14: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/14.jpg)
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
![Page 15: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/15.jpg)
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.
![Page 16: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/16.jpg)
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
![Page 17: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/17.jpg)
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
![Page 18: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/18.jpg)
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
![Page 19: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/19.jpg)
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
![Page 20: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/20.jpg)
-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
![Page 21: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/21.jpg)
NPC1 + 1M Thaps 1h
Con + 1M Thaps 1hCon Con + 25M W7
NPC1 + 25M W7 + 1M Thaps 1h
NPC1
Cholesterol transport is Ca2+/Calmodulin dependent
![Page 22: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/22.jpg)
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**
![Page 23: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/23.jpg)
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?
![Page 24: Identifying the key events behind Niemann-Pick type C1 disease pathology, cholesterol (for once) is not a killer Emyr Lloyd-Evans](https://reader036.vdocuments.net/reader036/viewer/2022062516/56649d8c5503460f94a748bf/html5/thumbnails/24.jpg)
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