Willie Girald-Rosa, PhD
MAP Research Fellow
Harvard Medical School
Department of Genetics
NAKED-MOLE RAT MITOCHONDRIAL-NUC...
INTRODUCTION OF THE NAKED MOLE-RAT (NMR)
HETEROCEPHALUS GLABER
HETEROCEPHALUS GLABER (NAKED MOLE-RAT)
• Ecology (distribution, habitat & roles)
• Native of East Africa (Ethiopia, Kenya,...
NMR IS USEFUL FOR BIOMEDICAL STUDIES: AGING AND CANCER
• Holds a record longevity of 32 years
• The longest-living rodent ...
NAKED MOLE-RAT RESISTANCE TO CANCER
• NMR cells were infected with SV40 Tag and Ras
(cancer-causing genes)
• Skin treatmen...
NMR FIBROBLASTS DISPLAY HYPERSENSITIVITY
TO CONTACT INHIBITION
• Contact inhibition (CI) is a key anticancer mechanism
tha...
INTRODUCTION TO MITOCHONDRIAL GENETICS
THE MITOCHONDRIAL GENOME
• Circular ds mtDNA molecule of 15,000-17,000 bp
• Heavy (H) strand and Light (L) strand
• H-stra...
SOME CONCEPTS USED IN MITO GENETICS
• Homoplasmy: is the state whereby all non-nuclear genomes
are the same whether wild t...
DIFFERENT LEVELS AT WHICH PURIFYING SELECTION
CAN OCCUR IN THE MATERNAL GERMLINE
• Genomes with mutations could be blocked...
MITOTIC SEGREGATION OF MTDNA MUTATION
• A particular mtDNA molecule may be replicated many times or not at all during a si...
HUMAN DISEASES CAUSED BY MITOCHONDRIAL
GENOME MUTATIONS
Examples of Diseases
in human
mtDNA gene single base
mutation or d...
REPORTED MTDNA MUTANT IN CULTURED MOUSE CELLS
Locus Nucleotide Change Amino Acid Change OXPHOS Phenotype References
16S T2...
MITOCHONDRIAL DNA MUTATIONS IN CANCERS
A Chatterjee et al., Oncogene , 2006.
• Mitochondrial alterations:
• Altered expres...
GOALS & OBJECTIVES
• Goals
• To design a complete genetic system to study NMR mitochondrial/nuclear dynamics
based on muta...
CANCER CELLS TENDS TO SELECT HIGHLY MUTATED MTDNA IMPLYING
A POSSIBLE SELECTION IN TUMORIGENESIS.
HYPOTHESIS:
IF THE NAKED...
WHAT WE NEED TO TEST THE HYPOTHESIS?
• Clonal selection of NMR cell lines with a very low level of heteroplasmy
• Culture ...
MULTIPLEX SYSTEM FOR NAKED MOLE-RAT MTDNA GENOME SEQUENCING
Amplicons Size Restriction
Enzyme
1
1100 XbaI or TaqI
2
1351 S...
ELECTROPHEROGRAM PATTERNS
FOR TGCE MULTIPLEX ANALYSIS
Mutation
2M
2C
2U
(M) Mix samples; (C) control; (U) unknown
135 247 ...
GENERATION OF NMR RHO0 CELLS
• Ethidium bromide treatment  effective at 20 ng/ml in mouse & human cells
• Generate ρ0 cel...
CELLS & CYTOPLASTIC HYBRID (CYBRID)
• A special case of cybrid formation involves the use of rho-zero cells (Rho0 or ρ0)
a...
CYBRID PRODUCTION
mtDNA donor cell line XX ES cell
Enucleated &
fuse with ρ0 cells
R6G treatment
ρ0 LMTK- cells
LMTK- cybr...
NMR XENOCYBRID & CYBRID PRODUCTION
mtDNA donor cell line
Enucleated &
fuse with LMTK- ρ0 cells
Xenocybrid
Select cybrids
(...
INTERSPECIES MITOCHONDRIAL TRANSFER
EXAMPLES OF XENOCYBRIDS
*Kenyon and Morales, 1997; *Dey et al., 2000; *Mackenzie & Tro...
OVERALL COMPARISON OF SOME PRIMATE MTDNAS
http://genomics.senescence.info/evolution/overall_stats.txt
NMR “HETEROPLASMIC COMPLEMENTATION” XENOCYBRID
Triple Selection & Double Enucleation Method
mtDNA donor cell line
LMTK- Cy...
INDUCTION OF CUTANEOUS TUMORS IN THE NAKED-MOLE-RAT BY UV TREATMENT
720 mJ
UVA
60 mJ
UVA
5 days/week/10weeks
Mouse
Jandova...
ACKNOWLEDGMENTS
Thank you ALL!
See the list @ http://arep.med.harvard.edu/gclab6.htm
Pete Wei
Yoav Sara ? Pedro
SusanGeorg...
IS IT POSSIBLE TO CREATE A HOMOPLASMIC
TRANS-MITO MOUSENMR-MTDNA BY ZONA-FREE SCNT?
COMING SOON…LAB RETREAT NEW HAMPSHIRE ...
GENERATION OF TRANS-MITOCHONDRIAL MOUSE
Adapted from Carl A. Pinkert & Ian A. Trounce, Methods 2002 348-357
ES Cell Transf...
IS IT POSSIBLE TO CREATE A HOMOPLASMIC TRANS-
MITO MOUSENMR-MTDNA BY ZONA-FREE SCNT?
+
Naked-mole rat
Oocyte donor
Somatic...
of 31

Naked Mole Rat Mitochondrial-Nuclear dynamics for LinkedIn

Published on: Mar 3, 2016
Source: www.slideshare.net


Transcripts - Naked Mole Rat Mitochondrial-Nuclear dynamics for LinkedIn

  • 1. Willie Girald-Rosa, PhD MAP Research Fellow Harvard Medical School Department of Genetics NAKED-MOLE RAT MITOCHONDRIAL-NUCLEAR DYNAMICS
  • 2. INTRODUCTION OF THE NAKED MOLE-RAT (NMR) HETEROCEPHALUS GLABER
  • 3. HETEROCEPHALUS GLABER (NAKED MOLE-RAT) • Ecology (distribution, habitat & roles) • Native of East Africa (Ethiopia, Kenya, & Somalia) • Clusters averaging 75 to 80 individuals live together in complex systems of burrows in arid deserts • Adapted for the limited availability of oxygen • Exhibit eusocial structure: the queen and one to three males reproduce • Unusual physical traits • Lack of pain sensation in its skin • Extreme hypoxia tolerance, that enable them to thrive in a harsh, underground environment
  • 4. NMR IS USEFUL FOR BIOMEDICAL STUDIES: AGING AND CANCER • Holds a record longevity of 32 years • The longest-living rodent with a 6-10 longer lifespan than expected based on its body size (mice and rats can only live up to 4-5 years) • It displays negligible senescence • Very slow changes in physiological parameters with age • Lack of an age-related increase in mortality rate • High fecundity rate until death • Shows an unusual resistance to cancer The Longevity of Heterocephalus Compared to other Mammals NMR weigh 30 to 35 grams Naked Mole-Rat Genome Resource 2011. http://naked-mole-rat.org
  • 5. NAKED MOLE-RAT RESISTANCE TO CANCER • NMR cells were infected with SV40 Tag and Ras (cancer-causing genes) • Skin treatment with chemical carcinogens • *7,12-dimethylbenz[α]anthracene (DMBA) • *12-O-tetradecanoyl-phorbol-13-acetate (TPA) • Pouring carcinogens down the mole rat’s throats to induce liver or mammary cancer • Chemotherapies, oxidative stressors, and heavy metals • **Gamma irradiation The mechanisms responsible for the cancer resistance of NMR were unknown *Interview by Daniel Engber to Buffenstein R. (http://www.slate.com/articles/health) *Buffenstein R . J. Endocrinol 1993 Jul;138(1):59-64 *Nazar Labinskyy et al. Am J Physiol Heart Circ Physiol 291:H2698-H2704, 2006
  • 6. NMR FIBROBLASTS DISPLAY HYPERSENSITIVITY TO CONTACT INHIBITION • Contact inhibition (CI) is a key anticancer mechanism that arrests cell division when cells reach a high density • In cell culture NMR fibroblasts arrest at much lower density than those from the mouse • Early Contact Inhibition (ECI) • The roles of of p16Ink4a & p27Kip1 in the control of contact inhibition became temporally separated • ECI is controlled by p16Ink4a & Regular Contact Inhibition by p27Kip1 Seluanov, A et al. PNAS.2009 Model comparing CI in NMR to mouse and human
  • 7. INTRODUCTION TO MITOCHONDRIAL GENETICS
  • 8. THE MITOCHONDRIAL GENOME • Circular ds mtDNA molecule of 15,000-17,000 bp • Heavy (H) strand and Light (L) strand • H-strand encodes 28 genes and the L-strand 9 genes • 37 genes are found in the mtDNA genome • 2 ribosomal RNAs, 22 tRNAs, and 13 polypeptides • 1500 proteins in the mitochondria are coded by nuclear DNA • Mitochondrial Targeting Sequence (MTS) • mtDNA Copies: • 2 -10 mtDNAs/mitochondrion • 100-10,000 mtDNA/cell • mtDNA is replicated by the gamma DNA polymerase complex • Mutation rate is 10-20 times greater than nuclear DNA
  • 9. SOME CONCEPTS USED IN MITO GENETICS • Homoplasmy: is the state whereby all non-nuclear genomes are the same whether wild type of mutated. • Heteroplasmy: mutation affect only a fraction of mtDNA copies. • Microheteroplasmy: is the presence of mutations levels of up to about 2−5% . Wild type All mutant Wt + Mut • Cytoplasmic hybrid (cybrid): eucaryotic cell line produced by the fusion of a whole cell with a cytoplast (enucleated cell) from the same specie. • Cytoplasmic xenohybrid (xenocybrid) Heteroplasmy mutation detection
  • 10. DIFFERENT LEVELS AT WHICH PURIFYING SELECTION CAN OCCUR IN THE MATERNAL GERMLINE • Genomes with mutations could be blocked from replication or selectively destroyed without the need for gene expression. The colors indicate mutant (Red) and wild-type (white) mtDNA (top); respiratory chain–deficient (red) and normal (white) mitochondria (middle); and respiratory chain–deficient (red) and normal (white) cells (bottom). Adapted from Chaen Bae Park and Nils-Goran Larsson, 2011 JCB vol. 193 no. 5 809-818 Level of Selection mtDNA Organelle Cell • Mitochondrion with deficient respiratory chain function, would lead to selection against and/or destruction of this organelle. • Cells with high levels of mutated mtDNA may fail to compete with respiratory chain–competent cells and may be selected against or undergo apoptosis.
  • 11. MITOTIC SEGREGATION OF MTDNA MUTATION • A particular mtDNA molecule may be replicated many times or not at all during a single cell cycle. • Repeated cell division will lead to mitotic segregation of normal and mutated mtDNA • mtDNA mutation must reach a threshold level of 60% or more to have a significant effect on the physiology of the cell. • NO synchronization between cell division and mtDNA replication mutated mtDNA normal mtDNA 0% 30% 50% 70% 100% Respiratory chain dysfunction • A single mutational event creates heteroplasmy in a cell, but the level of mutated mtDNA is very low in comparison with normal mtDNA.
  • 12. HUMAN DISEASES CAUSED BY MITOCHONDRIAL GENOME MUTATIONS Examples of Diseases in human mtDNA gene single base mutation or deletion Leber’s hereditary optic neuropathy (LHON) Cytb, ND (1,4 ) Mitochondrial Encephalomyopathy with lactic acidosis & stroke-like episodes (MELAS) tRNA Leu Myoclonic epilepsy with ragged red fibers (MERRF) tRNA Lys Neurogenic weekness with ataxia & retinitis pigmentosa (NARP) ATP6 Chronic progressive external ophtalmoplegia (CPEO) Large heteroplasmy deletions
  • 13. REPORTED MTDNA MUTANT IN CULTURED MOUSE CELLS Locus Nucleotide Change Amino Acid Change OXPHOS Phenotype References 16S T2432C A2381T - - Moderate Complexes I, III and IV defects Blanc et al.,1981b; Howell & Lee, 1989; Howell & Nalty, 1987; Kearse & Craig, 1981 ATP6/16S T8563A C2380T Val  Glu - Not reported Slott et at., 1983 Cyt b G14830A G14251T G14563C C14578T A15020T G15263A Glu 231  Asp Gly 38  Val Gly 142  Ala Thr 147  Met Leu 294  Phe E373K Not reported Not reported Not reported Not reported Not reported Severe Howell and Gilbert , 1988; Howell et al., 1987 Acín-Pérez et al., 2004 ND6 13879-13884 Cins Frameshift Severe Complex I defect Acín-Pérez et al., 2003; Bai and Attardi, 1998 ND5 C12081A Frameshift Severe Complex I defect Bai et al., 2000 COI C6063A T6589C Leu 246  Lle Val 421  Ala ~50% complex IV defect Acín-Pérez et al., 2003
  • 14. MITOCHONDRIAL DNA MUTATIONS IN CANCERS A Chatterjee et al., Oncogene , 2006. • Mitochondrial alterations: • Altered expression & activity of respiratory chain subunits • High frequency mtDNA mutations • Homoplasmic in nature • Posible explanation for homoplasmic mtDNA point mutations in cancer: • Mutant mtDNA might alter mitochondrially mediated apoptotic pathways to escape cell death. • Alternatively, mtDNA changes may endow the tumor cell with a selective growth advantage directly or in combination with acquired nuclear-encoded mutations.
  • 15. GOALS & OBJECTIVES • Goals • To design a complete genetic system to study NMR mitochondrial/nuclear dynamics based on mutation screening methods, cell line generations and cybrid development. • To compare levels of homoplasmy and heteroplasmy in NMR cells. • To study how mitochondria selection take place in NMR cells. • Objectives: • Characterization of mtDNA mutations in the NMR cell lines • Characterize OXPHOS phenotype from cytoplasmic hybrids harboring specific mtDNA mutation. • To understand the selection dynamics in vitro of mitochondria isolated from cancer cells in the NMR cell line.
  • 16. CANCER CELLS TENDS TO SELECT HIGHLY MUTATED MTDNA IMPLYING A POSSIBLE SELECTION IN TUMORIGENESIS. HYPOTHESIS: IF THE NAKED MOLE-RAT CELLS SHOW AN UNUSUAL RESISTANCE TO CANCER DEVELOPMENT THEN THEIR CELLS TEND TO SELECT NORMAL MITOCHONDRIA.
  • 17. WHAT WE NEED TO TEST THE HYPOTHESIS? • Clonal selection of NMR cell lines with a very low level of heteroplasmy • Culture conditions: 32ºC, 3 to 5% O2 and 5% CO2 • Pre-sequencing Multiplex System for NMR mtDNA mutation screening • Generation of Naked Mole-Rat mtDNA-less cells (NMR ρ0 cells) • Ethidium bromide treatment (0.1-2 μg/ml) • Endonuclease targeting mtDNA genome • A good selection system for cells harboring specific mtDNA mutations • NMR cells resistance to rotenone • Thymidine Kinase deficient cells (TK- cells) • Chloramphenicol & doxycycline: inhibits mitochondrial translation • Generation of cybrids and xenocybrids
  • 18. MULTIPLEX SYSTEM FOR NAKED MOLE-RAT MTDNA GENOME SEQUENCING Amplicons Size Restriction Enzyme 1 1100 XbaI or TaqI 2 1351 SspI 3 1339 HindIII 4 1300 BclI or SspI 5 431 None 6 1269 EcoNI or TaqI 7 1226 EcoNI 8 894 None 9 1248 PstI 10 421 None 11 1297 BamHI 12 1160 EcoRI 13 1051 TaqI 14 646 None 15 1163 TaqI 16 1154 TaqI Amplicons mtDNA wild type Amplicons Posible mtDNA mutation Haploid System Heteroduplexes Diploid System Homoduplexes Allele A Allele B A T G C Wt + Mt A T G C A C G T HEAT Slow Cool Temperature Gradient Capillary Electrophoresis TGCE • TGCE • Identifies DNA variations in ethidium bromide stained heteroduplexes • Programmable thermal gradient • Laser-induced fluorescence detection system
  • 19. ELECTROPHEROGRAM PATTERNS FOR TGCE MULTIPLEX ANALYSIS Mutation 2M 2C 2U (M) Mix samples; (C) control; (U) unknown 135 247 396 531 pb Samples
  • 20. GENERATION OF NMR RHO0 CELLS • Ethidium bromide treatment  effective at 20 ng/ml in mouse & human cells • Generate ρ0 cells in months of treatment • Endonuclease (Kukat, A et al., 2008) • Generate ρ0 cells in 3-5 days COX VIII MTS EGFP EcoRIN C DsRed1-Mito MTS-EGFP-EcoRI Overley Detail
  • 21. CELLS & CYTOPLASTIC HYBRID (CYBRID) • A special case of cybrid formation involves the use of rho-zero cells (Rho0 or ρ0) and other type of cells. • Rho0 cells are depleted of their own mtDNA • Retain functional mitochondria (except lacking OXPHOS) • Can grow in rich culture medium with certain supplements (selection regime) • ATP production is 100% from glycolisis • Naked Mole-Rat (NMR) Rotenone (ROT) resistance cells • Inhibits the transfer of electrons from iron-sulfur centers in complex I to ubiquinone • LMTK- cells • Murine Fibroblast • Thymidine kinase deficient
  • 22. CYBRID PRODUCTION mtDNA donor cell line XX ES cell Enucleated & fuse with ρ0 cells R6G treatment ρ0 LMTK- cells LMTK- cybrid Enucleate & fuse Select cybrids (BrdU to eliminate any TK+ donor cells) ES cell cybrid TK- Mouse cell line TK+ Mouse ES cell Same nuclear background
  • 23. NMR XENOCYBRID & CYBRID PRODUCTION mtDNA donor cell line Enucleated & fuse with LMTK- ρ0 cells Xenocybrid Select cybrids (BrdU ) Mouse Cancer cell line NMR ρ0 cell TK+ OXPHOS Phenotype NMR ROTR cell NMR mtDNA donor cell line High level of heteroplasmy deletions NMR ρ0 NeoR cell Enucleated & fuse with ρ0 cells Select cybrids (ROT & Neo) Xenocybrid CybridDifferent nuclear background Same nuclear background
  • 24. INTERSPECIES MITOCHONDRIAL TRANSFER EXAMPLES OF XENOCYBRIDS *Kenyon and Morales, 1997; *Dey et al., 2000; *Mackenzie & Trounce 2000; * Mackenzie et al. 2003, 2004 ρ0 Cell mtDNA donor cell Succeded xenocybrid + + Human Chimpanzee + Human Gorilla Human Orangutan * * * Yes NO; Defective Complex I activity Primate mtDNA donors Yes + Yes Mus spretus (Rodent)Mouse* Ratus norvegicus (Rodent) + Murid mtDNA donors Mouse*** NO Hamster(Rodent) + Mouse* NO Mild complex I and IV defects emerged in intermediate divergence xenocybrids, but a severe Comlex III defect was evident in the most divergent xenocybrids. **
  • 25. OVERALL COMPARISON OF SOME PRIMATE MTDNAS http://genomics.senescence.info/evolution/overall_stats.txt
  • 26. NMR “HETEROPLASMIC COMPLEMENTATION” XENOCYBRID Triple Selection & Double Enucleation Method mtDNA donor cell line LMTK- Cybrid OXPHOS Phenotype NMR ROTR cell NMR ρ0 NeoR cell 9821insA , mt-Tr Enucleation Di-hybrid xenocytoplast Naked Mole-Rat Cell Enucleation Fuse with NMR ρ0 cells Tracking Mutant Mitochondria Final Goal: production of di-hybrid xenocybrid cell Select cybrids (BrdU & Neo)
  • 27. INDUCTION OF CUTANEOUS TUMORS IN THE NAKED-MOLE-RAT BY UV TREATMENT 720 mJ UVA 60 mJ UVA 5 days/week/10weeks Mouse Jandova J et al. 2011 Tumor No Tumor mtDNA screening for point mutations & deletions Time
  • 28. ACKNOWLEDGMENTS Thank you ALL! See the list @ http://arep.med.harvard.edu/gclab6.htm Pete Wei Yoav Sara ? Pedro SusanGeorge Marc Po-Yi
  • 29. IS IT POSSIBLE TO CREATE A HOMOPLASMIC TRANS-MITO MOUSENMR-MTDNA BY ZONA-FREE SCNT? COMING SOON…LAB RETREAT NEW HAMPSHIRE 2012 Other Possible Projects to consider in a near future
  • 30. GENERATION OF TRANS-MITOCHONDRIAL MOUSE Adapted from Carl A. Pinkert & Ian A. Trounce, Methods 2002 348-357 ES Cell Transfer ES Cell Culture R6G Treatment Cytoplast Fusion Mutant Mitochondria/ Donor Cell Transfection Clonal Selection Superovulate Donor Harvest Ova Co-culture Transgenic Chimeras Transfer to Recipient Transfer to Recipient Injection Mitochondrial Microinjection Mitochondrial Isolation (+/- Transfection) Superovulate Donor Harvest Ova Mitochondrial Injection Transfer to Recipient Transgenic
  • 31. IS IT POSSIBLE TO CREATE A HOMOPLASMIC TRANS- MITO MOUSENMR-MTDNA BY ZONA-FREE SCNT? + Naked-mole rat Oocyte donor Somatic cell Nucleus donor Cloned Trans-mito mice NMR mtDNA+ Foster mother Embryo culture Somatic cell Electrofusion Cumulus Cells Zonae Pellucidae Vortex Demecolcine (1-2h) Pronase Extrusion cone Enucleation Karyoplasts with Chromating discarded Cytoplast Phyto- hemagglutinin Fusion Chamber Chemical Activation Reconstructed Embryo Emerging Blastocyst

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