cellule ips
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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
3
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
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