Parkinson's and Alzheimer's disease are neurodegenerative diseases that affect 40 million people worldwide. Adult neural stem cells (NSC) have great potential as cell replacement therapies. The adult NSC microenvironment is likely highly dynamic, with signaling molecules presented at modulating intensities and durations. The canonical Wnt signaling pathway, whose activation involves -catenin stabilization, is involved in regulating NSC behavior, including neurogenesis and apoptosis. To mimic in vivo dynamic signaling, our lab has developed a tunable optogenetic system to modulate -catenin signaling through Cry2 oligomerization of the LRP6 intracellular domain. Canonical Wnt pathway activation via Wnt3a can lead to robust neurogenesis, and I have observed that NSCs undergo neuronal differentiation in a signal dosage-dependent manner in the presence of low level fluctuations of dark and light cycles. In contrast, we observed that brie stabilization of - catenin followed by extended signal withdrawal induces apoptosis, a novel outcome that may act to eliminate incompletely differentiated neurons. Specifically, catastrophic signal loss was induced by the total withdrawal of light, and increased apoptosis was observed in cells exposed to light for less than 3 days. Under low intensity and varying light cycle conditions, a decrease in total photons also led to increased apoptosis. However, cells rescued with light within 24 hrs did not have increased apoptosis whereas those without light for greater than 24 hrs exhibited increased apoptosis. In addition, I show a loss-of-signal `buffer region, within which the neurogenic fates of NSCs were relatively insensitive to signal fluctuation, whereas, outside of the `buffer', NSC fate is significantly shifted towards apoptosis. Therefore, dynamic -catenin signaling likely directs NSCs towards differentiation or apoptosis.
In Aim 1, we explore the dynamics of -catenin (de-) stabilization, with our unique optogenetic system, through the on/off kinetics, transcriptional activity, and downstream effects on NSC fate. The kinetic response of -catenin to signal fluctuations and cell fate decisions will be correlate to the levels of -catenin phospho- species and other key proteins to gain insight into the molecular mechanism(s) involved.
In Aim 2, we will determine the molecular mechanism(s) involved in the apoptosis cell fate. In biased strategies, we are exploring known protein pathways involved in apoptotic activities. Complementary, unbiased searches with ChIP-Seq and RNA-Seq will identify potential transcriptional regulation of apoptotic processes. Notably, ChIP-seq of -catenin throughout differentiation will be broadly useful for studies of Wnt signaling and neurogenesis. The precise control of signal induction offered by our optogenetic system allows for unprecedented exploration of -catenin dynamics. The observed apoptotic outcome may represent an important regulatory step for in vivo neurogenesis and determination of downstream signaling effectors will yield more complete molecular mechanisms by which canonical Wnt signaling regulates NSC fate. Thus, this work will advance our understanding of Wnt signaling and the efforts to harness NSCs for neuroregeneration.
Progressive neurodegenerative diseases, such as Alzheimer's and Parkinson's, affect more than 40 million people worldwide with no known reparative treatment; however, adult neural stem cells within the brain undergo processes of survival (or loss), proliferation, and differentiation that contribute to active neurogenesis that persists throughout adulthood and could potentially be harnessed for neuroregeneration. To understand how the dynamic signaling environment of these cells regulates their behavior, we propose to harnesses a novel optogenetic system to investigate how Wnt signaling dynamics may regulate two alternate cell fates, differentiation vs. apoptosis, as well as elucidate downstream signaling effectors and mechanisms. The proposed integration of optogenetics, biochemical approaches, and genome-wide analysis will yield knowledge that will benefit both basic biology and regenerative medicine efforts to harness these stem cells as novel therapeutic treatments.
