Rho GTPases Mediate the Mechanosensitive Lineage Commitment of Neural Stem Cells

Abstract

Adult neural stem cells (NSCs) play important roles in learning and memory and are negatively impacted by neurological disease. It is known that biochemical and genetic factors regulate self-renewal and differentiation, and it has recently been suggested that mechanical and solid-state cues, such as extracellular-matrix (ECM) stiffness, can also regulate the functions of NSCs and other stem cell types. However, relatively little is known of the molecular mechanisms through which stem cells transduce mechanical inputs into fate decisions, the extent to which mechanical inputs instruct fate decisions versus select for or against lineage-committed blast populations, or the in vivo relevance of mechanotransductive signaling molecules in native stem cell niches. Here we demonstrate that ECM-derived mechanical signals act through Rho GTPases to activate the cellular contractility machinery in a key early window during differentiation to regulate NSC lineage commitment. Furthermore, culturing NSCs on increasingly stiff ECMs enhances RhoA and Cdc42 activation, increases NSC stiffness, and suppresses neurogenesis. Likewise, inhibiting RhoA and Cdc42 or downstream regulators of cellular contractility rescues NSCs from stiff matrix- and Rho GTPase-induced neurosuppression. Importantly, Rho GTPase expression and ECM stiffness do not alter proliferation or apoptosis rates indicating that an instructive rather than selective mechanism modulates lineage distributions. Finally, in the adult brain, RhoA activation in hippocampal progenitors suppresses neurogenesis, analogous to its effect in vitro. These results establish Rho GTPase-based mechanotransduction and cellular stiffness as biophysical regulators of NSC fate in vitro and RhoA as an important regulatory protein in the hippocampal stem cell niche.

Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells

“…Because traction forces are known to vary with substrate stiffness, an important modulator of differentiation in several different stem cell types (7,8), we asked whether Piezo1 activity varies with substrate stiffness. We performed TIRFM imaging of spontaneous Ca 2+ transients of hNSPCs grown on highrefractive-index Qgel silicone elastomers of varying stiffness (41), fabricated as described in SI Methods.…”

“…In rodent neural stem cells, substrate stiffness was found to have the opposite effect, with soft substrates (<1 kPa) supporting more neurogenesis than stiffer substrates (>5 kPa) (8,16,17). Because we performed our differentiation assays on Qgel-based substrates, whereas earlier studies used polyacrylamide or methacrylamide chitosan as supporting material (8,16,17), we wondered whether this difference could be responsible for the discrepant results. However, when we differentiated rat adult hippocampal neural stem cells [rahNSCs, which generated more neurons on soft polyacrylamide substrates (17)] on Qgels (0.7 kPa vs. 750 kPa), we similarly observed more neurogenesis on softer substrates (Fig.…”

“…Earlier studies on rodent neural stem cells have shown the importance of matrix elasticity in lineage specification (8,16,17). However, the effect of substrate stiffness on hNSPC differentiation has not been investigated previously.…”

“…In particular, we focused here on cationic stretch-activated channels (SACs) because they are known to detect mechanical forces with high sensitivity and broad dynamic range and because they are permeable to Ca 2+ , an important second messenger implicated in cell fate (14,15). We examined the role of SACs in neural stem cells, for which mechanical cues influence specification along the neuronal-glial lineage (8,16,17). We find that human neural stem/progenitor cells (hNSPCs) express the SAC Piezo1 and that the activity of the channel is mediated by cellgenerated traction forces.…”

“…Studies in mesenchymal and neural stem cells have revealed the involvement of focal adhesion zones and cytoskeletal proteins, such as integrins, nonmuscle myosin II (7), Rho GTPases (8)(9)(10), and vinculin (11), that participate in the generation of cellular traction forces. Recent work also has identified the nucleoskeletal protein lamin-A (12) and the transcriptional coactivators Yap (Yesassociated protein) and Taz (transcriptional coactivator with PDZbinding motif) (13) in mechanotransduction in mesenchymal stem cells.…”