Progetto Mitofusina



Rizzo F, Ramirez A., Ronchi R., Salani S, Nizzardo M, Fortunato F, Bordoni A, Bresolin N, Comi GP, Corti S.
Dino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation(DEPT), Univerity of Milan, Neurology Unit, IRCCS Fondation Cà Granda Ospedale Maggiore Policlinico, Milan, Italy

The disease type 2A Charcot-Marie-Tooth (CMT2A) is a sensorimotor polyneuropathy, characterized by the death of motor neurons and sensory, which results in progressive limb weakness, muscle atrophy and loss of sensory. The disease is due to mutations in the gene Mitofusina2 (MFN2), which encodes a protein localized at the mitochondria, organelles very important for the survival of the cells and their proper functioning. To date, unfortunately, there are no conclusive therapies for this condition.
The reprogramming of these mature cells in induced pluripotent stem cells (iPSC) offers the possibility of obtaining patient-specific cells, such as human motor neurons and sensory neurons, typically affected in the disease, but impossible to obtain directly from patients with other strategies. In the study published online August 9, 2016, on Human Molecular Genetics, the research team of the Centro Dino Ferrari, University of Milan, Fondazione IRCCS Ca 'Granda Ospedale Maggiore Policlinico , differentiated motor neurons (MN) from iPSCs derived from biopsy skin of patients with CMT2A. In this way, we have generated an in vitro model of the disease, currently not available. The cells thus obtained showed some typical aspects of the disease, such as reducing the amount of mitochondria and changes in their location, with no significant differences in survival. These defects are most apparent in neuronal cells results compared to skin fibroblasts, in agreement with the neuronal specificity of the disease. Overall, these data suggest that the reduction of mitochondria in motor neurons that express the mutated form of MFN2 , is not the result of a reduced production of mitochondria, but more likely the result of the death of these organelles. These mechanisms represent possible new molecular therapeutic targets for the development of an effective treatment for this disease.

Last updated on Oct 27, 2016


Dr. Federica Rizzo, Dr. Stefania Corti, Prof. Giacomo P. Comi
Centro Dino Ferrari Università degli Studi di Milano

Charcot-Marie-Tooth disease type 2A (CMT2A) is a sensory-motor polyneuropathy, characterized by the involvement of motor and sensitive neurons, resulting in progressive weakness, limb muscle atrophy and loss of sensitivity. Mitofusin2 (MFN2) gene has been identified as causative of the disease. The MFN2 protein,
located in the outer mitochondrial membrane, is involved in the mitochondrial functions. A treatment for CMT2A is not currently available. In our project, we aim to develop an effective therapy, based on the understanding of the disease molecular mechanisms, helpful not only to identify new therapeutic targets, but also to define specific disease hallmarks.
We first generated fibroblasts from CMT2A patients with different MFN2 mutations. The reprogramming of mature somatic cells into induced pluripotent stem cells (iPSCs) provides the derivation of disease-specific cell types, such as motor and sensitive neurons, affected in the disease. Based on this method, we successfully generated human iPSCs from a CMT2A patient and demonstrated their differentiation into motor neurons. In particular, we observed an alteration in mitochondria localization, a reduction in the amount of mitochondrial DNA and a dysfunction of the mitochondrial respiratory chain, identifying specific hallmarks of the disease phenotype. These defects are more evident in neuronal cells compared to fibroblasts, in agreement with neuronal specificity of the disease. In addition in vitro models, the analysis of these aspects has been conducted in the only currently available mouse model of CMT2A (MitoCharc 1) to extend its characterization, searching for biomarkers of disease phenotype.

We aimed to develop a therapeutic approach for this disorder. We silenced endogenous MFN2 gene by short harpin RNA (shRNA) in CMT2A fibroblasts. At the same time, in order to restore correct MFN2 protein levels, we transfected a MFN2 c-DNA modified to be resistant to shRNA-mediated silencing. The results of this strategy were very promising in CMT2A fibroblasts, and preliminary data were also obtained in the CMT2A mouse model.

This study contributed to deepen the knowledge about disease molecular mechanisms, generating an in vitro model of CMT2A by patient-specific iPSCs, and to identify a possible therapeutic strategy for CMT2A.

The future developments of our research project will be:
- to increase the number of iPSC lines obtained from CMT2A patients and to differentiate them into neurons
-to test our RNAi therapeutic strategy in CMT2A neurons
-to apply our strategy in CMT2A mouse model, using adeno-viral type 9, capable of transferring our constructs into neuronal cells.

Recently, we decide to test CRISPR-CAS9 genome editing system to correct the disease MFN2 gene mutation directly at the DNA level. We will establish gene-editing complexes that include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, guiding Cas9 in its cutting. At the same time, we will also deliver a DNA template strand. Cells repair the damage produced by Cas9 copying from the template and thus introducing new genetic material into the genome, which results in the correction of MFN2 mutation. The CRISPR system is considered a gold standard strategy for genome editing, such as demonstrated by positive results obtained in other pathologies.

Updated in Jan 2015


Corti S., Rizzo F., Ronchi D., Del Bo R., Nizzardo M., Comi G.P.

Charcot-Marie-Tooth disease type 2A (CMT2A), the most common type of CMT2, is caused by mutations in the mitofusin 2 gene (MFN2). For CMT2A treatment, no effective therapy is available. In this project, we propose two different approaches as a possible therapeutic strategy: one based on cell therapy and second on Morpholino antisense oligomer. To test these strategies, we will use MitoCharc1 mouse model, characterized by R94Q (Arginine to Glutamine) amino acid substitution to mimic the most common mutation found in CMT2A (Cartoni R et al.; 2010).

Regarding cell therapy strategy, a possible approach is to perform transplantation of stem cells to deliver growth factors which could have a therapeutic role on CMT2A neurons. In fact we have previously demonstrated that neural stem cell (NSC) transplantation can improve the phenotype in motor neuron disease (MND) mouse models (Corti S. et al.; 2006-2010). Growth factors such as Glia-derived neurotrophic factor (GDNF) are known to have neuroprotective effects in vitro and in vivo models of neurodegenerative diseases (Henderson CE et al.; 1994; Suzuki M et al.; 2007; 2008). Based on these data, we will evaluate the therapeutic potential of human NSCs and Myogenic Stem Cells (MySCs) obtained by patient specific Induced Pluripotent Stem Cells (iPSCs), alone or combined with growth factors delivery. Our goal is to isolate and characterize NSCs and MySCs from iPSCs, to genetically manipulate them to overexpress GDNF and to define an adequate transplantation protocol for their delivery into MitoCharc1 mice (intrathecal of NSCs and intramuscular of MySCs). Once determined survival and differentiative fate of NSCs/MySCs following transplantation, we will investigate the potential of wild-type and genetically modified NSCs/MySCs in protecting neurons and neuromuscular junctions improving neuromuscular functions.

Concerning Morpholino antisense oligomer strategy, we would like to use phosphorodiamidate morpholino (MO), third-generation backbone-modified antisense oligonucleotides (ASO), is designed to reduce human MNF-2 protein level. The use of MO can offer great promise for treatment of human disease, based on data recently described about motor neuron disease mouse models (Porensky PN et al.; 2011). In this project, we want to transfer this approach into CMT2A field. In particular we will test MO sequences, capable to silence MNF2 protein, in human or mouse cell lines to evaluate in vitro protein level reduction. Then we define the best MO administration protocols into MitoCharc1 mice, determining the dose and modalities of injection, considering the local (intracerebroventricular and/or intratechal) and/or systemic (intravenous) approaches. We will study the biodistribution of MO and their capacity to decrease mRNA and protein MNF-2 level in the mice by gene expression and protein analysis. We will also evaluate therapeutic efficacy of MO in improving neuromuscular phenotype, delaying disease progression and providing a survival benefit in treated MitoCharc1 mice. Our intention is to define a pre-clinical protocol with morpholino to high reduction of MNF2 level with minimum toxic consequences, offering a therapeutic clinical realistic approach.

Updated in Nov 2013