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Keywords:

  • Dopamine;
  • Sympathetic neurons;
  • Apoptosis;
  • Semaphorin;
  • Collapsin-1

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Rescuing of chick collapsin-1 by RT-PCR
  5. DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342
  6. Expression of semaIII
  7. RESULTS
  8. Induction of CRM during neuronal apoptosis
  9. SemaIII induces apoptosis in neuronal cells
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Abstract : Shrinkage and collapse of the neuritic network are often observed during the process of neuronal apoptosis. However, the molecular and biochemical basis for the axonal damage associated with neuronal cell death is still unclear. We present evidence for the involvement of axon guidance molecules with repulsive cues in neuronal cell death. Using the differential display approach, an up-regulation of collapsin response mediator protein was detected in sympathetic neurons undergoing dopamine-induced apoptosis. A synchronized induction of mRNA of the secreted collapsin-1 and the intracellular collapsin response mediator protein that preceded commitment of neurons to apoptosis was detected. Antibodies directed against a conserved collapsin-derived peptide provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. Moreover, neuronal apoptosis was inhibited by antibodies against neuropilin-1, a putative component of the semaphorin III/collapsin-1 receptor. Induction of neuronal apoptosis was also caused by exposure of neurons to semaphorin III-alkaline phosphatase secreted from 293EBNA cells. Anti-collapsin-1 antibodies were effective in blocking the semaphorin III-induced death process. We therefore suggest that, before their death, apoptosis-destined neurons may produce and secrete destructive axon guidance molecules that can affect their neighboring cells and thus transfer a “death signal” across specific and susceptible neuronal populations.

Apoptosis of neuronal cells has been suggested to have a role in several progressive neurodegenerative disorders (Bredesen, 1995 ; Johnson et al., 1995 ; Thompson, 1995 ; Charriaut-Marlangue et al., 1996). Although a major line of research has been devoted to the final steps common to various types of cells undergoing apoptosis, the cascade of molecular events that precede these final pathways in neuronal cells is largely unknown.

One of the early morphological changes accompanying cell death in cultured neuronal cells is a retraction of the neuritic network. Axonal damage has been observed even before the emergence of the typical morphological hallmarks of apoptosis (Deckwerth and Johnson, 1993 ; Wakade et al., 1995). Typically, axonal degeneration is manifested by irregular blebbing of neurites with thinning and fragmentation, followed by retraction and collapse of the axonal network.

The molecular and biochemical mechanisms involved in the retraction and collapse of the axonal network during neuronal apoptosis are still unknown. It is not clear whether the axonal damage is an outcome of the death process occurring within the cell body or whether damage to the axonal network per se can serve as a trigger for apoptosis of the whole neuron. The dymaic process of axon pathfinding during neurodevelopment has been shown to result from the combined action of attractive and repulsive cues, either long or short range, provided by various axon guidance molecules (for review, see Goodman, 1994, 1996 ; Kolodkin, 1996 ; Tessier-Lavigne and Goodman, 1996). The secreted and highly conserved axon guidance molecule collapsin-1 isolated from chick brain (Luo et al., 1993) acts as a repulsive cue in axonal pathway formation during neuronal development (Kolodkin et al., 1993 ; Luo et al., 1995). Collapsin-1 belongs to the semaphorin family of proteins. These were suggested to have a functional role in neuronal development by inhibiting growth cone extension toward unwanted directions in a receptor-mediated process (Luo et al., 1995 ; Goodman, 1996 ; Kolodkin, 1996). Several distinct members of the semaphorin family exist within the same organism and display differential distribution in specific areas of the developing nervous system (Luo et al., 1995 ; Adams et al., 1996 ; Hamajima et al., 1996 ; Kolodkin, 1996 ; Puschell et al., 1996 ; Tessier-Lavigne and Goodman, 1996). All members of this family (both intra- and interspecies) share a highly conserved semaphorin domain. In particular, 93% amino acid homology in the semaphorin domain has been reported for chick collapsin-1 and its human paralogue, semaphorin III (semaIII). A family of receptors (or components of receptors) for semaphorins, the neuropilins, has been recently described (Chen et al., 1997 ; He and Tessier-Lavigne, 1997 ; Kolodkin et al., 1997). These receptors are differentially expressed during development and were suggested to interact with various collapsins through two independent binding sites : one that signals the biological response and one that potentiates the response (Koppel et al., 1997). It was further proposed that an additional component of the receptor complex may exist that regulates the binding specificity of the receptor to various semaphorins (Chen et al., 1997 ; Feiner et al., 1997). Nevertheless, a role for semaphorins in neuronal cell death has not yet been suggested.

We have studied neuronal apoptosis in a model system of postmitotic neurons undergoing apoptotic cell death induced by dopamine (DA) (Ziv et al., 1994 ; Shirvan et al., 1997 ; Zilkha-Falb et al., 1997). DA has been shown to trigger apoptosis in both neuronal (Tanaka et al., 1991 ; Mytilineou et al., 1993 ; Ziv et al., 1994 ; Masserano et al., 1996 ; Zilkha-Falb et al., 1997 ; Luo et al., 1998) and nonneuronal (Offen et al., 1995) cell types in a nonreceptor-mediated way (Masserano et al., 1996). The toxic effects of DA can be prevented by overexpression of bcl-2 (Offen et al., 1996b ; Ziv et al., 1997b). In our experimental model of DA neurotoxicity in cultured sympathetic neurons from chick embryos, the neuronal death process is characterized by a fast decline in cell viability that occurs 12-14 h after exposure to DA (Zilkha-Falb et al., 1997). Such neurons become irreversibly committed to die 8-10 h after initiation of DA treatment (Shirvan et al., 1997). The early molecular events that lead the neuronal cells toward commitment to the self-destructive program are still unknown. Therefore, identifying a profile of mediators that are involved in the very early stages of the cascade culminating in neuronal apoptosis may lead to the development of novel neuroprotective therapeutic approaches. We have taken an unbiased approach to screen for changes in gene expression that occur as a function of neuronal apoptosis and therefore applied the differential display (DD) method (Liang and Pardee, 1992 ; Bauer et al., 1993), which is a comparative and non-hypothesis-bound approach, to cultured chick sympathetic neurons before and after receiving the apoptotic trigger DA.

We report here that one of the genes that is upregulated during the early stages of DA-induced neuronal apoptosis encodes for collapsin response mediator protein (CRM ; CRMP-62), a molecule involved in axon guidance signaling that has been shown to be essential for collapsin-1 activity (Goshima et al., 1995 ; Wang and Strittmatter, 1996). We therefore followed the expression pattern of collapsin-1 and CRM during DA-induced neuronal apoptosis and present evidence for a functional role of collapsin-1 and human semaIII in mediating the early stages of neuronal apoptosis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Rescuing of chick collapsin-1 by RT-PCR
  5. DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342
  6. Expression of semaIII
  7. RESULTS
  8. Induction of CRM during neuronal apoptosis
  9. SemaIII induces apoptosis in neuronal cells
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Cell cultures and treatments

Chick embryo sympathetic neurons.

Tissue cultures from paravertebral sympathetic ganglia were prepared according to previously described methods (Greene, 1977 ; Shirvan et al., 1997 ; Zilkha-Falb et al., 1997).

Mouse cerebellar granule neurons.

Cultures of highly enriched granule neurons were isolated from cerebella of 8-day-old BALB/c mice and were prepared according to the technique of Nardi et al. (1997).

Treatments

DA (no. CH8502 ; Sigma, St. Louis, MO, U.S.A.) was dissolved directly in the proper culture medium. Sympathetic neurons (day 4-5 in culture, in the presence of nerve growth factor) were treated for a given time with 300 μM DA [determined earlier as the most potent concentration following detailed dose-response studies (Ziv et al., 1994 ; Zilkha-Falb et al., 1997)]. Plates maintained in identical conditions but without exposure to DA served as controls.

Antibody treatments.

Partially purified antibody was dissolved directly in the proper medium at concentrations between 0.1 and 1 mg/ml. In cases when neuronal viability was evaluated at 48 and 72 h following DA treatment, the antibody-containing medium was replaced every 24 h with fresh medium with antibodies. Anti-neuropilin-1 (anti-NP-1) antibodies were a generous gift of Dr. M. Tessier-Lavigne and were used at a concentration of 5 μg/ml. The antibodies were added to the cultures concomitantly with addition of DA.

Neuronal viability.

Neuronal survival was determined by Alamar blue (Alamar Biosciences, U.S.A.). Evaluating the death process as an apoptotic process was as described in our earlier works (Ziv et al., 1994 ; Zilkha-Falb et al., 1997).

DD and northern blot analysis

DD experiments were done according to the procedure of Shirvan et al. (1997). Chick sympathetic neurons were treated with DA (300 μM) for 6 h. The primers used were dT12GT and the arbitrary primers P5 (5′-GGTCTCCAGG) and P6 (5′-CTGGAGGATGG) (Shirvan et al., 1997).

Rescuing of chick collapsin-1 by RT-PCR

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Rescuing of chick collapsin-1 by RT-PCR
  5. DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342
  6. Expression of semaIII
  7. RESULTS
  8. Induction of CRM during neuronal apoptosis
  9. SemaIII induces apoptosis in neuronal cells
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

A 1,057-bp fragment of collapsin-1 was prepared by RT-PCR from sympathetic neurons treated with DA for 6 h, using sequence-specific primers located in the semaphorin domain. The sense primer was 5′-TTGTACTCTGGCACAGCAGCAGACTTCATG, and the antisense primer was 5′-ATCCCAGGCGCAGTAGGGGTCTCTTGG. Reactions were done using the access RT-PCR kit from Promega. The amplified fragment was cloned into pGEM-T vector (Promega) and sequenced to verify its identity.

Analysis of morphological changes

Serial phase-contrast micrographs were obtained of fields neurons maintained in culture for 4 days before and after exposing the neurons to different treatments as indicated (Olympus IX70 camera with Trimax film 100 ASA).

DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Rescuing of chick collapsin-1 by RT-PCR
  5. DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342
  6. Expression of semaIII
  7. RESULTS
  8. Induction of CRM during neuronal apoptosis
  9. SemaIII induces apoptosis in neuronal cells
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

4′,6-Diamidino-2-phenylidole staining was performed as described by Nardi et al. (1997). For Hoechst staining, 1 μg/ml Hoechst 33342 (Molecular Probes) was added to growing cultures. Cells were incubated at 37°C for 20 min and then visualized under UV light microscopy.

Antibody preparation and function-blocking assay

Two peptides were chosen for antibody preparation : Pep 1A corresponds to amino acids 363-380 from chick collapsin-1, and pep 2A corresponds to amino acids 258-275 (Luo et al., 1993). Synthetic peptides were obtained from GeneMed (U.S.A.) and purified to homogeneity on an RP-18 column (Vydac, U.S.A.) using a Gilson model 303 HPLC system. Rabbits were immunized by subcutaneous injections of 0.1 mg of peptide-keyhole limpet hemocyanin conjugates. Both the immune and preimmune sera were fractionated using 40% saturated ammonium sulfate and dialyzed against phosphatebuffered saline. The repulsion assay was performed exactly as previously described (Messersmith et al., 1995 ; He and Tessier-Lavigne, 1997).

Western blot analysis

Western blot analysis was performed as described (Shirvan et al., 1997) using 11% polyacrylamide gels.

Expression of semaIII

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Rescuing of chick collapsin-1 by RT-PCR
  5. DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342
  6. Expression of semaIII
  7. RESULTS
  8. Induction of CRM during neuronal apoptosis
  9. SemaIII induces apoptosis in neuronal cells
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Expression of human semaIII in 293EBNA cells was performed according to the procedure of He and Tessier-Lavigne (1997). 293EBNA cells expressing the empty vector were used as control. Alkaline phosphatase (AP) assay was carried out and quantified colorimetrically as previously described (Cheng and Flanagan, 1994).

Induction of CRM during neuronal apoptosis

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Rescuing of chick collapsin-1 by RT-PCR
  5. DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342
  6. Expression of semaIII
  7. RESULTS
  8. Induction of CRM during neuronal apoptosis
  9. SemaIII induces apoptosis in neuronal cells
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

To isolate genes whose expression is transcriptionally regulated during neuronal apoptosis, we used the DD approach (Liang and Pardee, 1992 ; Bauer et al., 1993) and compared the mRNA repertoire of sympathetic neurons undergoing DA-triggered apoptosis with that of control nontreated cells. Apoptosis was induced by exposing the neurons to 300 μM DA, which has been determined previously as the potent apoptotic triggering concentration for sympathetic neurons (Zilkha-Falb et al., 1997). This concentration is within the estimated range (0.1-1 mM) of its normal levels in dopaminergic neurons (Lichtensteiger, 1970 ; Jonsson, 1971 ; Michel and Hefti, 1990). At 6 h after DA administration, cells are still in a reversible stage : They are not fully committed to the apoptotic process and can be rescued from their death on removal of DA from the tissue culture medium (Shirvan et al., 1997). Yet, differences in the expression pattern of several genes were detected at this stage. Some of these changes are presented in one representative DD reaction (Fig. 1) and exhibit either up- or down-regulation as a function of DA treatment. Only molecules that repeatedly responded to DA treatment were considered for isolation and identification. One of the genes that was found to be induced after 6 h (indicated as a 420-bp cDNA molecule by an arrow in Fig. 1) showed a 100% identity to chick CRM (CRMP-62), a molecule involved in axon guidance signaling (Goshima et al., 1995). CRM has been suggested to regulate a G protein-coupled cascade of events initiated by collapsin-1 (Goshima et al., 1995). The activity of both proteins is needed for mediating the repulsive guidance assay that defines collapsin-1 function (Goshima et al., 1995). However, the possible involvement of these molecules in neuronal cell death is unknown, and therefore the induction of CRM during DA-induced apoptosis of sympathetic neurons was intriguing and challenged us to investigate further this phenomenon.

image

Figure 1. Differential expression of CRM during DA-induced neuronal apoptosis. Transcriptionally regulated genes, associated with the early stages of DA-induced apoptosis, were identified by applying the DD method (Liang and Pardee, 1992 ; Bauer et al., 1993) to chick embryo sympathetic neurons that had been treated with 300 μM DA for 6 h. Reaction conditions were as previously described (Shirvan et al., 1997). One DD reaction was carried out with the arbitrary primers p5 and p6. Several up- and down-regulated cDNA molecules can be observed, an one of them, which was repeatedly up-regulated in several independent experiments, is indicated by an arrow and represents the cDNA encoding for chick CRM. Sequence from the above band (from 1 to 420 bp) revealed a 100% identity with chick CRM, spanning 420 bp from 1,661 to 2,080 (GenBank accession no. U17277).

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Synchronized expression of axon guidance molecules before the commitment to apoptosis

To follow the expression pattern of both CRM and collapsin-1 in the sympathetic neurons as a function of the death process, we used as probes the cDNA of CRM isolated by the DD procedure and a chick 1,057-bp fragment from the semaphorin domain of collapsin-1 that was rescued by RT-PCR from neurons treated with DA for 6 h. Total RNA was extracted from cells exposed to DA for different durations (0.5-12 h) and used for northern blot analysis. The expression of both collapsin-1 and CRM was monitored over the course of the DA-induced death process (Fig. 2). Overall, low expression levels of both axon guidance molecules were observed in the sympathetic neurons. However, expression of both molecules accumulated and peaked at 6 h after DA exposure, followed later by a steep decline (Fig. 2). Quantitative analysis of the signal showed a sixfold increase for collapsin-1 and a fourfold increase in CRM (Fig. 2B). It is interesting that the increases in collapsin-1 and CRM expression were simultaneous and occurred immediately before the irreversible commitment point of cells to the death process, which is indicated by the bar at the bottom of Fig. 2B. The commitment point was determined as the time point following DA administration from which 50% of the neurons cannot be rescued upon DA removal and are irreversibly committed to die (Shirvan et al., 1997). The high expression levels of the secreted protein collapsin-1 that coincided with the death commitment point raised the possibility that this molecule may have a functional role in neuronal apoptosis. Nonneuronal glial cells differ from neurons in their response to DA treatment and show remarkable resistance to a DA concentration of 1 mM, which is extremely toxic to sympathetic neurons. Collapsin-1 induction by DA (at 300 μM) occurred only in neuronal but not in glial cells, whereas a twofold induction of CRM expression was observed in glial cells (data not shown). These results indicate that in response to the apoptotic trigger DA, coupling of the expression of collapsin-1 and its mediator CRM occurred only in neuronal but not in glial cells.

image

Figure 2. Expression of collapsin-1 and CRM in sympathetic neurons as a function of DA-induced neuronal apoptosis. Northern blot analysis of total RNA was used for monitoring the expression of collapsin-1 and CRM during DA-induced neuronal apoptosis. A : A typical blot. The probes used were (a) a 1,057-bp fragment of collapsin-1 that was prepared by RT-PCR from RNA isolated from sympathetic neurons treated for 6 h with DA, using sequence-specific primers located in the semaphorin domain, and (b) a 420-bp fragment encoding for CRM, isolated in the DD procedure as described in the legend to Fig. 1. Sympathetic neurons (106 cells) were treated with DA for 0.5-12 h, and at different time points, total RNA was prepared for northern blot analysis. Nontreated cultures served as controls. As a control for gel loading and transfer, membranes were stripped and rehybridized with an oligonucleotide of 18S rRNA. Experiments were repeated four times, and a representative blot is shown. B : Intensity of bands was monitored by both soft laser scanning densitometer and by a digital camera-based software (Kodak). RNA quantities in each lane were normalized against 18S rRNA levels and are presented as percentages of control untreated cells. Data are mean ± SD (bars) values of four independent whole experiments, starting with the preparation of the cultures ; we show the SD only when it is greater than the size of the symbols. The solid bar at the bottom represents the commitment of neurons to the apoptotic process.

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Preparation and characterization of antibodies against collapsin-1-derived peptides

Antibodies to collapsin-derived peptides were prepared to assess the possible relevance of collapsin-1 induction during neuronal apoptosis. Polyclonal antibodies against two collapsin-derived peptides were raised in rabbits. One type of antibodies was directed against a sequence conserved peptide NYQWVPYQGRVPYPRPGTC (pep 1A) from chick collapsin-1 (Luo et al., 1993). The sequence of this peptide is identical between collapsin-1 and its mammalian paralogues and is very highly conserved among all the secreted types of semaphorins. Another polyclonal antibody was raised against a different and less specific collapsin-derived peptide, GKATHARIGQICKNDFGG (pep 2A), of chick collapsin-1 (Luo et al., 1993).

Anti-pep 1A antibodies were able to immunoprecipitate a recombinant form of the protein, which was translated in vitro. Immunoprecipitation was obtained only with anti-pep 1A but not with the preimmune serum (data not shown).

The anti-peptide collapsin antibodies were evaluated for their potential to block the functional repulsive activity of semaIII. The human semaIII protein, as well as its related chick collapsin-1 homologue, has been shown to induce a repulsive effect on nerve growth factor-responsive dorsal root ganglion axons in collagen gel cultures (Luo et al., 1993 ; He and Tessier-Lavigne, 1997). Sympathetic neurons exhibited a similar reaction to semaIII (Puschel et al., 1996), indicating that both types of neurons respond to the chemorepulsion signal of semaIII. The effect of anti-pep 1A antibodies on the repellent assay was therefore tested. Application of antibodies (at a concentration of 25 μg/ml) to cocultures of dorsal root ganglion explants and aggregates of Cos cells secreting semaIII-AP protein (He and Tessier-Lavigne, 1997) exhibited a drastic inhibition of the chemorepulsive activity of semaIII (Fig. 3, panel 3). However, addition of equal amount of the preimmune IgG fraction resulted in a minimal function-blocking effect (Fig. 3, panel 2). These results indicate the usefulness of our anti-collapsin-1 antibody preparation as a function-blocking reagent.

image

Figure 3. Characterization of the antibodies against collapsin-derived peptides. Anti-pep 1A antibodies inhibit the repellent activity of semaIII protein. Embryonic day 14 rat dorsal root ganglion explants were cultured in collagen gels with 25 ng/ml nerve growth factor to elicit outgrowth of semaIII-responsive axons. Explants were cocultured with aggregates of COS cells secreting SemaIII-AP protein in the absence of anti-pep 1A (panel 1) or in the presence of 25 μg/ml anti-pep 1A IgG (panel 3) or 25 μg/ml preimmune IgG (panel 2) for 40 h. The explants were then fixed and visualized by whole-mount immunostaining with the anti-neurofilament antibody NF-M. The panels shown represent several independent experiments (n = 7).

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Induction of collapsin-1 protein in neurons undergoing apoptosis

To follow the expression of collapsin-1 on the protein level as a function of DA treatment, sympathetic neurons were exposed to DA for different durations, and crude extracts of whole cells were subjected to western blot analysis, using anti-collapsin antibodies (anti-pep 1A). A major band of 65 kDa was up-regulated at 6-7 h following DA exposure and then decreased (Fig. 4). A highersize protein of 100 kDa (which is the expected size of the full-length collapsin-1) was much less abundant (data not shown). The 65-kDa protein presumably represents a processed form of the protein containing within it the sequence of pep 1A. Recent evidence indicates that the semaphorin proteins (such as semaphorin D and collapsin-1) are proteolytically processed and that their repulsive activity is regulated by such processing (Adams et al., 1997 ; Klostermann et al., 1998). One of the major cleavage fragments is of 65 kDa, which represents the amino-terminal fragment of the semaphorins and includes also the sequence of pep 1A. The collapsin-1 protein has been shown to have a homodimer structure within the cell (Klostermann et al., 1998 ; Koppel and Raper, 1998). It is possible, however, that the 100-kDa monomer is less stable within the cell, and therefore its identification in a crude cell lysate is difficult. Indeed, previous studies (Shepherd et al., 1997) demonstrated difficulty obtaining anti-collapsin-1 antibodies that can recognize collapsin-1 in a nonconcentrated source. Therefore, we anticipate that the 65-kDa protein recognized by the anti-pep 1A antibodies represents the cleavaged form of collapsin-1 and that this product is induced within neurons undergoing apoptosis. In accordance with the northern blot studies, the time table of the protein expression pattern correlates with the mRNA expression pattern (Figs. 2 and 4).

image

Figure 4. Up-regulation of collapsin 1 protein during DA-induced apoptosis of sympathetic neurons. Sympathetic neurons were treated with DA for different durations (4-9 h).Cells were harvested, and DA-treated and control, nontreated cell extracts were loaded on the gel and blotted onto a polyvinylidene difluoride membrane. Blots were stained with Ponceau to verify loading and transfer of equal amountsof proteins on each well. Anti-pep 1A antibodies were used to probe the membrane. Although equal amounts of protein were loaded on each lane, an induction of a 65-kDa protein (indicated by an arrow) is evident at 6-7 h after DA treatment, followed by a decrease. The same band was recognized by serum prepared from two rabbits immunized against pep 1A. This protein probably represents a truncated and processed form of collapsin-1 protein.

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Antibodies directed against collapsin-derived peptide protect neuronal cells from DA-induced apoptosis

Coupling of neuronal apoptosis to mRNA induction of axon guidance molecules pointed out a possible linkage between the axonal damage observed during neuronal cell death and the inhibitory and destructive effect of these molecules on axon growth cones during development. Such association suggests a possible functional role for collapsin-1 in the damage, collapse, and shrinkage of axons during the process of neuronal apoptosis and may distinguish this protein as a potent mediator of neuronal cell death.

We therefore examined whether antibodies raised against collapsin-1 were capable of attenuating the DA-induced apoptosis in chick sympathetic neurons. DA and anti-pep 1A antibodies were added to the cells, and neuronal survival was monitored. In the first 16 h, the antibodies were able to provide a marked (90-100%) protection from DA-induced cell death, as compared with the effect of the preimmune serum (Fig. 5A). At 24 h after DA administration, the survival rate of neurons cotreated with the antibody was 75%, whereas the viability of neurons treated by DA alone was only 24%. At 72 h after DA addition, the antibody-treated cells still exhibited high levels of survival (65% as compared with 22% of the cultures treated with DA alone). These results indicate that the antibody-treated cells can be protected from DA-induced cell death for at least 3 days. Inhibition of DA-induced apoptosis by these antibodies was dose-dependent, reaching maximal protective capacity at concentrations between 0.4 and 1.2 mg/ml. Treatment of cells with the polyclonal antibody raised against pep 2A of chick collapsin-1 (Luo et al., 1993) had no protective effect from DA-induced cell death (data not shown). This finding suggests that pep 1A (but not pep 2A) may be involved in the death-promoting activity of collapsin-1. Protection of the axonal network by the antibodies was also observed in morphological studies (Fig. 5B). At 24 h after DA treatment, neurons had clearly passed their irreversible commitment to apoptosis and exhibited remarkable retraction of the axonal network. However, cotreatment with the antibody protected both the cell bodies and the axonal network and prevented its collapse, thus extending neuronal survival despite the continued presence in the medium of the strong apoptotic insult DA. The effect of anti-pep 1A antibodies is not simply by buffering the oxidized effects of DA, because a depleted IgG fraction of this antibody was incapable of providing protective effects to cultured sympathetic neurons (data not shown). Moreover, oxidation of DA (manifested as formation of a distinctive dark color in the medium) was observed in both antibody-treated and non-treated cultures.

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Figure 5. Functional involvement of collapsin in neuronal death. A : An anti-collapsin antibody inhibits DA-induced neuronal cell death. Polyclonal antibodies directed against a collapsin-derived peptide (pep 1A) that were raised in rabbits neutralized the death process in sympathetic neurons treated with 300 μM DA for 1-72 h. Non-treated cells served as control. The time course of inhibition of DA-induced cell death by these antibodies (at a concentration of 0.4 mg/ml total IgG) is demonstrated : DA-treated cells (●), and cells treated with both DA and preimmune serum (▴) and with DA and anti-collapsin antibodies (▪). Two rabbits were immunized against pep 1A, and the ammonium sulfate-precipitated immune sera from both rabbits exhibited similar protective effects from DA-induced apoptosis. Cell viability was determined at different time points by the fluorescent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Alamar). Treatment with ammonium sulfate-precipitated preimmune serum from each one of the rabbits (at the same concentration as the immune serum) had no protective effect on cell viability. B : Effect of anti-collapsin antibodies on sympathetic axonal integrity. Control nontreated sympathetic neurons grown in culture (Control) are compared with neurons treated with 300 μM DA for 24 h (DA). Axonal fragmentation and blebbing, as well as decreased sprouting, were observed in DA-treated neurons. Cotreatment with anti-collapsin antibodies at a concentration of 0.8 mg/ml (DA + anti-Col Ab) prevented DA toxicity, and the cells manifested a healthy axonal network like in the control culture. Bar = 50 μm. C : Anti-collapsin antibodies can rescue cerebellar granule neurons (CGN) from apoptosis induced by DA. Chick sympathetic neurons (SYM) were induced to undergo apoptosis by DA (300 μM for 16 h). DA treatment reduced cell viability to 35%. However, anti-collapsin antibodies (Ab ; anti-pep 1A at a concentration of 0.8 mg/ml) were capable of protecting the SYM from DA-induced apoptosis, and their survival rate was 88%. Mouse CGN were treated with DA (300 μM for 6 h). Following treatment, cell viability was 31% for DA, and addition of Ab raised the survival rates to 75%, indicating that it provided protection from cell death.

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The specificity of the phenomenon to the cell type was evaluated by extending the study to other types of cultured neurons. Treatment of mouse cerebellar granule neurons with anti-collapsin antibodies rescued the cells from DA-induced apoptosis, and 74% survival rates were monitored in the presence of both DA and the antibody, as compared with only 31% in the presence of DA alone (Fig. 5C). Our results therefore demonstrate that induction of neuronal apoptosis in two different neuronal cell types by DA may be regulated by collapsin-1.

Antibodies directed against NP-1 can attenuate DA-induced neuronal apoptosis

Recently, NP-1 was identified as a high-affinity cell surface receptor for the secreted semaIII protein (He and Tessier-Lavigne, 1997 ; Kolodkin et al., 1997). NP-1 is expressed on semaIII-responsive neurons, including sympathetic neurons (Kawakami et al., 1996). Antibodies directed against NP-1 were shown to inhibit the repellent activity of semaIII on nerve growth factor-dependent dorsal root ganglion neurons in culture, thus emphasizing its role in mediating the signaling pathway induced by the secreted semaphorins (He and Tessier-Lavigne, 1997 ; Kolodkin et al., 1997). We have shown that our anti-pep 1A antibodies were capable of inhibiting the same repellent activity of semaIII (Fig. 3), as well as inhibiting the DA-induced apoptotic activity in sympathetic neurons (Fig. 5A and B). Whether the signaling pathway mediating these two activities is similar is still unclear. However, it prompted us to evaluate the possible involvement of the semaIII receptor NP-1 in the death-inducing activity of collapsin-1. We therefore tested the effect of anti-NP-1 antibodies on the DA-induced apoptosis in chick sympathetic neurons. Addition of anti-NP-1 antibodies to the culture medium inhibited the neurotoxicity of DA (Fig. 6), and neuronal viability in the presence of DA and anti-NP-1 was 80%. Therefore, protective levels obtained by anti-collapsin-1 and anti-NP-1 antibodies were similar. These results indicate that both the collapsin-1 protein and its receptor have a role in determining the fate of neurons receiving a strong apoptotic insult and suggest that they participate in the same signaling pathway.

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Figure 6. Anti-NP-1 antibodies inhibit DA-induced neuronal apoptosis. Sympathetic neurons treated with 300 μM DA were incubated with or without anti-NP-1 antiobdies for 48 h. Antibody concentrations were 5 μg/ml. Neuronal viability in the presence of DA was 30%, whereas in the presence of anti-NP-1, protection levels reached 75%. Data are mean ± SD (bars) values of three independent experiments.

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SemaIII induces apoptosis in neuronal cells

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Rescuing of chick collapsin-1 by RT-PCR
  5. DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342
  6. Expression of semaIII
  7. RESULTS
  8. Induction of CRM during neuronal apoptosis
  9. SemaIII induces apoptosis in neuronal cells
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

As collapsin-1 was found to be up-regulated during neuronal apoptosis and as the anti-collapsin antibodies inhibited the death process, we hypothesized that the semaphorin family of proteins may mediate neuronal apoptosis. We therefore examined whether semaIII, the human homologue of collapsin-1, is capable of inducing neuronal apoptosis.

SemaIII-AP protein was expressed in human kidney 293EBNA cells as a secreted protein, fused to AP. This fusion protein has been characterized before as having both a repellent and collapsing activity on dorsal root ganglion axons and is capable of binding to NP-1. AP alone, also expressed in these cells, did not have any repellent or collapse activity (He and Tessier-Lavigne, 1997). Conditioned medium from semaIII-AP transfected cells was collected and applied to cultured sympathetic neurons, and cell survival was monitored 48 h later. AP assay was used to determine the concentration of semaIII-AP in the conditioned medium.

Neurons exposed to semaIII-AP-containing medium exhibited up to 50% reduction in cell survival as compared with neurons exposed to conditioned medium from control 293EBNA cells transfected with an empty vector (Fig. 7A). Morphological studies of neurons stained with Hoechst 33342 revealed a typical apoptotic pycnotic nuclei and chromatin fragmentation in cells treated with semaIII-AP but not in control cells (Fig. 7B). Anti-pep 1A antibodies were capable of inhibiting the semaIII-AP-induced cell death, and neurons exposed to both the conditioned medium and the antibodies exhibited 50% higher survival rates than neurons exposed to semaIII-AP only (Fig. 7A). Although the anti-collapsin-1 antibodies did not provide full protection from cell death, protection levels of 50% were consistently obtained. Because the semaIII-AP is applied as a partially purified protein contained within the conditioned medium, it is possible that some nonsemaphorin toxic component of this medium is responsible for neutralizing the protective effects of the antibody. The death-inducing activity of semaIII-AP on neuronal viability was dose-dependent. However, a saturable effect of 50% cell death was observed when the protein concentrations were >0.75 μM. Protein concentrations needed to induce neuronal death were within the micromolar range (0.75-2 μM), which is substantially higher than the concentrations of SemaIII-AP needed for the collapse activity [0.5 nM, as described by He and Tessier-Lavigne (1997)]. These results may indicate that at low concentrations, semaphorins act together with other axon guidance molecules to affect axon pathway formation, whereas at much higher concentrations they are toxic and can induce severe axonal damage and neuronal apoptosis.

image

Figure 7. Induction of apoptosis in sympathetic neurons by semaIII-AP. A : Conditioned medium was collected from 293EBNA cells expressing and secreting semaIII-AP, grown for 72 h in culture. Conditioned medium from 293EBNA cells transfected with an empty vector served as control. The semaIII-AP concentration was determined by AP assay. The conditioned medium was applied, at various concentrations, to sympathetic neurons grown in culture for 2 days. Neuronal survival was determined 48 h later. Anti-pep 1A antibodies (Ab) were used at a concentration of 0.8 mg/ml and were added to the neuronal cultures concomitantly with the semaIII-AP conditioned medium. Experiments were repeated four times, and a representative experiment is presented. Data are mean ± SD (bars) values. B : Cells treated with semaIII-AP conditioned medium or control medium were stained with Hoechst 33342 and photographed under UV light microscopy. Some of the apoptotic cells with condensed and fragmented chromatin are indicated by arrows. Bar = 50 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Rescuing of chick collapsin-1 by RT-PCR
  5. DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342
  6. Expression of semaIII
  7. RESULTS
  8. Induction of CRM during neuronal apoptosis
  9. SemaIII induces apoptosis in neuronal cells
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Our study provides cumulative evidence linking induction of axon guidance molecules with repulsive cues to the early stages of neuronal apoptosis caused by DA. We have shown that mRNA expression of collapsin-1 and the CRM protein is induced during neuronal apoptosis and that this induction precedes the time point of commitment of neurons to the death process. Our data further indicate that collapsin-1 is not only induced but may have an active role in determining the fate of neurons that are exposed to an oxidative stress-inducing apoptotic trigger. Antibodies directed against a collapsinderived peptide are capable of conferring protection against the oxidative stress-inducing agent DA. The anticollapsin antibodies prolonged cell survival in the presence of an apoptotic trigger for at least 72 h, indicating that long-term survival can be achieved by neutralizing collapsin-1. Both anti-collapsin-1 antibodies and anti-NP-1 antibodies were capable of inhibiting the semaIII-mediated repulsion of axonal growth cones in culture and exhibited similar protective effects on survival of cells exposed to DA. These results demonstrate the correlation between the death-inducing activity and the repulsive activity on axonal growth cones of collapsin-1. The results also suggest that both activities may be mediated by the same signaling pathway and substantiate the relevance of axonal signals in the determination of cell fate. We have shown that attenuation of neuronal apoptosis by the anti-collapsin antibodies occurred in neurons derived from both the CNS (cerebellar granule neurons) and the PNS (sympathetic neurons). Glial cells are much less susceptible to the apoptotic triggering potential of DA, and, accordingly, they induce only CRM but not collapsin-1 levels in response to DA challenge. We therefore suggest that mediation of apoptosis by collapsin-1 may be neuronal-specific, although not restricted by the neuronal cell type.

We have shown that in the absence of other noxious stimuli, a secreted semaIII-AP fusion protein can promote neuronal apoptosis, therefore supporting a functional role for semaphorins in mediating neuronal cell death. We have found that the full protein semaIII-AP, expressed and secreted from 293EBNA cells, was active as a neuronal death inducer at concentrations between 0.75 and 2 μM. Under in vitro assay conditions, the whole protein collapsin-1 or the semaIII-AP fusion protein exerts its collapsing activity on target axons at concentrations that are within the picomolar range (Luo et al., 1993 ; Koppel et al., 1997). The effective concentrations of the protein during neuronal embryogenesis, where a subtle interplay between several axon guidance molecules may determine the fate of the growing axons, are still unknown. During maturation of the developing brain, ~50% of its neurons are lost by an apoptotic mode of death (Oppenheim, 1991 ; Naruse and Keino, 1995 ; Jacobson et al., 1997). It is also not known whether axon guidance molecules with repulsive cues such as collapsins or other semaphorins are involved in the apoptotic process of the developing nervous system, although the results presented in this communication may be suggestive of such a role. We propose that in such a case, substantially higher concentrations of semaphorins would be needed to promote neuronal apoptosis, rather than collapse of the axon growth cone. Alternatively, it is possible that unique mechanisms may function during induction of apoptosis by DA in neurons that are not activated during mouse development.

The anti-collapsin antibodies were able to inhibit cellular apoptotic processes that were initiated by the DA-induced oxidative stress. Thiol-containing antioxidants are capable of blocking the DA-induced apoptosis (Offen et al., 1996a ; Zilkha-Falb et al., 1997). The extension of neuronal survival under constant conditions of DA-induced oxidative stress obtained by cotreatment with the antibodies to collapsin-1 suggests that the DA-generated toxic free radicals per se may not suffice for initiating and maintaining the apoptotic cascade of events. This cascade, however, is blocked at its very early phases via a different mechanism than free radical scavengers.

Because the semaphorins exert their action in a receptor-mediated mechanism (Tessier-Lavigne and Goodman, 1996 ; Feiner et al., 1997 ; He and Tessier-Lavigne, 1997 ; Kolodkin et al., 1997 ; Koppel et al., 1997), our findings suggest a possible involvement of receptor-mediated axonal signaling pathways in determining the survival of the neuronal cell body under oxidative stress conditions. Hence, extracellular signals, either secreted by the cell body itself or provided by the surroundings, may be detected by axon guidance receptors and have a direct and immediate role in regulating neuronal survival.

Prior results of semaIII/semaphorin D genetic deletion (Behar et al., 1996 ; Taniguchi et al., 1997) demonstrate perturbations in axonal and organ development and exhibit some disorientation of neurons. Incorrect sprouting of nerve fibers was also detected in loss of function of NP-1 in mice (for review, see Fujisawa et al., 1997). However, no increase in cell survival or other obvious apoptotic defects were reported, as might be predicted from our studies. In the CNS, semaIII expression was found in the cerebellum, cerebral cortex, and midbrain. Nevertheless, no obvious abnormalities in any of the major CNS axon projections in semaIII knockout mice were detected, suggesting no absolute requirement of semaIII for neurodevelopment (Catalano et al., 1998). However, in these systems, as in any other transgenic animal system, compensatory mechanisms may exist and fulfill, at least partially, several functions of axon guidance. It is therefore suggested that during embryogenesis, a delicate balance between all components of the axon guidance system may be responsible for normal development of the nervous system. It is still unknown whether collapsin-1/semaIII molecules have an active role in determining cell survival during neuromal development through their repulsive action on axonal growth cones. It is possible, however, that in adulthood, dys-regulation of such a balance by unscheduled induction of a repulsive cue may lead susceptible neuronal populations into cell death.

Recent evidence shows that adult sensory neurons retain the ability to respond to semaIII (Tanelian et al., 1997). Moreover, expression of semaIII was recently demonstrated in selective adult brain tissues (Giger et al., 1998). This indicates that repulsive cues are not restricted to the early stages of neurodevelopment but may have a role in the response of axons to their environment during adulthood as well. Several studies point at the possible involvement of semaphorins in determining cell fate : One line of evidence suggests that human semaphorins IV and V are possible tumor suppressor genes that may be involved in promoting small-cell lung cancer (Sekido et al., 1996). An opposite role is suggested for the secreted human semaphorin E, as a protein associated with increased resistance of cancer cells to chemotherapy (Yamada et al., 1997). A similar role is also observed for CD100, a nonneuronal member of the semaphorin family, which improves viability of B cells (Hall et al., 1996). Further evidence for the possible correlation between neuronal cell survival mechanisms and axonal integrity comes from studies with the bioactive peptide mastoparan, which is an inducer of both neuronal apoptosis (Lin et al., 1997) and growth cone collapse (Goshima et al., 1997). Moreover, a recent report demonstrated that DCC, a receptor of the axon guidance molecule netrin-1, is involved in regulation of apoptotic process during tumor growth (Mehlen et al., 1998). Thus, a possible link may exist between various cell death mechanisms and the destructive effects of different semaphorins on axons. Because viability of both neuronal and nonneuronal cells seems to be affected by different members of the semaphorin family, it is still unclear whether regulation of cell survival is mediated via axonal-specific or cell-specific receptors, and this question awaits further investigation.

In our studies, the function-blocking anti-collapsin-1 antibodies were able to block the DA-induced neuronal apoptosis in culture and also to prevent the disintegration of the axonal network. Cell survival and axonal maintenance are considered to be two distinct processes that may be regulated via different mechanisms (Sagot et al., 1997). Several in vivo studies indicate that bcl-2 over-expression in various models of transgenic mice can protect the neuronal cell body from apoptosis but is unable to prevent axonal degeneration. A similar phenomenon was observed in cultured sympathetic neurons (for review, see Sagot et al., 1997). The long-term fate of a neuronal cell that has been rescued from apoptosis but still possesses injured neurites is not clear. Moreover, although abortion of neuronal death could be achieved by caspase inhibitors, the surviving neurons appear atrophic and metabolically hypoactive and fail to maintain normal growth (Deshmukh et al., 1996). Therefore, development of additional tools that will act to protect both the cell body and the neuritic network from apoptotic damage may be of great benefit. In that respect, the neutralizing anti-collapsin-1 antibodies can serve as a model system for modulation of both the apoptotic process of the cell body and the integrity of the axonal network.

Collapsin-1 and its mammalian paralogues are secreted proteins. Therefore, in cultured neurons, collapsin induction before the execution of the death process suggests that apoptosis-destined neurons may affect their still intact neighboring cells and transfer a “death signal” along a specific and susceptible neuronal population.

Although caution should be practiced when evaluating the relevance of our cellular model to neurodegenerative diseases, it is tempting to speculate that in pathological apoptotic cell death, dying neurons, before their death, may secrete axon guidance molecules with repulsive cues that can further affect and harm the neighboring neurons. In that respect, the target specificity displayed by different semaphorin molecules may have a role in the selective degeneration of neurons in several human neurological disorders. In support of this view is the recent finding of CRM-2 protein in neurofibrillary tangles in Alzheimer’s disease (Yoshida et al., 1998). Further studies are now in progress to evaluate the possible clinical relevance of semaphorin involvement in neuronal cell survival in pathological conditions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Rescuing of chick collapsin-1 by RT-PCR
  5. DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342
  6. Expression of semaIII
  7. RESULTS
  8. Induction of CRM during neuronal apoptosis
  9. SemaIII induces apoptosis in neuronal cells
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

This work was supported, in part, by the National Parkinson’s Disease Foundation, U.S.A. The authors wish to thank Dr. E. M. Johnson for critically reviewing the manuscript and Dr. Marc Tessier-Lavigne for his generous gift of anti-NP-1 antibodies and the semaIII-AP expression vector. The authors appreciate the help of Dr. Karin Halevi with the immunoprecipitation experiments.

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  2. Abstract
  3. MATERIALS AND METHODS
  4. Rescuing of chick collapsin-1 by RT-PCR
  5. DNA staining with 4′,6-diamidino-2-phenylinodole and Hoechst 33342
  6. Expression of semaIII
  7. RESULTS
  8. Induction of CRM during neuronal apoptosis
  9. SemaIII induces apoptosis in neuronal cells
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES
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