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Asymmetric membrane ganglioside sialidase activity specifies axonal fate

Nature Neuroscience volume 8, pages 606–615 (2005)Cite this article

Abstract

Axon specification triggers the polarization of neurons and requires the localized destabilization of filamentous actin. Here we show that plasma membrane ganglioside sialidase (PMGS) asymmetrically accumulates at the tip of one neurite of the unpolarized rat neuron, inducing actin instability. Suppressing PMGS activity blocks axonal generation, whereas stimulating it accelerates the formation of a single (not several) axon. PMGS induces axon specification by enhancing TrkA activity locally, which triggers phosphatidylinositol-3-kinase (PI3K)- and Rac1-dependent inhibition of RhoA signaling and the consequent actin depolymerization in one neurite only. Thus, spatial restriction of an actin-regulating molecular machinery, in this case a membrane enzymatic activity, before polarization is enough to determine axonal fate.

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Figure 1: Early segregation of PMGS to a single neurite correlates with axonal fate and reduced F-actin content.
Figure 2: PMGS activity specifies the single axon by reducing local F-actin concentration.
Figure 3: NGF signaling requires PMGS activity.
Figure 4: PMGS leads to localized activity of TrkA receptors.
Figure 5: PMGS inactivates the small GTPase RhoA.
Figure 6: PI3K and Rac1 couple TrkA and RhoA in PMGS-induced signaling.
Figure 7: PMGS activity modulates the ROCK/profilin IIa actin-regulating complex.

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Acknowledgements

We thank B. Hellias and E. Cassin for technical assistance; M. Giustetto and H. Vara for help with the local perfusions. J.S.S. is supported by an FCT/PRAXIS XXI scholarship (Portuguese Ministry of Science and Technology). Part of this work is supported by EU Grant Apopis (FP6-2002–LIFESCIHEALTH).

Author information

Authors and Affiliations

  1. Cavalieri Ottolenghi Scientific Institute, University of Turin, Turin, 10043, Orbassano, Italy

    Jorge Santos Da Silva, Carlos G Dotti & Jose Abad-Rodriguez

  2. Division of Biochemistry, Miyagi Prefectural Cancer Center, 47-1 Nodayama, Medeshima-Shiode, Natori, 981-1293, Miyagi, Japan

    Takafumi Hasegawa & Taeko Miyagi

  3. Center for Human Genetics, Catholic University of Leuven and Flanders Interuniversitary Institute for Biotechnology, Leuven, 3000, Belgium

    Carlos G Dotti

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Correspondence to Carlos G Dotti or Jose Abad-Rodriguez.

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Supplementary information

Supplementary Fig. 1

(a) Quantification of percentage of cells with an obvious polarization of PMGS to one neurite tip (defined as having 100% more PMGS fluorescence than any other cell area). The bias of PMGS distribution to one process is always significant and is gradually more marked as the future axon extends and neurons reach stage 3 and full morphological polarization. (Neurons of sister cultures were used for each individual experiment; stage 2, n = 40; stage 2+, n = 37; stage 3, n = 31; Data are mean + SD values in percentage of each stage population; *P <0.001, comparison with stage 2; ** P < 0.001, stage 3 in comparison with stage 2+). (b)Correlation of PMGS fluorescence and F-actin accumulation within the tips of a representative example of a stage 2+ neuron. The same shift to a lower actin filament content is observed in the tip with higher PMGS accumulation (similarly to stage 2 cells, see Fig. 1A).( c )Representative cells from the stage 3 population used in this study also show the same inversed correlation of F–actin and PMGS distribution, as observed in stage 2 (see Fig. 1A) and stage 2+ neurons (see panel B). Scale bar, 10µm. (d) Scatter plots of stage 2 neurons correlating PMGS and F–actin fluorescence for each neurite tip of each cell in the population. The example shown in Fig. 1A is marked with an arrow. This cell represents the population. (e) Same as in D for stage 2+ neurons. The example shown in B is marked with an arrow. This cell represents the population. (f) Same as in D and E for stage 3 neurons. The example shown in C is marked with an arrow. This cell represents the population. (g) Scatter plots of neurons at 48 hours in culture treated with PMGS inhibitor (NeuAc2en) correlating PMGS and F-actin fluorescence for each neurite tip of each cell in the population. The example shown in Fig. 2B is marked with an arrow. This cell represents the population. (PDF 4229 kb)

Supplementary Fig. 2

(a) After 30 hours in culture, control (upper panels) and PMGS over-expressing hippocampal neurons present enriched PMGS labeling in early tau1-positive axons (arrowheads). Tau-negative neurites are mostly devoided of PMGS labeling (arrows). (b) Consistently, PMGS labeling (arrowheads) is absent from the somato-dendritic compartment defined by MAP2 staining (arrows). (c) In the population of HA-PMGS over-expressing cells PMGS co-accumulates preferentially and significantly with Tau1 (black bar) but not with MAP2 (white bar). (d) After 30 hours in culture, control (upper panels) constitutively active RhoA (left panel, mycRhoAL63), dominant negative Rac1 (central panel, FlagRacN17) or PI3K inhibitor (right panel, wortmannin) preclude the formation of tau-positive neurites (early axons) as shown for control or NGF-treated neurons (bottom panels). (e) The membrane protein Transferrin receptor (TfR) does not show any significant local enrichment in stage 2 neurons (right panel), in contrast with PMGS (left panel, arrowhead). On average there is a slightly higher level of TfR in the same growth cone where PMGS accumulates (black bars) although not significantly different from other neurites when considering the whole population (white bars). (f) Laminin coating in parallel stripes was performed on PLL-pretreated coverslips using silicon matrices with parallel micro-channels as described by Baier, H. and Klostermann, S (1994). Briefly, PLL-covered coverslips were placed onto the silicon matrices and the micro-channels were injected with laminin solutions (1 mg/ml) supplemented with FITC (1 µg/ml). As control we used a solution of FITC at the same concentration. After three hours incubation, the coverslips were washed and placed in multiwell plates with appropiate culture media. Primary hippocampal neurons were then plated, fixed after 24 hours and immunolabeled with anti-PMGS antibodies. Local accumulation of PMGS (as determined by endogenous PMGS immunoreactivity) to one neurite of stage 2 neurons (stage 2) occurs independently of contact with Laminin (left panel) or PLL (central and right panel). Local accumulation of PMGS to one neurite of stage 2+ (stage 2+) occurs independently of contact with Laminin (left panel) or PLL (central and right panel). Scale Bar, 10µm. (PDF 12256 kb)

Supplementary Fig. 3

Examples of stage 2 and 2+ neurons followed by time–lapse microscopy during 30 minutes and then fixed and labeled against PMGS and F-actin. The area of all the growth cones (marked individually from 1 to 5) was measured and plotted against time (scatter plots). (a) Individual time–lapse frames composing Supplementary Video 1. Of all neurites, similar in length, neurite 1 (arrow) shows future axon characteristics since it displays higher average growth cone area, has higher motility (indicated by the amplitude of growth cone area variation over time; graphic, upper line) and accumulates less actin filaments (F-actin, arrow). Subsequent analysis shows that the neurite identified as the future axon significantly accumulates more PMGS (PMGS, arrow). (b) Individual time-lapse frames composing Supplementary Video 2. Neurite 1 (arrow) shows future axon characteristics since it displays longer length, higher average growth cone area, has higher motility (indicated by the amplitude of growth cone area variation over time; graphic, upper line) and accumulates less actin filaments (F-actin, arrow). Subsequent analysis shows that the neurite identified as the future axon significantly accumulates more PMGS (PMGS, arrow). (PDF 12941 kb)

Supplementary Fig. 4

(a) Over-expression of PMGS showing that the protein (PMGS, as detected by the specific antibody) is greatly enriched in the prematurely specified axon. This indicates the high protein levels do not affect distribution. Functionally, more GM1 (the product of PMGS enzymatic activity) is detected in the areas where more PMGS accumulates (arrows, the specified axon and the cell body). (b) An example of a PMGS over–expressing cell with low levels of ectopic protein as judged by the weaker signal detected with ant-HA antibody (compare with other over–expressing cells shown in Fig.s 2, 3, 4, 5, 6 and 7). Despite the lower expression efficiency the PMGS over–expression phenotype is still observed (specified axon at 24 hours). Importantly, the enrichment of PMGS in the specified axon (arrow versus arrowheads) is not affected by ectopic expression (HA) and this correlates with the signal detected with the specific PMGS antibody (PMGS). (c) Cells over–expressing HA-PMGS were labeled against tubulin and either HA (HA–PMGS) or PMGS (PMGS protein). The relative fluorescence of each signal was calculated taking into account the total fluorescence and background of each channel of each image, as well as the distribution of tubulin to control for volume, since in some case,s the specified axon was thicker than the other processes (d). Both HA and PMGS significantly enrich in the specified axon (Axons) in comparison to the remaining neurites (Neurites). The averages of both signals are very similar when either axons or the remaining neurites are considered. (Neurons of sister cultures were used for each individual experiment; HA-PMGS, n = 64; PMGS protein, n = 57; Data are mean + SD values of relative fluorescence; * P < 0.001, comparison with HA–PMGS Axons; ** P < 0.001, in comparison with PMGS protein Axons). (e) Percentage of the different populations of cells in 48 hours in vitro hippocampal neurons. At this time in culture most cells (80 ± 4%) are polarized stage 3 neurons. Scale bar, 10µm. (PDF 12052 kb)

Supplementary Fig. 5

Hippocampal neurons were transfected with specific iRNA targeted against PMGS (iRNA), with non–targeting iRNA (nt–RNA) or in the absence of any oligonucleotide (Mock). (a) Western blot analysis shows that the expression of PMGS in cell membranes is precluded by iRNA transfection, while non–targeting iRNA or Mock transfected cells show normal levels of the enzyme. (b) Supporting the former result, slot blot analysis shows much lower levels of the ganglioside GM1 (the main product of PMGS activity) in the membrane of iRNA transfected cells than in the two control cases. (c) To prove biochemically that PMGS enzymatic activity is reduced in the membrane after PMGS knocking down by iRNA, SK-N-MC cells 46–48 were transfected as indicated in the case of hippocampal neurons. After 48 hours in culture, membrane–associated PMGS activity was tested using a mixture of the gangliosides GT1b and GM1 as substrate, and analyzing the products of the reaction by TLC. As shown in the panel, membranes of NT–iRNA and Mock transfected cells transform part of the GT1b in GM1 (compare upper bands of GM1 and lower bands of GT1b between Mock, NT-iRNA and the original quantity in the standard mix). On the contrary, membranes of iRNA-transfected cells are not able to digest GT1b and GM1 is not produced, indicating that PMGS activity has been extensively reduced. (d) In comparison with buffer-treated controls, neurons added with BDNF or NT-3 (for 24 hours) are not polarized. This indicates that these neurotrophic factors, under our experimental conditions and within this experimental time span, do not induce axon specification in backgrounds with endogenous levels of PMGS, similarly to what was observed in backgrounds with increase PMGS activity (PMGS over-expression, see Fig. 3A). Images were taken at the same magnification as in Fig. 3A. (e) HA-PMGS transfected hippocampal neuron extracts were immunoprecipitated (IP) with anti–HA antibody and western blotted with anti–TrkA, TrkB and TrkC antibodies. Total lysates were probed for HA to assure the homogeneity of the HA-PMGS overexpression (lines 2, 3 and 4, bottom panel). Only a low amount of TrkA (line 2, upper panel), but no TrkB or C (line 2, central panels), is immunoprecipitated in HA-PMGS overexpressing cells. The presence of NGF enhances the quantity of immunoprecipitated TrkA (line 3, upper panel). Neither in this case TrkB and C are immunoprecipitated (line 3, central panels). (PDF 9021 kb)

Supplementary Fig. 6

(a) Local buffer perfusion of a single growth of a stage 2+ neuron (likely future axon, as judged by length,*) does not alter general growth cone structure (see insets,*), actin filament content (lower than in other cones, F-actin, *) or PMGS polarized distribution (PMGS, *). (b) Local NeuAc2en perfusion of a single growth cone of a stage 2+ neuron (likely future axon, as judged by length,*) induces growth cone area reduction (see insets, *) and favors local actin polymerization (F-actin, *). PMGS polarized distribution is not affected (PMGS, *). (c) Area variation of growth cones perfused with buffer (upper graphic) and of the non–perfused growth cones of the same cells (lower graphic) over the perfusion period (15 minutes). Both populations do not display any significant change of growth cone area between time 0 and time 15 minutes. (d) Area variation of growth cones perfused with NeuAc2en (upper graphic) and of the non–perfused growth cones of the same cells (lower graphic) over the perfusion period (15 minutes). While non-perfused cone do not display any significant change of growth cone area between time 0 and time 15 minutes (lower graphic), local PMGS inhibition induces growth cone retraction over the perfusion period (upper graphic). The populations shown in C and D comprise stage 2 and 2+ cells (including the examples shown in Fig. 2C and D and in panels A and B). The average growth cone area variation as well as the standard deviations of these populations were summarized in Fig. 2E. Scale bar, 10µm. (PDF 12888 kb)

Supplementary Fig. 7

Lines with pre-defined width (10 pixels) were drawn to tangentially intersect the tips of all neurites in individual neurons. The intensity of each pixel width of individual tangential lines was measured (pixel intensity) and plotted to the respective distance from the starting point of the tangential line. Epitomized here are the same examples shown in the respective Fig.s 5 and 6. (a) While constitutively active RhoA is evenly distributed in all identified peaks (black line; numbers correspond to tip numbers indicated in the fluorescent image panels), HA-PMGS (grey line) still preferentially polarized to one tip (4). However, this increased local activity of PMGS does not produce axon specification, indicating that the incapacity to induce RhoA inactivation in this neurite (4), but not in others (1,2,3 and 5) does not allow PMGS-directed neuronal polarization. (b) Wortmannin bath application does not preclude preferential accumulation of HA-PMGS in one neurite tip (3). Thus, PI3K activity does not influence PMGS distribution. (c) Flag-RacN17 distributes equally in all process tips (black line) but such changes in Rac1 activity do not modify the polarization of HA–PMGS to one neurite (2). Lack of Rac1 activity in this one neurite where PMGS accumulates hinders the capacity of such localized increased of PMGS activity to favor initial outgrowth. Scale bar, 10µm. *, peak is significantly higher than other peaks in the corresponding plot. (d) Scatter plots of neurons at 24 hours co-transfected with HA-PMGS and mycRhoAL63 correlating HA and myc fluorescence for each neurite tip of each cell in the population. The example shown in A is marked with an arrow. This cell represents the population. (e) Scatter plots of HA fluorescence of neurons at 24 hours transfected with HA-PMGS and treated with Wortmanin for each neurite tip of each cell in the population. The example shown in B is marked with an arrow. This cell represents the population. (f) Scatter plots of neurons at 24 hours co-transfected with HA-PMGS and FlagRacN17 correlating HA and Flag fluorescence for each neurite tip of each cell in the population. The example shown in C is marked with an arrow. This cell represents the population. (g) Neuronal cultures treated with PMGS inhibitor NeuAc2en (left panel) present a retardation in axonal determination as shown in Fig.s 2, 3 and 4. Inactivation of ROCK (Y-27632, central panel) or PIIa (ASPIIa, right panel), reverts the polarity arrest induced by PMGS inactivation (tau-1 staining, central and right panel; quantification of polarized cells in the bar graphic). (PDF 3699 kb)

Supplementary Fig. 8

(a) Quantification of non-axonal neurite length in different experimental conditions. OE PMGS neurons do not show any difference in non-axonal neurite length when compared to controls or to any of the experimental perturbation indicated in the graphic. (b) Constitutively active RhoA alone precludes axon formation (lower panel) as observed when co-transfected with PMGS (upper panel). (c) Dominant negative Rac prevents axon specification(lower panel), similarly to when it is co-expressed with HA-PMGS (upper panel). (d) Quantification of the effects in B and C. No difference in the number of polarized cells is observed when RhoAL63 and RacN17 are expressed alone or with HA-PMGS. Since HA-PMGS expression alone favors axon formation, the polarization arrest is specific for the alterations in RhoA and Rac activities. (e) Neuronal cultures 3 (stage 1) and 6 (stage 1+) hours after plating, fixed and labeled for PMGS and F-actin. In some stage 1 neurons accumulations of PMGS (left-upper panel, arrowhead) could be detected in coincidence with zones of low F-actin content (right-upper panel, arrowhead). In slightly more developed neurons (stage 1+) the cells initiate to create defined sprouts. As shown in the left-lower panel (arrowhead) PMGS tends to accumulate in one of the sprouts, the one with less F-actin (right-lower panel, arrowhead). (f) pTrkA accumulates in axons (arrowheads) but not in dendrites (arrows) of stage 3 neurons (right panel) in coincidence with the distribution of endogenous PMGS (central panel). *, **p<0.001. Scale Bars, 10µm. (PDF 92 kb)

Supplementary Fig. 9

TrkA, but not TrkB or TrkC, accumulates in early axons of hippocampal neurons. The future axon of stage 2, 2+ and 3 neurons was identified by low local F-actin staining or by high PMGS local staining. These cells were also labeled against Trk receptors. The ratios between fluorescence signals in early axon (axon*) and other neurites (dendrite*) were plotted for each neuronal stage (bar plots). (a) At all stages, TrkA accumulates in neurite tips with less F-actin (arrowheads in panels; ratios significantly different from 1 in lower logarithmic bar plots). (b) At all stages, TrkB labeling is evenly distributed to all neurites independently of F-actin content (arrowheads in panels; ratio not significantly different from 1 for TrkB in lower logarithmic bar plots, black bars). (c) At all stages, TrkC labeling is evenly distributed to all neurites independently of PMGS content (arrowheads in panels; ratio not significantly different from 1 for TrkC, in lower logarithmic bar plots, white bars). Scale Bar, 10µm. (PDF 179 kb)

Supplementary Fig. 10

Model of PMGS–dependent axon specification in hippocampal neurons. Stage 2 neurons. PMGS (red bar) is polarized to one neurite (B, axonal tip). Increased PMGS concentration, and thus increased GM1 concentration, induces local recruitment of activated NGF receptor TrkA (pTrkA) and spatial recruitment the downstream effectors PI3K and Rac1. These results in local dissociation of RhoA from the membrane dissociation (RhoA inactivation), the consequent disassembly of thr ROCK/PIIa complex and thus local actin depolymerization. In A (dendritic tip), low PMGS levels would induce local GM1 production albeit not sufficient to induce robust recruitment of the PMGS/TrkA downstream machinery, which is preferably recruited to B. Overall, PMGS enrichment in B favors this neurite for rapid initial polarized outgrowth, thus triggering axon formation. (PDF 54 kb)

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Da Silva, J., Hasegawa, T., Miyagi, T. et al. Asymmetric membrane ganglioside sialidase activity specifies axonal fate. Nat Neurosci 8, 606–615 (2005). https://doi.org/10.1038/nn1442

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