Kawalec 2016 Praca doktorska

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Kawalec MM (2016) Badanie komórkowych skutków mutacji w genie mitofuzyny 2. Próba zaproponowania testu patogenności mutacji. Praca doktorska p86.

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Kawalec MM (2016) Praca doktorska

Abstract: Charcot Marie Tooth diseases (CMT) are heterogeneous group of inherited neuropathies characterized by progressive muscle weakness and atrophy affecting especially lower limbs and distal parts of the body. CMT neuropathies occur with the prevalence of 1 in 2500 patients, which makes them the most common inherited neurological disorders. In majority CMT neuropathies are caused by nerve demyelination (CMT1), however there is also a large group of polineuropathies caused by axonal degeneration (CMT2). Among axonal types of CMT, the most common form, CMT2A, is caused by mutations in mitofusin 2 gene (Mfn2). Although there are over a hundred known pathogenic mutation in mitofusin 2 gene, genetic examination may not be sufficient for precise diagnosis. Mfn2 gene variants with unrecognized pathogenic effect are still indentified. Different time of CMT2A onset and highly polymorphic character of Mfn2 gene complicate diagnosis based on genetic and neurological examination. Therefore, finding a biochemical method to estimate potential pathogenic effect of Mfn2 mutations seems crucial for CMT2A diagnosis and prognosis.

To achieve this aim the following steps were planned: (1) selection of potential parameter for Mfn2 mutation testing on the basis of comparative characteristic of three Mouse Embryonic Fibroblasts types: wild type MEFs (wtMEFs, ATCC-CRL-2991), Mfn2-null MEFs (ATCC-CRL-2993) and Mfn1/Mfn2-null MEFs (ATCC-CRL-2994); (2) selection of cell type for Mfn2 mutation testing after comparative Mfn1/Mfn2 expression studies in particular mouse tissues and organs and (3) evaluation of chosen parameter after over-expression of wild type Mfn2 and Mfn2 with CMT2A mutations, according to the assumption that over-expression of wild type Mfn2 (wtMfn2) will restore chosen parameter while re-expression of mutated Mfn2 gene will not influence the parameter as significantly as wtMfn2.

Mfn2 is nuclear-encoded GTPase, located mainly in mitochondria and together with its analogue, Mfn1, promotes fusion of the outer mitochondrial membrane. It was shown that the disturbances in Mfn2 expression cause fragmentation of the mitochondrial network, mitochondria aggregation, as well as metabolic changes. Mfn2 is also present on the endoplasmic reticulum (ER) surface, participating in mitochondria-ER tethering, calcium release/accumulation in the ER and influencing Ca2+-dependent processes. Further studies shown Mfn2 participation in mitochondrial trafficking and cell cycle.

Labels: MiParea: Respiration, mtDNA;mt-genetics, nDNA;cell genetics, Genetic knockout;overexpression  Pathology: Inherited, Other 

Organism: Mouse  Tissue;cell: Fibroblast  Preparation: Intact cells 

Coupling state: LEAK, ROUTINE, ET 

HRR: Oxygraph-2k 


Abstract continued

Considering Mfn2 function and localization in the cell, cellular bioenergetics as well as mitochondrial features were investigated. It was observed that both, Mfn2 and Mfn1/2 gene knockout in MEFs causes metabolic switch from oxidative phosphorylation to glycolysis. However, this phenomenon was much more significant in Mfn1/2-null MEFs, which seem to synthesize ATP almost exclusively by glycolysis. This conclusion was based on the observation of the elevated lactate synthesis and significant ATP decrease in the presence of glycolysis inhibitor, sodium iodoacetate. Moreover Mfn1/2 gene knockout inhibited MEFs proliferation and decreased cellular respiration. Mitochondrial content and the expression of characteristic mitochondrial proteins in Mfn1/2-null MEFs were also reduced, as well as mtDNA/nDNA ratio. On the other hand, Mfn2 knockout alone resulted in increased proliferation MEFs rate, which was inhibited by re-expression of wild type Mfn2. Increased Mfn2-null MEFs proliferation was accompanied by increased cellular respiration. Elevated level of particular proteins: Tom20, Tfam and selected proteins of OXPHOS complexes indicated increased mitochondrial mass and activity in this cells.

As mtDNA/nDNA ratio was significantly reduced in Mfn1/2-null MEFs in comparison to wild type MEFs and restored by over-expression of wtMfn2 up to 75% of its value in MEFwt, mtDNA/nDNA ratio was chosen for further studies on Mfn2 gene mutation character. Moreover, a method for mtDNA/nDNA ratio measurement is well established, quantitative and reproducible.

The mtDNA/nDNA ratio was measured in Mfn1/2-null MEFs, in the absence of Mfn1, due to positive verification of the hypothesis concerning low Mfn1 gene expression in peripheral nerves. Mfn1/Mfn2 ratio was measured in particular mouse organs. Peripheral nervous system was represented by dorsal root ganglia (DRG). Both, mRNA comparative analysis as well as protein immunoreactivity confirmed that Mfn1/Mfn2 ratio was the lowest in DRGs in comparison to other nervous system samples (brain cortex, cerebellum, spinal cord) and organs (heart, liver, kidney, skeletal muscle and skin). Low Mfn1/Mfn2 ratio observed for DRG supports the assumption, that effects of Mfn2 gene mutation are more severe in absence of Mfn1 and seems to support the thesis that peripheral nerves are more sensitive to Mfn2 mutation due to the lack of Mfn1 compensatory effect.

The mtDNA/nDNA ratio was investigated in MEFs after over-expression of wt-Mfn2 gene and Mfn2 with mutations recognized in Polish families with CMT2A: c.281G>A (Arg94Gln, R94Q), c.748C>T (Arg250Trp, R250W) and c.1198C>T (Arg400X, R400X). R94Q is one of the most common Mfn2 gene mutations observed in unrelated CMT2A patients. R94Q and R250W are point mutations located close to the GTPase domain of Mfn2 and within, respectively. The R400X mutation is also a point mutation, however it results in premature stop-codon. Therefore Mfn2R400X lacks transmembrane motifs as well as HR2 domain, which participates in mitofusins tethering during mitochondrial fusion. Polish patient carrying both mutation R250W/R400X developed CMT2A symptoms, while his parents and sibling, who carry only one mutation, have no clinical symptoms so far.

Over-expression of wtMfn2 in Mfn1/2-null MEFs restored mtDNA/nDNA ratio from 0.43 ± 0.11 to 0.74 ± 0.04 and this result seems to confirm presumed connexion between Mfn2 and mtDNA in the cell. While over-expression of Mfn2R94Q and Mfn2R250W increased mtDNA/nDNA ratio to 0.64 ± 0.11 and 0.65 ± 0.24 respectively, Mfn2R400 did not changed mtDNA/nDNA ratio in Mfn1/2-null MEFs. As over-expression of mutated Mfn2 gene in Mfn1/2-null MEFs did not influenced mtDNA/nDNA ratio (Mfn2R400X) or not as significantly as wtMfn2 (Mfn2R94Q, Mfn2R250W), mtDNA/nDNA ratio seems to be a potential supplementary parameter for Mfn2 mutation pathogenicity testing and further studies on diagnostic test based on mtDNA measurements seem reasonable.