MITOCHONDRIA FUNCTIONALITY AND GENETICS

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1 MITOCHONDRIA FUNCTIONALITY AND GENETICS 3 RD INTERNATIONAL WORKSHOP ON HIV AD AGING Andrea Cossarizza Dept. Biomedical Sciences Univ. of Modena & Reggio Emilia BALTIMORE, NOVEMBER 5, 2012

2 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?

3 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

Mitochondrial genetic diseases Mitochondrial dysfunctions, secondary to DNA mutations, have been associated with a wide spectrum of diseases LHON Leber Hereditary Optic Neuropathy MM Mitochondrial

4 Mitochondrial genetic diseases Mitochondrial dysfunctions, secondary to DNA mutations, have been associated with a wide spectrum of diseases LHON Leber Hereditary Optic Neuropathy MM Mitochondrial Myopathy AD Alzeimer's Disease LIMM Lethal Infantile Mitochondrial Myopathy ADPD Alzeimer's Disease and Parkinsons's Disease MMC Maternal Myopathy and Cardiomyopathy NARP Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa FICP Fatal Infantile Cardiomyopathy 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

5 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.

6 Mitochondrial Eva and evolutionarly acceptable mutations

7 Association between mtdna and multifactorial diseases or aging Haplogroup H Alzheimer disease, Carrieri et al Parkinson disease, Huerta et al. 2005; van der Walt et al 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 Haplogroup T Bipolar disorders, McMahon et al Sperm motility, Ruiz-Pesini et al Wolfram Syndrome, Hofmann 1997

8 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.

10 Methods Variables: ### mtdna haplogroups ### lipidic and glucidic parameters ### acid-base balance parameters ### disease classifications (CDC, MACS) ### antropometric measures ### exposure to drugs

11 Results HAPLOGROUPS ALLELES FREQUENCIES W 2 0,57% I 5 1,43% X 15 4,3% V 6 1,72% OTH; 32; 9,17% H ,5% J 42 12,03% K 16 4,58% T 50 14,33% U 50 14,33% European Haplogroups = 91,83% Other origins Haplogroups = 8,17%

12 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).

13 Data as revealed by Principal Component Analysis

14 Data as revealed by Principal Component Analysis

15 AIDS 2008, 22:

16 A visual heat plot display of P values for genetic association for each of the 34 mtdna genotypes Patients: 615

17 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.

18 September 2010 Volume 5 Issue 9 e12862

19 1455 European American patients from 5 US cohorts

21 J Acquir Immune Defic Syndr Volume 53, Number 4, April 1, 2010 Patients: 298

22 Patients: 218

23 Differences among different haplogroups

24 Frequency of HIV/hepatitis C virus coinfected patients (N= 231) separated by liver fibrosis stage within each mitochondrial DNA haplogroup.

25 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.

26 J Acquir Immune Defic Syndr 2012;59: A total of 187 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 = ], 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.

27 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.

29 Relationship between adipose tissue mtdna/ndna and selected CD8+ T-lymphocyte subsets

30 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.

31 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

32 The immune system triggers inflammation after the encounter with PAMPs (Pathogen Associated Molecular Patterns), Danger-AMPs or ALARMINS Features of DAMPs: Rapidly released following nonprogrammed cell death; Can be produced and released by cells of the immune system without dying; They recruit and activate receptor expressing cells of the innate immune system Molecule HMGB1 S100s HDGF HSPs IL 1a Uric Acid Cathelicidins Defensins Galectins Thymosins Annexins Role in inflammation/immunity Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

33 High plasma levels of mtdna in patients with trauma compared with volunteers

34 Mitochondrial DAMPs (MTD) activates polymorphonuclear neutrophils (PMN)

35 Mitochondria are thought to have originated as symbiotic bacteria They use common mechanisms to trigger INNATE IMMUNE RESPONSES to injury and infection

37 Plasma levels of mtdna (free or protein bound) in HIV+ patients (but not LTNP) are significantly higher than healthy controls Log 10 mtdna copies/ml * CTRL AHI NAIVE LTNP * Cossarizza et al. Mitochondrion, 2011

38 Plasma mtdna levels significantly correlates with viral load Log 10 mtdna copies/ml p= Log10 pvl (copies/ml) Cossarizza et al. Mitochondrion, 2011

39 The levels of scd14 are not correlated with plasma levels of mtdna Log 10 mtdna (copies/ml) scd14 (mg/ml) p=0.381

40 The levels of scd14 are not correlated with plasma levels of mtdna Log 10 mtdna (copies/ml) scd14 (mg/ml) p=0.381 Thus, plasma mtdna levels might be independent from the microbial translocation that causes macrophage activation

41 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?

42 Antiretrovirals can cause oxidative stress SW872 adipocytes were treated for 14 h with 100 μm stavudine and stained with four dyes. Cossarizza A. et al., Nature Prot. 2009

43 ROLE OF MITOCHONDRIAL ROS IN AUTOPHAGY ROS STRESSOR Apoptotic threshold APOPTOSIS DEATH

44 ROLE OF MITOCHONDRIAL ROS IN AUTOPHAGY ROS Autophagic threshold AUTOPHAGY STRESSOR Massive Autophagy Apoptotic threshold APOPTOSIS AUTOPHAGIC CELL DEATH DEATH

45 ROLE OF MITOCHONDRIAL ROS IN AUTOPHAGY ROS Autophagic threshold AUTOPHAGY ADAPTATION SURVIVAL STRESSOR Mutual inhibition Massive Autophagy AUTOPHAGIC CELL DEATH Apoptotic threshold APOPTOSIS DEATH

46 HAART AND AUTOPHAGY

47 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 (n=100) Names with letters A and B only

48 Mizushima et al., Cell 2010 DETECTION OF AUTOPHAGY

Using a variety of sophisticated technologies, which include polychromatic flow cytometry, image stream cytometry, multiphoton confocal microscopy and electron microscopy, along with

49 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, and have started to characterize the phenomenon.

50 Cell model and drugs SW872 preadipocytic liposarcoma human cells Drugs: Ritonavir (RTV) Amprenavir (APV) Atazanavir (ATV) Drug concentrations used: M Incubation times: 6, 16, 24 hours

51 Apoptosis (as revealed by FCM) CTRL Necrosis Late APV Early TOPRO (cell permeability) ATV ANX-V (PS exposure) RTV

ATV increases mitochondrial superoxide levels 4 3 1: ANXV-/DAPI- 2: ANXV+/DAPI- 1 2 3: ANXV+/DAPI+ DAPI 4:

52 ATV increases mitochondrial superoxide levels 4 3 1: ANXV-/DAPI- 2: ANXV+/DAPI : ANXV+/DAPI+ DAPI 4: ANXV-/DAPI+ ANXV Relative mto 2 - level * * * * CTR 10uM 50uM 100uM 200uM 0 6h 16h 24h

53 ATV depolarizes mitochondrial membrane potential, but only at high doses CTR 10 μm 50 μm 100 μm 200 μm H I/L JC 1 monomers 16h 24h * * 20 CTR 10 um 50 um 100 um 200 um 0 I/L H % cells I/L H % cells 20 CTR 10 um 50 um 100 um 200 um 0 JC 1 aggregates

54 Starvation induces the formation of autophagosomes CTR EBSS EBSS Cytosolic LC3 LC3 punctae EBSS: Earle s Balanced Salt Solution, used to induce nutrient deprivation (starvation) in egfp-lc3 SW872 cells

55 ATV induces the formation of autophagosomes CTR EBSS EBSS Cytosolic LC3 LC3 punctae CTR ATV 10 μm ATV 200 μm

56 The membrane bound lipidated form of LC3 (LC3 II) increases in the presence of 50 or 200 μmatazanavir GAPDH LC3-I LC3-II 37kDa 16kDa 14 kda CTR 10uM 50uM 100uM 200uM CTR 10uM 10uM 50uM 100uM 200uM EBSS 6 h 16 h

57 Autophagosomes colocalize with mitochondria and lysosomes (MITOPHAGY) hmit= anti human mt mab; LAMP 2= lysosome associated membrane protein 2 LC3 = enhanced GFP LC3; DAPI= DNA content DAPI LC3 hmit DAPI LC3 LAMP 2 LC3 LAMP 2 hmit 50μM ATV CTR A B C F G H

58 Autophagosomes colocalize with mitochondria and lysosomes Colocal. map LC3 hmit D I CTR ATV ** Colocal. map LC3 LAMP 2 E J CTR ATV 0.0 ** 50μM ATV CTR EGF LC3 EGF LC3 Pearson's correlation coefficent Pearson's correlation coefficent

59 In ATV treated cells, mitochondria are enclosed into autophagosomes

60 ACKNOWLEDGEMENTS University of Modena and Reggio Emilia, Italy Dr. Lara Gibellini Dr. Sara De Biasi Dr. Marcello Pinti Dr. Milena Nasi Prof. Anto De Pol Dr. Massimo Riccio Prof. Giovanni Guaraldi Prof. Cristina Mussini Prof. José Enrique O Connor Dr. Guadalupe Herrera Dr. Francisco Sala

61 SATELLITE MEETING ON IMMUNOLOGICAL AGING

An Introduction to mitochondrial disease.

9 th September 2017 An Introduction to mitochondrial disease. Dr Andy Schaefer Consultant Neurologist and Clinical Lead NHS Highly Specialised Rare Mitochondrial Disease Service and Wellcome Trust Centre