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Cellule indotte pluripotenti (iPS): un nuovo strumento di studio per latrofia ottica di Leber e altre malattie mitocondrialiValeria TirantiUnit of Molecular Neurogenetics

Convegno MitoconRoma 26-28 maggio 2016

Mitochondrial optic neuropathiesLebers hereditary optic neuropathy (LHON) is due to maternally inherited mtDNA point mutations affecting complex I.

Dominant optic atrophy (DOA) is determined by heterozygous mutations in the nuclear gene OPA1, encoding a mitochondrial protein crucially involved in mitochondrial dynamics and maintenance.

Both LHON and DOA are characterized by the selective degeneration of retinal ganglion cells (RGCs), the retinal neurons that project their optic nerve-forming axons to the brain.

Normal retina

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4

Defective complex I function with reduced bioenergetic efficiency

Increased oxidative stress

Defective dynamics of the mitochondrial network

Increased predisposition to apoptotic cell death

Increased levels of basal autophagy

Compensatory activation of mitochondrial biogenesis, which partially rescues the mitochondrial dysfunction Common pathogenic pathways

cytochalasin B+spin

Et-Br + uridine143BTk- cells143BTk- r

Depletion of mtDNA

CybridPEG

Patient fibroblastsCytoplastsenucleation

Cellular models: Cybrids

100% wild-type clone50% mutant clone100% mutant clone

Investigation of biochemical basis of LHON

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Open questionsWhy only specific complex I gene mutations present with optic atrophy?Why RGC and the optic nerve are preferentially affected? What is the relationship between the severity of the mutation and the different consequences for the optic nerve?

A great advantage is currently offered by the possibility of reprogramming primary cells (fibroblasts) from patients to differentiate neurons, and possibly RGCs, through the induced pluripotent stem cells (hiPSCs) strategy Several difficulties in generating mouse models for mtDNA mutations

There are several anatomical and functional differences between the mouse and human eye

Mice models of OPA1-mutant DOA have been generated and studied, revealing some unexpected details on RGCs degeneration, such as dendritic involvement, thus emphasizing the importance of studying directly the target tissue New approaches to study mitochondrial optic neuropathies

induced Pluripotent Stem cells

Pluripotent reprogramming and therapeutic applications

FibroblastsModified from Yamanaka., Nat. Med., 2009 Oct4

Sox2

Klf4

c-Myc

Patient specific neurons

Healthy donorneurons

iPS cellHealthy donors and disease patients:LHONm.11778/MT-ND4m.3460G>A/MT-ND1DOAp.R445H/OPA1 Morphological phenotypeDifferentiation potentialFunctional activityPathophysiological mechanismsSendai virus non-integrating vectorsDrug screening

This new technology has paved the way to generate autologouspluripotent cells from any individual with a straightforward approach providing a valuable alternative to the use of human ES cells which suffered from severe ethical concerns. In principle, individual-specific iPS cells can be turned into a particular somatic cell type both for in vitro studies as disease-model or eventually grafted to the patientin an autologous sort of transplantation.

Nanog Tra 1-60ABCControl hiPSC

Nanog Tra 1-60LHON hiPSC

Nanog Tra 1-60DOA hiPSCDEFIGH

OCT4SOX2

NANOG

Ctr iPSCBlkCtr ES

fibroblasts

GAPDHREX1

DOA iPSC

LHON iPSCCharacterization of pluripotency state of iPSC linesRT-PCR of pluripotency associated genesBrightfield image Alkaline phosphataseImmunofluorescenceSegnali, unpublished

GAPDHPAX6SOX1OCT4NESTIN

Ctr NPCBlkFibroblasts

DOA NPCLHON NPCCtr ESGeneration of NPC from hiPSC of controls, LHON and DOA patients

Sox1 NestinControl NPC

LHON NPC (A3460G)

DOA NPC(R445H)Embryoid Bodies RosettesStable NPC Immunofluorescence for NPC markersRT-PCR of neuroectodermal genesSegnali, unpublished

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Day7

Day14Day31

Ctr NeuronsDOA NeuronsLHON Neurons

Time points of neurons differentiationSegnali, unpublished

TO-PRO3 Beta-Tubulin

Map2 NeuN

TO-PRO3 Beta-Tubulin20X63 XGeneration and characterization of neuronsBrightfield image 63XImmunofluorescence for Map2/NeuN Immunofluorescence for Beta-Tubulin expression in two magnificationsSegnali, unpublished

OCRMicro-oxygraphy analysis on LHON and DOA NPC Reduced OCR was observed in LHON patients: two mutants A11778G; one mutant A3460G

Increased OCR was observed in the DOA R445H mutation after uncouplingSegnali, unpublished

Maresca & Segnali, unpublished

Ctr NeuronsLHON NeuronsMitotracker LysotrackerMitotracker LysotrackerEvaluation of Autophagy in neurons*********Evaluation of mtDNA content

Ctr Neurons

LHON Neurons

DOA NeuronsAnalysis of mitochondria movements in neuronsMitotrackerMitotrackerMitotrackerSegnali & Cordiglieri, unpublished

Modeling of LHON and DOA in neurons and RGCs derived from patients fibroblasts through the generation of hiPSCs, will clarify in the target cells the pathogenic pathways relevant for mitochondrial optic neuropathies.

Drugs tackling these pathways will be tested to providing a strong pre-clinical evidence of efficacy for rapid translation into patients therapy.

These studies, performed in the context of patients's genetic backgrounds, will pave the way to the identification of possible therapeutic targets and to the evaluation of the in vitro efficacy of specific compounds

Future Plans

UO Molecular NeurogeneticsAlice SegnaliCamille PeronSabrina DusiPaola VencoFederica InvernizziCostanza LampertiSilvia MarchetBarbara GaravagliaUO Neuroimmunology and neuromuscular diseasesChiara CordiglieriFulvio Baggi

THANKS TO!

Vania BroccoliAngelo IannelliValerio CarelliAlessandra Maresca

Paolo PintonSimone PatergnaniAlberto Danese