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    From Neurodegeneration to Infertility and Back - Exploring Functions of Two Genes: ARMC4 and TARDBP: A Dissertation

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    Authors
    Cheng, Wei
    Faculty Advisor
    Zuoshang Xu, MD, PhD
    Academic Program
    Interdisciplinary Graduate Program
    UMass Chan Affiliations
    Biochemistry and Molecular Pharmacology
    Document Type
    Doctoral Dissertation
    Publication Date
    2014-01-10
    Keywords
    Dissertations, UMMS
    Amyotrophic Lateral Sclerosis
    DNA-Binding Proteins
    Armadillo Domain Proteins
    Gudu protein, Drosophila
    Drosophila Proteins
    Spermatogenesis
    Amyotrophic Lateral Sclerosis
    DNA-Binding Proteins
    Armadillo Domain Proteins
    Drosophila Gudu protein
    Drosophila Proteins
    Spermatogenesis
    Biochemistry
    Cellular and Molecular Physiology
    Developmental Biology
    Developmental Neuroscience
    Nervous System Diseases
    Reproductive and Urinary Physiology
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    Abstract
    Amyotrophic Lateral Sclerosis (ALS) is an adult-onset progressive neurodegenerative disease that causes degeneration in both upper and lower motor neurons. ALS progresses relentlessly after the onset of the disease, with most patients die within 3-5 years of diagnosis, largely due to respiratory failure. Since SOD1 became the first gene whose mutations were associated with ALS in 1993, more than 17 ALS causative genes have been identified. Among them, TAR DNA-binding protein (TARDBP) lies in the central of ALS pathology mechanism study, because TDP43 proteinopathy is observed not only in familial ALS cases carrying TARDBP mutations, but also in most of the sporadic ALS cases, which account for 90% of the whole ALS population. Several TDP43 overexpression mouse models have been successfully generated to study the gain-of-toxicity mechanism of TDP43 in ALS development, while the investigation of loss-of-function mechanism which could also contribute to ALS still awaits a proper mouse model. The major difficulty in generating TARDBP knock out mouse model lies in the fact that TARDBP is a development essential gene and complete depletion of TDP43 function causes embryonic lethality. In chapter I, I reviewed the recent advances in ALS study. Emphasis was given to ALS mouse models, especially TARDBP ALS mouse model. In Chapter II, I made a Tet-responsive construct that contains mCherry, a fluorescent protein, as an indicator for the expression of the artificial miRNA (amiTDP) residing in the 3’UTR of mCherry and targeting TARDBP. The construct was tested in NSC34 cells and TRE-mCherry-amiTDP43 transgenic mouse was generated with this construct. Crossing TRE-mCherry-amiTDP43 mouse with mPrp-tTA mouse, mCherry expression was successfully induced in mouse forebrain and cerebellum, but not in other tissues including spinal cord. By quantitative real-time PCR, amiTDP43 expression was confirmed to be coupled with mCherry expression. Fluorescent immunostaining revealed that mCherry was expressed in neurons, but not in astrocytes or microglia cells, and that in mCherry positive cells, TDP43 was significantly knocked down. Results from Nissl staining and GFAP immunostaining suggested that decrease of TDP43 in forebrain neuron only was not sufficient to cause neurodegeneration and neuron loss. In chapter III, I investigated the function of Armadillo Containing Protein 4 (ARMC4), which was originally considered ALS causative gene. Our study of the function of CG5155, the possible homolog of ARMC4 in Drosophila, indicated that CG5155 is a male fertility gene that is involved in spermatogenesis. Therefore, we have named this gene Gudu. The transcript of Gudu is highly enriched in adult testes. Knockdown of Gudu by a ubiquitous driver leads to defects in the formation of the individualization complex that is required for spermatid maturation, thereby impairing spermatogenesis. Furthermore, testis-specific knockdown of Gudu by crossing the RNAi lines with Bam-Gal4 driver is sufficient to cause the infertility and defective spermatogenesis. Since Gudu is highly homologous to vertebrate ARMC4, also an Armadillo-repeat-containing protein enriched in testes, our results suggest that Gudu and ARMC4 is a subfamily of Armadillo-repeat containing proteins with an evolutionarily conserved function in spermatogenesis.
    DOI
    10.13028/M2JS4J
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/32051
    Rights
    Copyright is held by the author, with all rights reserved.
    ae974a485f413a2113503eed53cd6c53
    10.13028/M2JS4J
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