The Neurofibromatosis 2 (NF2) disorder affects approximately l in 40,000 individuals. The primary physical manifestations of the disease are formation of bilateral vestibular schwannomas that lead to hearing and balance impairment (Evans et al 1992). Mutations in the ef2 gene, located on chromosome 22 in humans, have been mapped to all 17 exons. The nj2 gene encodes merlin, a 595 amino acid tumor suppressor protein that is believed to regulate mitogenic signaling in Schwann cells (SCs ). An in-frame deletion of exon 2 has been shown to lead to a more severe form of the disorder (Baser et al 2005). Exon 2 encodes amino acids 40-80 that serve as a paxillin binding domain l (PBD l) that is necessary for merlin's localization to the plasma membrane (Fernandez-Valle et al 2002). An nf2flox2/flxo2 mouse model was developed for conditional deletion of nj2 exon 2 using the Cre-Lox system (Giovannini 2000). These mice developed schwannomas by l 0 months of age. The goal of this thesis was to develop an in vitro model for studying the loss of merlin expression in SCs. SCs were removed from nf2flox2/flxo2 mice, and were infected in vitro with an adenovirus expressing Cre-Recombinase (Adeno-Cre) to excise exon 2, or with an adenovirus expressing B-Gal (Adeno-B-Gal) as a control. SCs were studied for nine days after infection. The nf2flox2/flxo2 SCs expressed nuclear CreRecombinase (Cre) by two days post-infection, and underwent a change in morphology beginning at day three. SCs transitioned from a typically bipolar shape when confluent to either a rounded and spread morphology or extended many processes becoming multipolar. Also by three days post-infection, there was a decrease in the expression of merlin, and a loss of its plasma membrane localization in ere-expressing SCs. Furthermore, there was a persistent increase in erbB2 expression at the plasma membrane in filopodia and other membrane protrusions in ere-expressing SCs. Finally, nf2flox2/flxo2 SCs expressing Cre displayed disordered growth by nine days post-infection in sub confluent cultures. These results suggest that Adeno-Cre viral infection of nf2flox2/flxo2 SCs in vitro leads to phenotypic changes observed in human schwannoma cells, and therefore is a suitable model for the study of loss of merlin expression in SCs.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:honorstheses1990-2015-1650 |
| Date | 01 January 2007 |
| Creators | McLain, John |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | HIM 1990-2015 |
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