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. 2019 Dec 10;13(6):1006-1021.
doi: 10.1016/j.stemcr.2019.10.008. Epub 2019 Nov 7.

Environmental Elasticity Regulates Cell-type Specific RHOA Signaling and Neuritogenesis of Human Neurons

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Environmental Elasticity Regulates Cell-type Specific RHOA Signaling and Neuritogenesis of Human Neurons

Robert H Nichol 4th et al. Stem Cell Reports. .
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Abstract

The microenvironment of developing neurons is a dynamic landscape of both chemical and mechanical cues that regulate cell proliferation, differentiation, migration, and axon extension. While the regulatory roles of chemical ligands in neuronal morphogenesis have been described, little is known about how mechanical forces influence neurite development. Here, we tested how substratum elasticity regulates neurite development of human forebrain (hFB) neurons and human motor neurons (hMNs), two populations of neurons that naturally extend axons into distinct elastic environments. Using polyacrylamide and collagen hydrogels of varying compliance, we find that hMNs preferred rigid conditions that approximate the elasticity of muscle, whereas hFB neurons preferred softer conditions that approximate brain tissue elasticity. More stable leading-edge protrusions, increased peripheral adhesions, and elevated RHOA signaling of hMN growth cones contributed to faster neurite outgrowth on rigid substrata. Our data suggest that RHOA balances contractile and adhesive forces in response to substratum elasticity.

Keywords: RHOA; adhesion signaling; axon outgrowth; cortical neuron; growth cone; iPSC; mechanotransduction; regeneration.

Figures

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Figure 1
Figure 1
Substrata Elasticity Influences Neuronal Morphogenesis in a Neuronal-type Specific Manner in Two-Dimensional Cultures (A–D) Low-magnification confocal images of iPSC-derived hMN neurospheres cultured on soft (0.5 kPa), intermediate (4 kPa), and rigid (25 kPa and 50 kPa) LN-coated PAA gels, and immunolabeled for acetylated tubulin (magenta) and F-actin (phalloidin, green). Note longer processes on rigid substrata compared with soft and intermediate. (E) hMN neurite lengths were measured on increasing PAA gel rigidities (0.5–125 kPa). Due to the density of neurites extending from neurospheres, the ten longest neurites were measured for this analysis and compared between experimental groups. hMNs extend greater neurite lengths on progressively more rigid substrata up to 25 kPa and fitted linear regression lines showed strong goodness of fit (R2 = 0.8952) and are significantly more sloped (p < 0.001) compared with hFB neurons (below). n ≥ 40 neurites from n = 4 experiments from n = 2 differentiations for each condition. (F–I) Representative images of hFB neurospheres cultured on soft (0.5 kPa), intermediate (4 kPa), and rigid (25 kPa and 50 kPa) LN-coated PAA gels, and immunolabeled for acetylated tubulin (magenta) and F-actin (phalloidin, green). Note similar neurite lengths on each elastic condition. (J) Quantification of the ten longest hFB neurite lengths on increasing PAA gel rigidities shows no significant trend across rigidities, and linear regression shows poor goodness of fit (R2 = 0.03432). n ≥ 50 neurites from n = 4 experiments from n = 2 differentiations for each condition. (K) Inverted contrast grayscale image of a representative labeled neurosphere used for Sholl analysis (ImageJ plugin, see Experimental Procedures) to measure neurite number and length. (L and M) Sholl analysis of all neurites confirms that there are more intersecting neurites at greater distances on more rigid substrata for hMNs (L), while hFB neurite lengths show a modest increased number of short processes on rigid substrata (M). Insets compare the mean number of total intersections, showing significant elasticity-dependent differences. n ≥ 13 neurons from n = 3 experiments from n = 2 differentiations for each condition. ∗∗∗p < 0.001, one-way ANOVA. It should be noted that elasticity differences in hFB neurite extension are limited to short processes, suggesting possible effects on neurite initiation. (N and O) Representative live-cell images over a 40-min time period of hMN neurites extending from neurospheres plated on soft and rigid PAA gels. (P) Quantification of rate of outgrowth from time-lapse movies confirms that neurite extension from hMNs is significantly faster on rigid substrata. n ≥ 362 neurites from n = 3 experiments from n = 2 differentiations for each condition. ∗∗∗p < 0.001, Student's t test. (Q and R) Representative live-cell images over a 40-min time period of hFB neurites extending from neurospheres plated on soft and rigid PAA gels. (S) Quantification of rate of outgrowth shows that neurite extension from hFB neurites is significantly faster on soft substrata. n ≥ 362 neurites from n = 3 experiments from n = 2 differentiations for each condition. ∗∗p < 0.01, Student's t test. Scale bars in (D) and (N), 100 μm (apply also to A–I, N, O, Q, R). See also Figures S1–S4.
Figure 2
Figure 2
Substrata Elasticity Influences Neuronal Morphogenesis in Dissociated Two-Dimensional Cultures (A and B) Dissociated hMNs on soft and rigid LN-coated PAA gels. Note that hMNs on 0.5 kPa (A) appear to have shorter, but more branched processes compared with 25 kPa (B). (C–G) While the longest neurite (C) and total neurite length (D) of hMNs are greater on rigid substrata, the number of neurites per neuron are greater (E and F) and neurites are more branched (G) on soft substrata. n ≥ 96 neurites from n = 3 experiments from n = 2 differentiations for each condition. ∗∗∗p < 0.001, Student's t test.
Figure 3
Figure 3
Substratum Elasticity Influences Axon Development in Three-Dimensional Collagen I (A–B′) Maximum projections of multi-photon SHG volumetric images (35 slices covering 50 μm) of self-assembled 1.5 mg/mL collagen I hydrogels untreated (A) and crosslinked with 1 mM genipin (B). (B′) SHG image of collagen (blue) merged with confocal genipin fluorescence image (excitation 620 nm, red). Note strong colocalization of the collagen fibrils with genipin and reduced fibril density compared with control. (C–E) The total area occupied by collagen fibrils (C) and fibril count (D) are significantly reduced in genipin-treated hydrogels compared with untreated, while mean SHG intensity (E) is increased. (F and G) Fibril orientation becomes less variable (F) and total fibril alignment (G) is increased by genipin treatment. All collagen fibril measurements were made using CT-FIRE software on three hydrogels per condition (see Experimental Procedures). (H) Elastic modulus measured using a nanoindentation device (see Experimental Procedures). n = 2–3 hydrogels for each condition. (I–L) Low-magnification confocal z-stack images of hMN (I and J) and hFB (K and L) neurospheres immunolabeled for acetylated tubulin (magenta) and F-actin (phalloidin, green). Neurospheres were cultured on untreated (I and K) and genipin-crosslinked collagen hydrogels (J and L) for 4 DIV. (M) Quantification of neurite lengths on control collagen and collagen with genipin for hMN and hFB. Note the opposite effect on neurite length for hMN and hFB on soft and stiff collagen. n ≥ 755 neurites from n = 3 experiments from n = 2 differentiations for each condition. (N) Sholl analysis of mean number of intersections shows that there are more intersecting neurites on more rigid substrata for hMNs. n ≥ 700 neurites from n = 3 experiments from n = 3 differentiations for each condition. ∗∗∗p < 0.001, Student's t test. Scale bars, 40 μm (A and B) and 100 μm (I–L).
Figure 4
Figure 4
The Stability of Lamellipodial Protrusions by hMN and hFB Growth Cones Depends on Substratum Rigidity (A) A GFP-expressing hMN growth cone on rigid LN-PAA. (B) Two-time-point image merge of growth cone in (A) at times indicated showing initial (red) and final (green) position. Yellow line indicates line used to produce kymograph from 5-min time-lapse sequence. (C) Kymograph constructed from region specified in (B) showing protrusion and retraction events. (D and E) Representative kymographs generated from hMN growth cone lamellipodial protrusions on soft (D) and rigid substrata (E). Note greater stability of leading-edge protrusions on rigid substratum. (F) Duration of protrusion and retraction events on soft and rigid substrata for hMN growth cones. (G) Rate of membrane protrusion and retraction of hMN growth cone leading edge on soft and rigid substrata. (H and I) Representative kymographs generated from hFB growth cone lamellipodial protrusions on soft (H) and rigid (I) substrata. Note unstable leading-edge protrusions on soft and rigid substrata. (J) Duration of protrusion and retraction events on soft and rigid substrata for hFB growth cones. (K) Rate of membrane protrusion and retraction of hFB growth cone leading edge on soft and rigid substrata. Note that protrusion durations and rates are significantly different between hMNs and hFB neurons. n ≥ 164 kymographs from n = 3 experiments from n = 2 differentiations for each condition. ∗∗∗p < 0.001, p < 0.01, Student's t test. Scale bars, 10 μm (A) and 5 um (B).
Figure 5
Figure 5
RHOA and MLC Activities Are Increased in hMNs on Rigid Substrata (A) Biochemical measurement of active RHOA in hMNs cultured on soft and rigid PAA hydrogels shows RHOA activity is higher in hMNs on rigid substrata. n = 9 replicates from n = 5 experiments for each condition. p < 0.01, Student's t test. (B–C′) Representative confocal fluorescent images of hMN neurites on soft (B and B′) and rigid (C and C′) LN-coated PAA hydrogels. Neurons were immunolabeled with phospho-specific MLC antibodies at the activation site Ser19 (green in merge) and co-labeled for F-actin (phalloidin, magenta). Note increased pS19-MLC labeling in axons and growth cones on rigid PAA hydrogels. (D–E′) Zoomed images (from boxes indicated in B′ and C′) showing hMN growth cones on soft (D and D′) and rigid (E and E′) substrata. (F) Quantification of pS19-MLC fluorescence intensities in hMN growth cones on soft and rigid PAA hydrogels. Measurements of pS19-MLC fluorescence are normalized to total protein labeling (see Experimental Procedures). n ≥ 50 growth cones from n = 3 experiments from n ≥ 2 differentiations for each condition. ∗∗∗p < 0.001, Student's t test. (G) Confocal images of growth cones of hMNs cultured on LN-coated glass and stimulated for 1 h with control medium (top), 100 nM LPA (middle), or 2 μg/mL Rho Inhibitor I (bottom), then immunolabeled for pS19-MLC. Note robust pS19-MLC labeling of growth cone treated with LPA. (H) pS19-MLC fluorescence intensity measurements of hMN growth cones. n ≥ 43 growth cones from n = 3 experiments from n ≥ 2 differentiations for each condition. ∗∗∗p < 0.001, Student's t test. Scale bars, 20 μm (B and C) and 10 μm (D, E, and G).
Figure 6
Figure 6
Activation of Adhesion Signaling and Scaffolding in hMN Growth Cones on Rigid Substrata (A–J′) Representative confocal images of hMN growth cones on soft (A, C, E, G, and I) and rigid (B, D, F, H, and J) LN-coated PAA gels. Neurons were immunolabeled with phospho-specific antibodies (green in merges) to the sites indicated and counterstained for F-actin (phalloidin, magenta). Scale bar, 5 μm. (K–O) Fluorescence intensity measurements for activation sites for FAK (y397) (K), SRC (y418) (L), and p130-CAS (y165 [N] and y410 [O]), and an inhibitory site of SRC (y529) (M). Measurements of phosphorylated adhesion proteins are presented as ratio against total protein labeling using SE647 (see Experimental Procedures) in hMN growth cones at specified phosphorylation sites, and ratio measurements of neurons on rigid substrata data are normalized to soft substrata. n ≥ 44 growth cones for each labeling from n = 3 experiments from n ≥ 2 differentiations for each condition. ∗∗∗p < 0.001, Student's t test. See also Figure S5.
Figure 7
Figure 7
Modulating RHOA Signals Switches the Effects of Elastic Substrata on hMN Neurite Outgrowth (A–F) Representative images of hMN neurospheres after 2 DIV on soft (A–C) and rigid (D–F) substrata. hMNs were treated with control medium (A and D), 100 nM LPA (B and E), or 2 μg/mL Rho Inhibitor I (C and F) for 24 h, then fixed and immunolabeled for acetylated tubulin (magenta) and F-actin (phalloidin, green). (G) Mean neurite lengths of hMNs upon chronic modulation of RHOA reveals opposing effects that depend on substrata elasticity. n ≥ 100 longest neurites from n = 3 experiments from n = 3 differentiations for each condition. ∗∗∗p < 0.001, one-way ANOVA. (H and I) Time-lapse differential interference contrast (DIC) images of live hMNs of soft (H) and rigid (I) substrata at time points indicated before and after treatment with 100 nM LPA. (J) Rate measurements show that LPA treatment significantly increases outgrowth on soft substrata, but reduces outgrowth on rigid substrata. n ≥ 116 neurites from n = 3 experiments from n = 2 differentiations for each condition. ∗∗∗p < 0.001, ∗∗p < 0.05, Student’s t test. (K and L) Time-lapse DIC images of live hMNs of soft (K) and rigid (L) substrata at time points indicated before and after treatment with 2 μg/mL Rho Inhibitor I. (M) Rate measurements show that RHOA inhibition significantly reduces outgrowth on soft substrata, but modestly increases outgrowth on rigid substrata. n ≥ 69 neurites from n = 3 experiments from n = 2 differentiations for each condition. ∗∗∗p < 0.001, Student's t test. (N–O′) Kymographs generated from GFP-expressing hMN growth cones on soft and rigid substrata before and after acute activation of RHOA with 100 nM LPA. Note increased stability of the leading edge (yellow line) on soft substrata after LPA treatment (N′). (P and Q) Leading-edge membrane protrusion duration (P) and retraction (Q) events on soft and rigid substrata for hMN growth cones before and after 100 nM LPA. Note significant increase in duration of protrusion and retraction after LPA on soft substrata but decrease on rigid substrata. n ≥ 141 kymographs from n = 3 experiments from n = 2 differentiations for each condition. ∗∗∗p < 0.001, Student's t test. Scale bars, 100 μm (A–F) and 10 μm (H, I, K, and L). See also Figure S6.

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