Mitochondria Functionality and Genetics

MITOCHONDRIA Andrea FUNCTIONALITY Cossarizza AND GENETICS Dept. Biomedical Sciences Univ. of Modena & Reggio Emilia 3RD INTERNATIONAL WORKSHOP ON HIV AD AGING

BALTIMORE, NOVEMBER 5, 2012 OUTLINE OF THE TALK

1. mtDNA genetics: why is it important? Which studies do we need? 2. mtDNA and its role in inflammation: not always a good guy. 3. Which mechanism are triggered in response to mt damages, namely is it better to eat or to die? OUTLINE OF THE TALK

LHON Leber Hereditary Optic Neuropathy MM Mitochondrial Myopathy Mitochondrial dysfunctions, AD Alzeimer's Disease LIMM Lethal Infantile Mitochondrial Myopathy secondary to DNA mutations, ADPD Alzeimer's Disease and Parkinsons's Disease MMC Maternal Myopathy and Cardiomyopathy have been associated with a NARP Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa FICP Fatal Infantile Cardiomyopathy wide spectrum of diseases MELAS Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes LDYT Leber's hereditary optic neuropathy and DYsTonia MERRF Myoclonic Epilepsy and Ragged Red Muscle Fibers MHCM Maternally inherited Hypertrophic CardioMyopathy CPEO Chronic Progressive External Ophthalmoplegia KSS Kearns Sayre Syndrome DM Diabetes Mellitus DMDF Diabetes Mellitus + DeaFness CIPO Chronic Intestinal Pseudoobstruction with myopathy and Ophthalmoplegia DEAF Maternally inherited DEAFness or aminoglycoside-induced DEAFness PEM Progressive encephalopathy SNHL SensoriNeural Hearing Loss mtDNA haplogroups

 mtDNA is inherited almost exclusively through the maternal line and is highly polymorphic – even if not as much as MHC.  Human populations can be divided into several mtDNA haplogroups on the basis of specific single nucleotide polymorphisms (SNPs) scattered throughout the mitochondrial genome, reflecting mutations accumulated by a discrete maternal lineage.  Each mtDNA haplogroup forms a mutually exclusive category, and among Caucasians, 95% of the population belongs to 1 of 10 main haplogroups: H, I, J, K, M, T, U, V, W, and X. “Mitochondrial Eva” and evolutionarly acceptable mutations Association between mtDNA and multifactorial diseases or aging

Haplogroup H Alzheimer disease, Carrieri et al. 2001 Parkinson disease, Huerta et al. 2005; van der Walt et al. 2003

Haplogroup J LHON (Leber Hereditary Optic Neuropathy), Hofmann 1997

Haplogroup J,K,U LOGNEVITY (model of CENTENARIANS) De Benedictis et al. 1999; Niemi et al. 2003; Capri et al. 2006; Santoro et al. 2006

Haplogroup T Bipolar disorders, McMahon et al. 2000 Sperm motility, Ruiz-Pesini et al. 2000 Wolfram Syndrome, Hofmann 1997 mtDNA haplogroups in HIV infection

In the last years, different groups have analyzed the distribution of mtDNA polymorphisms in different types of HIV+ patients, and have studied their correlation with a variety of parameters.

Methods Variables: ### mtDNA haplogroups

### lipidic and glucidic parameters

### acid-base balance parameters

### disease classifications (CDC, MACS)

### antropometric measures

### exposure to drugs Results

HAPLOGROUPS ALLELES FREQUENCIES

V OTH; 32; 6 9,17% X 1,72% W 15 H 2 4,3% 131 0,57% 37,5% I 5 1,43%

J 42 12,03%

K 16 4,58%

T 50 U 14,33% 50 14,33%

European Haplogroups = 91,83% Other origins Haplogroups = 8,17% Results

Parameters related to lipidic metabolism (total, HDL and LDL-cholesterol, tryglicerides, lipoprotein lipase, apolipoprotein A1 and B) did not differ among the haplogroups (p>0.05).

The same result was found analyzing glucose metabolism (insuline, blood glucose, glycosylated hemoglobin, C-peptide, incidence of diabetes), antropometric parameters (body mass index, waist-to-hip ratio and Homeostasis Model Assessment) and viro-immunological data (CD4 nadir, CD4+ T cell count and Viral Load). Data as revealed by Principal Component Analysis Data as revealed by Principal Component Analysis AIDS 2008, 22:2429–2439 A visual heat plot display of P values for genetic association for each of the 34 mtDNA genotypes

Patients: 615 Representative Kaplan–Meier plots for significant results

Mitochondrial DNA haplogroups J and U5a were elevated among HIV- 1+ people who display accelerated progression to AIDS and death.

Haplogroups Uk, H3, and IWX appeared to be highly protective against AIDS progression. September 2010 | Volume 5 | Issue 9 | e12862 1455 European American patients from 5 US cohorts

J Acquir Immune Defic Syndr Volume 53, Number 4, April 1, 2010

Patients: 298 Patients: 218 Differences among different haplogroups Frequency of HIV/hepatitis C virus coinfected patients (N= 231) separated by liver fibrosis stage within each mitochondrial DNA haplogroup. In HIV/HCV coinfected patients, the cluster or major haplogroup HV was significantly associated with reduced odds ratios (OR) for advanced fibrosis (P:0.009), cirrhosis (P:0.007), or high fibrosis progression rate (P:0.015). Within the major haplogroup HV, haplogroup H was significantly associated with an absence of advanced fibrosis, cirrhosis, or high FP.

A significant association was present with increased odds of cirrhosis (P:0.003) in the closely related major haplogroup U.

These data show that mtDNA haplogroup may play a significant role in liver fibrogenesis during HCV infection. J Acquir Immune Defic Syndr 2012;59:113–120

-Atotalof187 HIV+ patients with LD followed for >5 years

- In multivariable models, patients with haplogroup K were at higher risk of any lipodystrophy [adjusted relative risk (aRR) 4.02, P = 0.0009], lipoatrophy (competing-risk aRR 2.42, P = 0.09; cause-specific aRR 2.99, P = 0.031), and fat accumulation (competing-risk aRR, 2.63, P = 0.11; cause-specific aRR 5.27, P = 0.019) than those with haplogroup H. August 27, 2012

Patients: 104

Among ART-naive non-Hispanic blacks, mtDNA haplogroup L2 was associated with baseline and 48-week change in T cell activation, and poorer CD4 cell recovery.

These data suggest mtDNA variation may influence CD4 T cell dynamics by modulating T cell activation.

Relationship between adipose tissue mtDNA/nDNA and selected CD8+ T-lymphocyte subsets Just a few comments

The associations found in different studies support functional explanation by which mtDNA variation among haplogroups, influencing ATP production, reactive oxygen species generation, and apoptosis, is someway correlated to disease progression or to other clinical situations related to HIV infection or its treatment.

Repeating these results in cohorts with different ethnic backgrounds, would be informative and is required.

The use of homogeneous techniques for haplotyping (classification in higher branch haplogroups) is also crucial.

More patients have to be studied: in these 8 studies, the total number is 3,456 – but 1,455 for nuclear genetic polymorhism – thus 2,001 mtDNA haploytpes only. OUTLINE OF THE TALK

1. mtDNA genetics: why is it important? Which studies do we need? 2. mtDNA and its role in inflammation: not always a good guy! . 3. Which mechanism are triggered in response to mt damages, namely is it better to eat or to die? “You” (cells) “are what eat.” Feuerbach The immune system triggers inflammation after the encounter with PAMPs (Pathogen Associated Molecular Patterns), Danger-AMPs or ALARMINS

Molecule Role in Features of DAMPs: inflammation/immunity HMGB1 Yes • Rapidly released following non‐ S100s Yes programmed cell death; HDGF HSPs Yes • Can be produced and released by IL‐1a Yes cells of the immune system Uric Acid Yes without dying; Cathelicidins Yes Defensins Yes • They recruit and activate Galectins Yes receptor‐expressing cells of the Thymosins Yes innate immune system Annexins Yes High plasma levels of mtDNA in patients with trauma compared with volunteers Mitochondrial DAMPs (MTD) activates polymorphonuclear neutrophils (PMN) Mitochondria are thought to have originated as symbiotic bacteria

They use common mechanisms to trigger INNATE IMMUNE RESPONSES to injury and infection

Plasma levels of mtDNA (free or protein bound) in HIV+ patients (but not LTNP) are significantly higher than healthy controls

13 * * copies/ml

12 mtDNA 10 Log 11 CTRL AHI NAIVE LTNP

Cossarizza et al. Mitochondrion, 2011 Plasma mtDNA levels significantly correlates with viral load

14 p=0.002

13 copies/ml

12 mtDNA 10 Log 11 2 3 4 5 6 7 Log10 pVL (copies/ml)

Cossarizza et al. Mitochondrion, 2011 The levels of sCD14 are not correlated with plasma levels of mtDNA

14 p=0.381

13 (copies/ml)

12 mtDNA

Log 11 1.5 2.0 2.5 3.0 3.5 sCD14 (mg/ml) The levels of sCD14 are not correlated with plasma levels of mtDNA

14 p=0.381

13 (copies/ml)

12 mtDNA

Log 11 1.5 2.0 2.5 3.0 3.5 sCD14 (mg/ml)

Thus, plasma mtDNA levels might be independent from the microbial translocation that causes macrophage activation OUTLINE OF THE TALK

SW872 adipocytes were treated for 14 h with 100 μM stavudine and stained with four dyes.

Cossarizza A. et al., Nature Prot. 2009 ROLE OF MITOCHONDRIAL ROS IN AUTOPHAGY

ROS STRESSOR Apoptotic threshold

APOPTOSIS

DEATH ROLE OF MITOCHONDRIAL ROS IN AUTOPHAGY

ROS STRESSOR Apoptotic Autophagic threshold threshold

AUTOPHAGY APOPTOSIS Massive Autophagy

AUTOPHAGIC DEATH CELL DEATH ROLE OF MITOCHONDRIAL ROS IN AUTOPHAGY

ROS STRESSOR Apoptotic Autophagic threshold threshold Mutual inhibition AUTOPHAGY APOPTOSIS Massive Autophagy ADAPTATION AUTOPHAGIC DEATH CELL DEATH SURVIVAL HAART AND AUTOPHAGY DETECTION OF AUTOPHAGY

Daniel J. Klionsky and 1,269 other Authors

Guidelines for the use and interpretation of assays for monitoring autophagy

Autophagy 2012, vol. 8 (4), pp. 445-544 (n=100) http://dx.doi.org.pros.lib.unimi.it/10.4161/auto.19496

Names with letters A and B only DETECTION OF AUTOPHAGY

Mizushima et al., Cell 2010 Using a variety of sophisticated technologies, which include polychromatic flow cytometry, image stream cytometry, multiphoton confocal microscopy and electron microscopy, along with classical cell biology methods, we investigated autophagy and mitophagy in human adipocytes treated with different protease inhibitors,andhavestartedto characterize the phenomenon. Cell model and drugs

SW872 preadipocytic liposarcoma human cells

Drugs:

 Ritonavir (RTV)  Amprenavir (APV)  Atazanavir (ATV)

Drug concentrations used: 10 – 200 M Incubation times: 6, 16, 24 hours Apoptosis (as revealed by FCM)

CTRL Necrosis Late APV

Early

ATV RTV TOPRO (cell permeability) (cell TOPRO ANX-V (PS exposure) ATV increases mitochondrial superoxide levels

4 3 1: ANXV-/DAPI-

2: ANXV+/DAPI-

12 3: ANXV+/DAPI+

4: ANXV-/DAPI+ DAPI ANXV

* * 500 CTR 10uM 400 * * level

- 50uM 2 300 100uM 200uM 200

Relative mtO Relative 100

h 0 h 4 h 6 2 6 1 I/L H

M μ

* M

200 0 *

0 5 doses membrane

0 1

100

R T

high C

0 80 60 40 20

100

at cells % M μ only

I/L H mitochondrial

but

200

M50 0 0 1

10 M

0 5 16h 24h

I/L potential, M

depolarizes

u 10 CTR H 0 ATV 80 60 40 20 100

monomers

CTR cells % 1 ‐

aggregates 1 ‐ JC Starvation induces the formation of autophagosomes

CTR EBSS EBSS

Cytosolic LC3 punctae LC3

EBSS: Earle’s Balanced Salt Solution, used to induce nutrient deprivation (starvation) in eGFP-LC3 SW872 cells ATV induces the formation of autophagosomes

CTR EBSS EBSS

Cytosolic LC3 punctae LC3 CTR ATV 10 μMATV 200 μM The membrane‐bound lipidated form of LC3 (LC3‐II) increases in the presence of 50 or 200 μMatazanavir

GAPDH 37kDa

LC3-I 16kDa LC3-II 14 kDa

CTR 10uM 50uM 100uM 200uM CTR 10uM 10uM50uM100uM 200uM EBSS

6 h 16 h 50μM ATV CTR with colocalize Autophagosomes hMIT= DAPI

anti LC3

human and

hMit LC3

lysosomes =

mAb;

A F enhanced

LAMP DAPI

GFP ‐ 2= LC3

lysosome LC3;

LAMP

(MITOPHAGY)

DAPI= ‐ 2

associated

DNA G B

content

membrane mitochondria LC3

LAMP ‐

2 protein hMit ‐ 2 H C

50μM ATV CTR

EGF‐LC3 EGF‐LC3 uohgsmsclclz with colocalize Autophagosomes mitochondria

and

lysosomes Pearson's correlation coefficent 0.0 0.2 0.4 0.6 0.8 1.0 LC3 C Colocal. T R

hMit

map

A ** T V D I

Pearson's correlation coefficent 0.0 0.2 0.4 0.6 0.8 LC3 map Colocal.

C T R LAMP ‐ 2

A ** T V E J In ATV‐treated cells, mitochondria are enclosed into autophagosomes ACKNOWLEDGEMENTS

University of Modena and Reggio Emilia, Italy

Dr. Lara Gibellini Prof. Anto De Pol Dr. Sara De Biasi Dr. Massimo Riccio Dr. Marcello Pinti Prof. Giovanni Guaraldi Dr. Milena Nasi Prof. Cristina Mussini

Prof. José Enrique O’Connor Dr. Guadalupe Herrera Dr. Francisco Sala SATELLITE MEETING ON IMMUNOLOGICAL AGING