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Caveolin-1 Phosphorylation Is Essential for Axonal Growth of Human Neurons Derived From iPSCs

Proper axonal growth and guidance is essential for neuron differentiation and development. Abnormal neuronal development due to genetic or epigenetic influences can contribute to neurological and mental disorders such as Down syndrome, Rett syndrome, and autism. Identification of the molecular targets that promote proper neuronal growth and differentiation may restore structural and functional neuroplasticity, thus improving functional performance in neurodevelopmental disorders. Using differentiated human neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs), the present study demonstrates that during early stage differentiation of human NPCs, neuron-targeted overexpression constitutively active Rac1 (Rac1CA) and constitutively active Cdc42 (Cdc42CA) enhance expression of P-Cav-1, T-Cav-1, and P-cofilin and increases axonal growth. Similarly, neuron-targeted over-expression of Cav-1 (termed SynCav1) increases axonal development by increasing both axon length and volume. Moreover, inhibition of Cav-1(Y14A) phosphorylation blunts Rac1/Cdc42-mediated both axonal growth and differentiation of human NPCs and SynCav1(Y14A)-treated NPCs exhibited blunted axonal growth. These results suggest that: (1) SynCav1-mediated dendritic and axonal growth in human NPCs is dependent upon P-Cav-1, (2) P-Cav-1 is necessary for proper axonal growth during early stages of neuronal differentiation, and (3) Rac1/Cdc42CA-mediated neuronal growth is in part dependent upon P-Cav-1. In conclusion, Cav-1 phosphorylation is essential for human neuronal axonal growth during early stages of neuronal differentiation.


INTRODUCTION
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(Head et al., , 2014Pantera 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 growthpromoting signaling complexes within MLRs and regulated neurotrophic receptor signaling pathways (Head et al., 2008(Head et al., , 2011Egawa 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 Mandyam et al., 2017). Past findings demonstrated that Cav-1 was widely involved in opioid-induced dendritic growth , 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/Cdc42mediated neuronal axonal growth and differentiation.

NPCs Culture and Differentiation
The use of human induced pluripotent stem cells (hiPSC) was approved by the UCSD IRB/ESCRO (project #:1616206ZX). Human differentiated neuronal progenitor cells (NPCs) were previously derived from hiPSC CVB line (RRID: CVCL_1N86, GM25430) (Young et al., 2015). NPCs were cultured on 20 mg/ml poly-L-ornithine (PLO) and 5 mg/ml laminin (both from Sigma) coated plates as previously described (Kassan et al., 2017). NPC medium [DMEM/F12/Glutamax media with N-2 supplement, B-27 supplement, Pen/Strep and basic Fibroblast Growth Factor (bFGF)] were used for culturing NPCs. The incubator was set at 37 • C in 5% CO 2 . Medium was changed every 2-3 days and cells were split with Accutase and Accumax (Innovative Cell Technologies) when arrived 100% confluency. bFGF was removed from the medium to induce differentiation. Medium was changed every 2-3 days.

Drug Application
After seven days of differentiation, neurons were treated with a small molecule [(#CN02-A) dissolved in NPC medium)] that directly activates Rac1/Cdc42 with various doses and time points (1, 2, and 3 units/ml; 5, 15, and 30 min). Untreated cells served as negative control. To measure related protein expression level change, cells were harvested directly after activator treatment and prepared for Western blot.

Western Blot
Cells were homogenized with cold RIPA lysis buffer followed by 3 cycles of 20-s bursts of sonication at 4 • C. The amount of protein per sample was determined using a dye-binding protein assay (Bio-Rad). Electrophoresis was performed on the samples using 4-12% or 10% acrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride membranes (Millipore) by electroelution. Membranes were blocked in blocking solution [20 mM TBS Tween (1%) (TBST) containing 3% bovine serum albumin (BSA)] and then incubated with primary antibodies overnight at 4 • C. After 3 washes with TBST, the membrane was incubated with secondary antibody for 1 h at room temperature (RT). Horseradish peroxidase (HRP)-labeled goat anti-rabbit FIGURE 2 | SynCav1 enhances axonal growth in differentiated NPCs derived from human iPSCs. Differentiated NSCs were transfected with lentiviral SynCav1 (2 × 10 9 IU/µl) (or SynGFP) on day 3 for 4 days, IF microscopy was performed at 1-3 weeks post transfection. Representative IF images are shown in (A). SynCav1 significantly enhanced axonal length (um) and volume (um 3 ) after 1 week (B) and increased both dendritic and axonal length (um) and volume (um 3 ) at 3 weeks (C) as measured by Autoneuron, a tracing algorithm that measures 3D image volume stacks (Head et al., 2011). NPCs were stained for the MAP2 (dendrites and magenta), SMI 31 (axons and green), and Cav-1 (red). 40× magnification, Nikon confocal microscope. Data are expressed as mean ± S.E (n = 3, * p < 0.05, t-test).
IgG (Santa Cruz Biotech) was used as secondary antibody. After another 3 times wash with TBST to remove the rest secondary antibody, membrane was incubated with ECL reagent (Amersham Pharmacia Biotech, Piscataway, NJ, United States) and prepared for imaging. Bands were compared to molecular weight standards (Santa Cruz Biotech). GAPDH served as the internal reference by which to standardize the other protein bands. The calculated ratio of the control group was normalized to 100%, and the comparisons of other groups to the control group were represented as percentages.

Immunofluorescence Confocal Microscopy
Neurons were fixed with 2% paraformaldehyde for 10 min at room temperature and then permeabilized in 0.1% Triton X-100 and blocked with 1% BSA/PBS/Tween (0.05%) for 60 min. Next, cells were incubated with primary antibodies in 1% BSA/PBS/Tween (0.05%) for 24-48 h at 4 • C followed by incubation with FITC or Alexa conjugated secondary antibody (1:600 to 1:800) for 1 h at room temperature (RT). Stained cells were mounted in mounting medium containing DAPI and imaged with Olympus confocal microscope system (Applied Precision, Inc., Issaquah, WA, United States) that included a Photometrics CCD mounted on a Nikon TE-200 inverted epifluorescence microscope. Exposure times were set to the same value for all groups every time. Quantitation of intensity of GTP-Rac1/GTP-Cdc42 was conducted by using Image J (NIH). Quantitation of axonal length and volume was conducted by using Autoneuron, which measures 3D image volume stacks (MBF Bioscience) generated by Neurolucida as previously described (Head et al., 2011); representative images and tracing image are shown in Supplementary Figure S1.

SynCav1 Increases Axonal Growth in Early Stage Differentiation of Human NPCs
As shown in Figure 1A, NPCs exhibited increased neuronal processes (i.e., increased axonal and dendritic growth as indicated by SMI 31 and MAP2 respectively, Figures 1B,C), while concurrently, the expression of Sox1, a stem cell marker, gradually decreased during this early stage of differentiation and were undetectable by day 7 days in vitro (DIV7) (Figures 1D,E). We have previously shown that SynCav1 increases dendritic growth and arborization in primary rodent neurons in vitro (Head et al., 2011), therefore we tested whether these effects on neuroplasticity from SynCav1 could be recapitulated in human NPCs. NPCs treated with SynCav1 on DIV3 exhibited significant axonal growth (Figure 2A) (quantitation of length and volume, n = 3, p < 0.05, Figure 2B), with no effect dendritic growth after 1 week (DIV7). However, by week 3 (DIV21, 17 days FIGURE 4 | SynCav1 increases T-Cav-1 but not P-Cav-1 in NPCs. (A) NPCs were treated with SynCav1 (2 × 10 9 IU/µl) for 72 hours followed by Western blot for P-Cav-1, T-Cav-1, and GAPH. Quantitation for P-Cav-1 and T-Cav-1 are shown in (B,C), respectively. Data are expressed as mean S.E (n = 3, * p < 0.05, t-test).
post SynCav1 treatment, Figure 2C) NPCs exhibited a significant increase in both MAP2-positive dendrites and SMI 31-positive axons (n = 3, p < 0.05, Figure 2D), akin to SynCav1treated primary rodent neurons in vitro (Head et al., 2011). These findings demonstrate for the first time that SynCav1 increases both dendritic and axonal growth in human neurons derived from iPSCs.
These results suggest that Rac1/Cdc42-mediated axonal growth is in part dependent upon P-Cav-1.

DISCUSSION
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(Head et al., , 2011Mandyam 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 FIGURE 6 | SynCav1 (Y14A) inhibits P-Cav-1 expression in NPCs. NPCs were incubated with SynRFP, SynCav1, or SynCav1(Y14A) (2 × 10 9 IU/µl) on day 3. On week 2, NPCs were lysed and probed for P-Src(Y416), T-Src, P-Cav-1(Y14), T-Cav-1, and GAPDH. Quantitation for P-Src and P-Cav-1 are shown in (B,C), respectively. Data are expressed as mean ± S.E (n = 3, * p < 0.05, * * p < 0.01, * * * p < 0.001, One-way ANOVA). (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 Cdc42mediated 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.
Our previous findings showed that SynCav1 increased dendritic arborization in hippocampal neurons in vivo , 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., 2016Egawa et al., , 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 . Although we did not test whether P-Cav-1 affects axonal transport of vesicles FIGURE 8 | SynCav1(Y14A) prevents SynRac1CA and SynCdc42CA-mediated increased P-Cav-1 expression in NPCs. NPCs were treated with vectors (2 × 10 9 IU/µl) as indicated on day 3 and WB was performed on week 2 to assay for P-Cav-1, T-Cav-1, and GAPDH. (A) Representative WB images of Ctrl, SynRac1CA, or SynRac1CA + SynCav1(Y14A) treated NPCs; Quantitation of P-Cav-1 and T-Cav-1 expression are shown in (B,C), respectively. (D) Representative WB images of Ctrl, SynCdc42CA, or SynCdc42CA + SynCav1(Y14A) treated NPCs; Quantitation of P-Cav-1 and T-Cav-1 expression are shown in (E,F), respectively. Data are expressed as the mean ± SE (n = 3, * p < 0.05, * * p < 0.01, One-way ANOVA). 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(Cao et al., , 2004Salanueva 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 FIGURE 9 | SynCav1(Y14A) suppresses SynRac1CA and SynCdc42CA-mediated axonal growth during NPC differentiation. NPCs were treated with vectors (2 × 10 9 IU/µl) as indicated on day 3 and IF was performed on week 2. (A) Representative images of Ctrl, SynRac1CA, or SynRac1CA + SynCav1(Y14A) treated NPCs. Quantitation of axonal length (um) and volume (um 3 ) are shown in (B,C), respectively. (D) Representative images of Ctrl, SynCdc42CA, or SynCdc42CA plus SynCav1(Y14A) treated NPCs. Quantitation of axonal length (um) and volume (um 3 ) are shown in (E,F), respectively. NPCs were stained for MAP2 (red), SMI 31 (green) and DAPI (blue). Data are expressed as the mean ± SE (n = 3, * p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001, One-way ANOVA).
(ischemic or traumatic) and in neurodegenerative diseases (AD, ALS, and multiple sclerosis).

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

AUTHOR CONTRIBUTIONS
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.

FUNDING
This work was supported by the Veteran Affairs Merit Award from the Department of Veterans Affairs BX003671 (BH) and BX001963 (HP), and the National Institutes of Health, Bethesda, MD, United States, NS073653 (BH) and HL091071 and HL107200 (HP).