Simultaneous Knockdown of Sprouty2 and PTEN Promotes Axon Elongation of Adult Sensory Neurons

Abstract

Sprouty2 (Spry2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are both well-established regulators of receptor tyrosine kinase (RTK) signaling, and knockdown of Spry2 or PTEN enhances axon regeneration of dorsal root ganglia (DRG) neurons. The major role of Spry2 is the inhibition of the rat sarcoma RAS/extracellular signal-regulated kinase (ERK) pathway, whereas PTEN acts mainly as an inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In non-neuronal cells, Spry2 increases the expression and activity of PTEN, and PTEN enhances the amount of Spry2 by the inhibition of the microRNA-21 (miR-21) that downregulates Spry2. Applying dissociated DRG neuron cultures from wild-type (WT) or Spry2 deficient mice, we demonstrate that PTEN protein was reduced after 72 h during rapid axonal outgrowth on the laminin substrate. Furthermore, PTEN protein was decreased in DRG cultures obtained from homozygous Spry2−/− knockout mice. Vice versa, Spry2 protein was reduced by PTEN siRNA in WT and heterozygous Spry2+/− neurons. Knockdown of PTEN in DRG cultures obtained from homozygous Spry2−/− knockout mice promoted axon elongation without increasing axonal branching. Activation of Akt, but not ERK, was stronger in response to PTEN knockdown in homozygous Spry2−/− DRG neurons than in WT neurons. Together, our study confirms the important role of the signaling modulators Spry2 and PTEN in axon growth of adult DRG neurons. Both function as endogenous inhibitors of neuronal growth factor signaling and their simultaneous knockdown promotes axon elongation more efficiently than the single knockdown of each inhibitor. Furthermore, Spry2 and PTEN are reciprocally downregulated in adult DRG neuron cultures. Axon growth is influenced by multiple factors and our results demonstrate that the endogenous inhibitors of axon growth, Spry2 and PTEN, are co-regulated in adult DRG neuron cultures. Together, our data demonstrate that combined approaches may be more useful to improve nerve regeneration than targeting one single inhibitor of axon growth.

Exosome-Mediated miR-21 Was Involved in the Promotion of Structural and Functional Recovery Effect Produced by Electroacupuncture in Sciatic Nerve Injury

“…In this study, we found that miR-21 showed a significant negative correlation with its target gene, SPRY2, during PNI and the repair process. miR-21 can promote the repair of nerve fibers by directly targeting SPRY2 [ 40 ], and the knockout of SPRY2 can enhance axonal regeneration of DRG neurons [ 95 ]. However, its in-depth mechanisms need to be further explored.…”

How miRNAs Regulate Schwann Cells during Peripheral Nerve Regeneration—A Systemic Review

“…All three are essential tumor suppressors promoting programmed apoptosis [ 79 , 80 , 81 ]. In particular, SPRY2 and PTEN act as inhibitors of the Ras/Raf/ERK and Pi3k/AKT/mTOR pathways [ 82 ]. Furthermore, PTEN is involved in the regulation of NGF.…”

“…The existence of PTEN and SPRY 2 is crucial for the regulation of axonal outgrowth and SC migration and proliferation in homeostasis. During the acute phase of nerve injury, high levels of PTEN and SRPY2 are unfavorable [ 82 , 102 ]. Consequently, a reduction of these two proliferation-inhibitory proteins by the application of miR-21 or miR-221/222 is necessary to accelerate nerve regeneration.…”

Neuronal activity-dependent ATP enhances the pro-growth effect of repair Schwann cell extracellular vesicles by increasing their miRNA-21 loading

“…Importantly, miRNA-21 contained in rSC-EVs showed a 1.7-fold increase in ATP-treated rSC compared to non-treated rSC (Figure 4C). miRNA-21 targets various genes involved in the regulation of neurite elongation and axonal growth, such as PTEN, SPRY2, and TIMP3 (Duraikannu et al, 2019;Jamsuwan et al, 2020;Borger et al, 2022); therefore, we evaluated whether ATP-rSCderived EVs regulate the expression of these genes in DRG neurons. Indeed, we observed a 70% decrease in the expression of PTEN, a 15% decrease in the expression of SPRY2, and a 30% decrease in the expression of TIMP3 in neurons treated with ATP-rSC-derived EVs compared to neurons treated with control rSC-derived EVs (Figure 4D).…”