Characterization of the mitofusin 2 R94W mutation in a knock‐in mouse model

Abstract

Charcot-Marie-Tooth disease (CMT) comprises a group of heterogeneous peripheral axonopathies affecting 1 in 2,500 individuals. As mutations in several genes cause axonal degeneration in CMT type 2, mutations in mitofusin 2 (MFN2) account for approximately 90% of the most severe cases, making it the most common cause of inherited peripheral axonal degeneration. MFN2 is an integral mitochondrial outer membrane protein that plays a major role in mitochondrial fusion and motility; yet the mechanism by which dominant mutations in this protein lead to neurodegeneration is still not fully understood. Furthermore, future pre-clinical drug trials will be in need of validated rodent models. We have generated a Mfn2 knock-in mouse model expressing Mfn2(R94W), which was originally identified in CMT patients. We have performed behavioral, morphological, and biochemical studies to investigate the consequences of this mutation. Homozygous inheritance leads to premature death at P1, as well as mitochondrial dysfunction, including increased mitochondrial fragmentation in mouse embryonic fibroblasts and decreased ATP levels in newborn brains. Mfn2(R94W) heterozygous mice show histopathology and age-dependent open-field test abnormalities, which support a mild peripheral neuropathy. Although behavior does not mimic the severity of the human disease phenotype, this mouse can provide useful tissues for studying molecular pathways associated with MFN2 point mutations.

Mice Hemizygous for a Pathogenic Mitofusin-2 Allele Exhibit Hind Limb/Foot Gait Deficits and Phenotypic Perturbations in Nerve and Muscle

“…We did not detect a reduction in axonal numbers, size or increase in g-ratio (Fig 7) to account for this phenotype. This finding is in contrast with a previously described study using hemizygous mice with the R94W mutation [29], in which axonal loss and a slight increase in g-ratio were reported in peripheral nerve. Also, in homozygous mice with the R94W mutation, they exhibited increased mitochondrial numbers in distal nerves [28].…”

“…[28]), whereas CMT2A is almost always dominantly inherited; this discrepancy in inheritance pattern suggests that the pathogenesis of neuropathy in these mouse models may differ from the presumptively dominant negative mechanism responsible for the human disorder. However, a recent study characterizing deficits associated with the Arg94Trp (R94W) knock-in mutation did find that these mutant mice exhibited decreased mobility in open-field testing but not other gaiting parameters, as well as a small but statistically relevant decrease in axon size and myelination in mice hemizygous for this mutation [29]. Another deficiency of currently available CMT2A models is that, in contrast to human CMT2A, expression of the pathogenic MFN2 allele in these mouse models is, by design, limited to neurons, therefore not permitting analysis of the role, if any, of other cell types including skeletal muscle expression of the mutant allele in the genesis of CMT2A phenotype.…”

Mitofusin gain and loss of function drive pathogenesis in Drosophila models of CMT 2A neuropathy

“…While studies in animal models revealed that DRP1 inactivation alters mitochondrial distribution in neurites [21] leading to mitochondrial depletion at synapses [22], the roles of mitochondrial fusion and MFNs in neurons are poorly documented. The study of transgenic mice expressing MFN2 mutations associated with CMT2A revealed animals with locomotor defects associated with axonal loss [26], or alterations of axonal width [27,28]. These models confirm the requirement of MFN2 and the relevance of mitochondrial fusion for neuronal function, but do not mimic the specific alterations of peripheral sensorimotor nerves observed in CMT2A patients.…”

Human biallelic MFN2 mutations induce mitochondrial dysfunction, upper body adipose hyperplasia, and suppression of leptin expression

“…Despite this largely murine evidence for important roles of MFN2 in several tissues, MFN2 loss-of-function mutations in humans have until recently been implicated solely in axonal sensorimotor neuropathy (Züchner et al, 2004), which is only weakly recapitulated in mouse models (Detmer et al, 2008; Strickland et al, 2014). Our findings, together with those of Sawyer et al (2015), now highlight a second tissue-selective phenotype in humans, namely severe upper body adipose overgrowth or ‘Multiple Symmetrical Lipomatosis’ (MSL).…”