Publication Date

2015-04-29

Availability

Open access

Embargo Period

2015-04-29

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Neuroscience (Medicine)

Date of Defense

2015-04-03

First Committee Member

Stephan L Zuchner

Second Committee Member

Miguel Perez-Pinzon

Third Committee Member

Carlos Moraes

Fourth Committee Member

Gavriel David

Fifth Committee Member

Michael Shy

Abstract

Charcot-Marie-Tooth disease (CMT) is a common, hereditary, length-dependent peripheral neuropathy roughly classified as either demyelinating (CMT1) or axonal (CMT2). CMT has no known cure, with patients relying on corrective surgeries and braces, as well as low-stress strength training, for support. Though the disease is highly heterogeneous, many of the most severe axonal cases are caused by dominant mutations in mitofusin 2 (MFN2). MFN2 (and its paralog, MFN1) reside in the outer mitochondrial membrane, where they participate in various mitochondrial functions, some of which are complimentary (including mitochondrial fusion and transport). The mechanism by which MFN2 mutations cause CMT are unknown. A robust animal model could inform our pathologic understanding of the disease and could be used in pre-clinical trials for novel therapies. We have characterized the first knock-in mouse model using a common mutation seen in human patients, Mfn2R94W (Chapter 2). Homozygous inheritance of the mutation was found to be lethal within a day of birth, with concurrent deficits in oxygen consumption, ATP production, and mitochondrial network morphology. Heterozygous animals showed only mild changes, including nerve morphology, age-dependent movement and rearing behaviors, and mitochondrial network morphology. In order to produce a more severely affected model, we took a number of approaches to stress the surviving heterozygous animals (Chapter 3, 4). Three different genetic approaches and two different chemical approaches (tested at multiple doses) were unable to elicit a pronounced neuropathic phenotype in these mice. One specific challenge, however, revealed decreased nerve regenerative capabilities: optic nerve crush with ciliary neurotrophic factor (CNTF) treatment (Chapter 4). In humans, the R94W mutation has been linked to optic nerve degeneration that presents after onset of peripheral neuropathy, which is an uncommon phenotype in CMT patients. Therefore we reasoned this tissue may be particularly susceptible to insult. Wild type animals that underwent nerve crush with CNTF treatment showed robust axonal regeneration that was not matched by heterozygotes. Over 80 genes are known to cause CMT, with wide-ranging cellular functions and proposed mechanisms. MFN2 is involved in mitochondrial fusion, axonal transport, mitochondrial tethering to the ER, mitophagy, and mitochondrial metabolism, further confounding pathological understanding of the disease. To better understand the pathways that might contribute to MFN2-induced neuropathy, we performed transcriptome analysis using RNA-sequencing data from neural tissue isolated from newborn mice (Chapter 5). Differential expression between genotypes did not include significant levels of genes involved in any known MFN2 functions, however, we did see significant downregulation of known CMT genes, of genes transcriptionally regulated by EGR2, and of genes differentially expressed in other mouse models of CMT and delayed peripheral nerve regeneration. In conclusion, we have described the first Mfn2 knock-in model of CMT, which displays homozygous lethality, mostly mild heterozygous phenotypes, and a unique vulnerability to optic nerve crush. Expression analysis revealed differential expression of known CMT genes and genes involved in Schwann cell functions, indicating cohesive forces among the broad genotypic and phenotypic variance of this disease. Future studies may reveal the nature of these interactions.

Keywords

Charcot-Marie-Tooth; CMT2; MFN2; peripheral neuropathy

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