Mitochondrial diseases are genetic disorders characterized by defects in oxidative phosphorylation caused by mutations in mitochondrial DNA, or in nuclear genes whose products are related to oxidative phosphorylation or mitochondrial biology. The first part of the project was focused on the generation and characterization of a mouse model of mitochondrial disease, Ttc19ko. Patients with mutations in TTC19 were characterized by neurological impairments and mitochondrial respiratory complex III deficiency. Ttc19 is a mitochondrial protein that seems to be associated to complex III assembly and/or stability. We showed that Ttc19ko mice have neurological symptoms, muscular weakness and reduction in spontaneous locomotors activity, clearly resembling the human disease. Brain also had neurological abnormalities with presence of ubiquitin and GFAP positive staining. Comprehensive lab animals monitoring system revealed a reduction in O2 consumption, CO2 production and energy expenditure in Ttc19ko mice, indicating an overall reduction of energy metabolism. Complex III activity was significantly reduced in tissues and this was linked to an increased ROS production. BNGE analysis of mitochondrial complex III showed a substantial reduction of the incorporation of the catalytic Rieske iron-sulfur protein into the fully assembled complex. A stable isotope labelling by amino acids in cell culture (SILAC) expressing TTC19-Flag followed by immunoprecipitation and mass spec analysis revealed a higher scored interaction between Ttc19 and the subunits of the pre-complexIII, and a lower scored interaction with Rieske protein and Uqcrh, both of them are late assembled subunits. We also demonstrated that Ttc19 is associated to the fully assembled complex III. Taken together, these results suggests that Ttc19 is an intrinsic assembly factor of complex III that interacts with the pre-complex III thus facilitating the incorporation of the late assembled Rieske protein. The second part of the project was focused on a gene therapy approach on a second mouse model of mitochondrial disease, MPv17ko. Mutations in hMPV17 cause a hepatocerebral form of mtDNA depletion syndrome hallmarked by early-onset liver failure, leading to premature death. Liver transplantation and frequent feeding using slow-release carbohydrates are the only available therapies, although surviving patients develop slowly progressive neuropathy. The physiological role of Mpv17 is still unclear. We showed that Mpv17 is part of a high molecular weight complex of unknown composition, which is essential for mtDNA maintenance in liver. On a standard diet, Mpv17ko mouse shows hardly any symptom of liver dysfunction, but a ketogenic diet leads these animals to liver cirrhosis and failure. However, when expression of human MPV17 is carried out by adeno-associated virus mediated gene replacement, the Mpv17ko mice are able to reconstitute the Mpv17-containing supramolecular complex, restore liver mtDNA copy number and oxidative phosphorylation proficiency and prevent liver failure induced by the KD. These results open new therapeutic perspectives for the treatment of MPV17-related liver-specific MDS.

Le malattie mitocondriali sono disturbi genetici caratterizzati da difetti di fosforilazione ossidativa causati da mutazioni nel DNA mitocondriale, o in geni nucleari i cui prodotti sono legati alla fosforilazione ossidativa o alla biologia mitocondriale. La prima parte del progetto è stata focalizzata sulla generazione e la caratterizzazione di un modello murino di malattia mitocondriale, Ttc19ko. I pazienti con mutazioni in TTC19 sviluppano danni neurologici e deficit di complesso III. Ttc19 è una proteina mitocondriale che sembra essere coinvolta nell’assemblaggio e/o stabilità del complesso III. Abbiamo dimostrato che il modello murino Ttc19ko ha sintomi neurologici, debolezza muscolare e riduzione dell’attività locomotoria spontanea, in analogia con la malattia umana. L’analisi istologica ha rivelato alcune anomalie neurologiche con presenza di accumuli di ubiquitina e GFAP. I topi Ttc19ko hanno una riduzione complessiva del metabolismo energetico, una diminuzione del consumo di O2 e di produzione di CO2. L’attività enzimatica del complesso III è significativamente ridotta nei tessuti e ciò è legato ad un aumento della produzione di ROS. L’analisi BNGE ha mostrato una riduzione della incorporazione della subunità catalitica Rieske nel complesso assemblato. L’immunoprecipitazione di TTC19-Flag in colture cellulari trattate con amminoacidi marcati ha rivelato una maggiore interazione tra Ttc19 e le subunità del pre-complesso III, e una minore interazione con proteine Rieske e Uqcrh, entrambe assemblate tardivamente. Abbiamo inoltre dimostrato che Ttc19 rimane associata al complesso III assemblato. Nell’insieme, questi risultati suggeriscono che Ttc19 è un fattore intrinseco di assemblaggio del complesso III che interagisce con il pre-complesso III facilitando così l'incorporazione della proteina Rieske. La seconda parte del progetto è stata focalizzata sulla messa a punto di una terapia genica su un altro modello murino di malattia mitocondriale, MPV17ko. Mutazioni in MPV17 causano una sindrome epatocerebrale con deplezione del mtDNA, insufficienza epatica a esordio precoce, gravi crisi ipoglicemiche e morte. Il trapianto di fegato e l'alimentazione frequente a base di carboidrati a lento rilascio sono le uniche terapie disponibili, anche se in seguito si sviluppano danni neurologici. Il ruolo fisiologico di MPV17 non è chiaro. Abbiamo dimostrato che MPV17 fa parte di un complesso ad alto peso molecolare a composizione sconosciuta, che è essenziale per il mantenimento del mtDNA nel fegato. In dieta standard, il topo MPV17-/- non mostra quasi alcun sintomo di disfunzione epatica, ma una dieta chetogenica porta questi animali a sviluppare cirrosi e insufficienza epatica grave. Tuttavia, quando l'espressione di MPV17 è ripristinata dalla somministrazione di virus adeno-associato, si assiste ad un ricostituzione del complesso supramolecolare contenente Mpv17, ad un ripristino completo del numero di copie di mtDNA, ed alla prevenzione dell’insufficienza epatica indotta dal dieta chetogenica. Questi risultati aprono nuove prospettive terapeutiche per il trattamento delle sindromi da deplezione del mtDNA indotte da mutazioni nel gene MPV17.

(2015). Mitochondrial diseases: from gene function to therapy. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).

Mitochondrial diseases: from gene function to therapy

BOTTANI, EMANUELA
2015

Abstract

Mitochondrial diseases are genetic disorders characterized by defects in oxidative phosphorylation caused by mutations in mitochondrial DNA, or in nuclear genes whose products are related to oxidative phosphorylation or mitochondrial biology. The first part of the project was focused on the generation and characterization of a mouse model of mitochondrial disease, Ttc19ko. Patients with mutations in TTC19 were characterized by neurological impairments and mitochondrial respiratory complex III deficiency. Ttc19 is a mitochondrial protein that seems to be associated to complex III assembly and/or stability. We showed that Ttc19ko mice have neurological symptoms, muscular weakness and reduction in spontaneous locomotors activity, clearly resembling the human disease. Brain also had neurological abnormalities with presence of ubiquitin and GFAP positive staining. Comprehensive lab animals monitoring system revealed a reduction in O2 consumption, CO2 production and energy expenditure in Ttc19ko mice, indicating an overall reduction of energy metabolism. Complex III activity was significantly reduced in tissues and this was linked to an increased ROS production. BNGE analysis of mitochondrial complex III showed a substantial reduction of the incorporation of the catalytic Rieske iron-sulfur protein into the fully assembled complex. A stable isotope labelling by amino acids in cell culture (SILAC) expressing TTC19-Flag followed by immunoprecipitation and mass spec analysis revealed a higher scored interaction between Ttc19 and the subunits of the pre-complexIII, and a lower scored interaction with Rieske protein and Uqcrh, both of them are late assembled subunits. We also demonstrated that Ttc19 is associated to the fully assembled complex III. Taken together, these results suggests that Ttc19 is an intrinsic assembly factor of complex III that interacts with the pre-complex III thus facilitating the incorporation of the late assembled Rieske protein. The second part of the project was focused on a gene therapy approach on a second mouse model of mitochondrial disease, MPv17ko. Mutations in hMPV17 cause a hepatocerebral form of mtDNA depletion syndrome hallmarked by early-onset liver failure, leading to premature death. Liver transplantation and frequent feeding using slow-release carbohydrates are the only available therapies, although surviving patients develop slowly progressive neuropathy. The physiological role of Mpv17 is still unclear. We showed that Mpv17 is part of a high molecular weight complex of unknown composition, which is essential for mtDNA maintenance in liver. On a standard diet, Mpv17ko mouse shows hardly any symptom of liver dysfunction, but a ketogenic diet leads these animals to liver cirrhosis and failure. However, when expression of human MPV17 is carried out by adeno-associated virus mediated gene replacement, the Mpv17ko mice are able to reconstitute the Mpv17-containing supramolecular complex, restore liver mtDNA copy number and oxidative phosphorylation proficiency and prevent liver failure induced by the KD. These results open new therapeutic perspectives for the treatment of MPV17-related liver-specific MDS.
ZEVIANI, MASSIMO
VISCOMI, CARLO
mitochondria, rare diseases, gene therapy
mitocondri, malattie rare, terapia genica
MED/03 - GENETICA MEDICA
English
27-nov-2015
Scuola di Dottorato in Medicina Traslazionale e Molecolare
SCUOLA DI DOTTORATO IN MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 72R
28
2014/2015
open
(2015). Mitochondrial diseases: from gene function to therapy. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/94380
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