Investigating the Molecular Mechanisms Involved in the Mechanosensation of Neural Stem Cell Differentiation

Since it was discovered that neural stem cells (NSCs) have the capacity to self-renew and produce progenitor cells beyond development, there has been increasing interest in harnessing these endogenous processes for repairing the injured or disease brain. Specifically, the hippocampus is one of the few areas of the brain that continue to produce neurons postnatally. A decrease in neurogenesis in this region of the brain is associated with not only aging, but also with a rise in neurodegenerative disorders such as Alzheimer’s disease. Elucidating the regulation of neurogenesis is crucial for understanding adult brain function, as well as for treating neurodegeneration using cell replacement therapy. The local microenvironment of the NSCs, also known as the neurogenic niche, is composed of a complex network of signaling mechanisms that strongly regulate NSC function, and recapitulating this network in vitro is a major barrier to neuronal cell replacement therapy. One such signaling mechanism is mechanotransduction of biophysical cues from the extracellular matrix (ECM) into cytoskeletal changes that influence whether NSCs differentiate into neurons, astrocytes, or oligodendrocytes. However, our understanding of the underlying mechanism still largely derives from focused studies on a limited set of molecular candidates. Therefore, the focus of my dissertation has been on probing mechanosensitive lineage commitment in a more unbiased-fashion using high-throughput sequencing technology. In the first chapter of this dissertation, I conducted whole-transcriptomic RNA sequencing to NSCs cultured on soft (500 Pa) versus stiff (73 kPA) substrates, which we previously showed bias NSCs towards neuronal and astrocytic fates, respectively. Importantly, we conducted our studies 12-36 hours after cells were seeded and exposed to differentiation cues, a window where we have shown NSC fate is maximally sensitive to mechanical cues. While we identified a large number of differentially expressed genes in NSCs cultured on soft vs. stiff substrates, eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) stood out in its high differential expression and established contributions to F-actin bundling and protein synthesis. To demonstrate the functional importance of eEF1A1 to mechanosensitive lineage commitment, we suppressed its expression with shRNAs, which resulted in an increase on both soft and stiff substrates. Rescue of full eEF1A1 on top of the knockdowns concomitantly reduced neurogenesis on both soft and stiff substrates. We further determined that eEF1A1 is regulating fate commitment by controlling Yes-Associated Protein (YAP) levels, and Rho-GTP levels. Thus, eEF1A1 is a novel mechanoregulators of NSCs that plays an important role in NSC fate commitment. In the second chapter, we used the RNA-sequencing results from Chapter 1 and determined that substrate stiffness is regulating the usage of different alternative polyadenylation sites in the 3’UTR of mRNA during NSC differentiation. This difference in usage results in different mRNA isoforms that have the same coding region but vary in 3’UTR length in NSCs differentiating on soft versus stiff substrates. Interestingly, we show that most of the 3’UTR isoforms identified in soft substrates have a longer 3’UTR relative to NSCs differentiating on stiff substrates. A longer 3’UTR results in a higher chance of the mRNA to degrade in the cytoplasm through microRNA interactions. Furthermore, we show that there are higher expression levels of the Cleavage Factor Im 25 (CFIm25) protein in NSCs differentiating on soft substrates relative to stiff, which may be the cause of the difference in 3’UTR length. Lastly, suppression of CFIm25 suppresses overall neurogenesis, thus establishing its important functional role in fate commitment. Overall, this work integrates systems level measurements with biophysical approaches to identify the novel roles of eEF1A1 and 3’UTR lengthening and shortening in controlling stem cell mechanosensitive lineage commitment.

Advisor: Kumar, Sanjay
Schaffer, David V