Mitochondrial DNA (Mtdna)

• a cellular organelle – in the cytosol of most nucleated cells • produces energy – by oxidising organic acids and fats with oxygen – process of oxidative phosphorylation • generates oxygen radicals as a toxic by‐product – Reactive Oxygen Species (ROS) The power plant of eukaryotic cells

• Like a power plant, the mitochondrion: – burns fuel (fat and organic acids) – produces energy (ATP) – emits pollution (ROS) Environmental Mitochondriomics

Pollutant sources

Release pollutants in the environment

Mitochondria

Release endogenous ‘pollutants’ within cells

Amplification of exposure effects Piece by Charles W. Schmidt Environmental Health Perspectives, July 2010 Mitochondrial DNA (mtDNA)

• Extranuclear genome – not part of the genetic code in the nucleus of your cells • Small DNA molecule – 16,569 bp • 37 genes – 13 for proteins (phosphorilation enzymes) [N.B., all other proteins coded in nuclear DNA] – 22 for tRNAs – 2 for rRNAs (12S, 16S) Unique characteristics of mtDNA

• Oxidative damage 5 to 10 times higher than nuclear DNA: – direct exposure to endogenous ROS – lacks protective histones – diminished DNA repair capacity • Damaged mitochondria burn fat and other energy substrates more inefficiently: – less energy – more ROS Presentation Outline

Mitochondrial Mitochondrial Mitochondria damage & & dysfunction Epigenetics Environmental Disease

investigating environmental mitochondriomics Mitochondrial damage & dysfunction

mitochondrial DNA copy number as an environmental biosensor Cell types, mitochondria and mtDNA

Cell type Mitochondrion mtDNA

X X X XX

 Red blood cell  0  Skin  few hundred  Lymphocyte  1,000  2 to >10,000  5,000  Heart /brain  >100,000 Mitochondrial and nuclear DNA

mtDNA nDNA

Size (bp) 16,569 ~3,000,000,000 DNA copies per cell 2 to more than 10,000 2 Genes 37 30,000 # of CpGs 435 >28,000,000 Introns No Yes Histones No Yes Oxidative stress x5 x1 DNA repair Absent or quite limited High Mutation rate x10 x1 Byun HM et al., Human Genetics 2014 Mitochondrial damage and copy number

Exposure Damage to nuclear DNA, RNA, proteins, and lipids

Oxidative stress mtDNA Increased damage ROS production Mitochondrial number increases Air Pollution – health effects & sources

• Epidemiology investigations: – air pollution exposure is associated with increased hospitalization and early death – Both acute and long‐term effects on cardiorespiratory disease, lung cancer, neurological effects • Traffic is primary source – traced by air benzene, black carbon • Proxidant exposure • Exposed individuals → high levels of oxidave markers Italian multi‐city benzene exposure study

• Benzene is a widespread pollutant associated with vehicular traffic emissions – Low‐level benzene may induce oxidative damage – No mechanistic biomarkers are available to detect biological dysfunction at low doses • To determine whether low‐level benzene is associated with increased blood mitochondrial DNA copy number (mtDNAcn). Italy multicity benzene exposure study median personal air benzene, by city and exposure group )

3 140 P<0.001 120 (µg/m 100 80 P<0.001 benzene 60 air P<0.001 40 20 Median 0

Genoa Milan Cagliari Carugno et al., Environ Health Perspect 2012 Relative mtDNA copy number (RmtDNAcn) analysis

• qPCR analysis on 384‐well plate format: – Mitochondrial gene (Mt reaction): mtND1 – Single copy nuclear gene (S reaction): β‐globin – Mt/S ration reflects MtDNAcn • Relative mtDNAcn – To avoid plate effects, MtDNAcn is calculated as relative difference to a standard DNA (run in each plate) – E.g. RmtDNAcn=1.24: the sample’s mtDNAcn is 24% higher than the standard DNA – CVs of 3‐5% on duplicate samples run on different days • Key features – Easy to measure – Reflects both damage and dysfunction Blood RmtDNAcn, by city and exposure group

City Group N RMtDNAcn (Unadjusted) RMtDNAcn (Adjusted*) Mean (95% CI) p Mean (95% CI) p

Genoa Referents 48 0.75 (0.65‐0.86) 0.75 (0.66‐0.85) Bus Drivers 151 0.90 (0.84‐0.97) 0.013 0.90 (0.84‐0.97) 0.019

Milan Referents 56 0.76 (0.68‐0.84) 0.75 (0.69‐0.82) Police Officers 77 1.14 (1.07‐1.22) <0.001 1.10 (1.01‐1.19) <0.001 Gas Attendants 76 0.86 (0.79‐0.94) 0.037 0.90 (0.83‐0.98) 0.005

Cagliari Distant 10 0.94 (0.59‐1.48) 0.90 (0.60‐1.41) Close 47 1.24 (1.01‐1.52) 0.215 1.25 (1.03‐1.51) 0.206 Petrochemical 24 1.64 (1.30‐2.07) 0.024 1.63 (1.22‐2.18) 0.041 *Geometric mean adjusted for age, sex, smoking habit, number of cigarettes/day

Carugno et al., Environ Health Perspect 2012 Blood RmtDNAcn vs. personal air benzene by city and in all subjects

Carugno et al., Environ Health Perspect 2012 Mitochondrial epigenetics

mtDNA methylation as environmental target Epigenetics

• Programming of gene expression that: – does not depend on the DNA code – (relatively) stable, i.e., replicated through: • cell mitosis • meiosis, i.e. transgenerational (limited evidence in humans) • Characteristics of epigenetic programming – Modifiable (can be reprogrammed) – Active or poised to be activated: • Potentially associated with current health states or predict future events DNA methylation inactive methylation DNA (more DNA

accurately: methylation

usually

suppresses

associated

RNA

with

expression

suppressed active demethylation DNA to

activated

poised

RNA)

Environmental exposures on nuclear DNA methylation Results from our labs in Milan and Boston • Air pollution (PM, foundry PM) • PAHs – Baccarelli, AJRCCM 2009; – Pavanello, Int J Cancer 2009; – Tarantini, EHP 2009; – Pavanello, Carcinogenesis 2010; – Dioni, EHP 2010; – Peluso, Int J Epidemiol; – Madrigano, EHP 2011; – Alegria, Torres Chemosphere 2012 – Hou, Part Fibre Tox 2011; • POPs and Pesticides – Bind, Epidemiol 2012; – Rusiecki, EHP 2008; – Madrigano, AJE 2012 – Zhang, Environ Mol Mutagen 2012; – Sofer, Epigenomics, in press – Zhang, Environ Tox Pharmacol 2012; • Metals – Villahur, in preparation. – Wright, EHP 2010; • Phsychosocial stress – Kile, EHP 2012; – Bollati, Chronobiol Int 2010; – Lambrou, Epidemiology 2012 – Rusiecki, Epigenomics 2012 – Byun, Part Fibre Tox, in press • Smoking and allergens – Guo, under review – Sordillo, Int Arch Aller Immun 2012 – Seow, in preparation – Wan, Hum Mol Gen 2012 • Benzene – Baccarelli, Epigenomics 2012 – Bollati, Cancer Res 2007; – Seow, WH PlosONE 2012; – Fustinoni, Med Lav 2012 Epigenetics of mitochondria

• Methylation of mtDNA has been widely overlooked – total absence of methylation reported in 1973 (Dawid et al, Science) – subsequent reports showed low methylation levels • Schock et al., PNAS 2010 – previous studies underestimated the level of cytosine modification in the mtDNA. – DNMT1 translocates to the mitochondria • driven by a mitochondrial targeting sequence immediately upstream of the commonly accepted translational start site. – mitochondrial DNMT1 • is upregulated in response to hypoxia • affects mtDNA gene expression mtDNA methylation in foundry workers

• Foundry workers are exposed to metal‐rich air particles (PM) • mtDNA methylation analysis of a sequence ajdjacent two genes key to mitochondrial protein translation – MT‐RNR1 : protein that facilitates formation of RNA secondary structures, assembly of the mitochondrial ribosome, and mitochondrial translation – MT‐TF gene: a mitochondrion‐specific transfer RNA • Blood DNA from 20 foundry workers with high PM exposure vs. 20 controls CpG sites in mtDNA

The outer ring (in black) shows the relative position of each of the 435 predicted CpGs

Chinnery et al, Int J Epidemiol 2012 in mtDNA

steel

workers MT-TF & MT-RNR1 methylation Methylation (%)

exposed P Controls =0.002 (n=20)

metal High-exposed steel workers Byun (n=20) ‐ rich et al,

air

Particle

particles

Fib

Tox,

(PM1)

2013 mtDNA methylation modeled dose‐response with PM1

P=0.02 for linear effect 1

(%)

RNR1 in ‐

MT 0

Change TF

‐ -1 MT-TF & MT-RNR1 & MT-RNR1 MT-TF Methylation % Methylation MT

-2

0.5 1.0 1.5 2.0 2.5

Log (PM1 exposure level)

Byun et al, Particle Fib Tox, 2013 mtDNAcn copy number and mtDNA methylation

3.0 r=0.36 P=0.02 2.5

2.0

1.5

1.0

0.5 Relative mitochondrial copy number

0.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 MT-TF & MT-RNR1 % Methylation Byun et al, Particle Fib Tox, 2013 Ongoing plans for mtDNA research at our lab

2012‐2015 • Blood buffy coat and buccal cells • mtDNA methylation (D‐loop promoter region and 12s rRNA) • Disease outcomes

2015‐onwards • Cell type specific • Purified mtDNA • Whole mtDNA seq/ bisulfite seq • And more

41 Cardiovascular disease pilot

• 10 patients with atherosclerotic D-loop Cytochrome b

16S rRNA NADH MT-ND5 cardiovascular disease Dehydrogenase subunits NADH • 15 healthy controls Dehydrogenase Mitochondrial genome tRNA-leucine MT-TL1 subunits NADH Dehydrogenase with similar age and subunits Cytochrome Cytochrome Oxidase Oxidase MT-CO3 sex distributions subunits ATP Synthasesubunits MT-CO1 subunits MT-CO2 • Isolation of mtDNA MT-ATP6 MT-ATP8 from platelets Isolation of mtDNA from human platelets

Why platelets?

“Cell type specific” Single blood cell type “Technically convenient” Anucleate cell with mitochondria Disorders in which platelets play a key role: Atherosclerosis, coronary artery “Disease related” disease, myocardial infarction, cerebrovascular disease, stroke, asthma etc. Isolation of mtDNA from human platelets

platelets 200 μl Plasma Centrifuge Buffy coat Erythrocytes

Lysis buffer AL Centrifuge DNase I (3hr) (Qiagen) mtDNA Isolating mtDNA from nuclear DNA

Total Cellular DNA

Mitochondrial DNA

X Nuclear DNA XX XX Nuclear DNA + Numts

Nuclear DNA sequences of Mitochondrial origin (Numts) Purity of isolated mtDNA by Real Time‐PCR

1 2 3

1. Positive control ‘chrM:3313-3322’ 2. Positive control HBB 3. Test sample ‘chrM:3313-3322’ 4. Test sample HBB

Byun HM et al. Methods Mol Biol submitted 2015 CVD patients have different mtDNA methylation

a)MT-CO1 b) MT-CO2 c) MT-CO3 d) MT-TL1 **** *** **** *** 30 8 2.5 6 7 25 2.0 5 6 20 4 5 1.5 15 4 3 3 1.0 10 2 2 5 0.5 1 1 % mtDNA methylation % mtDNA % mtDNA methylation % mtDNA % mtDNA methylation % mtDNA % mtDNA methylation % mtDNA 0 0 0.0 0 Healthy CVD Healthy CVD Healthy CVD Healthy CVD (n=12) (n=10) (n=12) (n=10) (n=15) (n=10) (n=10) (n=9)

g) e) MT-ATP6 f) MT-ATP8 MT-ND5

5 5 12 10 4 4 8 3 3 6 2 2 4

1 1 2 % mtDNA methylation % mtDNA % mtDNA methylation % mtDNA % mtDNA methylation % mtDNA 0 0 0 Healthy CVD Healthy CVD Healthy CVD (n=9) (n=9) (n=12) (n=10) (n=9) (n=10) Predictive value of mtDNA methylation

a) b) c) d) MT-CO1 MT-CO2 MT-CO3 MT-TL1 100 100 100 100 90 90 90 90 80 80 80 80 70 70 70 70 60 60 60 60 50 50 50 50 40 40 40 40 Sensitivity% Sensitivity% 30 Sensitivity% 30 Sensitivity% 30 30 20 20 20 20 10 10 10 10 0 0 0 0 0 102030405060708090100 0 102030405060708090100 0 102030405060708090100 0 102030405060708090100 100% - Specificity% 100% - Specificity% 100% - Specificity% 100% - Specificity%

AUROC: 0.99 AUROC: 0.94 AUROC: 0.96 AUROC: 0.97 Sensitivity: 100% Sensitivity: 100% Sensitivity: 100% Sensitivity: 100% Specificity: 90% Specificity: 70% Specificity: 70% Specificity: 78% PPV%: 92% PPV%: 80% PPV%: 82% PPV%: 83% NPV%: 100% NPV%: 100% NPV%: 100% NPV%: 100% mitochondria & environmental disease

Mitochondrial haplogroup clusters and cognitive aging Air pollution and age‐related cognitive decline

• >10% of individuals >65 years and 50% of those ≥85 years have some form of cognitive impairment • Environmental exposures that augment systemic oxidative stress have been shown to hasten cognitive aging by as much as 5 years – PM from vehicular traffic associated with lower mini– mental state examination (MMSE) in the Normative Aging Study (Powers, EHP 2012) – Results consistent with data from the NHANES (Chen, Neurotoxicology 2009), China (Zheng AJPH 2010) and Germany (Rantf Environ Res 2008) • Rare mitochondrial DNA mutations/deletion produce neurocognitive phenotypes Air pollution, age related cognitive loss and mitochondria in the NAS

• The Normative Aging Study – ongoing longitudinal cohort study of ~700 elderly men – followed up every 3‐5 years from 1996 to date – mean age 73 years (range 55‐100) • Haplogroups measured for most of the individuas. • Exposure to Black Carbon – a tracer of particulate air pollution from traffic – validated spatio‐temporal land‐use regression model – 1‐year average the participant’s address prior to the date of the first cognitive assessment mtDNA mutations/deletions are associated with neurological phenotypes

• Leber’s Hereditary Optic Neuropathy • Mitochondrial encephalopathy, Lactic Acidosis and Stroke‐like apisodes (MELAS) • Kearns‐Sayre syndrome • Progressive encephalopathy Super‐haplogroup clusters in the Normative Aging Study (n=616)

Fronto‐temporal degeneration Clusters of haplogroups created using Cluster 4 phylogenetic network and evolutionary tree AD Haplotypes N % Cluster 1 (J or T) 111 18.0 Cluster 1 haplogroup J 52 8.4 haplogroup T 59 9.6 Cluster 2 (H or V) 314 51.0 haplogroup H 53 8.6 haplogroup V 261 42.4 Cluster 3 (K or U) 126 20.5 haplogroup K 60 9.7 haplogroup U 66 10.7 Cluster 4 (I, W or X) 65 10.6 haplogroup I 32 5.2 Cluster 3 haplogroup W 10 1.6 haplogroup X 23 3.7 Cluster 2 Mini‐mental state examination (MMSE)

• 30‐point questionnaire test of cognitive function • Commonly used to screen for dementia. • Score<25 → low MMSE

Possible Category Description points Orientation to From broadest to most narrow. Orientation to time has been 5 time correlated with future decline. Orientation to From broadest to most narrow. This is sometimes narrowed 5 place down to streets, and sometimes to floor. Registration 3 Repeating named prompts Serial sevens, or spelling "world" backwards It has been Attention and 5 suggested that serial sevens may be more appropriate in a calculation population where English is not the first language. Recall 3 Registration recall Language 2Naming a pencil and a watch Repetition 1Speaking back a phrase Complex 6Varies. Can involve drawing figure shown. commands Estimated Risk of low MMSE due to traffic PM

8.00 Risk for a doubling in 1‐yr black carbon at baseline

P=0.01 for interaction 4.00 between exposure and clusters CI)

2.00 (95%

1.00

0.50 All subjects Cluster 1Cluster 2Cluster 3Cluster 4 Adjusted for education, alcohol, physical activity, diabetes, dark fish consumption, computer experience, first language, non‐white census tract percentage, college degree census tract percentage, first cognitive assessment, part time residents, matrilineal ethnicity. Colicino et al., Environmental Health, 2014 Summary of our findings

• Effects of air pollution exposure on: – mtDNA copy number – mtDNA methylation • Opportunities for disease investigations – Cardiovascular disease – Air pollution acceleration of cognitive aging • Questions and future directions – Relevance of mtDNA copy numbers and mtDNA methylation to human disease? – Novel techniques, e.g., deep sequencing – Need for longitudinal prospective studies linking past exposures→mtDNA markers →phenotypes Acknowledgments

Harvard Environmental Epigenetics Lab Collaborators on mtDNA researcg • Golareh Agha • Pier Bertazzi, Milan • Kasey Brennan • Michele Carugno, Milan • Juan Carmona • Miriam Hoxha, Milan • Akin Cayir • Valentina Bollati, Milan • Elena Colicino • Deep Deb • Valeria Motta, Harvard/Milan • Alexandra Dereix • Laura Dioni, Milan • Hanine Haji • Mindy Powers, Johns Hopkins • Allan Just • Marc Weisskopf, Harvard • Oskar Karlsson • Joel Schwartz, Harvard • Hannah Laue • Pan Vokonas, VA Boston • Rosie Martinez • Hyang‐Min Byun, Newcastle • Jamaji Nwanaji‐Enwerem • Cheng Peng Funding • Diddier Prada • Rodos Rodostensis • R01ES021733 • Marco Guerra Sanchez Molecular and Epigenetic • Letizia Trevisi • Yan Zhao Mitochondriomics of Air Particles, • Jia Zhong Lead and Cognition Andrea Baccarelli ‐ Harvard School of Public Health Environmental Epigenetics Lab [email protected]