The CLN5 and CLN8 proteins as Ceramide Synthases or Their Modulators

1. 3-Complementation
studies in patient cell
lines
1- CLN5 sequencing Exon
3
2-Western blot showing
the presence of a
truncated CLN5 protein
in patient cell lysates.
4- Measurement of
Ceramide leves
7
5-Perturbation of
sphingolipid metabolism
in mouse mnd
fibroblasts.
KEY PROJECTS 6-Ceramide and
dhCeramide changes in
CLN5 and CLN8-deficient
Cells
WHAT THIS
MEANS FOR
THERAPY
Conclusion
CLN5
Neurogenetics lab:Neurogenetics lab:
The CLN5 (aka CLN9) and CLN8 proteins as Ceramide Synthases orThe CLN5 (aka CLN9) and CLN8 proteins as Ceramide Synthases or
their Modulatorstheir Modulators
Boustany R-MBoustany R-M11
, Saria El Haddad, Saria El Haddad11
, Marwan Khoury, Marwan Khoury11
,Mohammad Daoud,Mohammad Daoud11
, Talal Mousallem, Talal Mousallem22
, Oscar Alzate, Oscar Alzate22
.. 11
Neurogenetics Program and Division ofNeurogenetics Program and Division of
Pediatric Neurology, Departments of Pediatrics and Biochemistry, American University of Beirut, Beirut, Lebanon.Pediatric Neurology, Departments of Pediatrics and Biochemistry, American University of Beirut, Beirut, Lebanon. 22
Duke University Medical Center, Departments ofDuke University Medical Center, Departments of
Pediatrics and Neurobiology, Durham, NC, USA. rb50@aub.edu.lbPediatrics and Neurobiology, Durham, NC, USA. rb50@aub.edu.lb
NEUROGENETICSPROGRAM
DEPARTMENTOFPEDIATRICS
NEUROGENETICSPROGRAM
DEPARTMENTOFPEDIATRICS
INTRODUCTION
(Laboratory Objectives)
Four patients with variant CLN9 NCL are reclassified as
CLN5 disease. CLN5-/- fibroblasts demonstrate adhesion
defects, increased growth, apoptosis and ↓ ceramide (Cer),
sphingomyelin, and glycosphingolipids. CLN8p corrects
growth/apoptosis in CLN5-/- cells. This study highlights role
of CLN5/8 as activators of (dihydro)ceramide synthase
(CerS). Homozygositymapping led to identifying the CLN5
gene. CerS activity and Cer are ↓ in CLN8-/- / CLN5-/-cells.
Normal vs. CLN5-/- CerS1-bound proteins by
immunoprecipitation, differential gel electrophoresis, and
mass spectrometry reveal absence of γ-actin. γ-actin gene
sequence is normal in CLN5-/-. γ-actin-bound proteins
vimentin and histone proteins H2Afz/H3F3A/Hist1H4 were
absent from the γ-actin protein complex. Interpretation:
functions of these proteins and failure to bind γ-actin explain
the CLN5 phenotype. We explore the role of CLN5/ CLN8 in
sphingolipid biosynthesis and suggest that CLN5/ CLN8 are
closely related.
Probands Mother
FatherNormal
Dendrogram of a stretch of nucleotides in Exon
3 revealing the presence of a substitution
mutation (red arrows) replacing cytosine by
thymine in DNA of probands. In the parents,
there is a clear super-position of a red a blue
peak reflecting presence of cytosine and thymine
denoting that both parents are carriers of the
mutated allele.
A. No band is seen using anti-CLN5 against the C-
terminus while. B. a smaller band is detected with the N-
terminus anti-CLN5 antibody. NFB= normal fibroblasts,
CLN5= CLN5-deficient fibroblasts.
Apoptosis rate of CLN5-deficient fibroblasts transfected
with emptypCMV(control) vs CLN5 pCMV (CLN5)
0
10
20
30
40
50
60
70
80
0 1 2 3 4
Time (days)
Apoptosisrate(%)
Control
CLN5
Growth curve of CLN5-deficient fibroblasts transfected with
empty pCMV (control) vs CLN5 pCMV (CLN5)
0
500000
1000000
1500000
2000000
0 1 2 3 4
Time (days)
Numberofcells/ml
Control
CLN5
Measurement of de novo C17 ceramide levels
generated over 4 hours reflecting CerS activity
in mnd cells in top panel. Ceramide levels were
measured by tandem mass spectrometry
(values from single measures are shown).
Ceramide species levels in mnd cells are
decreased compared to normal mouse
fibroblasts (lower panel). This is mostly seen in
C16, C20 and C 24:1 carbon length of
ceramide species.
Ceramide (A), glucosylceramide (B),
gallceramide (C), lactosylcer (D), ceramide
trihexoside (E), globoside (F) levels , are
decreased in mnd cells compared to normal
fibro-blasts measured by 14 C-palmitate (A)
γ-
actin
NF CLN5 Merge NFCLN5 Merge
A B C
A. 1DGel of proteins from Normal (NF) and CLN5
deficient (CLN5) fibroblasts after
immunoprecipitation with LASS1 antibody. Overlay
samples display a strong red band present in NF
sample but absent in CLN5.This band identified as γ-
actin by mass spec. The gene ACTG1 coding for γ-
actin was normal in CLN5-/- Fibroblasts. B. 1DGel
of NF and CLN5 protein samples after
immunoprecipitation with ACTG1 antibody. It is
clear that there is a difference in the bands between
the 2 cell lines. C. Gel bands present in NF, absent in
CLN5-/- were cut and analyzed by Mass Spec.
8
Ceramide and Dihydroceramide species in CLN5-
deficient cells
Sphingolipid abnormalities
Ceramide species in CLN5
Overexpression of CLN8 in normal Fibroblasts
Overexpression of CLN8 in normal Fibroblasts: effect
on Ceramide Species
Sphingolipid abnormalities
Ceramide species in CLN5
Overexpression of CLN8 in normal Fibroblasts
Sample
(Band/
Spot
On gel)
Protein Name Protein Function
Correlation with
CLN9 phenotype
1
No Significant
Hits
3
Glycerol-3-
phosphate
dehydrogenase
Ubiquitous
protein involved
in respiration and
metabolism
Unknown
2 and 4 Vimentin
Imparts
Resilience to cell
Body. Maintains
cell shape. Binds
Globoside
Adhesion
defect Rounded
morphology
Low Globoside
level
5
No Significant
Hits
6
Histone
H2AFZ
DNA binding
Gene expression
DNA repair
Mitosis
Aneuploidy
after several
culture
passages
accelerated
growth and
apoptosis rates
Histone
H3F3A
7
Histone1H4H
Histone H2A
type 2-C
Metaphase spread: passage #22 CLN5-/-
fibroblasts demonstrating aneuploidy. This
was shown in 2 different cell lines from two
unrelated families.
Acknowledgements:
1. Patient families for their support.
2. The Tarailo family for their funding and support
(American Serbian Orthodx church).
3. The Batten Disease Research and Support Foundation
(BDRSA).
4. The MPP /URB AUBMC fund.
This study highlights the close interaction
between CLN5/CLN8 proteins, and their role
in sphingolipid metabolism. Our findings
suggest that CLN5p/CLN8p are positive
modulators of CerS. CLN5/CLN8p defects
affect metabolism of C24/C24:1 ceramide
species and CLN8p corrects CLN5p defects.
There is a strong possibility that these
proteins are functionally related. CLN8p and
NCL proteins cross-correcting the CLN8-/-
phenotype, such as CLN3p and CLN6p, could
form a dynamic protein complex, that could
include CLN5p, modulating CerS.
Phosphorylation is a major post-translational
modification that affects CerS function
Similarly, CLN5p and CLN8p could impact
CerS function through a mechanism still to be
determined. Vimentin and several histone
proteins, crucial to cell cycle regulation and
cellular morphology, may fail to function due
to CLN5p defects, in part explaining the
CLN5 cellular phenotype.