Category Archives: Thesis Seminars
“Computational Approaches to the Study of Human Trypanosomatid Infections”
“Advances in Understanding the Genetic Architecture of Cleft Lip and Palate Disorders”
Dr. Jeff Murray
Advisers: Diane Slusarski and Val Sheffield
Place: Braley Auditorium (Pomerantz Family Pavilion)
Elucidating a role for BBS3 in syndromic and non-syndromic retinal disease
Bardet-Biedl Syndrome (BBS, OMIM) is a syndromic form of retinitis pigmentosa, characterized by retinal degeneration, obesity, learning disabilities, congenital abnormalities and increased incidences of hypertension and diabetes. Individuals with BBS are blind by the third decade of life. At least fourteen genes are reported to individual cause BBS. This thesis focuses on BBS3, with the overall goal of characterizing the function of BBS3 in terms of both syndromic and non-syndromic retinal degeneration.
A member of the Ras family of small GTP-binding proteins, BBS3 is postulated to play a role in vesicular transport. A second highly conserved transcript of BBS3, BBS3L, is expressed in the mouse and zebrafish eye. Histological analysis of Bbs3L knockout mice at 9 months reveals disorganization of the inner segments, indicative of retinal degeneration. To further evaluate the functional effects of BBS3 deficiency in the eye, an antisense oligonucleotide (Morpholino) approach was utilized to knockdown bbs3 gene expression in zebrafish. Consistent with an eye specific role, knockdown of bbs3L results in mislocalization of the photopigment green cone opsin and reduced visual function, but not abnormalities of the Kupffer’s vesicle or delays in intracellular trafficking of melanosomes, both cardinal features of BBS in the zebrafish. To dissect the individual functions of BBS3 and BBS3L, BBS3 or BBS3L RNA was co-injected with the bbs3 morpholinos. BBS3L RNA, but not BBS3 RNA, restores green opsin localization and vision. Moreover, only BBS3 RNA is sufficient to rescue melanosome transport. Together these data demonstrate that BBS3L is necessary and sufficient for retinal function and organization.
This work was extended to humans by characterizing the A89V missense mutation in BBS3 identified in retinitis pigmentosa patients. To evaluate the in vivo function of the A89V missense mutation in non-syndromic retinal degeneration and BBS, rescue experiments were performed in the zebrafish. Unlike BBS3L RNA, BBS3L A89V RNA does not rescue the vision defect seen with loss of bbs3 in zebrafish; however, BBS3 A89V RNA suppresses melanosome transport delays. These data demonstrate that the mutation identified in patients with non-syndromic retinal degeneration is critical and specific for the vision defect.
Place: 2117 MERF
Developing RNAi Therapy for DYT1 Dystonia
DYT1 (OMIM) is the most common inherited dystonia, a disabling movement disorder with no effective treatment caused by a dominantly inherited mutation in the protein torsinA. Our group and others have previously demonstrated the therapeutic potential of RNAi for DYT1 dystonia by achieving potent shRNA-mediated silencing of mutant torsinA in cultured cells. In this project we had two main aims: one- to see if we could identify phenotypes that could be utilized to evaluate therapeutic efficacy in preclinical trials and two- to determine whether RNAi therapy could be successfully employed to treat DYT1 murine models. In an effort to identify novel phenotypes we examined whether the presence of mutant torsinA was sufficient to induce changes in transcriptional regulation, miRNA expression or produced an abnormal motor behavior in DYT1 murine models.
During our studies we found that abnormal accumulation of mutant torsinA at the nuclear envelope was not sufficient to induce transcriptional dysregulation in vitro or in vivo, despite its interaction with nuclear envelope proteins. We did find several miRNAs that had significantly altered expression in the cerebellum which may implicate a role for this brain region in DYT1 dystonia. And finally we found no gross motor abnormalities in heterozygous DYT1 knockin (KI) mice or in a novel DYT1 transgenic model indicating that these mice may better embody nonmanifesting DYT1 carriers.
Our preclinical trial to test the feasibility of employing RNAi therapy in DYT1 murine models presented some unexpected results. We found that intrastriatal injections of AAV2/1 vectors expressing different shRNAs, whether targeting torsinA expression or mismatched controls, resulted in significant toxicity with progressive weight loss, motor dysfunction and animal demise. Toxicity was not observed in animals that received control AAV2/1 encoding no shRNA. Interestingly, the different genetic background of both mouse models influenced toxicity, being earlier and more severe in 129/SvEv than C57BL/6 mice, perhaps explained by the lower levels of exportin5 expression observed in 129/SvEv mice. In conclusion, our studies demonstrate that expression of shRNA in the mammalian brain can lead to lethal toxicity. Furthermore, the genetic background of rodents modifies their sensitivity to this form of toxicity, a factor that should be taken into consideration in the design of preclinical therapeutic RNAi trials.
In summary, the studies completed in this thesis contribute important information to the fields of dystonia pathogenesis and therapeutics, and more broadly pertain to the development of therapeutic gene silencing for neurological disease.
The Modulation of ROS by Short Telomere Signaling
Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal the cell to cease division and enter a cell fate termed senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in the telomerase complex or telomere-related genes give rise to the premature aging disorder Dyskeratosis Congenita (DC). DC provides a unique model system to study human aging in relation to telomerase insufficiency and the subsequent accelerated telomere attrition. In this thesis, cells were analyzed from patients with Autosomal Dominant Dyskeratosis Congenita (AD DC, OMIM) caused by a single allele mutation in the telomerase RNA component (TERC) that leads to telomerase haploinsufficiency. These cells were determined to have a severe proliferative defect and extremely short telomeres. It is demonstrated that the short telomeres in AD DC cells initiate a DNA damage response transduced by the p53/p21WAF/CIP pathway which mediate an elevation in steady-state levels of mitochondrially-derived superoxide and oxidative stress. Exogenous expression of the catalytic reverse transcriptase component of telomerase (TERT) activated telomerase in DC fibroblasts but resulted in reduced activity (~50% compared to control fibroblasts); however telomeres were successfully maintained, albeit at a short length. Simultaneous expression of both TERT and TERC led to robust telomerase activity and elongation of telomeres, indicating that TERC haploinsufficiency is a rate-limiting step in telomere maintenance in DC cells. Reconstitution of telomerase activity in AD DC cells ameliorated the proliferative defects, reduced the p53/p21WAF/CIP response and decreased oxidative stress. Increased superoxide and slow proliferation found in DC cells could also be mitigated by inhibiting p21WAF/CIP or by decreasing the oxygen tension to which the cells are exposed. Together, these results support the hypothesis that the insufficient telomerase leads to critically short telomeres which signal the activation of p21WAF/CIP, leading to increased steady-state levels of mitochondrial superoxide and metabolic oxidative stress as a means to engage senescence. These studies provide important insight into mechanisms whereby shortened telomeres lead to premature aging in a human model and point to potential strategies to reduce the effects of tissue dysfunction in DC patients.