Abstract
The cellular prion protein (PrPC) is a highly conserved glycosylphosphatidylinositol (GPI)-anchored membrane protein that is involved in the signal transduction during the initial phase of neurite outgrowth. The Ras homolog gene family member A (RhoA) is a small GTPase that is known to have an essential role in regulating the development, differentiation, survival, and death of neurons in the central nervous system. Although recent studies have shown the dysregulation of RhoA in a variety of neurodegenerative diseases, the role of RhoA in prion pathogenesis remains unclear. Here, we investigated the regulation of RhoA-mediated signaling by PrPC using both in vitro and in vivo models and found that overexpression of PrPC significantly induced RhoA inactivation and RhoA phosphorylation in hippocampal neuronal cells and in the brains of transgenic mice. Using siRNA-mediated depletion of endogenous PrPC and overexpression of disease-associated mutants of PrPC, we confirmed that PrPC induced RhoA inactivation, which accompanied RhoA phosphorylation but reduced the phosphorylation levels of LIM kinase (LIMK), leading to cofilin activation. In addition, PrPC colocalized with RhoA, and the overexpression of PrPC significantly increased neurite outgrowth in nerve growth factor-treated PC12 cells through RhoA inactivation. However, the disease-associated mutants of PrPC decreased neurite outgrowth compared with wild-type PrPC. Moreover, inhibition of Rho-associated kinase (ROCK) substantially facilitated neurite outgrowth in NGF-treated PC12 cells, similar to the effect induced by PrPC. Interestingly, we found that the induction of RhoA inactivation occurred through the interaction of PrPC with RhoA and that PrPC enhanced the interaction between RhoA and p190RhoGAP (a GTPase-activating protein). These findings suggest that the interactions of PrPC with RhoA and p190RhoGAP contribute to neurite outgrowth by controlling RhoA inactivation and RhoA-mediated signaling and that disease-associated mutations of PrPC impair RhoA inactivation, which in turn leads to prion-related neurodegeneration.
Highlights
The activity of Rho GTPases (Rho, Rac, and Cdc42) is controlled by regulatory proteins that cycle between an inactive GDP-bound state and an active GTP-bound state
To further investigate the signaling pathway of Ras homolog gene family member A (RhoA) regulated by PrPC expression, we determined whether PrPC modulates the RhoA-Rho-associated kinase (ROCK)-LIM kinase (LIMK)-cofilin pathway
PrP knockout and siRNA-mediated knockdown of endogenous mouse PrP (mPrP) cells exhibited less phosphorylated RhoA at Ser[188] (p-RhoA), which negatively regulates RhoA activity by enhancing its interaction with RhoGDI and translocates RhoA from the membrane to the cytosol[27] with increases in phospho-LIMK1/2 (p-LIMK1/2) and phosphocofilin (p-cofilin) (Figures 2a and b). Supporting these results, the re-introduction of mPrP reversed the changes in the levels of phosphorylated RhoA at Ser188 (p-RhoA), p-LIMK1/2, and p-cofilin compared with Zpl cells expressing the empty vector alone, yielding a result similar to that observed for the ZW cells (Figure 2c)
Summary
The activity of Rho GTPases (Rho, Rac, and Cdc42) is controlled by regulatory proteins that cycle between an inactive GDP-bound state and an active GTP-bound state. GTPase-activating proteins (GAPs), which stimulate Rho GTPase activity, and Rho guanine nucleotide dissociation inhibitors (GDIs), which inhibit the exchange of GDP for GTP in the cytoplasm by forming a Rho–RhoGDI complex, induce inactivation state of these GTPases.[1,2] the Rho–RhoGDI complex needs to be dissociated by GDI displacement factor (GDF) before Rho GTPases are activated by GEFs.[3] Activated Rho GTPases stimulate effector proteins, such as Rho-associated kinase (ROCK), mDia, and p21-activated kinase (PAK). The functional interaction between PrP and RhoA-related signaling molecules remains unknown
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