Vaibav Ramu
Research Mentor(s): Huiyuan Wu
Mentor Department: Department of Pediatrics
Authors: Vaibav Ramu, Xinyue Zhang, Jeremy Spenler, Huiyuan Wu
Session: Session 3 (11:00am – 11:50am)
Presentation Type: Poster 28
Abstract
Childhood-Onset Neurodegeneration with Cerebellar Atrophy (CONDCA) is a very rare human disease stemming from biallelic pathogenic variants of the AGTPBP1 gene, characterized by cerebellar atrophy and other biological anomalies. The AGTPBP1 gene encodes the Nna1/CCP1 (cytosolic carboxypeptidase 1) enzyme, which regulates the post-translational modification, specifically deglutamylation, of alpha- and beta-tubulins. When the CCP1 gene is mutated, tubulins remain hyperglutamylated, resulting in toxically lengthy glutamate chains. Ultimately, it causes cell death in sites such as the cerebellar Purkinje cells, retinal photoreceptors, and male germinal epithelium (affecting spermatogenesis). This disease is well modeled by the Agtpbp1 mutant Purkinje Cell Degeneration (pcd) mouse. Previous research has shown that knocking out certain enzymes from the tubulin tyrosine ligase-like (TTLL) family, such as TTLL1 and TTLL4 can result in significant rescue to Purkinje cells and photoreceptors in pcd. WL-4 was found to act as an inhibitor to the TTLL4 enzyme, improving cell survival. This project aims to further investigate WL-4’s efficacy in a two-phase approach. First, plasmids for recombinant TTLL4 and TTLL6 (a TTLL4 homolog) expression were constructed using blunt-end digestion and subsequent ligation independent cloning (LIC), followed by protein expression and purification. Then crystallographic analysis will be conducted to document the physical interaction between these enzymes and the WL-4 compound. The second phase of the project aims to reaffirm the impact of the WL-4 compound on Purkinje cell rescue in pcd mice, while determining the optimal dosage to promote maximal cell rescue. WL-4 is administered to mice every three days beginning P7 (postnatal day 7) till P25. Post-treatment, they are subjected to transcardiac perfusion, their brains collected, and immunohistochemical (IHC) analysis is performed to determine whether Purkinje cells were rescued at each dosage. Findings from this study may contribute to therapeutic strategies for CONDCA and related ciliopathies.
