"Le neuropatie ottiche mitocondriali: oltre LHON e DOA"

Valerio Carelli MD, PhD

IRCCS Istituto di Scienze Neurologiche di Bologna,

Ospedale Bellaria, Bologna, Italia

Unita’ di Neurologia, Dipartimento di Scienze Biomediche e NeuroMotorie (DiBiNeM), Universita’ di Bologna, Bologna, Italia

NUCLEAR DNA and ENVIRONMENTAL

MODIFIERS in LHON

Leber’ hereditary optic neuropathy (LHON)

FIELD INVESTIGATIONS (2001-2011, 2013, 2014)

THE EXTENDED SOA-BR LHON PEDIGREE

THE ITALIANBRANCH (LB10)

BSL07

Nuclear families: we separated those having an affected mother from those descending from an unaffected female.

General penetrance (affected over the total number of offspring)

Penetrance by sex (affected males or females over total number of male or female siblings).

PENETRANCE

Penetrance: 46.5% (20/43)61.9% in males (13/21)31.8% in females (7/22)

I

II

1II

III

2

III

IV

3III

IV

4 III

IV

5

IV

V

6 IV

V

7

Penetrance: 23.1% (9/39)40.9% in males (9/22)0% in females (0/17)

III

IV

12III

IV

11IV

V

13

IV

V

14

IV

V

17

IV

V

15 IV

V

16

*

We proposed that estrogens are protective in LHON females by activating mitochondrial biogenesis, providing a possible explanation for

male prevalence

mtDNA copy number and mito-biogenesis modulate the pathogenic

potential of LHON mutations determining the variable penetrance

Confounding factors for genetics:environmental triggers

(tobacco smoking)?

We believe there is a relevant role for nuclear background in

LHON penetrance

Differences in affected with LHON: genetic versus environmental triggered disease

ITALIAN COHORT: INDICATION OF TWO LHON PHENOTYPES – GENETIC vs ENVIRONMENTAL

LHON affected (n=134) and unaffected mutation carriers (n=126) from 75 Italian pedigrees re-investigated with the Kirkman criteria

TOBACCO smoke is associated with affected and lowers mtDNA copy number

TOBACCO EXTRACTS DECREASES mtDNA COPY NUMBER - CARRIERS ARE STILL ABLE TO INCREASE MITOCHONDRIAL BIOGENESIS

Luca Giordano, PhDUniversity of Bari

Multilayered strategy to identify candidate genes or pathways relevant to penetrance in LHON (Telethon project #GGP11182 to Valerio Carelli):

• Affimetrix microarray expression analysis comparing muscle biopsies and fibroblasts cultured in glucose and galactose from discordant brothers

• MitoExome analysis of LHON 27 discordant sib-pairs (in collaboration with Vamsi Mootha)

• Linkage analysis and Genome Wide Association studies

• Direct sequencing of candidate genes

CHASING THE NUCLEAR MODIFYNG GENES IN LHON:

CANDIDATE GENES UNDER VALIDATION AND FURTHER INVESTIGATIONS

TFB1M (mitochondrial transcription factor B1): strongly associated in the Brazilian family, not in the Italian cohort check-point for mitochondrial ribosome biogenesis and translation efficiencyE2F1 (transcription factor): associated only in the Italian cohort overexpressed is thought to promote apoptosis, underexpressed upregulates mitochondrial biogenesis SOD2 (manganese superoxide dismutase): association in both cohortssuperoxide detoxification, retrograde signaling to nucleus for mitochondrial biogenesisSGIP1 (SH3-domain GRB2-like (endophilin) interacting protein 1): associated in both cohorts overexpressed in the hypothalamus of an obesity mouse model, highly expressend in brain, strong interaction with endophillins, regulators of clathrin mediate endocytosis, considered important for energy homeostasisNNT (Nicotinamide Nucleotide Transhydrogenase): associated in the Brazilian family located in the inner mitochondrial membrane catalyzing reduction of NADP+ at the expence of NADH oxidation with H+ reentry in the matrixSPATA18 (MIEAP): associated in the Brazilian family involved in a bizarre form of mitochondrial quality control (intramitochondrial lysosome-like organella)

Switching gears: news from mitochondrial

“Fission/Fusion”

OPA3: A FISSION PROTEIN?Alessandra Maresca, PhD

OPA1

OPA3B

ACO2

TOM70

TIM23

NaCl SDSOPA1

OPA3B

ACO2

TOM70

TIM23

P SN P SN P SN

- + + + + Digitonin- 10 25 50 50 Trypsin- - - - + SDS

OPA3A

OPA3A

OPA3AA

B

C

OPA3A

OPA3B

OPA3B

OPA3 is a transmembrane protein localized in the inner mitochondrial

membrane

YFP ATP synthase MERGE

OPA

3AO

PA3B

YFP

Overexpression and silencing of OPA3 variants in HeLa cells

Scra

mbl

e si

RNA

siEx

2 (O

PA3A

)

siE

x2b

(OPA

3B)

siEx

1 (O

PA3A

+OPA

3B)

scramble siEx1 siEx2 siEx2b0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%Mitochondrial morphology

% c

ells

FRAGMENTED

INTERMEDIATE

FUSEDHYPERFUSED

T0 Glu

Gal 48h

T0 Glu

Gal 48h

WT FIBROBLASTS Q105E

WT p.Q105E0.00

10.00

20.00

30.00

40.00

50.00

60.00Glucose

cells

%

WT p.Q105E0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00Galactose 48h

cells

%

ctrl p.Q105E0.000.200.400.600.801.001.201.401.601.802.00

Gene expression

OPA1MFN1MFN2

mRN

A re

lativ

ve co

nten

t

ctrl p.Q105E0.000.200.400.600.801.001.201.401.601.802.00

Gene expression

DRP1FIS1

mRN

A re

lativ

e co

nten

t

20 kDa90 kDa

84 kDa

86 kDa82 kDa

17 kDa

10 kDa

50 kDa

FRAGMENTED

INTERMEDIATE

FUSEDHYPERFUSED

UGO1-like protein (SLC25A46) in humans: recessive mutations associated with optic atrophy, CMT (axonal) and cerebellar atrophy

Claudia Zanna, PhD

Alessandra Maresca, PhD

In collaboration with Stephan Zuchner, Miami

CONTROL

UGO-LIKE SILENCING

UGO1 is a known mitochondrial fusion factor in Saccharomyces cerevisiae with a hitherto missing ortholog in metazoans, but in humans the UGO-like protein behaves like a fission factor

NEW OPA1 PHENOTYPE: CPEO WITH PARKINSONISM AND DEMENTIA

LeonardoCaporali, PhD

Maria LuciaValentino, MD

ChiaraLa Morgia, MD, PhD

AlessandraMaresca, PhD

LuisaIommarini, PhD

ClaudiaZanna, PhD

ValentinaDel Dotto, PhD

Subclinical optic atrophy

COX negative fibers with accumulation of mtDNA multiple deletions

Abnormal mitophagy in OPA1 mutant fibroblasts

CPEO/Parkinson/dementia with OPA1 mutations:

• mtDNA multiple deletions in postmitotic tissues

• hyperfragmented mito-network

• increased basal mitophagy, but impairedmitophagy response after metabolic stress

OPAthies

Parkinsonism/Dementia

Thanks for your attention and to the many collaborators:• Alfredo A. Sadun, USC, Los Angeles• Rubens Belfort Jr, UNIFESP, Sao Paolo• Carla Giordano, University “La Sapienza”, Rome• Palmiro Cantatore, University of Bari, Bari• Anna Ghelli, University of Bologna, Bologna• Pio D’Adamo, University of Trieste, Trieste• Vamsi Mootha, Harvard, Boston• Massimo Zeviani, MRC, Cambridge• Patrick Chinnery and Patrick Yu-Wai-Man, University of Newcastle, Newcastle• Piero Barboni, San Raffaele, Milano

• ………and may lab: