Effects of Altered TPP1 Expression in Human Neural Progenitor Cells

Examining enzyme replacement therapy using a human neural progenitor cell model of CLN2 disease. Alana M. Williams 1, Aisha Y. Abdool 1, Diane-Marie Brache-Smith 2, James R. Munoz 1. 1 Nova Southeastern University, 2 Miami-Dade College North Campus. Neuronal ceroid lipofuscinosis type 2 (CLN2) is an autosomal recessive, neurodegenerative lysosomal storage disorder due to a deficit of the metabolic enzyme tripeptidyl peptidase (TPP-1). Due to the lack of TPP-1, an accumulation of lysosomal waste leads to cell death. The disease is characterized by language delays, seizures, cognitive and motor decline, blindness and early death. Currently a clinical trial of the experimental treatment Brineura® (cerilponase alfa) is the only approved treatment for CLN2. The clinical trial involves an intracerebroventricular infusion of recombinant TPP-1. Diffusion models suggest the protein will spread along a concentration gradient through the brain. It is unclear how altered concentrations of TPP-1 will effect ongoing neurogenesis in the subventricular zone. The goal of this study is to examine proliferation, cell cycle kinetics, differentiation, and cell death in human neural stem/progenitor cells (hNPCs) following knockdown, overexpression, or exposure to human recombinant TPP-1. Altered levels of TPP-1 will be assessed using western blot analysis. Proliferation and differentiation will be assessed using immunostaining, fluorescent microscopy, and NIH Image J analysis. Cell cycle kinetics will be assessed using EdU-incorporation assays. Cell death will be assessed using TUNEL assays. We anticipate two 2-way ANOVAs will be used to assess differences between treatment conditions. These results may have implications in clinical trials using intracerebroventricular infusion for enzyme replacement therapies. The original research summarized in the following was supported, in part, by U.S. Department of Education grant award P031C160143 (STEM EnginE). Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the respective funding agency