Basic Biology IV
$1 755 000
Experiments with mammalian embryonic stem cells (ESCs) have clearly demonstrated their capacity to replicate continuously. We hypothesized that the immortality of these cells depends in part upon an increased ability to protect themselves from the accumulation of damaged proteins normally seen with age. We found that stem cells had a six-fold higher proteasome activity than their differentiated counterparts. Notably, stem cell populations exhibit a proteasome activity that is correlated with increased levels of one proteasome subunit, PSMD11, and increased assembly of the proteasome. FOXO4, an insulin/IGF-1 responsive transcription factor associated with long lifespan in invertebrates, regulates proteasome activity by modulating the expression of PSMD11 in human embryonic stem cells (hESCs) and is necessary for hESC differentiation into neuronal lineages. Our results establish a novel regulation of proteostasis in hESCs that links longevity in invertebrates with hESCs function and identity. Manipulation of PSMD11 allows us to genetically induce proteasomal activity, providing a unique method for manipulating protein degradation in stem cells. We will take advantage of these findings to promote our understanding of exactly how stem cells are protected from damage. Moreover, we hypothesize that the activity and expression of the proteasome may be key determinants in our capacity to reprogram somatic cells and in mitigating the effects of age-onset protein misfolding diseases.
Statement of Benefit to California:
The number of aging Californians diagnosed with protein misfolding diseases, including Alzheimer’s disease, is currently undergoing exponential growth: within the next 20 years, well over a million Californians are expected to be diagnosed with Alzheimer’s. The cost of care and treatment for these individuals reaches into the 100’s of billions of dollars within California alone and could eventually undermine the economic and social stability of the state. Tragically, in such diseases, diagnosis usually occurs after wide spread neuronal death has already occurred. One of the more promising therapeutic options for patients with protein misfolding diseases is stem cell therapy, which hopes to replace lost neurons with ones generated from stem cells. However, we do not yet understand much of the basic biology of how stem cells maintain their health, including how they can protect themselves from the accumulation of damaged proteins -- especially in an older individual who is rapidly succumbing to proteotoxic disease in other cells. This research is designed to address a basic and often overlooked question about stem cell health: what machinery does the stem cell employ to guarantee the health of its proteome, and how is this related to the aging process? This research will provide fundamental insights into the mechanisms of protein homeostasis within the stem cell, findings that can be immediately applied by those searching for therapeutic options for these diseases.
This project aims to understand the mechanisms involved in the regulation of protein degradation and homeostasis in stem cells. Preliminary results indicate that proteasome activity decreases during differentiation of embryonic stem cells (ESCs) to specific cell types such as neurons. Furthermore, a transcription factor called FOXO4 regulates proteasome activity during this process by modulating the expression of the proteasome subunit PSMD11, which correlates with proteasome activity. Four specific aims have been proposed. The first specific aim proposes to examine the effects of PSMD11 or FOXO4 activity on protein expression levels and half-life. Aim 2 will study the activity and composition of the proteasome in ESCs. The third specific aim will use novel reporter assays and examine the differentiation potential of ESCs with high or low proteasome activity. The last specific aim will examine whether proteasome activity is affected during the process of making induced pluripotent cells (iPSCs), and any impact that decreasing the expression of PSMD11 or FOXO4 may have on the iPSC generation. Significance and Innovation - Although reviewers agreed that there are novel components in this proposal that could lead to improved methods of reprogramming, they felt that the studies do not directly address whether proteasome activity is required for stem cell function. - Reviewers agreed that the preliminary results presented in this proposal lacked sufficient maturity to support the proposed specific aims. Feasibility and Experimental Design - Reviewers raised major concerns that other than correlative results, there is no direct evidence to link the knock-down of PSMD11 expression with a loss of pluripotency. - Reviewers raised concerns that none of the proposed studies examine whether the impact of FOXO4 loss of neural cell differentiation is actually due to its involvement in PSDM11 expression. - Some reviewers expressed concerns that morphology will be used in lieu of specific molecular markers, and argued that this is not the most reliable marker of cell fate. - One reviewer suggested that given that proteasome activity is regulated by reactive oxygen species, the role of oxidative stress should have been addressed in the proposal. Principal Investigator (PI) and Research Team - Reviewers noted that the PI is a highly accomplished investigator with an impressive track record in hESC. - The team and collaborators were judged appropriate with the right expertise to carry out the proposed experiments. Responsiveness to the RFA - The proposed research was viewed as responsive to the RFA.