Proper axonal growth and guidance are crucial for development of functional neuronal networks. Neuronal growth cones, located at the tip of emerging axons, transduce extracellular growth, and inhibitory cues to the underyling cytoskeleton within the axon (Head et al., 2014). Growth cones are enriched in MLRs, discrete plasmalemmal microdomains enriched in cholesterol, glycosphingolipids, and the scaffolding protein caveolin (Cav) (Sekino et al., 2007; Grider et al., 2009; Whitehead et al., 2012). Cav-1 is a cholesterol binding and scaffolding protein within MLRs that organizes signaling complexes such as Src family kinases (SFKs), cytoskeletal tethering proteins, and Rho GTPases (RhoA, Cdc42, and Rac1) that regulate cytoskeletal dynamics (Head et al., 2008, 2014; Pantera et al., 2009; Berta et al., 2011; Stuermer, 2011). Loss or disruption of MLRs from the leading edge results in growth cone collapse and inhibition of neuritogenesis (Whitehead et al., 2012). Previous research from of our group showed that Cav-1 organized growth-promoting signaling complexes within MLRs and regulated neurotrophic receptor signaling pathways (Head et al., 2008, 2011; Egawa et al., 2017; Mandyam et al., 2017). Specifically, neuron-targeted overexpression of Cav-1 (SynCav1) enhanced MLR formation, receptor-mediated cAMP production, TrkB receptor expression and signaling, and augmented dendritic arborization in primary neurons in vitro (Head et al., 2011). In addition, in vivo delivery of SynCav1 to the hippocampus of adult and aged mice enhanced both MLR-localized TrkB and structural neuroplasticity, improving hippocampal-dependent memory (Egawa et al., 2017; Mandyam et al., 2017). Past findings demonstrated that Cav-1 was widely involved in opioid-induced dendritic growth (Cui et al., 2017), further demonstrating a regulatory role of Cav-1 on various forms of neuroplasticity.

Modulation of cytoskeletal dynamics is important for proper neuronal differentiation. Rho GTPases (RhoA, Cdc42, and Rac1) are key regulators of cytoskeletal dynamics (Hall and Lalli, 2010; Dent et al., 2011). Dysregulation of cytoskeletal dynamics can lead to growth cone collapse and aberrant synaptic connections that occur in many neurodegenerative conditions such as Alzheimer’s disease (AD) (Heredia et al., 2006; Penzes and Vanleeuwen, 2011) and Down syndrome (Jabbour et al., 1992). While RhoA regulates actin stress fiber assembly and focal adhesion sites; Rac1 controls actin filament accumulations and plasmalemmal protrusion in the form of lamellipodia; Cdc42 produces filopodia and neurite outgrowth. Previous work from others showed that Cav-1 modulates Rho GTPase signaling in non-neuronal cells (Baltierrez-Hoyos et al., 2012; Lim et al., 2014). Because Cav-1 has been shown to colocalize and modulate Rho GTPase activity in non-neuronal cells (Cheng et al., 2010; Bassi et al., 2011; Diaz et al., 2014; Williamson et al., 2015; Coelho-Santos et al., 2016; Xu et al., 2016), the present study tested whether Cav-1 was also involved in Rho GTPase-mediated axonal growth and neuronal differentiation in human neurons derived from induced pluripotent stem cells (iPSCs). A better mechanistic understanding of this interplay within neurons could yield potential targets for promoting functional neuroplasticity.

Kawauchi and colleagues recently demonstrated that Cav-1 promoted early neurite maturation in an endocytic-dependent manner (Shikanai et al., 2018). However, whether Cav-1 phosphorylation was necessary for neurite maturation, specifically axonal maturation and guidance, has never been investigated. Using human neuron progenitor cells (NPCs) derived from iPSCs, we first tested the effects of neuron-targeted Cav-1 expression and Rac1/Cdc42 activation on neuronal growth during differentiation. Furthermore, by using a Cav-1 mutant construct SynCav1 (Y14A), that prevents Cav-1 phosphorylation at tyrosine (Y) 14, we demonstrated that Cav-1 phosphorylation was required for both SynCav1-mediated and Rac1/Cdc42-mediated neuronal axonal growth and differentiation.

The present study used differentiated human NPCs derived from iPSCs to demonstrate that (1) neuron-targeted Cav-1 (i.e., SynCav1) augments axonal growth during early stages of neuronal differentiation, a finding that extends previous work that SynCav1 promotes neuroplasticity in vitro and in vivo (Head et al., 2010, 2011; Mandyam et al., 2017), (2) Cav-1-mediated axonal growth is abolished by inhibition of Cav-1 phosphorylation (Y14), and (3) axonal growth induced by Rac1/Cdc42 activation is in part dependent upon Cav-1 phosphorylation (Y14). In conclusion, Cav-1 phosphorylation is necessary for human neuronal axonal growth during early stages of neuronal differentiation.

Rho GTPases (e.g., RhoA, Rac1, and Cdc42) are key regulators of cytoskeletal dynamics, growth cone motility, and axonal guidance (Hall and Lalli, 2010; Dent et al., 2011) and are shown to regulate these processes at the plasma membrane (Baltierrez-Hoyos et al., 2012; Lim et al., 2014). While RhoA activation results in axonal growth retraction, Rac1, and Cdc42 activation promotes axonal growth. Dysfunctional Rac1/Cdc42 has been associated with aberrant synaptic plasticity and intellectual disability (Jiang et al., 2010; Tejada-Simon, 2015; Chen et al., 2016; Pengelly et al., 2016). On a subcellular level, evidence from non-neuronal cells has shown that Rac1 and Cdc42 co-localize and interact with Cav-1 at the plasma membrane microdomains (Baltierrez-Hoyos et al., 2012; Lim et al., 2014). Polarization of active Rac1 was lost in fibroblasts lacking Cav-1 (Williamson et al., 2015), and Cdc42 knockdown prevented ApoA-I-mediated increased Cav-1 in astrocytes (Kheirollah et al., 2014), findings which indicate an interplay between Cav-1, Rac1 and Cdc42-mediated signaling, and associated cellular effects. The present study shows that, in addition to increasing T-Cav-1, Rac1/Cdc42 activation also increases P-Cav-1 in NPCs 3 days post-treatment. Surprisingly, we did not observe elevated P-Cav-1 in SynCav1 treated cells [which still increased T-Cav-1 as early as 24 hours post treatment (Supplementary Figure S2)]. The possible reason may be that increased P-Cav-1 occurs early and transiently during activated signaling events such as measured with constitutively Rac1(CA) and Cdc42(CA). Although not observed in the present study, SynCav1-mediated changes in P-Cav-1 may be detected immediately after receptor agonism or other activated signaling events (e.g., integrin activation, osmotic stress, and shear stress). However, the blunting of SynCav1-mediated axonal growth in the presence of P-Cav-1(Y14A) indicates that SynCav1-mediated axon growth is dependent upon its phosphorylation at Y14. These results lead us to postulate that P-Cav-1 may occur early and transiently following SynCav1 treatment, resulting in the profound neuroplastic changes observed 1 and 3 weeks later. The present study builds upon these previous findings and is the first to definitively show in human neurons that activating signaling events (e.g., Rac1/Cdc42) that increase axonal growth is dependent upon P-Cav-1.

Previous research showed that Cdc42 activation enhances P-Src, while silencing Cdc42 prevents P-Src (Shen et al., 2008). Other work also found that Rac1 activates Src signaling in non-neuron cells (Fang et al., 2016). Because P-Cav-1(Y14) is mediated by Src family kinases (SFKs) (Li et al., 1996), the significant enhancement of P-Cav-1 with constitutively active Rac1/Cdc42 or with the Rac1/Cdc42 small molecule activator observed in the present study may be caused by the persistent activation of SFKs (Grande-Garcia et al., 2007). Therefore, we hypothesized that activation of Rac1/Cdc42 is dependent upon SFK-mediated P-Cav-1(Y14). By using SynCav1(Y14A), we found that loss of P-Cav-1(Y14) blunted P-Src (Y416) Rac1/Cdc42-mediated axonal growth, indicating that Rac1/Cdc42-mediated axonal growth in early stages of differentiation is in part dependent on P-Src and P-Cav-1.

Our previous findings showed that SynCav1 increased dendritic arborization in hippocampal neurons in vivo (Mandyam et al., 2017), which is consistent with the present study that SynCav1 promotes dendritic growth in human neurons in vitro. Moreover, we also detected increased axonal growth as early as 1 week post SynCav1 transfection, the first evidence that Cav-1 also regulates axonal growth. These findings extend previous work by others that MLRs and MLR-associated proteins (such as Cav-1) provide subcellular and molecular signaling complexes essential for axonal growth and guidance (Guirland and Zheng, 2007; Yamashita and Kuruvilla, 2016; Batty et al., 2017). The present study attempted to better elucidate the molecular mechanism(s) underlying Cav-1-mediated neuronal growth. Previous research already demonstrated that even subtle changes in axon connectivity and neuronal network formation may cause various neurological disorders ranging from Down Syndrome to Autism. Because Cav-1 phosphorylation is essential for SynCav1-promoted axonal growth in early-stage of differentiation, disruption or imbalances in P-Cav-1 may have implications for neurological diseases or during aberrant neuronal development.

Endocytosis and intracellular trafficking are critical for neuritogenesis, axonal guidance and maturation (Winckler and Yap, 2011), and are dependent upon membrane lipid composition (Lauwers et al., 2016). Emerging evidence shows that MLRs and Cav-1 are necessary for neuritogenesis (Shikanai et al., 2018), axonal guidance, and synaptogenesis (i.e., formation of new synapses) (Head et al., 2014; Egawa et al., 2016, 2017). We have previously demonstrated that SynCav1 delivery to the hippocampus increased total excitatory type I synapses, multiple synapse forming boutons, LTP, and axonal myelination, all of which are ultrastructural indicators of axonal function and synaptic plasticity (Egawa et al., 2017). Although we did not test whether P-Cav-1 affects axonal transport of vesicles and/or cellular cargoes, the present study does show that NPCs treated with SynCav1(Y14A) resulted in profound impaired axonal growth in contrast to SynCav1, which significantly augmented axonal growth. Recent work demonstrated that P-Cav-1 facilitates microRNA insertion into extracellular vesicles by regulating RNA binding proteins in non-neuronal cells (Lee et al., 2019). Because P-Cav-1(Y14) and Src activation are linked to actin remodeling, endocytosis (Shi and Sottile, 2008), vesicular transport (Coelho-Santos et al., 2016), and trafficking (Cao et al., 2002, 2004; Salanueva et al., 2007; Chen et al., 2014) in non-neuronal cells, we are currently testing the hypothesis that P-Cav-1(Y14) may facilitate early stage signaling events necessary for axonal transport of vesicles to and from the presynaptic active zone as well as secretion of pro-growth neuronal microvesicles, subcellular events which are necessary for axonal growth and guidance, and synaptic maintenance.

In summary, these results show that Cav-1 phosphorylation is critical for axonal growth. Furthermore, because increased Cav-1 phosphorylation occurred with constitutively active Rac1 and Cdc42, but not with SynCav1 alone, suggests that this post-translational event may occur early and transiently (i.e., signal transduction) in response to extracellular pro-growth signaling cues. The present study also demonstrated that SynCav1-promoted axonal growth is dependent upon P-Cav-1(Y14). Moreover, inhibition of endogenous phosphorylation of Cav-1 even in the presence of increased T-Cav-1 (i.e., SynCav1) results in aberrant and blunted axonal growth. In conclusion, targeting Cav-1, specifically Cav-1 phosphorylation in neurons, may serve as a novel therapeutic target to promote axonal growth during early-stage differentiation in the setting of neurodevelopmental disorder such as Down Syndrome or serve to promote functional neuroplasticity after CNS injury (ischemic or traumatic) and in neurodegenerative diseases (AD, ALS, and multiple sclerosis).

The datasets generated for this study are available on request to the corresponding author.

SW wrote the manuscript and carried out the cell culture, immunofluorescence microscopy, Western blot, and data analyses. ZZ carried out the cell culture and Western blot. AA-Q provided the NPCs and assisted in cell culture. JL assisted in cell culture and Western blot. CD generated and provided the Rac1 and Cdc42 constructs. DR, PP, and HP assisted in editing and in experimental design. BH conceived the study, assisted in data analyses, provided the phospho-mutant Cav1 construct, and wrote the manuscript.

HP and BH are scientific founders of CavoGene LifeSciences LLC and hold equity interest in the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.