Heparan Sulfate Proteoglycan Agrin in CNS Development and Aging

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Title: Heparan Sulfate Proteoglycan Agrin in CNS Development and Aging
Author: Liu, I-Hsuan
Advisors: Philip L. Sannes, Committee Member
Jane L. Lubischer, Committee Member
Jeffrey A. Yoder, Committee Member
Robert R. H. Anholt, Committee Chair
Abstract: Agrin is an extracellular matrix heparan sulfate proteoglycan and is best known for its role as the organizer of the neuromuscular junction. Emerging in vitro evidence shows that it plays many possible roles in the central nervous system. The studies present in the Chapter 1 examine the hypothesis that agrin also plays a role in Parkinson's disease. In support of the hypothesis, this study demonstrates that agrin binds to α-synuclein via its heparan sulfate glycosaminoglycans chains, potentiates conformational changes in α-synuclein into β-sheet structure, and enhances insolubility of α-synuclein. Furthermore, agrin is also found colocalized with α-synuclein in neuronal Lewy bodies in the substantia nigra of Parkinson's disease human brain. These results suggest that agrin is capable of potentiating the formation of α-synuclein amyloidosis in Parkinson's disease brain and may indicate shared molecular mechanisms leading to the pathophysiology in Alzheimer's disease and Parkinson's disease, two most common neurodegenerative diseases. Zebrafish has been an attractive animal model for neural development. To begin to address agrin's function in central nervous system, a study on agrin knockdown in zebrafish is present in Chapter 2. Agrin mRNA is detected as a maternal message in embryonic zebrafish, and is expressed in the developing central nervous system and in nonneural structures such as somites and notochord. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon–Beard sensory neurons are also observed, which included truncation of axons and increased branching of motor axons, suggesting roles of agrin in both axon outgrowth and guidance. Moreover, agrin morphants exhibit significantly inhibited tail development, as well as defects in the formation of the midbrain–hindbrain boundary and reduced size of eyes and otic vesicles. These results show that agrin plays an important role in the development of both peripheral and central nervous system in zebrafish. Among the phenotypes that result from agrin knockdown using morpholino antisense oligonucleotides is reduced eye size in agrin morphants. The studies presented in Chapter 3 show that retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted in a dose-dependent manner following agrin morpholino treatment. Pax6.1 gene expression, a marker of eye development, is markedly reduced in agrin morphants, providing support for agrin's role in retinal development. Increased apoptosis is detected suggesting the reason for microphthalmia. Both pax2a and atoh7 gene expressions are decreased suggesting that both FGF8 and sonic hedgehog signaling are affected. Confocal micrographs of HuC-GFP transgenic zebrafish and immunostaining showed impaired differentiation and disorganization in all three cellular layers in the retina in agrin morphants. MHB formation, and expression of mRNAs in this organizer region, is disrupted in agrin morphants. The retinotectal topographic projection to the optic tectum is also perturbed in agrin morphants suggesting the role of agrin for FGF8 signaling. Collectively, these phenotypes in agrin morphants provide support for a crucial role of agrin in retinal development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.
Date: 2008-04-06
Degree: PhD
Discipline: Comparative Biomedical Sciences
URI: http://www.lib.ncsu.edu/resolver/1840.16/5644


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