In previous work in collaboration with Dr. Minoru Ko (LG-NIA),we were able to establish a stem-cell differentiation assay based on cell morphology alone, using phase-contrast imaging without specific markers. This year we were able to extend this work in a collaboration with Noriko Saitoh of Kumamoto University in Japan to characterize the quality of human induced pluripotent stem (iPS) cells. As part of the same collaboration, we also published a study where we used pattern recognition in fluorescence microscopy of cultured human cels to show that the actin-related protein ARP6 affects the structure of the nucleolus. We have continued work characterizing the molecular basis of morphological age-state transitions during the C. elegans life-span. In published work we used WND-CHARM to identify distinct morphological aging states in C. elegans. We used this technique to sort worms based on their age state during a transition period where an aging population is evenly divided between individuals in Stage I and Stage II. The worms were identical genetically, by chronological age, by growth conditions, and by visual appearance, and could only be sorted into age-states using WND-CHARM. Micro-array experiments performed on these two sub-populations revealed several hundred genes with significantly altered expression profiles. By comparing our gene lists with those from other aging studies in C. elegans, we were able to identify several gene families and functional groups that were unique to our study. A prevailing theme of the aging genes uniquely identified in this study were those involved in targeted proteolysis, which appears to be a hallmark of this first aging state transition. In previous work, we have developed an age-state classifier that enables us to more robustly identify the aging state occupied by individual worms. More recently we have shown that this state-classifier is robust enough to identify these states in other strains of worms. We used this classifier to manually sort several thousand worms by age-state in order to study gene expression patterns specific to these aging states. Currently, we have completed work on expression profiling of all four observed aging states and transitions in C. elegans. Our results indicate that there are many reproducible differences in gene expression during the transitions between age-states, and we've generated lists of genes that are differentially expressed in these states. Many of these genes have been previously found to be regulated with aging, but the unique properties of our assay allowed us to find new patterns of expression. One of the gene families we've focused on are the small heat-shock proteins (HSPs). We have characterized this family exhaustively both with microarrays and pPCR. Previously, it has been reported that expression of these genes declines with age, along with a corresponding decline in heat-shock response. This has been termed proteostasis collapse. In contrast, we find that it may more accurate to think of each aging-state as proteostasis collapse followed by a new proteostasis in the subsequent state. This is supported by our finding that expression levels of some heat-shock proteins indeed decrease during state transitions, and that several others increase their expression. One HSP appears to pulse in concert with age transitions. Our assay's ability to measure physiological age in individual worms has allowed us to observe effects that have been previously masked by observations made in mixed populations. This work is being prepared for publication. We have begun to characterize the effect of RNAi knockdown on the genes identified as differentially expressed in the first age-state transition. A group of genes producing secreted proteins with cysteine-rich regions appears to contain genes that can either advance or delay the first transition. In collaboration with Dr. Bohr's group (LMG) we have also examined the effect of gene mutations as well as drug treatments on the timing of these age-state transitions. This work has been submitted for review.
