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Date of Award
Doctor of Philosophy (PhD)
The vertebrate nervous system requires myelinating glia for the fast propagation of action potentials, as well as for vital trophic support to axons. Myelinating glia produce myelin, which is a lipid-rich, multi-lamellar sheath that surrounds axons and allows for rapid electrical signaling. In the central nervous system (CNS), myelin is produced by oligodendrocytes, while in the peripheral nervous system (PNS), Schwann cells perform this function. Although glia have historically been understudied compared to neurons, recent research has uncovered critical roles for glia in nervous system development and disease. Disruption to myelin or to the glial cells that generate myelin can have severe consequences for human health, as demonstrated by the debilitating symptoms of multiple sclerosis (MS) and Charcot-Marie-Tooth disease (CMT). In order to improve patient health, it is necessary to determine the etiology of demyelinating diseases, which in turn requires a comprehensive understanding of glial cell development and myelination. The scientific advances made in our understanding of myelinating glial cell development will be discussed in Chapter 1 of this dissertation. Although great progress has been achieved, our understanding of the molecular mechanisms that regulate myelination is incomplete.
The zebrafish has emerged as an important model organism for studying myelin. In particular, the ability to perform forward genetic screens in zebrafish has greatly increased our understanding of the individual genes involved in myelination in both the CNS and the PNS. Although several myelin-related forward genetic screens have been previously performed in zebrafish, these screens were not done to saturation, potentially leaving essential genes unidentified. Our lab therefore performed a large scale forward genetic screen to uncover new players in myelin development. The screen was a collaborative effort between students in the Monk lab and members of the Solnica-Krezel lab. The myelin screen was highly successful, uncovering 31 mutants. The set-up and outcome of the screen is described in more detail in Chapter 2.
One of the mutants identified in the screen was found to be the result of a mutation in the gene actin related protein 10 (actr10). Actr10 (or Arp11) is a component of the dynactin complex, which is necessary for retrograde transport of cargo by cytoplasmic dynein. Two alleles of actr10 zebrafish mutants, actr10stl83 (the allele originally identified in the screen) and actr10nl15 (a presumptive null generously shared by the Nechiporuk lab) exhibited reduced myelin in the CNS and in the PNS, as well as a punctate expression of myelin basic protein (mbp) in the hindbrain. Mbp has important roles in myelin compaction as well as in initiating wrapping of myelin around axon segments. Initial characterization of actr10nl15/nl15 mutants revealed a reduction in oligodendrocyte precursor cells, fewer myelinated axons by ultrastructural analysis, and increased cell division in mutant oligodendrocytes. Moreover, the punctate mbp phenotype was reminiscent of another zebrafish mutant in the anterograde kinesin motor kif1b. Importantly, mbp mRNA is transported and translated locally at the developing myelin sheath. I hypothesized that dynein/dynactin regulates anterograde transport of mbp mRNA in oligodendrocytes. To test this, I collaborated with another lab and found that indeed, mbp mRNA transport was arrested/perturbed in both rat oligodendrocyte cell culture and zebrafish in response to dynein inhibition, demonstrating a previously unknown role for dynein/dynactin in mbp transport. This published work can be found in its entirety in Chapter 3.
In addition to myelin defects, actr10 zebrafish mutants exhibited axonal swellings in both the CNS and in the PNS. Electron microscopy revealed neurofilament accumulation in the axons of mutant animals, which is a hallmark of many neurodegenerative disorders. We therefore wondered whether ACTR10 might have a role in human disease. In collaboration with a neurologist at Washington University, several patients diagnosed with amyotrophic lateral sclerosis (ALS), distal myopathy and CMT were also found to have mutations in ACTR10. Using genome editing technologies in zebrafish, we generated a line of zebrafish containing the CMT2 patient ACTR10 mutant single nucleotide polymorphism (SNP), thereby generating a patient specific disease zebrafish model. Current work is ongoing to characterize the zebrafish mutant and future experiments could include drug screens to identify compounds that may ameliorate CMT mutant phenotypes. Generation of the this CMT zebrafish line and future directions for this project are described in Chapter 4.
From a forward genetic screen to identify novel regulators of myelination to generating patient specific mutations in zebrafish, my dissertation has involved a broad range of genetic and molecular techniques in the study of nervous system development in general, and myelinating glial development in particular. The identification of Actr10 as a player in oligodendrocyte development and myelination, as well as a potential regulator of a major human demyelinating disorder, demonstrates the power of zebrafish to address both basic and biomedical questions directly relevant to human patients.
Chair and Committee
Kelly James R. Monk Skeath
Aaron DiAntonio, Charles Kaufman, Lila Solnica-Krezel,
Herbert, Amy, "The Role of Actr10 in Nervous System Development and Disease" (2018). Arts & Sciences Electronic Theses and Dissertations. 1535.
Available for download on Wednesday, April 24, 2019