One puzzling aspect of CMT2A is that the symptoms of the disease are restricted to very specific tissues or cell types, despite the fact that mutant MFN2 alleles are expressed in every cell. Thus, in addition to discovering the molecular defects caused by MFN2 mutations, it will be essential to uncover the basis for the selective effects of the mutations in peripheral nerves. While animal models will be useful for deciphering some features of CMT2A pathogenesis, it is also necessary to study mitochondrial function in affected and unaffected cells from patients. Here, we have expanded upon a recent study of CMT2A fibroblasts by evaluating mitochondrial phenotypes, including direct assays of mitochondrial fusion and mitofusin protein abundance, in patient fibroblasts harboring five MFN2 mutations not examined previously. We found that, regardless of the location of the Mfn2 mutation, CMT2A fibroblasts are as robust as Control cells, with normal mitochondrial fusion and function. Below, we summarize current molecular models for CMT2A pathology based on studies in cell cultures, mouse models, and patient-derived cells. We discuss our data in the context of these earlier findings and propose a unifying model that incorporates all available data.
The polarity and length of peripheral nerve axons has led to speculation that axonal degeneration in CMT2A results from failure to deliver functional mitochondria to nerve terminals. To test whether mutant Mfn2 proteins interfere with mitochondrial transport in axons, Baloh and colleagues overexpressed CMT2A alleles in primary rat DRG cells (Baloh, et al., 2007
). Axonal mitochondria in WT cells were simple tubules and fragments of variable length. In cells over expressing the mutant Mfn2 proteins, mitochondria were clustered in the cell body and very few organelles were seen in the axons. Based on these results, the authors proposed that mutant Mfn2 proteins interfere with an undefined role of Mfn2 in mitochondrial transport, which helps direct mitochondria down the long axons of peripheral nerves. However, interpretation of these studies is difficult because phenotypes were generated by overexpression of mutant Mfn2 protein in the presence of endogenous WT mitofusins. Overexpression of both WT and mutant Mfn2 (as well as other mitochondrial membrane proteins) has been shown to cause morphological changes (typically mitochondrial aggregation or clustering), often in a dose-dependent manner (Detmer and Chan, 2007a
, Eura, et al., 2003
, Huang, et al., 2007
, Kimura and Okano, 2007
, Rojo, et al., 2002
, Stojanovski, et al., 2004
, Yano, et al., 1997
). Since expression levels of the mutant Mfn2 proteins were not documented in the Baloh et al. study, it is difficult to predict how substantial mitochondrial aggregation and transport defects might be in neurons expressing endogenous levels of the mutant Mfn2 protein. In addition, mitochondrial accumulation and impaired movement prevented direct analysis of mitochondrial fusion. Thus, the investigators could not determine whether mutant Mfn2 proteins directly impaired mitochondrial transport defect or whether defective movement was a secondary consequence of a change in fusion activity. Our findings that both mitochondrial fusion and distribution are normal in CMT2A patient fibroblasts (no mitochondrial clustering) would suggest that Mfn2 protein-related transport defects may be specific to neurons.
Another study directly tested the fusion function of CMT2A mutant Mfn2 proteins in the presence and absence of other mitofusins (Detmer and Chan, 2007a
). Using this approach, the authors established that the majority of the mutant Mfn2 proteins (including the T105M mutant protein analyzed here) were non–functional for fusion when expressed alone or in the presence of WT Mfn2. However, when the mutant proteins were expressed together with Mfn1, both tubular mitochondrial morphology and normal fusion activity were restored, suggesting that fusion competent complexes were formed between Mfn1 and mutant Mfn2. The finding that Mfn1 can correct the fusion defect of mutant Mfn2 proteins suggested a model for the tissue-specificity of the disease. As proposed by Chan and colleagues, peripheral nerves may be specifically affected in CMT2A patients because they express low levels of Mfn1 (or no Mfn1) and rely primarily on Mfn2 for fusion. By contrast, mitochondrial fusion should be normal in cells that express Mfn1 at levels sufficient to compensate for the Mfn2 defect (Detmer and Chan, 2007a
, Detmer and Chan, 2007b
). Consistent with this model, we found that CMT2A patient fibroblasts express Mfn1, and that mitochondria in these cells fuse normally. In principle, siRNA knockdown of Mfn1 could be used to reveal fusion defects associated with the Mfn2 mutations in CMT2A patient fibroblasts. However, the results of such studies would be difficult or impossible to interpret due to mitochondrial fusion deficiencies and fragmentation caused by Mfn1 knockdown alone (Eura, et al., 2003
). Ultimately, validation of this model will require analysis of Mfn1 and Mfn2 expression levels in a collection of human tissues with an emphasis on the affected neuronal cell types.
Although the creation of a mouse model for CMT2A would greatly facilitate studies of disease pathology and tissue-specificity, initial studies indicate that mice heterozygous for a common CMT2A allele (MFN2+/R94Q
) do not exhibit neurological phenotypes (Detmer and Chan, 2007a
). In addition, conditional Mfn2 knockout mice experience neurodegeneration and exhibit mitochondrial abnormalities in Purkinje cells, but the mouse phenotype does not mimic CMT2A disease (Chen, et al., 2007
). Finally, a transgenic mouse expressing the T105M MFN2
mutation directly in motor neurons was recently described (Detmer, et al., 2007
). These mice display axonal loss, muscle atrophy, and hind-limb gait defects resembling CMT2A, however, the phenotype could only be generated upon significant overexpression of the transgene. Together, these observations suggest that development of a CMT2A mouse model may not be straightforward, and emphasize the importance of carrying out future studies in human stem-cell derived or primary neuronal cultures as well as in CMT2A patient cells and tissues.
Prior to our study, mitochondrial function had only been examined in CMT2A fibroblasts carrying three of the approximately 40 known MFN2
lesions (Loiseau, et al., 2007
). This study, like ours, reported essentially normal mitochondrial morphology, mtDNA content, respiration rates, and respiratory complex activity in patient fibroblasts. In addition, the authors found no significant differences in ATP production, ROS generation, or susceptibility to apoptosis-inducing agents. However, they did report statistically significant increases in oligomycin-insensitive respiration as well as decreases in mitochondrial membrane potential (ΔΨ) and mitochondrial coupling efficiency ([ATP]/oxygen consumed). These results are surprising because respiratory chain dysfunction is often associated with altered mitochondrial morphology, mtDNA integrity, and defects in electron transport components. In addition, the severity of the reported coupling defects is not consistent with the clinical severity of patient symptoms. The authors speculated that CMT2A symptoms may result from neuronal sensitivity to these subtle changes in mitochondrial energetics (Loiseau, et al., 2007
), however, it is not yet clear whether the coupling defects reported by Loiseau et al
. are a direct consequence of the MFN2
mutations or whether these defects do in fact contribute to CMT2A pathology.
While no single model discussed above fully explains the published data, the combined results can be accommodated by a synthesis of all the models. Specifically, the data suggest that fibroblasts and other unaffected CMT2A patient cells express sufficient levels of Mfn1 to allow the formation of functional fusion complexes that maintain mitochondrial integrity. In contrast, peripheral nerves are more sensitive to the presence of mutant Mfn2 proteins due to low levels of Mfn1 (Detmer and Chan, 2007a
, Detmer and Chan, 2007b
). The formation of mostly non-functional Mfn2-Mfn2mutant
fusion complexes (Detmer and Chan, 2007a
) in an environment with little to no Mfn1 would reduce the frequency of efficient fusion and may generate an energetically heterogeneous population of mitochondria in the affected cells (Chen, et al., 2005
, Loiseau, et al., 2007
). It has been shown that axonal transport of mitochondria depends on mitochondrial function, such that energetically compromised mitochondria are preferentially transported from the synapse back to the cell body (Miller and Sheetz, 2004
). As a result, mitochondria are in short supply at the nerve terminal where they are needed to provide energy essential for synaptic function (Verstreken, et al., 2005
). Alternatively, axonal transport defects could arise from mitochondrial aggregation (Baloh, et al., 2007
, Detmer and Chan, 2007a
), perhaps caused by mutant fusion complexes that prolong, or make permanent, the tethering of adjacent mitochondria (Eura, et al., 2003
, Huang, et al., 2007
, Koshiba, et al., 2004
, Rojo, et al., 2002
). In either case, depletion of mitochondria from peripheral nerve axons and terminals could cause progressive degeneration of these structures, producing the CMT2A symptoms observed in the patients. If this cascade of cellular events can be experimentally validated, it is possible that up-regulation of MFN1
expression or delivery of Mfn1 protein to peripheral nerves (Detmer and Chan, 2007a
) might slow or block the progression of clinical symptoms in individuals diagnosed with CMT2A.