The Differential Regulation of Adult Neural Stem Cells by Beclin1 and Atg5

Abstract

Adult hippocampal neurogenesis is orchestrated by neural stem cell (NSC) activity. Some associations exist between autophagy and neurogenesis, yet much remains unknown about autophagic regulation of adult neurogenesis. This thesis interrogates the requirement and role of Beclin1 and Atg5, two regulators of autophagy, in the formation of adult hippocampal neurons. To examine adult brain NSCs, the experiments presented in the first objective of this thesis test the ability to isolate adult NSCs using flow cytometry and a DNA-binding dye, DyeCycleViolet. While adult NSCs could not be isolated from the adult neurogenic niches using this methodology, it was effective in isolating endothelial cells. This provided valuable insight on the use of DNA-binding dyes and a new method for isolation of brain endothelial cells. The next objective determines the role of Beclin1 in adult NSCs and their progeny using an inducible model. Beclin1 loss in Nestin-expressing hippocampal NSCs resulted in reduced proliferation, autophagy, and adult neurogenesis within one month. Single-cell RNA sequencing and other methods illuminated that loss of Beclin1 resulted in mitosis reduction, disrupted mitotic regulation of chromatin maintenance, and induction of DNA damage. The final objective first tests whether Beclin1 loss results in similar deficits within GLAST-expressing NSCs and progeny. This model mirrored neurogenesis deficits and requirement of Beclin1 in mitosis and DNA maintenance. Next, to test whether this phenotype occurs with other autophagy proteins, Atg5 was removed from GLAST NSCs. This resulted in reduced autophagy and a transient decrease in neurons in the absence of any effect on NSC proliferation. Thus, proliferation deficits are unique to Beclin1 loss and do not underlie reduced adult hippocampal neurogenesis after Atg5 removal. This work demonstrates a novel discovery of mitosis regulation in adult NSCs by Beclin1, and individual roles of Beclin1 and Atg5 in neurogenesis.