Rac1/Cdc42‐activation enhances expression of total and phosphorylated Cav‐1 in differentiated human neurons derived from induced pluripotent stem cells (iPSCs)
Authors: Wang Shanshan , Zhang Zheng , Illum Ben , Head Brian , Patel Piyush 
Published: November 2023

Background

Identifying molecular pathways that promote neuroplasticity may yield new therapeutic targets to reverse neurodegenerative deficits. One pathway involves Rho GTPases (RhoA, Cdc42, and Rac1). Rac1 and Cdc42 activation promotes axonal growth, while RhoA results in axonal growth retraction. However, It is still unknown how Rho GTPases are organized at the membrane. Our previous work has shown that caveolin‐1 (Cav‐1) regulates neuronal signaling and neuroplasticity. Because Cav‐1 has been shown to regulate Rho GTPase activity in non‐neuronal cells, the present study tested whether Cav‐1 is involved in Rho GTPase‐mediated neuronal growth. Using a human neuron model, we tested constitutive activated (CA) Rho constructs on pro‐growth signaling and whether Rac1/Cdc42‐mediated neuronal growth is in part dependent upon Cav‐1.

Methods

Human differentiated neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSC) were cultured in DMEM/F12/Glutamax media with N‐2 supplement, B‐27 supplement, Pen/Strep, and basic Fibroblast Growth Factor (bFGF). bFGF was removed to induce differentiation, which was confirmed by immunofluorence (IF) using MAP2 (microtubule‐associated protein and SMI‐31 (neurofilament), a dendritic and axonal marker respectively as shown in Figure 1. After 3 days of differentiation, neurons were transfected with viral vectors containing synapsin driven constructs as indicated in each figure legend: Rac1Q61L (constitutively active Rac1), Cdc42Q61L (constitutively active Cdc42), or Cav‐1 (SynCav1). Antibodies to GTP‐bound Rac1 or Cdc42 were used to confirm activation. Neuronal cell lysates were immunobloted for total Cav‐1 (T‐Cav‐1), phosphorylated Cav‐1 (P‐Cav‐1) and P‐cofilin, a downstream signaling component of Rho GTPases. Neurons were additionally treated with the small molecule that directly activates Rac1/Cdc42 (2units/ml, EGF).

Results

SynCav1 significantly enhanced dendritic length, branching (i.e., arborization), area and volume 3 weeks post transfection. Rac1 and Cdc42 activation resulted in downstream phosphorylation (i.e, inactivation) of P‐cofilin, a protein that directly regulates actin polymerization. The same increase in P‐cofilin was shown in neurons stimulated with Rac1/Cdc42 activator. Rac1CA and Cdc42CA also increased expression of T‐Cav‐1 and P‐Cav‐1. However, increasing T‐Cav‐1 expression with SynCav1 did not increase P‐Cav‐1 after 3 days, suggesting that P‐Cav‐1 levels may depend upon a transiently activated pro‐growth signaling pathway.

Conclusion

The present study used a translational human neuronal cell model to better understand the mechanisms of Rho GTPase and Cav‐1‐promoted neuron growth and plasticity. Rac1/Cdc42 activation increased P‐cofilin, T‐Cav‐1 and P‐Cav‐1, which is also found in neurons treated with the Rac1/Cdc42 activator. As such, increased phosphorylation of Cav‐1, which is involved in Rho GTPase signaling, may potentially be a novel molecular target for promoting neuroplasticity after injury or in neurodegenerative disease. Further research will test a SynCav1(Y14A) mutant, which lacks the phosphorylation residue, on Rho GTPase‐mediated neuronal growth.