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Telethon Institute of Genetics and Medicine
Genetics and Medicine
MISSION: understanding the mechanisms of genetic diseases to develop preventive and
genetic diseases to develop preventive and therapeutic strategies
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Molecular
Molec
lar
analysis
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9
Metabolic
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pathway
involved
Mechanisms
12
of cell
damage
13
Tissue/
14
organ
dysfunction
Protein5
function/
dysfunction
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Lysosomal Storage Diseases (LSDs)
A group of approximately 50 inherited
diseases characterized by progressive
intracellular storage caused by a defect in
the lysosomes, the organelles that are
responsible for cellular clearance
Frequency as a group: 1:7.000-1:10.000
A normal human cell
A t h
Autophagy
Golgi
apparatus
Phagocytosis
Primary
lysosomes
Secondary
lysosomes
Early
endosome
Endocytosis
Late
endosome
Exocytosis
A cell from a patient with a
Lysosomal Storage Disease
A t h
Autophagy
Golgi
apparatus
Phagocytosis
Primary
lysosomes
Secondary
lysosomes
Early
endosome
Endocytosis
Late
endosome
Exocytosis
LYSOSOMAL STORAGE
Lysosomal storage in
p
y
and macrophages
p g
hepatocytes
from a patient with Multiple
Sulfatase Deficiency
Mental
retardation
Visceromegaly
g
Haematological
abnormalities
Bone
dysplasia
Facial
dysmorphisms
Ocular
abnormalities
Molecules accumulating in LSDs
ceramide
Gm1 ganglioside
galactocerebroside
g
glucocerebroside
Heparan sulfate
sfingomyelin
S
glycogen
Sulfogalactosylceramide (sulfatide)
30 years of research on
Lysosomal Storage Diseases (LSDs)
G
E
N
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P
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Researcher
1
11
2
3
Molecular
Molec
lar
analysis
4
10
9
Metabolic
8
pathway
involved
Mechanisms
12
of cell
damage
13
Tissue/
14
organ
dysfunction
Protein5
function/
dysfunction
7
15
6
The lysosome:
the cell
”waste
te basket"
b ket"
THE LYSOSOMES
Most cells of our body contain hundreds of
lysosomes, the core stations for the degradation and
recycling of cellular waste ((“cellular
cellular incinerators
incinerators”))
N l i
Nuclei
Lysosomes
Incinerator in Vienna
THE LYSOSOME
The lysosome is made of several elements:
Membrane
• Membrane: it separates the lysosome
from the rest of the cell
• Hydrolases: these are enzymes that
degrade specific molecules
• Protonic pump: the machinery for the
acidification of the organelle
Hydrolases
Cellular waste
• Transport proteins: they handle the
trafficking of material across the
lysosomal membrane
TRANSPORT OF CELLULAR WASTE TO LYSOSOMES
TRANSPORT OF CELLULAR WASTE TO LYSOSOMES
Molecules
M
l
l to be
b degraded
d
d d and
d recycled
l d are carried
i d
to the lysosomes through different processes
Autophagy
Nucleus
• Endocytosis: molecules
coming from other cells
Lysosomes
Phagocytosis
Endocytosis
• Phagocytosis:
microorganisms (pathogens)
or cellular debris
• Autophagy: material deriving
from cellular metabolism
THE LYSOSOMES AND DISEASES
Di
Disease
St
Storage
material
t i l
Lysosomal storage diseases
Mucopolysaccharidoses
mucopolysaccharides
Lipidoses
lipids
Gl
Glycogenoses
glycogen
l
Lipofuscinoses
proteins
Neurodegenerative disease
Alzheimer
β-amyloid, tau
P ki
Parkinson
α-synuclein
l i
Huntington
huntingtin
Others
Prion disease, parasitic infections,….aging
Studying lysosome biology using a
genomic
i approach:
h “LYSOSOMICS”
Marco Sardiello
OUR HYPOTHESIS
Is there a genetic program coordinating the activity
of lysosomes (i.e. regulating cellular clearance) ?
Lysosomal enzymes
COORDINATION
Lysosomal acidification
L
Lysosomal
l biogenesis
bi
i
I there
Is
th
a lysosomal
l
l
gene network?
Why doAwe
need
to look at more
GENE
NETWORK
than one gene?
?
TRANSCRIPTION FACTOR
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture
picture.
The p63 NETWORK (from Diego Di Bernardo, TIGEM)
Co-expression
H
How
can we id
identify
tif gene networks?
t
k ?
The “Systems Biology” approach
C di i
Conditions
M
Measure
response
C
Co-expression
i
Gene network
A MASTER GENE FOR LYSOSOMAL FUNCTION
The TFEB gene coordinates lysosomal activity
Lysosomal enzymes
TFEB
Lysosomal acidification
Lysosomal biogenesis
TFEB: A “REMOTE CONTROL”
TFEB
TFEB: A “REMOTE CONTROL”
TFEB
Do promoters of lysosomal
genes share regulatory
elements?
Most lysosomal promoters share a E-box type regulatory motif
Promoter analysis
PWM
(position
weight matrix)
consensus
logo
a
c
g
t
| 3 2 8 2 1 43 6 2 0 2 39 3 2 2
| 9 9 9 5 47 1 40 2 6 0 4 30 37 36
| 36 37 30 4 0 6 2 40 1 47 5 9 9 9
| 2 2 3 39 2 0 2 6 43 1 2 8 2 3
GGGTCACGTGACCC
Most lysosomal promoters share a E-box type regulatory motif
Promoter analysis
Example: LAMP1
TCAGACTTCTAAAATGCAGGAGCCAGGCGCCGTGGCTCAGGGCTGTAATCTCAGCACTTTGGGAGGCCGAGGCGGGTGGATTGCTTGAG
GTCAGGAGTTCGAGACCAGCCTGGCCAACTGGTGAAACCCCGTCTCTACTCAACATACAAAATCAGCCGGGCGTGGTGGCGCACGCCTG
TAATCCCAGCTACTTGGGAGGCTGAGGCAAGAGAATCGCTTGAACCCGGGAGGCGGAGGTTACAGCGAGCCGAGATCGCGCCACTGCAC
TCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAACAAAGTGCTCGACAGGAACTTTCTGTAAGCCAAAGAGTATCGCCA
TCGCACCTTCACCTTCACCTTGGGCAGCCTGGTGCCGTCTTCCCTCAGCCCGCGATTAACGAGCGGAAGGCCGCACTGACCCATTTCAG
ATCCCTTATTTCCTTCCTAAAACCACTCAAGAGTTTGGGCACAGTGGCCTCCCTGTGTGAGGAACTTGCTTCCCACTCACCAAAAGACA
AACCCTTCGCCCATCCCTGGCCGCGCACCCGGCCGGTCACCCGCGCTGCCACCCACACCACCCTCCTGTCCATAAAGAGCCCGCGTTTG
GGACGCCTCGCAGGCTGAAGGGAGGCCTGGGCGTCCGCGATCCGTCTGCCCTTTCTCCCCTCGCGGGCTTCCTCTTTCCTCACTTTCTC
CCGCCACTACTCTTCCTCTTCCTTTTCCGCGAACCCAGCCCACTTCCCGTTTCAGGACCTCGGCTCGGCCCCAGTCCCCCGGACCAGGC
CGGGTCACGTGGGTCCAGGGTCACGTGCCGCTGCGGGTCACGTGCCGCTGCGGGTCACGTGTCGGCCTGCATCACGCGTGAGGGGGCGC
GCGGTGCTGGAAGCTGCCGCACCTGCGGGGAGCCGAGCCGCCGGCGCTCGACGCGCGCGCTCTCGCGAGACCCGCGGGATCACGTGACG
CCCGGGCGCGGCGCAGCTCAC
TSS
Most lysosomal promoters share a E-box type regulatory motif
5 UTR
5’UTR
coding
introns
flanking genes
A gene network regulating lysosomal biogenesis and function
Coordinated
Lysosomal Expression
And Regulation
(CLEAR)
TFEB modulates the expression of CLEAR genes
T
Transient
i t TFEB modulation
d l ti iin H
HeLa
L
TFEB
TFEB
TFEB induction is specific to lysosomal functions
C t
Categories
i off CLEAR genes upregulated
l t db
by TFEB
• Lysosomal
L
lh
hydrolases
d l
• Lysosomal membrane proteins
• Lysosomal accessory proteins
• Cytoplasmic proteins protecting from lysosomal hydrolases (e.g. cystatin B)
• Transporters of lysosomal proteins residing at the TGN (M6PRs)
• Proteins involved in lysosomal acidification (proton pump subunits)
• Proteins involved in autophagy (UVRAG, VPSs)
Can the CLEAR network be
“predictive” for the
identification of novel
no el
lysosomal proteins?
Discovery of novel lysosomal proteins by testing
members of the CLEAR network
C1orf85 has CLEAR sites in its promoter,
is upregulated following TFEB overexpression
and displays a lysosomal localization
How is TFEB activated?
TFEB is induced by lysosomal sucrose storage
L
LAMP1
The addition of sucrose in cell’s culture medium causes lysosomal enhancement
S
Sucrose
added
dd d (2 weeks)
k )
Control
Fold
d activation
n
4.0
3.5
3.0
HEXA
PSAP
CTSD
CTSF
MCOLN1
ATP6V1H
ALN
TFEB
2.5
2.0
1.5
1.0
0.5
0.0
0
10
20
30
40
50
60
Time (hrs)
70
80
90
100
TFEB ACTIVATION
TFEB is activated by storage of molecules inside the cells
TFEB activation is associated to its nuclear translocation
Can we modulate lysosomal
activity and cellular clearance
b acting
by
ti on TFEB ?
TFEB OVEREXPRESSION INCREASES THE NUMBER
OF LYSOSOMES IN THE CELL
Endogenous levels of TFEB
Overexpression of TFEB
TFEB OVEREXPRESSION INCREASES THE NUMBER
OF LYSOSOMES IN THE CELL
Endogenous levels of TFEB
Overexpression of TFEB
Can we use the discovery of
a lysosomal gene network to
develop novel therapeutic
strategies?
POSSIBLE THERAPEUTIC STRATEGIES FOR DISEASES
DUE TO THE ACCUMULATION OF TOXIC MOLECULES
1) Inhibit the production
2) Increase the degradation
TFEB PROMOTES THE DEGRADATION OF THE PROTEIN RESPONSIBLE
FOR HUNTINGTON DISEASE (HUNTINGTIN)
TFEB
TFEB PROMOTES THE DEGRADATION OF THE PROTEIN RESPONSIBLE
FOR HUNTINGTON DISEASE (HUNTINGTIN)
GREEN = HUNTINGTIN
TFEB PROMOTES THE DEGRADATION OF THE PROTEIN RESPONSIBLE
FOR HUNTINGTON DISEASE (HUNTINGTIN)
TFEB
TFEB promotes the degradation of mutated HTT
HTT
HD43 cells were electroporated with
3xFLAG-TFEB with a bicistronic vector
containing GFP. Cells were sorted for GFP
for WB analysis.
analysis
3xFLAG-TFEB
Empty
vector
t
TFEB/
GFP -
TFEB/
GFP +
HTT
DAPI
ACTIN
1.2
HTT/ACTIN
1
0.8
MERGE
0.6
0.4
0.2
0
Empty vector
TFEB/GFP -
TFEB/GFP +
Glial cells derived from NPCs
WT
Glial cells derived from MSD NPCs
MSD
TFEB over-expression decreases GAGs accumulation and restores
normal morphology in glial cells derived from MSD-NPCs
MSD + TFEB
MSD
MSD + TFEB
20
18
16
14
vacuoles per cell
v
12
10
8
6
4
2
0
MSD
MSD+TFEB
THE TFEB TEAM
Thi discovery
This
di
is
i dedicated
d di t d to
t the
th memory off Susanna
S
Agnelli
A
lli