Kellie Schaefer and Tyson Fuller to present at Genetics Student Seminar 6/13/2016
Mutation in regulatory protease, CAPN5, results in eye disease
Uveitis (intraocular inflammation) causes significant visual morbidity and blindness in all ages, yet it has proven refractive to current therapies, underscoring the critical need for new clinical approaches. Nevertheless, a major barrier to developing new medicines has been the lack of any specific molecular cause for the disease. To gain insight into the molecular mechanisms driving uveitis, we sought the disease-associated gene in kindreds with a Mendelian form—Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV). Our studies showed the underlying cause of ADNIV was mutations in CAPN5 (calpain-5), making CAPN5 the first nonsyndromic uveitis gene discovered. This discovery provides an unprecedented starting point for investigating the substantial gap in our understanding of the molecular basis of ocular inflammation.
CAPN5 is a calcium-activated, intracellular protease expressed at photoreceptor synapses. Similar to the function of other calpains, we believe that proteolysis by CAPN5 is a tightly controlled, post-translational regulatory mechanism that deploys protein fragments with altered activity. Although it is a member of a relatively large, well-studied family, little is known regarding the CAPN5 natural substrates. One exception to this is CAPN5 autoproteolysis, which uncovers one natural substrate of CAPN5: CAPN5 itself. We aim to identify the sequences in CAPN5 that are the targets of autoproteolysis and also to determine the CAPN5 cleavage site in a substrate we recently identified, platelet-derived growth factor B (PDGFB). PDGFB was previously identified as a protein cleaved into an active angiogenic form by an unknown protease, and PDGFB is active in a number of similar eye diseases, including AMD, PVR and diabetic retinopathy. The insight gained from these studies may eventually be used to design therapies for a variety of previously untreatable eye diseases.
Functional Characterization of Epilepsy Related Genes in Zebrafish
TD Fuller1,2, TA Westfall1, DC Slusarski1
1Department of Biology, University of Iowa, Iowa City, IA 52242, 2Interdisciplinary Graduate Program in Genetics
Statement of Purpose: Epilepsy is a chronic condition of recurrent seizures which affects approximately one percent of the general population. Though several causative genes have already been identified, these account for only a small percentage of all the genetically caused cases of epilepsy. Further, even when genes are identified, we lack tractable animal models to rapidly translate these findings into mechanistic insights and ultimately new anti-epileptic therapies. For this reason, the zebrafish is increasingly being used as a model of epilepsy due to its high genetic and physiologic homology to humans and its seizure-like behavior in response to various pharmacological and genetic manipulations. Using High throughput sequencing, a significant number of gene variants are being identified, yet their role in the disease state remain unknown. My project utilizes the zebrafish and focuses on characterizing the functional role of 15 genes in the NIH Undiagnosed Diseases Program for which mutations have been associated with epilepsy, and for which zebrafish orthologues have been identified. Methods: I isolated the zebrafish orthologues and characterized gene expression patterns by RTPCR and whole mount in situ hybridization techniques. We previously demonstrated that knockdown of Prickle (PK), a gene associated with human epilepsy, sensitizes zebrafish to seizure-inducing drugs through the use of larval motility assays. To facilitate high-throughput in vivo screens, I adapted this approach and developed a code to rapidly and efficiently analyze the generated data sets. This allowed for the characterization of the 15 candidate genes in the context of seizure sensitization. Results: Of the fifteen candidates, I found five: syne1b, sms, ccdc89, wscd1, and nid2a, to result in seizure sensitization when knocked down in the zebrafish. I show that each of these genes is expressed in specific regions in the brain during critical times of neuronal development. Further, I find genes expressed in the retina result in axon defects when knocked down.