Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Neurosciences

Language

English (en)

Date of Award

1-1-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Robert Baloh

Abstract

The ubiquitous finding of axonal degeneration in a number of the most prevalent neuropathologies marks the importance of understanding axonal biology and the axonal self-destruct mechanism. Though our understanding of axonal degeneration remains largely incomplete, several down-steam steps of the molecular cascade have been elucidated. While this insight has emerged from models of axon degeneration following physical injury or toxic insult, a more comprehensive understanding of the upstream events may be gained from studying primary axonopathies with defined genetic causes. This dissertation aims to elucidate a molecular mechanism underlying the loss of axons in Charcot-Marie-Tooth Disease type 2A, which is caused by mutations in the mitofusin 2: MFN2) gene. Utilizing an in vitro culture system, we find that CMT2A associated MFN2 mutants disrupt the transport of axonal mitochondria in DRG neurons. Though MFN2 has a previously defined role in facilitating mitochondrial fusion, we propose a direct role for MFN2 in mediating transport based on its interaction with key components of the mitochondrial transport apparatus and perturbation of transport in MFN2 null DRG neurons. MFN2 does not provide a direct link between mitochondria and microtubule based motors, but is poised to mediate transport by a still undefined mechanism. The ability of MFN2 to mediate transport is separate from its ability to mediate fusion as MFN2 disease mutants, that have been shown to retain their ability to fuse mitochondria, cannot rescue the transport deficit in MFN2 null neurons. Additionally, loss of mitochondrial fusion by knockdown of opa-1 is not sufficient to disrupt mitochondrial transport despite reduced mitochondrial function. These findings may explain why mutations in or haplo-insufficiency of opa-1 leads to Dominant Optic Atrophy: DOA) but not degeneration of long peripheral axons, highlighting the potential importance of mitochondrial transport for axon integrity. To further test our hypothesis that mitochondrial transport is critical for the integrity of axons, we expressed MFN2 mutants in cultured DRG neurons and looked for signs of degenerating axons. High levels of Ca2+€€ or reactive oxygen species: ROS) delineated a population of degenerating axons that we not observed in opa-1 knock down cultures. Mitochondria in MFN2 mutant expressing cells showed little change in membrane potential compared to a significant change in the mitochondrial membrane potential of opa-1 silenced cells; however, both groups of cultured neurons upregulated glycolysis and were sensitive to treatment with 2DG. We hypothesize that these changes in the MFN2 mutant expressing cells could be explained by lack of mitochondrial flux across segments of axon which must resort to use of glycolysis. The absence of mitochondria could cause segments of axon to become vulnerable to local perturbations in energy or Ca2+ levels and explain the axonal degeneration observed in culture. In this way, disrupted redistribution of mitochondria in CMT2A patients would put the longest axons at the highest risk due to the probability of incurring at least one insult along its length for which mitochondria could not compensate. Finally we attempted to study an animal model of CMT2A to see if our in vitro findings we recapitulated in vivo. To this end we obtained a mouse line in which the R94Q mutation had been knocked in to the endogenous allele. To accurately mirror conditions in CMT2A patients we chose to analyze heterozygous mice. Though homozygous mice die by the third postnatal week, heterozygous mice are phenotypically normal showing no signs of axon loss or muscle denervation. Differential expression of the MFN2 homologue MFN1, absolute length of axons or absolute time to disease may account for the discrepancy between the mouse model and human patients. Hopefully this work will help elucidate the molecular mechanisms underlying CMT2A and contribute toward a more general understanding of why axons degenerate.

Comments

Permanent URL: http://dx.doi.org/10.7936/K70863B0

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