Studies suggest neurodegenerative injury in Charcot-Marie-Tooth disease, says researcher

October 1, 2008

Research using a conditional knockout mouse model of the mitofusin 2 gene are providing insights into the optic nerve damage sometimes associated with Charcot-Marie-Tooth disease. Results to date show degenerative changes in the retrobulbar optic nerve suggesting a response to a neurodegenerative injury.

Key Points

Los Angeles-Studies in a mouse model with a mutation in the mitofusin 2 (MFN2) gene are providing information about optic nerve damage associated with Charcot-Marie-Tooth (CMT) disease. Mild to moderate degenerative changes in the retrobulbar optic nerve have been found, suggesting a response to a neurodegenerative injury, said Fred N. Ross-Cisneros.

The MFN2 gene is required for normal mitochondrial fusion; if mutated, it can lead to neurologic disorders involving neuropathy and optic atrophy, including CMT disease.

The mouse model could serve as a tool for understanding how the mitofusins contribute to optic atrophy and other disorders associated with dysfunctional mitochondrial fusion, said Ross-Cisneros, laboratory supervisor in the neuro-ophthalmology lab of Alfredo Sadun, MD, PhD, Doheny Eye Institute, Keck School of Medicine, University of Southern California (USC), Los Angeles.

The Sadun lab worked with several other labs to develop a different mouse model with a conditional knockout (CKO) gene for MFN2. Yasuhide Furuta, PhD, and colleagues at the University of Texas, Houston, used the homeobox SIX3 gene to insert MFN2 into the eye, optic nerve, and parts of the brain only. This approach enabled the mice to survive, which in turn permitted researchers to study the visual system in adult animals.

Others involved in the development of the mouse model and breeding of the animals included David Chan, MD, PhD, and Hsiuchen Chen, PhD, of the California Institute of Technology, and Yi-Hsin Liu, PhD, of USC.

"This animal model gives us further clues as to what may be going on in Charcot-Marie-Tooth disease in a patient who has optic atrophy," Ross-Cisneros said. The research is supported by grants from Research to Prevent Blindness and the National Eye Institute.

According to Ross-Cisneros, an understanding of mitochondria is the basis of the MFN2 research.

Mitochondria are double membrane-bound organelles found in most eukaryotic cells. They maintain their own DNA and RNA and produce most of the cells' energy source, adenosine triphosphate. They also have other functions, including cell signaling, differentiation, and apoptosis.

Mitochondria constantly remodel and reshape through fission and fusion, forming a network throughout the cell, primarily around the nucleus. Fusion, which creates large, thread-like networks, involves mitochondrial proteins MFN1, MFN2, and OPA1. Impairment of this process causes increased mitochondrial fragmentation. Mutations are associated with neurodegenerative disorders such as autosomal dominant optic atrophy (linked to OPA1) and CMT disease, which is linked to MFN2 as well as many other mutations.

Using the new CKO mouse model of MFN2, Ross-Cisneros and colleagues examined tissues at the ultrastructural level with the transmission electron microscope (TEM). Two-month-old MFN2 CKO mice and controls were euthanized, and the optic nerves were dissected and fixed, embedded in plastic, and sectioned and stained.

Axonal and glial changes

Examination with the TEM revealed that the control mice appeared normal, whereas the CKO mice demonstrated several axonal and glial changes. An increased number of mitochondria, both normal and degenerative, were observed in the large axons, as was condensation of axoplasm. Changes in the small and medium axons included both demyelination and remyelination. The glial changes observed were degeneration of oligodendrocytes (apoptosis), activation of astrocytes (evidence of mitochondrial fission or fragmentation), giant mitochondria, and an increased number of microglia. The giant mitochondria have difficulty fusing with other mitochondria and are very inefficient at producing energy.

The significant changes to the mitochondria, axons, and their supporting glia are typical of the response to a neurodegenerative injury.

In the future, Ross-Cisneros said, he hopes to evaluate the ultrastructural features of the optic nerves in younger mice and particularly in animals older than the 2-month-old mice used in the recent study. Preliminary evaluation of a group of mice that are about a year old indicates that their optic atrophy has worsened.

"Often, with mitochondrial disorders, as you get older the mutation causes further damage to tissue," he explained.

This study was done of the retrobulbar area of the eye, and plans also call for examination of the axons that are entering the optic nerve head. This location is where the axons become myelinated and, therefore, are more susceptible to damage. Future studies also will include morphometric analysis of nerve size, axonal number, and density and measurement of the mRNA of MFN2 proteins to evaluate gene expression at different ages.

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