Centre for Neuroscience

Oligodendrocyte differentiation and the myelination of axons are crucial aspects of vertebrate central nervous system development and function. The processes of oligodendrocyte differentiation and the expression of these myelin proteins are tightly controlled by a number of transcription factors. Although the transcription factors required for the generation of oligodendrocytes and CNS myelination during development have been relatively well established, it is not known whether continued expression of the same factors is required for the maintenance of myelin in the adult. Myelin Regulatory Factor (MyRF), a putative transcription factor, is critical for the generation of mature oligodendrocytes and the initiation of myelination during development. It has however not been investigated whether MyRF is also required for the ongoing maintenance of myelin once oligodendrocyte differentiation is complete. Moreover, whether it is really a transcription factor has also been unclear, given it contains a putative transmembrane domain and the ability of the MyRF protein to interact with DNA has not been demonstrated. The aims of this thesis were therefore to investigate the roles of MyRF in the mature CNS, and to determine the molecular mechanisms by which it promotes myelination. To investigate whether ongoing expression of MyRF is required to maintain a mature myelinated central nervous system, an inducible conditional knockout strategy was used to ablate MyRF specifically in mature oligodendrocytes of adult mice. This approach resulted in a rapid down-regulation of key myelin genes, followed by an eventual death of many of the recombined oligodendrocytes and a delayed, but severe, CNS demyelination. As such, MyRF has clear roles in regulating myelination beyond the initial phase of oligodendrocyte differentiation. At the molecular level, it was found that MyRF is a novel example of a membrane-tethered transcription factor which undergoes cleavage via a unique autoproteolytic mechanism previously undescribed in eukaryotic cells. This mechanism relies on a protein domain previously only reported in bacteriophage tailspike proteins. This cleavage of MyRF allows the N-terminal cleavage product to translocate to the nucleus, where it directly binds DNA via evolutionary conserved residues and directly promotes expression of myelin genes. Together, these data provide crucial insights into the biological roles of MyRF, establishing it as a novel type of transcription factor with a direct role in regulating the expression of genes required for myelination in both the developing and mature CNS.

Schwann cells are specialised glial cells of the peripheral nervous system responsible for producing the myelin that ensheaths axons enabling salutatory conduction and providing neuroprotective and trophic support. In recent years, significant progress has been made into understanding the function of myelin and Schwann cells especially during development and in health, but several important questions concerning the mechanisms by which the peripheral nervous system responds to specific insults upon this myelinating cell population during adulthood remain unanswered. Currently, most experimental approaches to assess the consequences of myelinating Schwann cell death during adulthood that can be applied to answer some of these questions, use mechanisms that are non-specific, poorly-understood, result in animal death, and are often complicated by immune cell involvement or developmental onset. Such caveats make it difficult to understand the intricate sequence of degenerative and regenerative events and precise glial/axonal interactions responsible for functional outcomes in adult disease contexts. To address these technical limitations, a series of transgenic mouse lines were generated in the Merson laboratory to enable the specific inducible ablation of myelinating Schwann cells and oligodendrocytes, myelinating glial cells of the peripheral and central nervous systems, respectively. MBP-DTR transgenic mice express the human diphtheria toxin receptor (DTR) under the control of the proximal promoter of the murine myelin basic protein (MBP) gene, which is expressed in myelinating Schwann cells and oligodendrocytes. Challenging MBP-DTR mice with diphtheria toxin (DT) results in the specific ablation of myelinating glia via DT-mediated induction of apoptosis in DTR-expressing cells. Using this approach we have already demonstrated in transgenic line MBP-DTR100A, that oligodendrocytes undergo apoptosis resulting in rapid progressive terminal functional deficits within weeks of one intraperitoneal (i.p.) DT injection (10µg/kg). Histological analysis revealed that clinical decline was associated with a reduced density of oligodendrocyte cell bodies, alterations in the molecular and structural composition of nodes of Ranvier but no evidence of overt demyelination. There was some moderate signs of pathology in the peripheral nervous system as well, but this was not thoroughly characterised (Oluich et al., 2012). The major objective of the present work was to characterise both degenerative and regenerative responses to myelinating Schwann cell ablation. To enable such analyses, I have optimised several conditions across several MBP-DTR lines to identify conditions that elicit high survival rates that are appropriate for assessing both the early and late consequences of targeted Schwann cell death. Firstly, this thesis presents results demonstrating that I can modulate clinical parameters across MBP-DTR mouse lines dependent on DTR expression profile, DT dose and sex. From these experiments I have identified one line and one DT dosing paradigm of interest, the MBP-DTR25 line given 10µg/kg DT (i.p.) that elicits moderate non-lethal functional deficits, which is followed by rapid clinical recovery. MBP-DTR25+DT mice develop hindlimb weakness and a reduction in coordination, which peaks within 22-25 days and recovers rapidly by 28 days post-DT. To our surprise, we found that by clinical peak in MBP-DTR25+DT mice, myelinating Schwann cells had undergone apoptosis in the peripheral nervous system, but there was no signs of oligodendrocyte apoptosis in the central nervous system—nor apoptosis in wild-type+DT or MBPDTR25+saline control mice in either nervous systems, as expected. Such findings, illustrate tremendous regional specificity of this line and DT dosing paradigm— an aspect of the system that has been exploited to assess the precise consequences of specifically targeting myelinating Schwann cells in the peripheral nervous system. I have shown that at clinical peak there is; a reduction in the density of mature Schwann cell bodies, an increase in the density of demyelinated axons, evidence of myelin membrane swelling, an increase in the density of macrophages, but no difference in neuron or axon density. In addition, I have shown that mitochondria, sodium channels and potassium channels become redistributed and/or lost along the length of demyelinated axons. To assess whether remyelination plays a role in recovery, I have assessed the extent of Schwann precursor cell proliferation and remyelination. In the peripheral nervous system at 21, 28 and 35 days post-DT, MBP-DTR25+DT mice exhibited an increase in proliferative cells compared to wild-type+DT mice. At these time-points, a subset of proliferative cells were immunoreactive for Sox-10, indicating Schwann precursor cells enter the cell cycle after DT induced ablation of mature Schwann cells. Electron microscopy revealed evidence of remyelination at 28 and 35 days post-DT suggesting that responsive Schwann precursor cells mature rapidly into myelinating Schwann cells by acute clinical recovery. In conclusion, I have shown the precise dynamics of structural, functional and molecular responses of axons and the surrounding environment to targeted myelinating Schwann cell apoptosis at several timepoints post Schwann cell death. For the first time this has been shown in a model where non-specific direct damage to the axon does not confound outcomes, making this model ideal for understanding the precise cause-and-effect relationship between the axon and myelinating Schwann cells in the adult peripheral nervous system.

Centre for Neuroscience - Theses

Permanent URI for this collection

Filters

Date

Author

Subject

Type

Settings

Sort By

Results per page

Statistics

Citations

Search Results