(287g) Cellular Signaling Dynamics Regulate Stem Cell Mediated Neurogenesis in the Adult and Aged Mammalian Brain

Adult neural stem cells (NSC) continuously contribute new neurons in specific regions of the brain - a process known as adult neurogenesis - to modulate learning and memory. As a result, they represent therapeutic targets due to their potential roles in age-related cognitive decline, in disease pathologies, and as endogenous sources for cell replacement therapies. The adult NSC microenvironment is highly dynamic, with signaling molecules likely varying in concentration and duration at timescales including rapid electrophysiological activity, circadian rhythm, environmental cues, and organismal aging. Wnt signaling activates β-catenin signaling, which we (Nature Neuroscience, 2012) and others have shown regulates the differentiation of adult hippocampal neural stem cells into neurons in vivo and in vitro, and can contribute to age-associated declines in neurogenesis.To address the question of how dynamics in signaling impacts cell function, our lab has developed a tunable optogenetic system to modulate β-catenin signaling via Cry2 oligomerization of the LRP6 intracellular domain (Nature Methods, 2013). Similar to Wnt3a activation of the canonical Wnt pathway, blue light illumination of Cry2-LRP6c expressing NSCs induces robust neuronal differentiation. This raises the question: do stem cells differentiate simply when the integral of a signal 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 progressive, saturable increase in neuronal differentiation from 5% to 60%. However, variation in signaling intensity over time yielded different results. Specifically, we observed that initial pulses of light for 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 exposure to the differentiation signal also rendered cells dependent upon it for survival, potentially offering a mechanism for removal of incompletely or poorly differentiated stem cells from tissue. Moreover, investigation of candidates emerging from RNAseq has provided insights into the molecular mechanisms underlying how dynamic signaling regulates two alternate cell fates. In sum, these results 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 translational potential of adult neurogenesis.