Lily Paemka and Yan Zhang to present their research on 9th August 2012

Background for Lily Paemka’s talk

EVALUATING PRICKLE MUTATIONS IN HUMAN MYOCLONIC EPILEPSY

Epilepsy is a costly, complex, disabling, genetic brain disorder characterized by seizures that afflicts ~50 million people worldwide. The underlying causes in over 70% of cases remain unknown; impeding effective diagnosis and treatment. There is therefore a critical need to comprehensively characterize their pathophysiology in order to complete known epilepsy-related pathways and develop new therapeutic approaches. Progressive Myoclonic Epilepsy (PME) is a rare epilepsy syndrome characterized by neurodegeneration, myoclonus, and generalized seizures. Mutations in Prickle1 and Prickle2 have been shown to be strongly associated with PME in families and the general population. Prickle proteins function in the non-canonical WNT pathway which regulates calcium release: Increase in intracellular calcium is a process critical for NFAT (Nuclear factor of activated T-cells) activation. Apart from its well characterized importance in immune response, recent undisputable evidence shows that NFAT/calcineurin pathway is important for axonal growth. NFAT mouse models have defects in sensory axon projection and commissural axon growth. Using reporter assays, we have shown that Prickle proteins significantly increase NFAT activity. Results also revealed that the C-termini of Prickle proteins are significantly more active in NFAT activation. To further characterize the effect of Prickle on NFAT activity, neurite extension, protein localization and neuronal differentiation, tetracycline regulatable PC6-3 stable lines expressing wild-type and mutant Prickle proteins have been established.

A yeast-two hybrid screen carried out revealed Prickle1 association with a 39-residue ‘Peptide-X’ that shared 100% identity to part of human 18S non-coding rRNA. Results from the yeast two-hybrid and Co-IPs suggested a specific association of this peptide with the PET/LIM domain of PK1 & PK2. A polyclonal antibody raised against this Peptide recognized a brain specific protein which immunoprecipitation, followed by mass spectrometry identified to be synapsin1: A brain specific phosphoprotein implicated in modulation of transmitter release, axonogenesis, and synaptogenesis. Mutations in SYN1 have been linked to epilepsy, ADS, and Rett Syndrome. In vivo Co-IPs have confirmed the association of Prickle 1 & 2 with Synapsin1 in the mouse brain. Further experiments are being carried out to confirm these interactions in cell lines.

Findings from these studies would clarify molecular mechanisms by which Prickle mutations alter neuronal function and help identify novel therapeutic targets.

 

Background for Yan Zhang’s talk

PHYSICAL AND GENETIC INTERACTIONS BETWEEN BBS GENES AND CEP290

Bardet-Biedl syndrome (BBS) is a heterogenous autosomal recessive inherited disorders with clinical features that include retinal degeneration, obesity and developmental anomalies. At least 17 BBS genes have been reported.  Seven BBS proteins form a molecular complex know as the BBSome, and three additional BBS proteins form a second complex known as the BBS chaperone complex based on the homology of these proteins to chaperonins. The chaperone complex is required for BBSome assembly.  However, the interaction or function of other BBS proteins remains to be investigated. Recent studies have shown that mutation of a novel centrosomal protein, Cep290, also called NPHP6 and BBS14, results in BBS and other ciliopathies, but the exact mechanism remains unknown. In this study, we examined the BBS-related function of Cep290 and investigated interactions between Cep290 and other BBS proteins. We demonstrate that Cep290 interacts with BBS4 and BBS9, which are components of the BBSome. In addition, we found that the BBSome is required for correct localization of Cep290 in RPE Cells. Furthermore, Cep290 is mislocalized in retina of BBS1M390R/M390R mice, which are homozygous for a methionine to arginine substitution at codon 390  of Bbs1, another component of the BBSome. Moreover, the body weight and leptin level of BBS4 +/- Cep290 +/- (double heterozygous) mice are higher than those of single heterozygous mice.  BBS4 +/- Cep290 +/- mice also show leptin resistance compared to single heterozygotes. These data strongly indicate that there is both physical and genetic interactions between Cep290 and the BBSome.

 

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Posted on August 8, 2012, in Student Seminar. Bookmark the permalink. Leave a comment.

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