Myelin and collapsin-1 induce motor neuron growth cone collapse through different pathways: inhibition of collapse by opposing mutants of rac1

Abstract

Precise growth cone guidance is the consequence of a continuous reorganization of actin filament structures within filopodia and lamellipodia in response to inhibitory and promoting cues. The small GTPases rac1, cdc42, and rhoA are critical for regulating distinct actin structures in non-neuronal cells and presumably in growth cones. Collapse, a retraction of filopodia and lamellipodia, is a typical growth cone behavior on contact with inhibitory cues and is associated with depolymerization and redistribution of actin filaments. We examined whether small GTPases mediate the inhibitory properties of CNS myelin or collapsin-1, a soluble semaphorin, in chick embryonic motor neuron cultures. As demonstrated for collapsin-1, CNS myelin-evoked growth cone collapse was accompanied by a reduction of rhodamine-phalloidin staining most prominent in the growth cone periphery, suggesting actin filament disassembly. Specific mutants of small GTPases were capable of desensitizing growth cones to CNS myelin or collapsin-1. Adenoviral-mediated expression of constitutively active rac1 or rhoA abolished CNS myelin-induced collapse and allowed remarkable neurite extension on a CNS myelin substrate. In contrast, expression of dominant negative rac1 or cdc42 negated collapsin-1-induced growth cone collapse and promoted neurite outgrowth on a collapsin-1 substrate. These findings suggest that small GTPases can modulate the signaling pathways of inhibitory stimuli and, consequently, allow the manipulation of growth cone behavior. However, the fact that opposite mutants of rac1 were effective against different inhibitory stimuli speaks against a universal signaling pathway underlying growth cone collapse.

Fig. 1.

Adenoviral-mediated expression of c-myc-tagged mutants of small GTPases in chick motor neurons. A, Motor neurons from 6-d-old chick embryos were plated on fibronectin and infected with a recombinant adenovirus carrying c-myc-tagged constitutively active rac1 under the viral E1A promoter (^V12rac1-Ad^E1A). Three days after infection, c-myc immunoreactivity is present in growth cones and cell bodies (data not shown) of motor neurons. B, In lacZ-Ad^E1A-infected cultures, c-myc immunostaining was very faint, indicating very little unspecific staining using the anti-c-myc antibody. C, Motor neuron cultures were infected with ^V12rac1-Ad^E1A using a multiplicity of infection (moi or ratio of virus particles to neurons) of 20, 50, and 100. Three days after infection, cultures were solubilized, and extracted proteins were separated by 15% SDS-PAGE. After transfer to PVDF membranes, the presence of^V12rac1 was demonstrated using a rac1-specific antibody.D, Rac1-immunoreactive bands were quantified by densitometric analysis and normalized to the values at 20 moi. Total rac1 immunoreactivity was proportional to the moi.

Fig. 2.

Mutants of small GTPases expressed via recombinant adenoviruses are functional. A, Motor neurons were infected with c-myc ^V12rac1-Ad^E1A (200 moi) and stained for c-myc using β-galactosidase-enhanced histochemistry. The percentage of c-myc-positive motor neurons is plotted against days after infection in c-myc^V12rac1-Ad^E1A-infected cultures (▪) compared with controls (▵). More than 90% of c-myc^V12rac1-Ad^E1A-infected motor neurons were c-myc positive 3 d after infection. B, Motor neurons were grown on FN and infected with^V12rac1-Ad^E1A (▪, 200 moi) or lacZ-Ad^E1A (○, 200 moi) at the time of plating. The longest neurite per neurons was measured, and their distribution in a motor neuron population was plotted. Only neuronal processes >10 μm long were considered neurites. Expression of ^V12rac1 attenuates β1 integrin-mediated neurite outgrowth on the FN substrate (2 day postinfection), which is consistent with previous findings using trituration loading of motor neurons with purified recombinant ^V12rac1 protein (Kuhn et al., 1998).

Fig. 3.

CNS myelin-associated growth inhibitors cause a rearrangement of actin filaments in motor neuron growth cones. Motor neurons were grown on FN for 2 d and then treated with PBS (A, B) or 100 μg/ml CNS myelin (C–F). Cultures were fixed and stained for actin filaments with rhodamine-labeled phalloidin (A, C, E) or for microtubules using a monoclonal anti-tubulin antibody followed by a fluorescein-conjugated secondary antibody (B, D, F). In controls, growth cones displayed many actin filament-rich filopodia and lamellipodia (A) with a dense bundle of microtubules defining the central region of the growth cones (B). On exposure to CNS myelin, growth cones retracted both lamellipodia and filopodia concomitant with a decrease in rhodamine fluorescence predominantly in the periphery.C, Often motor neuron growth cones responded to CNS myelin by retracting the entire peripheral region using the tubulin staining as our criteria. E, In some cases, motor neuron growth cones displayed stubby filopodial remnants with decreased rhodamine fluorescence in the peripheral region. Images were acquired using identical parameters. Scale bar, 10 μm.

Fig. 4.

CNS myelin-associated growth inhibitors signal a disassembly of actin filaments. A, Motor neurons were grown on FN for 2 d, treated with PBS (open bars) or 100 μg/ml CNS myelin (filled bars), and actin filaments were visualized with rhodamine-labeled phalloidin. Growth cones were divided into a peripheral region, a central region defined by intense microtubule staining, and 15 μm of the proximal neurite. Images were acquired using identical parameters, and the total fluorescence intensity was analyzed in each growth cone region on a pixel-by-pixel basis after background subtraction. All values were normalized to the total fluorescence intensity of the peripheral region in control growth cones. CNS myelin caused a significant reduction of rhodamine fluorescence in the peripheral region (*p< 0.001) and also in the central region (*p < 0.001) and the proximal neurite (*p < 0.001). The loss of rhodamine fluorescence suggests a net disassembly of actin filaments signaled by CNS myelin as reported for collapsin-1 (Fan et al., 1993). B, Freshly prepared, intact growth cone particles were incubated with 100 μg/ml CNS myelin (My), 15 μg/ml enriched collapsin-1 (CLP), a mixture of 2 mm CaCl₂and 10 μm A23187 (A23), 50 μg/ml laminin (LN), or PBS (Con). After 1 hr, samples were solubilized and separated into a cytoskeletal fraction and cytosol fraction by high-speed centrifugation (100,000 ×g_max). Proteins were separated on 10% SDS-PAGE and blotted onto PVDF membranes. The percentage of total actin in each fraction was determined by densitometry. Plotted is the percentage of total actin immunoreactivity in the cytoskeletal fraction, most likely actin filaments of various lengths, for each treatment. A significant decrease in filamentous actin occurred during incubation with CNS myelin (*p < 0.0001) or enriched collapsin-1 (*p < 0.01). As our control, large increases in the free intracellular Ca²⁺concentration results in an almost complete depletion of actin filaments (*p < 0.0001). In contrast, incubation with LN caused an increase in actin immunoreactivity in the cytoskeletal fraction, suggesting a net polymerization of actin filaments (**p < 0.01).

Fig. 5.

Dose-dependent CNS myelin-induced collapse of motor neuron growth cones is abolished by expressing^V12rac1 or ^V14rhoA. A, Motor neurons were grown on FN for 2 d and then treated with CNS myelin (open bars) or a protein extract obtained from CNS myelin (hatched bars). In the case of CNS myelin, concentration of 20 μg/ml or higher achieved significant numbers of collapsed growth cones (*p < 0.01), whereas as little as 10 μg/ml CNS myelin protein extract induced significant growth cone collapse (**p < 0.01). Importantly, a basal level of collapsed growth cones existed in these motor neuron cultures. B, Motor neurons were infected at the time of plating with recombinant adenovirus carrying mutants of small GTPases and grown for 3 d. Cultures were treated with 100 μg/ml CNS myelin (stippled and filled bars) or with PBS (open bars). The percentage of collapsed growth cones was determined as a function of small GTPase mutants that were expressed. Only expression of constitutively active rac1 (^V12rac1, filled bar) and rhoA (^V14rhoA, filled bar) inhibited CNS myelin-induced growth cone collapse (*p < 0.01). Expression of other small GTPase mutants or lacZ was ineffective (stippled bars). It is noteworthy that a fraction of motor neuron growth cones exhibited a collapsed morphology regardless of proteins expressed (open bars). C, Motor neurons were plated on FN-coated dishes containing stripes of CNS myelin (100 μg/ml), infected with recombinant adenovirus carrying mutants of small GTPases, and grown for 3–4 d. The length of neurites grown entirely on CNS myelin stripes or grown into CNS myelin stripes was measured. Neurite length per 500 μm² CNS myelin is plotted as a function of small GTPase mutants expressed. Expression of either ^V12rac1 or ^V14rhoA (open bars) resulted in considerable neurite outgrowth on CNS myelin (*p < 0.01) compared with lacZ or the other small GTPase mutants (filled bars). Con, PBS; lacZ, reporter gene coding β-galactosidase; ^V12rac1, constitutively active rac1; ^N17rac1, dominant negative rac1; ^V12cdc42, constitutively active cdc42; ^N17cdc42, dominant negative cdc42;^V14rhoA, constitutively active rhoA.

Fig. 6.

Motor neurons only establish neurites on CNS myelin when expressing ^V12rac1. Motor neurons were plated on FN-coated dishes containing stripes of CNS myelin (100 μg/ml) and infected (200 moi) with ^V12rac1-Ad^E1A(A, B) or ^N17rac1-Ad^E1A(C, D). A, Three days after infection, motor neurons expressing ^V12rac1 exhibit neurites grown entirely (asterisk) on CNS myelin-coated areas (My) as well as crossing into CNS myelin stripes (arrowheads). Dotted line marks the FN–CNS myelin border. B, Neurites grown exclusively on FN, however, are much longer and more branched (same cultures as inA), forming a rather intricate network.C, Despite ^N17rac1-expressing motor neurons extending neurites toward CNS myelin stripes (My), neurite growth into or entirely on CNS myelin stripes was only marginal (arrowhead) or even absent. The dashed line indicates the border between CNS myelin and FN.D, Nevertheless, on FN (same culture as inC), ^N17rac1-expressing motor neurons established a network of neurites comparable to that in^V12rac1-infected cultures. Scale bar, 20 μm.

Fig. 7.

^N17rac1 and^N17cdc42 protect motor neuron growth cones from collapsin-1-induced collapse and support neurite outgrowth on a collapsin-1 substrate. A, Motor neurons grown on FN for 2 d were treated with increasing concentrations of enriched collapsin-1, and the number of collapsed growth cones was determined. Growth cone collapse was dose-dependent. A minimal concentration of 50 μg/ml was required for a significant number of collapsed growth cones (*p < 0.01). B, Motor neurons were plated on FN and infected with recombinant adenovirus carrying mutants of small GTPases. Three days after infection, cultures were treated with purified recombinant collapsin-1 (75 ng, 150 ng/ml). The percentage of collapsed growth cones is plotted as a function of the small GTPase expressed. Only ^N17rac1 or^N17cdc42 reduced the fraction of collapsed growth cones (*p < 0.01) (filled bars), whereas other small GTPase mutants and lacZ expression were ineffective (open bars). In particular, the protective effect of^N17rac1 has been reported previously in DRG neurons (Jin and Strittmatter, 1997). Even in the absence of collapsin-1, a fraction of motor neuron growth cones was collapsed, independent of proteins expressed as shown for CNS myelin in Figure 5B.C, In a similar experiment, motor neurons were plated on poly-d-lysine-coated dishes containing collapsin-1 stripes and infected with recombinant adenovirus carrying mutants of small GTPases. The neurite length of the longest neurite per neuron was measured on polylysine alone (open bars) or on collapsin-1 (hatched bars and filled bars), and the average length was plotted as a function of expressed proteins. Neurite outgrowth on polylysine was comparable among conditions (open bars). In particular, expression of ^N17rac1 or ^N17cdc42 supported neurite outgrowth on collapsin-1 (filled bars) that was indistinguishable from growth on polylysine alone (*p < 0.001). Neither of the other small GTPase mutants that were tested significantly increased neurite length on collapsin-1 (hatched bars). It is noteworthy that motor neurons exhibited a basal outgrowth on collapsin-1.lacZ, Control; ^V12rac1, constitutively active rac1; ^N17rac1, dominant negative rac1; ^V12cdc42, constitutively active cdc42; ^N17cdc42, dominant negative cdc42;^V14rhoA, constitutively active rhoA.

Fig. 8.

Dominant negative mutants of rac1 and cdc42 abolish the growth inhibitory effect of collapsin-1. Motor neurons expressing ^V12rac1 (A, D),^N17rac1 (B, E), or ^N17cdc42 (C, F) were plated on polylysine-coated dishes containing collapsin-1 stripes. A–C, On polylysine, motor neurons formed neurites (arrows) regardless of expressing ^V12rac1 (A),^N17rac1 (B), or ^N17cdc42 (C). D–F, On collapsin-1, expression of ^V12rac1 (D) achieved only basal outgrowth (asterisk), whereas both^N17rac1 (E) and ^N17cdc42 (F) supported neurite outgrowth (arrowheads) comparable to levels observed on polylysine alone. Scale bar, 30 μm.