Sara Pintwala, PhD Candidate
Peever Lab
ABSTRACT
Narcolepsy is a sleep disorder caused by the loss of orexin neurons in the lateral hypothalamus. This results in symptoms such as excessive daytime sleepiness and cataplexy, a sudden and involuntary loss of muscle tone during wakefulness. The objective of cell replacement therapy is to treat disease by replacing lost neurons. However, testing this for narcolepsy is primarily dependent on the availability of a reliable source of orexin neurons. Here, we describe the character of a novel immortal orexin cell line and determine the outcome on behaviour when these cells are transplanted into a mouse model of narcolepsy. The first major aim of this project was to validate the phenotype of a novel immortal cell line: the mHypoA-ORX/GFP4 cell line. To do this, we performed immunocytochemistry and a live cell secretion assay to detect the expression and secretion of orexin by live cells. Next, we engineered these cells to express the excitatory Designer Receptor Exclusively Activated by a Designer Drug (DREADD) hM3Dq, enabling us to remotely control their activity in vitro. Finally, we transplanted cells to the dorsal raphe in a mouse model of narcolepsy (orexin-knockout, KO) and determined the outcome on behaviour, specifically cataplexy. Using immunostaining, we found that a majority of cultured cells expressed orexin. When live cells were stimulated under physiological conditions there was significant increase in orexin release by cultured cells when compared to basal neurotransmitter release. When cultured cells were exposed to a viral vector carrying hM3Dq we found efficient transduction of hM3Dq. When exposed to the ligand clozapine-N-oxide (CNO), we found a significant increase in neuronal activation and orexin release by cultured transfected cells. Cultured orexin cells were then transplanted to the dorsal raphe of orexin-KO mice, as this nuclei is involved in the generation of cataplexy. Post-transplant we found that the number of cataplexy episodes was significantly reduced. In these subjects, many orexin cells were present in the dorsal raphe. This project demonstrates the potential of cell replacement therapy as a novel therapeutic strategy for narcolepsy and the potential of using immortal cell lines in cell replacement therapy for a variety of diseases and disorders.