Biochemical Purification of a Mammalian Slit Protein as a Positive Regulator of Sensory Axon Elongation and Branching

Kuan Hong Wang, Katja Brose, David Arnott, Thomas Kidd, Corey S. Goodman, William Henzel, and Marc Tessier-Lavigne.  Cell, Vol. 96, 771-784, March 19, 1999.

Reviewed by Melanie Majure

The authors of this paper attempted to identify an extracellular factor that regulates the branch initiation and extension of spinal sensory neurons.  This paper specifically looks at small diameter nerve growth factor (NGF)- responsive thermoceptive and nociceptive neurons with cell bodies located in dorsal root ganglia (DRG) and axons that project into the spinal cord.  The results indicate that an amino-terminal fragment of Slit2 protein may control collateralization.

Although much research has clearly described the branching patterns of sensory neurons, the authors examined their morphology in vitro with a 3D collagen gel assay that provided a clear view of individual neurons. Figures 1 (A, C, and D) address growth up until embryonic day 14 (E14) and figures 1 (B, E, and F) address growth up until E17.  The illustrations in figures 1A and 1B diagram the stages of extension and branching.  At E12 axons from the DRG reach the dorsal root entry zone (DREZ) of the spinal cord.  Each axon then splits into 2 branches that run along the spinal cord in opposite directions (figure 1A).  Figures 1C,D, E, and F are transverse and parasagittal  sections of DRG axon branches into the spinal cord.  DRG neurons were removed from E14 and E17 embryos and cultured for two days with NGF to promote the survival of small diameter neurons only.  (Other DRG neurons do not respond to NGF).  The neurons were immunostained with an antibody against neurofilament-M after the two day incubation [on E16 (figure 1G) and on E19 (figure 1H)].  Figure 1G, a collagen gel with stained neurons, shows individual neurons with simple axons, while figure 1H shows branching and elongation of axons.  These results suggest that NGF causes neuronal complexity to increase with time.  NGF promotes survival  (figure 1G), but does not appear to effect branching until E18 or E19 (figure 1H).

Collateralization in vivo begins at E16-E17; therefore a factor other than NGF must regulate this earlier branching.  E14 DRG neurons were cultured in a collagen gel with only NGF present for 24 hours.  Next, this medium was replaced with various tissue extracts.  Forty-eight hours later the cultures were fixed and immunostained with neurofilament-M (figures 2A-2H). No activity was detected when the original medium was replaced with fresh control medium (figure 2A, the negative control).  Axon length and branch number greatly increased with an E17 rat spinal cord extract (figure 2C), while less growth was detected with the E13 extract (figure 2B).  The authors claim that the E13 "extract contained 4-fold less total protein mass per embryo than the E17 extract".  However, figures 2B and 2C do not appear to be different.  Significant activity was also observed with E17 calf brain extract (figure 2D).  Comparison of high power magnifications of the control (figures 2E and 2F) and the calf brain extracts (figures 2G and 2H) emphasizes the effect of the extract’s activity on collateralization.  A dose-response curve quantifies the effect of the E17 calf brain extract on total axon length and branchpoints (figure 2I).  The authors described an activity to be equivalent to the "lowest amount of extract in a 2-fold dilution series that" results in branching when added to 1 ml of medium.

Table 1 lists other factors that were also added to E14 DRG neurons in a similar manner.  No branching response was stimulated by these factors.  Since calf brain extract can be bought cheaply and is available in large quantities, it was used to identify the activity.

Figure 3A is a flowchart that diagrams the biochemical purification of the activity.  A small amount of the main activity was recovered in the eluate of the WGA (wheat germ agglutin) affinity column.  In order to determine if the activity split on the column, the flow-through and eluate were added to E14 DRG neurons that were cultured on a collagen gel.  Figures 3 (B, C, D, and E) are the collagen gels immunostained with an antibody against neurofilament-M.  Figure 3B, a negative control, shows E14 neurons that were assayed with the control medium.  No branching was observed.  No activity was detected when the neurons were assayed with the WGA flow-through (figure 3C).  Activity was greater when the flow-through and eluate were added together (Figure 3E) than when only the eluate was added. (figure 3D).  Figures 3C and 3D appear to have an equivalent amount of branching.  The authors state that at higher concentrations, the flow-through never showed branching activity, while the eluate did have a stimulating effect.  No figures are provided to support this statement.

In order to see if a protein co-fractionates with the activity observed in calf brain extracts, activity was compared to protein bands on a SDS-PAGE gel.  The activity from the WGA eluate was purified with four columns in parallel: heparin-Sepharose high performance (figure 4A), hydroxylapatite (figure 4B), gel filtration (figure 4C), and Mono-S (figure 4D).  For example, the activity was fractionated on a heparin column (figure 4A).  The activity of each of the eleven fractions from the column was determined by a sample of its effect on E14 DRG neurons. Another sample was loaded in a SDS-PAGE gel to identify any proteins that co-fractionated with the activity. The graph at the top of figure 4A is an activity profile.  The vertical bars correspond to the lanes of the SDS-PAGE below them.  The fifth eluate had the greatest activity at 4 U/ml.  Several proteins co-purified with this activity.  After each of the four columns were run, a comparison was made between the greatest activity in each column and proteins in the corresponding lanes across all the columns.  A band of 140 kDa was observed to occur most frequently with the greatest activity.  The p140 band was identified by Coomassie staining and then sequenced.

Figure 5 is the amino acid sequence of human Slit-2 (MW 170 kDa).  Slit 1, 2, and 3 are drosophila, human, and rat homologs, respectively.  Slit proteins have already been seen to negatively regulate axon guidance by preventing midline axon crossing.  Underlined and bolded sequences correspond to ten peptide sequences obtained through Edman degradation of p140.  The ten sequences only mapped to the amino-terminus of human Slit-2 protein.  Expression of recombinant human Slit-2 in COS cells produces three protein products: Slit 2 (~190 kDa), Slit 2-N (~140 kDa), and Slit 2-C (~60 kDa).  p140 is probably the amino-terminus product of the cleavage of Slit 2.  The bolded sequence in figure 5 is a possible site for this cleavage.

Although Slit 2-N cofractionated with the activity, the actual function of this protein had not yet been examined.  Recombinant hSlit2-N was expressed in COS cells, and then removed by WGA chromatography.  Nickel affinity chromatography was used to separate Slit2-N, in the flow-through, from Slit-2 in the eluate. (Slit-2 remained in the eluate because of a histidine tag in the C-terminal.)  Cos cells that were transfected with control vectors were also run through the WGA and nickel affinity chromatography.  Figure 6 is a silver-stained SDS-PAGE gel with four lanes.  Negative controls are provided in lanes 1 and 3. The control flow-through from the nickel column was loaded in lane 1 and the control eluate was loaded in lane 3.  There was no evidence of bands in these lanes.  Lane 2 was loaded with the experimental flow-through.  The single band appeared around 140 kDa and corresponded to hSlit-2N.  Lane 4 was loaded with the experimental eluate and a single band appeared around 190 kDa; this lane contained hSlit2.

The purified flow-through and eluate from the nickel column were then added into culture mediums of E14 DRG neurons.  hSlit2-N (figure 7B) had an axon elongation and branch promoting activity, while h-Slit2 (figure 7D) had no such effect.  The negative controls that were used in lanes 1 and 3 of figure 6 were also added to the culture mediums (figures 7A and 7C).  The controls and the h-Slit2 look similar, which suggests that full length Slit-2 has no stimulating effect. Comparison of high power magnifications of the control (figures 7E and 7F) and hSlit-2-N (figures 2G and 2H) emphasizes the effect of hSlit-2-N on collateralization.  Figure 8 quantifies the qualitative effects observed in figure 7.  These graphs present the effect of increasing concentrations of control and experimental flow-through and eluate on total neurite length per neuron (figure 8A) and total number of branch points per neuron (figure 8B).  The authors concluded that Slit-2-N promotes collateralization in E14 DRG extracts.

In situ hybridization was performed on rat spinal cord at E14 and E17 in order to identify areas in vivo that express Slit proteins at these times. hSlit rat cDNA probes were utilized to locate Slit mRNA. Transverse sections of spinal cord at the forelimb level are provided in figures 9 (A, C, E, G, and I) from E14 and figures 9 (B, D, F, H, J) from E17.  Slit 1 and Slit 3 are expressed at low concentrations in the DRG during E14  (figures 9A and 9E).  High concentrations of Slit1 (figure 9B) and moderate levels of Slit2 (9D) are seen at E17, while there is no increase in the expression of Slit3 (figure 9F). The authors did not examine the expression of Slit2-N, which seems strange since it is the focus of the paper. Previous experiments show that Robo proteins and Slit proteins interact with each other.  Robo1 is not expressed during E14 or E17 (figures 9G and 9H), while Robo2 is expressed at both embryonic stages (figures 9I and 9J).

The authors of this paper identified a protein, Slit-2-N, that promotes branching and elongation of small diameter thermoceptive and nociceptive neurons of the DRG at E14.  Such a conclusion opens the door for much more research.  A greater understanding of axonal collateralization in embryos may lead to ways to enhance the effects of sprouting in adults who have suffered brain injury.

Which factor, NGF or Slit-2-N, contributes more to neuronal outgrowth?  Does developmental stage regulate these factors?  It is evident that each of these factors contributes to the branching and elongation of neurons.  The results of this paper suggest that NGF promotes branching around E19, while Slit-2-N stimulates such activity at E16.  The axons observed in this paper depend on NGF for their initial survival, it seems difficult to culture them without NGF present.  The cultures used to discover the activity that promotes collateralization (figure 2) were incubated with NGF during the entire assay.  Is NGF required for neuronal survival beyond E14?  One experiment would be to incubate NGF with DRG neurons for the first 14 embryonic days, remove it, and then assay with either NGF or Slit-2-N.  If neurons can survive without NGF beyond this point, then the effects of NGF and Slit-2-N could be  quantified and compared.  Two experimental incubations, one with NGF after E14 and another with Slit-2-N after E14, would be performed in a manner similar to those described above.

Does branching reflect defasciculation of parent axon shaft from preexisting branches or actual collateralization? The 3D collagen gel was used so that individual neurons could be viewed separately from others.  This was necessary so that actual branching was observed instead of the interaction between several neurons.  This interaction might consist of two axons lying adjacent to one another for a distance, but at some point separating and running in different directions.  Instead, an actual interstitial bud branching off of the axon as a growth cone should be observed.  The authors suggest that electron microscopy be used to gain a clearer image of the morphogenesis.  Another suggestion is to directly apply Slit-2-N to isolated axons and determine if any stimulating effects are present.

What is the relationship between full length Slit-2, Slit-2-N, and Slit-2-C, as well as Slit 1, 2, and 3?  When full length Slit-2 and Slit-2-N were purified from cos cells, the mixture of both proteins failed to produce any stimulating activity.  It is possible that Slit-2 inhibited the activity of Slit-2-N.  In vivo Slit-2 is probably present at the same time that Slit-2-N stimulates collateralization.  One step is to examine protein-protein interactions through immunoprecipitation assays.  This method could be used to determine if Slit-2 binds with Slit-2-N.  The authors suggest that in the full length protein, the carboxyl terminus may interfere with the amino terminus.  This interference may keep the full length protein from stimulating collateralization.  One way to test this would be through site directed mutagenesis of the carboxyl terminus of the full length protein.  It may be possible that by slightly changing the carboxyl terminus, the inhibitory effect would be terminated, and the full length protein could stimulate the activity of interest.  Furthermore, chimeric cDNAs could be used to see if a portion of the Slit-2 protein (but more than the Slit-2-N) is sufficient for activity.  Finally, the authors used in situ hybridization to show that all three Slit proteins are expressed at E17 in the DRG.  Immunoprecipitation experiments need to be performed to see with what these proteins interact.

What is the synergizing activity that potentiated the effect of the WGA eluate during the biochemical purification of Slit-2-N?   The flow-through that was described in figure 3 should be purified and sequenced.  The assays that were performed to identify the co-fractionating activity (figure 4) could be applied to the synergizing activity.

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