Optogenetic Investigation of Effects of Beta-Catenin Signaling Dynamics on Neural Stem Cell Differentiation

Adult neural stem cells (NSC) continuously generate new neurons in specific regions of the brain throughout adulthood to modulate learning and memory, and represent therapeutic targets due to their potential roles in the pathology of diseases and promise in cell replacement therapies. In general, stem cell niches are likely highly dynamic, and the NSC niche in particular likely presents signaling molecules that vary in concentration and duration at timescales ranging from electrophysiological activity, to circadian rhythm, to organismal behavior and environment. Wnt signaling activates β-catenin to induce neuronal differentiation of adult hippocampal neural stem cells in vivo and in vitro. To address the question of how dynamics in signaling impacts cell function, our lab developed a tunable optogenetic system to modulate
β-catenin signaling via Cry2 oligomerization of the LRP6 intracellular domain. Similar to responses observed upon Wnt3a ligand addition, blue light illumination of NSCs expressing Cry2-LRP6c induces neuronal differentiation. This outcome enables one to pose many questions related to signaling dynamics, including: do stem cells differentiate when the integral of signaling during a given temporal window exceeds a key threshold, or do dynamics in signal presentation matter? Continuous illumination at different light intensities in vitro resulted in a saturable dose response in which neuronal differentiation progressively increased from 5% to 60%. However, variation in signaling intensity over time yielded different results. Specifically, we observed that light initial pulses of variable duration, or oscilllating illumination at frequencies >12 hours, yielded considerably less neuronal differentiation than in cells that received the same overall signal dosage but with continuous illumination. Furthermore, this signal stimulation followed by signal loss led to increased apoptosis, indicating that exposure to this signal rendered cells dependent upon it not only for differentiation but also for survival, potentially offering a general mechanism for removal of incompletely or poorly differentiated stem cells from tissue. Whole transcriptome sequencing (RNA-seq) revealed a several candidate genes that may play significant roles in differentiation and survival, and overexpression of one cell cycle regulatory factor, whose endogenous expression is lost upon Wnt signal withdrawal, significantly decreased apoptosis and rescued
differentiation upon signal loss. These results indicate potentially critical roles for both cell cycle exit and prevention of aberrant cell cycle re-entry upon loss of β-catenin activity. In sum, we harness optogenetics to demonstrate that not only the overall dosage of a signal but temporal dynamics in its presentation can exert a strong impact on stem cell behavior, work that offers further insights into the biology and potential translational potential of adult neurogenesis.