MISSION: understanding the mechanisms of therapeutic strategies
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2 Telethon Institute of Genetics and Medicine MISSION: understanding the mechanisms of genetic diseases to develop preventive and therapeutic strategies
3 G E N O T Y P E
4 G E Researcher N O T Y P E
5 G E Researcher N 1 2 Molecular lar 3 4 Protein 5 O T Y P E 11 analysis function/ dysfunction 10 9 Metabolic8 7 pathway involved 6 Mechanisms of cell damage Tissue/ organ dysfunction
6 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: :10.000
7 A normal human cell Golgi apparatus Primary lysosomes Autophagy Secondary lysosomes Phagocytosis Early endosome Endocytosis Late endosome Exocytosis
8 A cell from a patient with a Lysosomal Storage Disease Golgi apparatus Primary lysosomes Autophagy Secondary lysosomes Phagocytosis Early endosome Endocytosis Late endosome Exocytosis
9 LYSOSOMAL STORAGE Lysosomal storage in hepatocytes and macrophages from a patient with Multiple Sulfatase Deficiency
10
11 Mental retardation Haematological abnormalities Facial dysmorphisms Visceromegaly Bone dysplasia Ocular abnormalities
12 Molecules accumulating in LSDs ceramide G m1 ganglioside galactocerebroside Heparan sulfate glucocerebroside sfingomyelin S glycogen Sulfogalactosylceramide (sulfatide)
13 30 years of research on Lysosomal Storage Diseases (LSDs)
14 G E Researcher N 1 2 Molecular lar 3 4 Protein 5 O T Y P E 11 analysis function/ dysfunction 10 9 Metabolic8 7 pathway involved 6 Mechanisms of cell damage Tissue/ organ dysfunction
15 The lysosome: the cell waste basket"
16 THE LYSOSOMES Most cells of our body contain hundreds of lysosomes, the core stations for the degradation and recycling of cellular waste ( cellular incinerators ) Nuclei Lysosomes Incinerator in Vienna
17 THE LYSOSOME The lysosome is made of several elements: Membrane Hydrolases Cellular waste 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 Transport proteins: they handle the trafficking of material across the lysosomal membrane
18 TRANSPORT OF CELLULAR WASTE TO LYSOSOMES
19 TRANSPORT OF CELLULAR WASTE TO LYSOSOMES Molecules l to be degraded d d and recycled are carried to the lysosomes through different processes Nucleus Lysosomes Endocytosis Autophagy Phagocytosis Endocytosis: molecules coming from other cells Phagocytosis: microorganisms (pathogens) or cellular debris Autophagy: material deriving from cellular metabolism
20 THE LYSOSOMES AND DISEASES Disease Storage material Lysosomal storage diseases Mucopolysaccharidoses mucopolysaccharides Lipidoses lipids Glycogenoses glycogen Lipofuscinoses proteins Neurodegenerative disease Alzheimer β-amyloid, tau Parkinson α-synucleinl i Huntington huntingtin Others Prion disease, parasitic infections,.aging
21 Studying lysosome biology using a genomic approach: LYSOSOMICS Marco Sardiello
22 OUR HYPOTHESIS Is there a genetic program coordinating the activity of lysosomes (i.e. regulating cellular clearance)? Lysosomal enzymes COORDINATION Lysosomal acidification Lysosomal biogenesis i
23 Is there a lysosomal l gene network?
24 Why do A we GENE need NETWORK to look at more than one gene? TRANSCRIPTION FACTOR QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.
25 The p63 NETWORK (from Diego Di Bernardo, TIGEM) Co-expression
26 How can we identify gene networks? The Systems Biology approach Conditions i Measure response Co-expression Gene network
27 A MASTER GENE FOR LYSOSOMAL FUNCTION The TFEB gene coordinates lysosomal activity Lysosomal enzymes TFEB Lysosomal acidification Lysosomal biogenesis
28 TFEB: A REMOTE CONTROL TFEB
29 TFEB: A REMOTE CONTROL TFEB
30 Do promoters of lysosomal genes share regulatory elements?
31 Most lysosomal promoters share a E-box type regulatory motif Promoter analysis PWM (position weight matrix) a c g t consensus GGGTCACGTGACCC logo
32 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
33 Most lysosomal promoters share a E-box type regulatory motif 5 UTR 5UTR coding introns flanking genes
34 A gene network regulating lysosomal biogenesis and function Coordinated Lysosomal Expression And Regulation (CLEAR)
35 TFEB modulates the expression of CLEAR genes Transient TFEB modulation in HeLa TFEB TFEB
36 TFEB induction is specific to lysosomal functions Categories of CLEAR genes upregulated by TFEB Lysosomal hydrolases 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)
37 Can the CLEAR network be predictive for the identification of novel lysosomal proteins?
38 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
39 How is TFEB activated?
40 TFEB is induced by lysosomal sucrose storage The addition of sucrose in cell s culture medium causes lysosomal enhancement LAMP1 Control Sucrose added d (2 weeks) 4.0 Fold activation Time (hrs) HEXA PSAP CTSD CTSF MCOLN1 ATP6V1H TFEB ALN
41 TFEB ACTIVATION TFEB is activated by storage of molecules inside the cells TFEB activation is associated to its nuclear translocation
42 Can we modulate lysosomal activity and cellular clearance by acting on TFEB?
43 TFEB OVEREXPRESSION INCREASES THE NUMBER OF LYSOSOMES IN THE CELL Endogenous levels of TFEB Overexpression of TFEB
44 TFEB OVEREXPRESSION INCREASES THE NUMBER OF LYSOSOMES IN THE CELL Endogenous levels of TFEB Overexpression of TFEB
45 Can we use the discovery of a lysosomal gene network to develop novel therapeutic strategies?
46 POSSIBLE THERAPEUTIC STRATEGIES FOR DISEASES DUE TO THE ACCUMULATION OF TOXIC MOLECULES 1) Inhibit the production 2) Increase the degradation
47 TFEB PROMOTES THE DEGRADATION OF THE PROTEIN RESPONSIBLE FOR HUNTINGTON DISEASE (HUNTINGTIN) TFEB
48 TFEB PROMOTES THE DEGRADATION OF THE PROTEIN RESPONSIBLE FOR HUNTINGTON DISEASE (HUNTINGTIN) GREEN = HUNTINGTIN
49 TFEB PROMOTES THE DEGRADATION OF THE PROTEIN RESPONSIBLE FOR HUNTINGTON DISEASE (HUNTINGTIN) TFEB
50 TFEB promotes the degradation of mutated HTT HD43 cells were electroporated with 3xFLAG-TFEB with a bicistronic vector containing GFP. Cells were sorted for GFP for WB analysis. HTT 3xFLAG-TFEB Empty TFEB/ TFEB/ vector GFP - GFP + HTT ACTIN DAPI HTT/ACTIN MERGE Empty vector TFEB/GFP - TFEB/GFP +
51 Glial cells derived from NPCs WT
52 Glial cells derived from MSD NPCs MSD
53 TFEB over-expression decreases GAGs accumulation and restores normal morphology in glial cells derived from MSD-NPCs MSD + TFEB
54 MSD MSD + TFEB cell 10 vacuoles per MSD MSD+TFEB
55
56 THE TFEB TEAM
57 This discovery is dedicated di d to the memory of Susanna Agnelli
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