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Sager, Jonathan (2012) USING ZEBRAFISH AS A MODEL SYSTEM FOR DYT1 DYSTONIA. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Dystonia is characterized by sustained involuntary muscle contractions producing repetitive twisting movements and abnormal postures. DYT1 dystonia, an early-onset primary dystonia, is caused by a trinucleotide deletion in the TOR1A gene, resulting in the loss of a single glutamic acid in the TorsinA protein. It is unknown how this mutation causes dysfunction of CNS motor circuits resulting in dystonia. The aims of this work were: (i) characterize the zebrafish homolog of human TOR1A in order to elucidate the functions of Torsins in vivo; (ii) generate transgenic zebrafish models of DYT1 dystonia suitable for mechanistic and drug discovery studies. An ancestral tor1 gene found in the genomes of several fish species was duplicated at the root of the tetrapod lineage. In zebrafish, tor1 is expressed as two isoforms with unique 5' exons. The amino acid sequences of both Torsin1 isoforms are 59% identical and 78% homologous to human TorsinA. A novel antibody was generated against Torsin1, and immunoreactivity was detected broadly in zebrafish CNS neurons. Introduction of ATP-hydrolysis abrogating mutations in the Walker B domain of Torsin1 caused a relocalization of the protein from the endoplasmic reticulum to the nuclear envelope in vitro, similar to findings with human TorsinA. Transient knockdown of tor1 expression during embryonic and early larval development did not produce a detectable cellular or behavioral phenotype, suggesting that essential functions of tor1 occur later in development, or that compensatory functions are provided by other Torsin family proteins. Co-expression of Torsin1 and the dystonia-associated human mutant TorsinA caused Torsin1 to relocalize to the nuclear envelope, strongly suggesting that human TorsinA and zebrafish Torsin1 interact. In view of this interaction, and the proposed dominant-negative mechanism whereby the DYT1 mutant causes clinical disease, we generated stable transgenic zebrafish in which the dystonia-related human TorsinA[ΔE] mutant was expressed in neurons of the zebrafish CNS. These transgenic animals exhibited a transient, juvenile-onset hypokinetic phenotype, beginning around one month of development and lasting for approximately one week. Future studies using these transgenic zebrafish will aim to elucidate the physiological and molecular basis of this phenotype and its relation to dystonia.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Sager, Jonathanjjs81@pitt.eduJJS81
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairLillien, Lauralillien@pitt.eduLILLIEN
Thesis AdvisorTorres, Gonzalogtorres@pitt.eduGTORRES
Committee MemberBurton, Edwardeab25@pitt.eduEAB25
Committee MemberHukriede, Neilhukriede@pitt.eduHUKRIEDE
Committee MemberMonaghan-Nichols, Paulamonaghan@pitt.eduMONAGHAN
Committee MemberOzelius, Laurielauri.ozelius
Date: 25 August 2012
Date Type: Publication
Defense Date: 12 June 2012
Approval Date: 25 August 2012
Submission Date: 17 August 2012
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 142
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Neurobiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Zebrafish Dystonia Animal Model
Date Deposited: 25 Aug 2012 15:57
Last Modified: 25 Aug 2017 05:15


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