Willie Girald-Rosa, PhD

MAP Research Fellow

Harvard Medical School

Department of Genetics

NAKED-MOLE RAT MITOCHONDRIAL-NUCLEAR DYNAMICS

INTRODUCTION OF THE NAKED MOLE-RAT (NMR) HETEROCEPHALUS GLABER

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

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

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

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 p16 Ink4a & p27Kip1 in the control of contact inhibition became temporally separated

• ECI is controlled by p16 Ink4a & Regular Contact Inhibition by p27Kip1

Seluanov, A et al. PNAS.2009

Model comparing CI in NMR to mouse and human

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-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

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

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.

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.

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

REPORTED MTDNA MUTANT IN CULTURED MOUSE CELLS

Locus Nucleotide Change Amino Acid Change OXPHOS Phenotype References

16S T2432CA2381T

--

Moderate Complexes I, III and IV defects

Blanc et al.,1981b; Howell & Lee, 1989; Howell & Nalty, 1987; Kearse & Craig, 1981

ATP6/16S T8563AC2380T

Val Glu-

Not reported Slott et at., 1983

Cyt b G14830AG14251TG14563CC14578TA15020TG15263A

Glu 231 AspGly 38 Val

Gly 142 AlaThr 147 MetLeu 294 Phe

E373K

Not reportedNot reportedNot reportedNot reportedNot reportedSevere

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 C6063AT6589C

Leu 246 LleVal 421 Ala

~50% complex IV defect Acín-Pérez et al., 2003

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.

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.

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.

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

MULTIPLEX SYSTEM FOR NAKED MOLE-RAT MTDNA GENOME SEQUENCINGAmplicons Size Restriction

Enzyme1 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

AmpliconsmtDNA wild type

AmpliconsPosible 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 GradientCapillary Electrophoresis

TGCE

• TGCE• Identifies DNA variations in ethidium bromide stained heteroduplexes• Programmable thermal gradient• Laser-induced fluorescence detection system

ELECTROPHEROGRAM PATTERNS FOR TGCE MULTIPLEX ANALYSIS

Mutation

2M

2C

2U

(M) Mix samples; (C) control; (U) unknown

135 247 396 531pb

Samples

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

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

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

NMR XENOCYBRID & CYBRID PRODUCTION

mtDNA donor cell line

Enucleated & fuse with LMTK- ρ0 cells

Xenocybrid

Select cybrids (BrdU )

Mouse Cancer cell line

NMR ρ0 cellTK+

OXPHOS Phenotype

NMR ROTR cell

NMR mtDNA donor cell lineHigh level of heteroplasmy deletions

NMR ρ0 NeoR cell

Enucleated & fuse with ρ0 cells

Select cybrids (ROT & Neo)

Xenocybrid CybridDifferent nuclear background

Same nuclear background

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

PrimatemtDNA donors Yes

+ YesMus spretus (Rodent)Mouse*

Ratus norvegicus (Rodent)

+Murid

mtDNA donorsMouse***

NOHamster(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.

**

OVERALL COMPARISON OF SOME PRIMATE MTDNAS

http://genomics.senescence.info/evolution/overall_stats.txt

NMR “HETEROPLASMIC COMPLEMENTATION” XENOCYBRIDTriple 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)

INDUCTION OF CUTANEOUS TUMORS IN THE NAKED-MOLE-RAT BY UV TREATMENT

720 mJ UVA

60 mJ UVA

5 days/week/10weeks

MouseJandova J et al. 2011

Tumor

No Tumor

mtDNA screening for point mutations

& deletions

Time

ACKNOWLEDGMENTS

Thank you ALL!See the list @ http://arep.med.harvard.edu/gclab6.htm

Pete Wei

Yoav Sara ? Pedro

SusanGeorge Marc

Po-Yi

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

GENERATION OF TRANS-MITOCHONDRIAL MOUSE

Adapted from Carl A. Pinkert & Ian A. Trounce, Methods 2002 348-357

ES Cell Transfer

ES Cell Culture

R6G TreatmentCytoplast Fusion Mutant Mitochondria/

Donor Cell Transfection

Clonal Selection

SuperovulateDonor

Harvest Ova

Co-culture

TransgenicChimeras

Transfer to Recipient

Transfer to Recipient

Injection

Mitochondrial Microinjection

Mitochondrial Isolation(+/- Transfection)

SuperovulateDonor

Harvest Ova

Mitochondrial Injection

Transfer to Recipient

Transgenic

IS IT POSSIBLE TO CREATE A HOMOPLASMIC TRANS-MITO MOUSENMR-MTDNA BY ZONA-FREE SCNT?

+

Naked-mole ratOocyte donor

Somatic cell Nucleus donor

Cloned Trans-mito mice NMR mtDNA+

Foster motherEmbryo culture

Somatic cell Electrofusion

CumulusCells

ZonaePellucidae

VortexDemecolcine

(1-2h)Pronase

Extrusion cone

Enucleation

Karyoplasts withChromating

discarded

CytoplastPhyto-

hemagglutinin

Fusion Chamber

ChemicalActivation

Reconstructed Embryo

EmergingBlastocyst