Charcot-Marie-Tooth disease (CMT) is a hereditary peripheral neuropathy resulting from demyelination and axon degeneration. Many cases of axon degeneration are caused by mutations in Mitofusin-2 (Mfn2), which is one of two dynamin-like proteins on the surface of mitochondria. Mitofusins primarily mediate mitochondrial outer membrane fusion, but Mfn2 can also promote association between ER and mitochondria in mitochondria associated membranes (the MAM). It is observed in spots that colocalize with the mitochondrial fission dynamin Drp1 and it was proposed to affect axonal transport of mitochondria, raising the possibility that CMT is caused by defects in one of these other functions of Mfn2. Preliminary data show that Mfn2 promotes constriction of mitochondria in Drp1?/? cells and fission in Drp1-Mfn1 DKO cells when treated with the fungal toxin PXA that causes the release of calcium from the mitochondrial matrix. Calcium release and the ability to constrict mitochondria was linked to Mfn2-mediated regulation of NCLX (a Ca/Na exchanger in the mitochondrial inner membrane). It is hypothesized that PXA activates NCLX and that Mfn2 is required for this activity. It is also hypothesized that NCLX-mediated calcium release causes mitochondrial constriction and that the effects of Mfn2 on mitochondrial fission are linked to axonal transport, a process that could be disrupted in CMT patients. These hypotheses will be tested by investigating three aims.
Aim 1. Investigate connections between PXA, NCLX, and Mfn2. These studies will include comprehensive tests whether PXA triggers calcium release from mitochondria by activating NCLX and investigates of the control of NCLX by Mfn2.
Aim 2. Investigate connections between Mfn2 and mitochondrial fission. Effects of Mfn2 and NCLX on mitochondrial fission will be tested with knockout cell lines and transfections of fission and fusion protein constructs followed by analyses with a range of imaging techniques.
Aim 3. Investigate the physiological consequences of Mfn2 and NCLX contributions to fission. Effects on mitochondrial transport proteins will be examined with kymographs of axonal processes in cultured neurons and zebrafish. Alternative functions, such effects on metabolism and a role in mitophagy, will also be considered. Together, these experiments will help establish NCLX as the target of PXA, assess the newly proposed role of Mfn2 in mitochondrial fission, and test possible downstream effects on transport or mitophagy. These experiments may therefore reveal a novel function for Mfn2 and shed new light on the underlying causes of CMT. Possible downstream effects of fission on axonal transport will change the understanding of the underlying causes of CMT and may suggest novel treatment strategies.
Charcot-Marie-Tooth disease (CMT) is a hereditary neuropathy caused by gene mutations that lead to demyelination and axon degeneration of peripheral neurons. This proposal follows through on data suggesting that mutations in one of the CMT genes called Mfn2 affects mitochondrial fission and axonal transport in unexpected ways. A successful outcome of this project can provide new insight into the underlying causes of the disease and it may point the way towards developing novel treatment strategies.