• brain-derived neurotrophic factor;
  • choline acetyltransferase;
  • neural cell adhesion molecule;
  • p75 neurotrophin receptor;
  • polysialic acid;
  • tropomyosin related kinase B.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine, is known to be activated by brain derived neurotrophic factor (BDNF). We found that the specific removal of the carbohydrate polysialic acid (PSA) significantly increased BDNF-induced ChAT-activity in embryonic septal neurons. Using a p75 neurotrophin receptor (p75NTR) function-blocking antibody and K252a, a-pan tropomyosin related kinase (Trk) inhibitor, we demonstrate that BDNF-induced ChAT activity requires the stimulation of p75NTR and TrkB. PSA removal drastically increased radioactive iodinated ([125I])BDNF's maximal binding capacity (Bmax), derived from concentrations of [125I]BDNF ranging from 1 pM to 3.2 nM. In the presence of unlabeled nerve growth factor to prevent the binding of [125I]BDNF to p75NTR sites, the impact of PSA removal on the binding capacity of [125I]BDNF was greatly reduced. In conclusion, PSA limits BDNF-induced ChAT activity and BDNF–receptor interactions. BDNF-induced ChAT activity is TrkB and p75NTR dependent, and upon PSA removal the additional binding of BDNF to its receptors, especially p75NTR, likely contributes to the maximal ChAT activity observed. In vivo, the ontogenetic loss of PSA in the postnatal period may allow more interactions between BDNF and its receptors to increase ChAT activity and assure the proper development of the cholinergic septal neurons.

Abbreviations used

brain-derived neurotrophic factor


maximal binding capacity


choline acetyltransferase


endoneuraminidase N


glyceraldehyde-3-phosphate dehydrogenase


apparent affinity


neural cell adhesion molecule


nerve growth factor


polysialic acid


p75 neurotrophin receptor


tropomyosin related kinase

Basal forebrain cholinergic neurons project to the cortex and hippocampal formation, releasing acetylcholine to mediate essential cognitive processes (Sarter and Parikh 2005). The enzyme catalyzing acetylcholine synthesis, choline acetyltransferase (ChAT; EC2.3.1.6), is critical for the proper functioning of cholinergic neurons (Dobransky and Rylett 2005).

In vitro, ChAT activity is increased by cell contact (Adler and Black 1985; Kessler et al. 1986). Polysialic acid (PSA), a large homopolymer of α-2,8 linked sialic acid found exclusively on the neural cell adhesion molecule (NCAM), has been well-characterized as a limiting factor of cell adhesion (Rutishauser and Landmesser 1996; Kleene and Schachner 2004; Johnson et al. 2005). The significance of PSA and NCAM in contact-dependent ChAT activity was investigated by Acheson and Rutishauser (1988). Their findings indicated that the addition of crude membrane fractions lacking PSA increased ChAT activity in sympathetic neurons. Membrane-cell contact was shown to be essential and mediated by both NCAM-dependent and independent adhesion mechanisms. Interestingly, membranes rich in PSA did not increase ChAT activity, even when NCAM-independent membrane-cell adhesion was provided. It was therefore suggested that PSA could prevent the transmission of ChAT-inductive signals, perhaps by acting as a screen to preclude certain ligands from interacting with their receptors. Congruent with the idea that PSA limits the activation of surface molecules, increased cell contact following PSA removal, triggers the neuronal differentiation of progenitor and neuroblastoma cells (Seidenfaden et al. 2003; Petridis et al. 2004).

In contrast to this shielding mode of action, other studies have suggested that PSA sensitizes the receptor tropomyosin related kinase B (TrkB) to its ligand, brain-derived neurotrophic factor (BDNF), thereby promoting long-term potentiation, neuronal survival and differentiation (Müller et al. 2000; Kiss et al. 2001; Vutskits et al. 2001). The mechanism by which this occurs is unknown and the impact of PSA on the other receptor for BDNF, the p75 neurotrophin receptor (p75NTR), has not been investigated.

All members of the neurotrophin family can increase ChAT activity (Alderson et al. 1990; Mobley et al. 1986; Nonner et al. 1996, 2000). Nerve growth factor (NGF) carries out this function by stimulating p75NTR and TrkA, and BDNF by activating p75NTR and TrkB (Alderson et al. 1990; Ernfors et al. 1992; Ringstedt et al. 1993; Knüsel et al. 1994; Nonner et al. 2000). Our study focuses on the influence of PSA on BDNF-induced ChAT activity in primary septal neurons, which express TrkB and p75NTR. Cultures of cholinergic septal neurons derived from the embryonic basal forebrain are ideal since they survive in defined conditions without the influence of glial cells and can be maintained in the absence of neurotrophins (Hefti et al. 1985; Nonner et al. 1992; Pongrac and Rylett 1998). Furthermore, primary cholinergic septal neurons respond to BDNF by increasing ChAT activity through the stimulation of TrkB and p75NTR (Nonner et al. 2000), making it possible to evaluate the effect of PSA on both receptors.

Our results demonstrate that PSA acts as a limiting factor for BDNF-induced ChAT activity in developing cholinergic neurons, most likely by preventing maximal BDNF–receptor interactions. BDNF-induced ChAT activity is TrkB and p75NTR dependent, and we suggest that the additional binding of BDNF to its receptors, especially p75NTR, upon PSA removal contributes to the maximal ChAT activity observed.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Time-pregnant Sprague Dawley rats were obtained from Charles River Laboratories (St. Constant, QC, Canada). They were housed individually and received food and water ad libitum for two days prior to embryo retrieval. All animal protocols were approved by the Animal Care Committee of Sunnybrook Health Sciences Centre and experiments were performed according to the guidelines set by the Canadian Council on Animal Care and the Animals for Research Act of Ontario.

BDNF was purchased from Alomone (Jerusalem, Israel). Endoneuraminidase N (endoN), a bacteriophage enzyme which specifically cleaves PSA from NCAM, was bought from AbCYS (Paris, France) and kindly donated by Dr Geneviève Rougon (CRNS, Marseilles, France). The REX antibody was generously supplied by Dr Louis Reichardt (UCSF, San Francisco, CA, USA). The TrkB antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PSA, NCAM and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies as well as TO-PRO-3 iodide were from Chemicon (Temecula, CA, USA). All secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA). K252a was bought from Calbiochem (La Jolla, CA, USA). [14C]Acetyl coenzyme A was purchased from Perkin Elmer (Boston, MA, USA). [125I]BDNF was custom prepared by GE Healthcare (Piscataway, NJ, USA). All other reagents were obtained from Sigma (Oakville, ON, Canada).

Cell Culture

Embryos at gestation day 17 were retrieved from Sprague Dawley rats and cells from the septal area of the basal forebrain were prepared according to the method of Hefti et al. (1989) with slight modifications (Pongrac and Rylett 1998). Briefly, septal cells were plated in 10% serum. After 1 h, the media containing unattached cells was removed and replaced by serum free media supplemented with N-2. Septal neurons were exposed to BDNF [3.7 nM] to stimulate ChAT activity, endoN [1 : 4,000] to remove PSA, K252a [50 nM,] to inhibit Trk signaling, and the REX antibody [1 µg/ml,] to block the function of p75NTR. In all conditions, we obtained a ∼98% pure neuronal culture after 4 days in vitro, the time at which all experiments were performed. None of the treatments affected the survival of septal neurons, which was determined using Trypan blue exclusion assays. Heat inactivated endoN was unable to remove PSA and it did not alter ChAT activity. Rabbit IgG antibodies were used as a negative control for p75NTR function blocking antibodies.


Cells were rinsed and incubated with an antibody directed against PSA [1 : 1,200] for 1 h at room temperature (25°C). Cells were then fixed with 4% paraformaldehyde and treated with blocking buffer containing 5% donkey serum and 0.25% Triton X-100 for 30 min. Secondary antibody, donkey anti-mouse IgM biotin, was added for 2 h at room temperature (25°C) followed by incubation with streptavidin-Alexa 488 and the nucleic acid-staining cyanine dye monomer TO-PRO-3 iodide (2 µM). Slides were rinsed and coverslipped with polyvinyl alcohol containing 2.5% 1,4-diazabicyclo-2,2,2-octane (PVA/DABCO). Fluorescent labeling was detected with a confocal microscope, equipped with argon and helium/neon lasers (Zeiss Axiovert 100M, LSM 510; Carl Zeiss, Toronto, ON, Canada). Wavelengths at 488 and 633 nm were used to detect Alexa 488 and TO-PRO-3, respectively.

ChAT Activity

[14C]Acetyl coenzyme A and choline were incubated with the culture samples for 50 min at 37°C following the method of Fonnum (1969). The reaction was stopped and the newly synthesized [14C]acetylcholine was extracted and counted in a Beckman liquid scintillation counter.

Acetylcholinesterase Histochemistry

Cells were fixed with 4% paraformaldehyde and incubated with acetylthiocholine iodide according to the method of Karnovsky and Roots (1964). The reaction product was visualized using diaminobenzidine and nickel chloride. At early stages of development in vitro, we and others have found that acetylcholinesterase histochemistry is more sensitive then immunocytochemistry for the quantification of cholinergic cells (Hefti et al. 1985; Hartikka and Hefti 1988; Mennicken and Quirion 1997). To assure the specificity of our acetylcholinesterase staining, we controlled for non-specific cholinesterase staining by using a butyrylcholinesterase inhibitor, tetra-isopropylpyrophosphoramide. Furthermore, we restricted the incubation time with acetylthiocholine iodide to 16 h, which provided positive staining in a subpopulation of primary septal neurons but not in cortical neurons. Cortical neurons were prepared and maintained in the same way as septal neurons and they were derived from the same animals.

Cholinergic cell bodies in primary septal cultures were counted using the StereoInvestigator software (MicroBrightfield Inc., Williston, VT, USA), which applies an unbiased (randomly chosen first field of view), systematic (subsequent fields of view are equally distributed) counting grid on the entire culture well. Sampling sites covered four percent of the entire well area, and therefore, the final counts obtained/well were multiplied by 25 to represent an estimate of the total number of cholinergic cells/well.

[125I]BDNF Binding Assay

The media containing BDNF was replaced with BDNF-free media, with or without endoN, for a 6 h washout period. Cells were then lysed and resuspended in Krebs-Ringer-HEPES solution containing 1 mg/ml bovine serum albumin to reduce the non-specific binding. Samples were incubated with [125I]BDNF at concentrations ranging from 1 pM to 3.2 nM at 4°C for 2 h with occasional mixing to prevent sedimentation. To identify non-specific binding, additional cells were incubated with [125I]BDNF in the presence of excess (1 µM) unlabelled BDNF. Cell bound radioactivity was collected by rapid centrifugation through a 10% glycerol solution. After centrifugation, the tubes were snapped frozen on dry ice and the bottom 6 mm of each tube was removed and counted in a Compugamma counter (LKB, Wallac, Finland). The specific binding was expressed as fmoles/mg protein. Values of maximal binding capacity (Bmax) and apparent affinity (Kd) were derived from a non-linear regression, using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA).

Western Blot Analysis

Cells were rinsed, lysed and analyzed for protein content. The protein samples were separated by 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was treated for 1 h in 5% skim milk, incubated at 4°C overnight with the primary antibody, and rinsed before the addition of the appropriate horseradish peroxidase conjugated antibodies for 2 h at room temperature (25°C). Immunoreactive signals were detected using the enhanced chemiluminescence system. We used primary antibodies directed against: PSA [1 : 500]; NCAM [1 : 1,000]; TrkB [1 : 100]; GAPDH [1 : 1,000]; and REX p75NTR[1 : 500]. Secondary antibodies conjugated to horseradish peroxidase were directed against mouse [1 : 10,000] and rabbit [1 : 100,000]. Western blots were quantified using Genetools Analysis Software (Syngene, Cambridge, UK).

Statistical Analysis and Presentation of the Data

GraphPad Prism 4 (GraphPad Software) was used for statistical analyses and for the presentation of bar graphs and binding curves. The mean values of three and more groups were compared with one-way ANOVA and Neuman-Keuls' post-tests. Mean values of Bmax and Kd were compared using student's t-test. In all cases, significance was noted at p < 0.05. Montages of the figures were made in Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, USA).


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

PSA expression by septal neurons in culture and its removal by endoN

PSA was clearly detected in embryonic septal neurons 2 h after plating (Fig. 1a, top panel) and it was strongly expressed at four days in vitro (Fig. 1a, bottom panel). Within 2 h and up to four days in vitro, the presence of endoN in the culture media completely removed PSA from septal neurons (Fig. 1a, inset). Western blotting analyses confirmed the presence of PSA in primary septal neurons and revealed that PSA's removal by endoN occurs on NCAM isoforms 140 and 180 kDa (Fig. 1b).


Figure 1.  PSA expression by septal neurons and its removal using endoN. (a) Embryonic septal neurons were labeled with PSA (green) and TOPRO-3 (blue). Top panel: At 2 h postplating at high-density, septal neurons expressed PSA. Short emerging neurites are PSA-positive (arrows). Inset: Exposure to endoN for 2 h completely removed PSA from septal neurons. Bottom panel: After 4 days in vitro, septal neurons plated at high-density have clustered, developed long neurites and they strongly expressed PSA. Similarly to the inset shown in (a), endoN present in the media for the entire 4-day period kept the neurons free of PSA. (b) Western blot using specific antibodies for PSA and NCAM together, confirmed the ability of endoN to remove PSA from NCAM isoforms. The control lane illustrates the typical thick band of NCAM in presence of PSA at high molecular weight. The endoN lane reveals NCAM in its 140 and 180 kDa isoforms upon PSA removal.

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PSA removal has no impact on basal ChAT activity in high-density cultures

When septal cells are plated at low-density (32 000 cells/cm2), the removal of PSA resulted in a significant 23% induction of ChAT activity (*p < 0.05) (Fig. 2a). To assess specifically the impact of PSA on BDNF-mediated ChAT activity, we optimized our experimental conditions so as to abolish the influence of PSA on basal ChAT activity. Septal cells, plated at high-density of 130 000 cells/cm2, generated cultures with comparable levels of aggregation with and without endoN present in the culture media (Fig. 2b). In these conditions, basal ChAT activity was three fold higher then observed at the low-density (***p < 0.001, Fig. 2a). Most notably, the addition of endoN did not influence ChAT activity in high-density cultures (Fig. 2a). Therefore, the use of high-density cultures in subsequent experiments allowed us to test specifically the impact of PSA removal on BDNF-induced ChAT activity. PSA removal with endoN had no effect on cell survival as determined by Trypan blue exclusion assays and illustrated for high-density cultures (Fig. 2c).


Figure 2.  PSA removal and its impact on ChAT activity in cultures plated at low- and high-densities after four days in vitro. (a) ChAT activity in septal neurons plated at low-density is significantly increased when PSA is removed with endoN (23% vs. control, *p < 0.05). ChAT activity in high-density cultures are three fold greater then in low-density (***p < 0.001), but is not affected by PSA removal by endoN. Values are the means ± SEM of three independent experiments, each performed in duplicates. (b) Differential image contrast photographs of septal cells plated at high-density and maintained for four days in vitro. Qualitative assessments revealed that PSA removal did not alter the general morphology and extent of cell aggregation. (c) EndoN treatments do not affect the survival of septal neurons, as illustrated here from Trypan blue exclusion assays in high-density cultures. Values are the means ± SEM of three independent experiments, each performed in duplicates.

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PSA removal significantly increases BDNF-induced ChAT activity

Exposure of septal neurons to BDNF caused the expected increase in ChAT activity (**p < 0.01, Fig. 3). Remarkably, the removal of PSA further increased BDNF-induced ChAT activity (100% over basal levels, ***p < 0.001; and 28% more then BDNF-stimulated levels, + p < 0.05; Fig. 3). PSA removal by itself did not significantly influence basal ChAT activity.


Figure 3.  PSA removal significantly increases BDNF-induced ChAT activity. Septal cells were plated at high-density. After four days in vitro, BDNF-induced ChAT activity reached 60% over control levels of basal ChAT activity, **p < 0.01). The removal of PSA in presence of BDNF further increased ChAT activity (28% greater then BDNF alone, + p < 0.05; 100% over control levels, ***p < 0.001). ChAT activity was not affected by PSA removal alone (EndoN). Values are the means ± SEM of six independent experiments, each performed in duplicate.

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As predicted, the number of detectable septal cholinergic neurons is approximately doubled in presence of BDNF compared to control conditions (**p < 0.01, Table 1). The removal of PSA had no impact on cholinergic cell numbers (Table 1). Therefore, the increase in BDNF-induced ChAT activity produced by the removal of PSA is due to a greater ChAT activity per cholinergic cell.

Table 1.   PSA removal does not affect the number of cholinergic cells in culture
 ControlEndoNBDNFBDNF + EndoN
  1. Unbiased semi-automated methods were used to provide a quantitative estimate of the number of cholinergic neurons in each condition. PSA removal using endoN did not affect cholinergic cell numbers, in absence or presence of BDNF. BDNF significantly increased the detection of cholinergic cells (**p < 0.01). Values are the means ± SEM of three independent experiments, each performed in triplicate.

Cholinergic cell number816 ± 86930 ± 882018 ± 132**1802 ± 101**

The maximal activation of ChAT by BDNF requires p75NTR and TrkB

In septal neurons, BDNF-induced ChAT activity was reported to require the activation of both p75NTR and TrkB (Nonner et al. 2000). To test whether PSA removal increases BDNF-mediated ChAT activity through the stimulation of the same receptors, we challenged septal neurons with an antibody that blocks p75NTR function, and with K252a, a pan-Trk inhibitor. Regardless of the presence of PSA, BDNF-induced ChAT activity was reduced to basal levels by the individual application of p75NTR function-blocking antiserum (Fig. 4a), or K252a (Fig. 4b). Trypan blue cell exclusion assays demonstrated that neither the p75NTR antiserum nor K252a affected cell viability (Fig. 4c). These results indicate that the increase in BDNF-induced ChAT activity caused by the removal of PSA is mediated by p75NTR and TrkB. Our data therefore suggest that PSA removal increases BDNF-induced ChAT activity by favoring the stimulation of p75NTR and TrkB.


Figure 4.  BDNF receptors, p75NTR and TrkB, are involved in the activation of ChAT. ChAT activity and survival assays were completed using septal neurons plated at high-densities and maintained for four days in vitro. BDNF [3.7 nM], p75NTR antibody [1 µM], K252a [50 nM] and endoN (1 : 4000) were added 1 h after neurons were plated and remained for the duration of the culture period. ChAT activity in untreated neurons (control) was not significantly affected by the addition of the p75NTR antibody (a) or K252a (b). ChAT activity induced by BDNF alone (160% of control levels, **p < 0.01) and by the removal of PSA in presence of BDNF (28% greater then BDNF alone, + p < 0.05; 200% over control levels, ***p < 0.001), was returned to normal by the addition of the p75NTR antibody (a) and K252a (b). (c) The total number of septal cells was unaltered in the presence of p75NTR antibody and K252a. Values are the means ± SEM of four (a, b) and three (c) independent experiments, each performed in duplicate.

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PSA removal increases the binding [125I]BDNF to its receptors

To examine the influence of PSA on BDNF–receptor interactions, radioligand binding assays were performed. [125I]BDNF's binding parameters were established in septal cells at concentrations ranging from 1 pM to 3.2 nM. Two saturable binding components were characterized, one of high- and the other of low-affinity (Figs 5a and b). As illustrated in Fig. 5(a), a representative experiment shows that the removal of PSA from septal cells does not impact [125I]BDNF binding at high-affinity sites. In contrast, at low-affinity sites, the removal of PSA significantly increased the amount of [125I]BDNF bound at concentrations greater than 1 nM (Fig. 5b). The mean Bmax and Kd values are provided in Table 2, confirming that [125I]BDNF binding parameters at high-affinity sites are unaltered by PSA removal (Table 2A). In contrast, at sites of lower affinities, the removal of PSA from septal neurons caused a drastic increase in the maximal number of [125I]BDNF specific binding sites (∼300%, *p < 0.05, Table 2B), and a significant increase in apparent Kd values (*p < 0.05, Table 2B). To evaluate the participation of p75NTR, we repeated the binding assays using [125I]BDNF (1 pM to 3.2 nM) with an excess of unlabelled NGF (3 nM) to saturate the p75NTR sites and render them unavailable for [125I]BDNF binding (Table 2C). In these conditions, no specific [125I]BDNF binding was detected above 80 pM, rendering the binding curves and parameters closer to the ones observed at sites of high-affinity. The impact of PSA removal on the Bmax and apparent Kd of [125I]BDNF receptor sites unoccupied by NGF was greatly reduced (Table 2C), indicating that p75NTR receptor sites are significantly contributing to the increase in Bmax observed after PSA removal.


Figure 5.  Binding curves of [125I]BDNF in septal neurons expressing PSA (Control) and upon PSA removal (EndoN). (a) Representative binding assay using 1–80 pM [125I]BDNF. At high-affinity sites, the removal of PSA by endoN did not significantly alter the binding curve. (b) Representative binding assay carried out at [125I]BDNF concentrations ranging from 1 pM to 3.2 nM. PSA removal by endoN caused a drastic change in the curve fitting at these lower-affinity sites. At each nM concentration of [125I]BDNF tested, PSA removal clearly increased the number of [125I]BDNF binding sites detected. All data points represent the mean value of duplicate measures at each concentration.

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Table 2.   Binding parameters of [125I]BDNF at high- and low-density and upon blocking p75NTR
  1. All Kd and Bmax values presented are means ± SEM of six independent experiments, each performed in duplicates. Bmax are in fmole/mg protein. (A) Binding parameters of [125I]BDNF at high-affinity sites are unaffected by PSA removal with endoN. (B) At lower affinity sites, PSA removal caused a significant increased in [125I]BDNF Bmax and apparent Kd (∼300% vs. control, *p < 0.05). (C) Blocking the p75NTR. receptor sites with NGF resulted in high-affinity [125I]BDNF binding and it significantly reduced the impact of PSA removal on [125I]BDNF binding parameters. Binding assays were performed as in B, with the modification that an excess of unlabelled NGF was added to occupy the p75NTR sites. In these conditions, specific [125I]BDNF binding at concentrations above 80 pM was abolished by the presence of NGF, and the appearance of these binding curves was similar to the one illustrated in Fig. 5(a).

A[125I]BDNFKd (pM)84 ± 17108 ± 29
(1–80 pM)Bmax0.32 ± 0.120.21 ± 0.10
B[125I]BDNFKd (nM)1.9 ± 0.66.6 ± 1.7 *
(1 pM – 3 nM)Bmax2.7 ± 0.59.4 ± 2.7 *
C[125I]BDNFKd (pM)25 ± 830 ± 14
(1 pM – 3 nM)Bmax0.46 ± 0.120.88 ± 0.12
+ 3 nM NGF   

Western blotting experiments confirmed, as previously reported (Elliott et al. 2001), that p75NTR expression was increased in the presence of BDNF, and we found that it was unaffected by the addition of endoN (Fig. 6). The amount of TrkB receptor protein was not significantly changed upon BDNF and/or endoN exposure (Fig. 6).


Figure 6.  PSA removal does not affect the amount of TrkB or p75NTR protein, and BDNF treatments increase the expression of p75NTR (a) Representative Western blot showing TrkB and p75NTR protein expression in the presence and absence of PSA and/or BDNF. (b) Quantification of immunoblots show that BDNF produced the expected increase in the levels of p75NTR (∼330% increase, *p < 0.05), but did not increase the amount of TrkB receptor. The amounts of TrkB and p75NTR receptor protein are unaffected by PSA removal using endoN. Values are the means ± SEM quantified from three independent Western blots and each BDNF receptor (BDNF Rc) protein band was normalized to GAPDH immunoreactivity and expressed as a percent of control.

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  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Our main goal was to evaluate the modulation of BDNF-induced ChAT activity by PSA in primary septal neurons. We established that the stimulation of ChAT activity by BDNF was significantly increased when PSA was removed from septal neurons. Mechanistically, we demonstrated that the activation of p75NTR and TrkB receptors is required to increase BDNF-related ChAT activity and PSA prevented the maximal binding of BDNF to its receptors. Finally, p75NTR sites are enriched in our culture conditions and they contribute significantly to the increase in [125I]BDNF binding observed upon PSA removal.

PSA was thought to prevent ChAT-inductive signals that are independent of adhesion and of NCAM (Acheson and Rutishauser 1988). We tested whether BDNF is one of these ChAT-inductive signals modulated by PSA. BDNF was chosen for two main reasons: (i) BDNF increases ChAT activity in septal neurons, and (ii) PSA increases BDNF's action on TrkB in non-cholinergic cortical and hippocampal neurons (Müller et al. 2000; Kiss et al. 2001; Vutskits et al. 2001). Therefore, we evaluated the impact of PSA on BDNF's effects on cholinergic neurons that express both p75NTR and TrkB.

Our first experiments were designed to establish the appropriate plating density of primary septal neurons, in which basal ChAT activity is not influenced by PSA removal. At low-densities of primary septal neurons under basal conditions (i.e. absence of BDNF), ChAT activity was significantly increased by PSA removal. ChAT activity is known to increase with the plating density of primary septal neurons (Hartikka and Hefti 1988; Mennicken and Quirion 1997), and this is attributed to an increase in cell contact rather than an enrichment of ChAT-inductive signals in the culture media (Adler and Black 1986; Acheson and Rutishauser 1988; Casper and Davies 1988). We suggest that PSA removal in low-density cultures facilitates cell contact-mediated ChAT activity. By increasing the number of septal cells plated, PSA removal no longer modulated basal ChAT activity. These conditions allowed us to address specifically the impact of PSA removal on BDNF-induced ChAT activity.

Interestingly, we found that BDNF-induced ChAT activity was significantly increased by PSA removal in high-density cultures of septal cells. The number of cholinergic neurons was unchanged when treated with endoN, indicating that PSA did not affect cholinergic survival or phenotypic maturation in these conditions. These results also indicated that the increase in ChAT activity due to PSA removal in presence of BDNF occurred on a per cell basis. Our data on the ability of BDNF to approximately double the detection of cholinergic neurons and to increase ChAT activity in septal cultures is consistent with previous reports (Alderson et al. 1990; Knüsel et al. 1991; Friedman et al. 1993; Nonner et al. 2000).

Using a p75NTR function-blocking antibody and K252a, we demonstrated that BDNF-induced ChAT activity in septal neurons occurs through the stimulation of both p75NTR and TrkB, supporting the results obtained by Nonner et al. (2000). With regard to the role of PSA in modulating BDNF responsiveness in cholinergic neurons, we found that upon PSA removal, BDNF-induced ChAT activity is also mediated through p75NTR and TrkB. Interactions between Trk and p75NTR can occur at the receptor level (e.g. by modifying the selectivity or affinity of neurotrophin binding) and via the modulation of intracellular signaling pathways (Roux and Barker 2002; Teng and Hempstead 2004). The nature of the interactions between TrkB and p75NTR in primary neurons is yet to be elucidated, and the specific events leading to the stimulation of ChAT activity by BDNF are unknown. Recently, it was demonstrated that NGF-induced ChAT activity in primary septal neurons involves the phosphatidylinositol-3′-kinase pathway (Madziar et al. 2005), mostly likely triggered by TrkA and p75NTR., since both receptors are required for ChAT stimulation by NGF (Nonner et al. 2000). We are currently investigating the signaling events associated with BDNF-induced ChAT activity.

We tested whether PSA influences BDNF's interactions with its receptors. The binding parameters of [125I]BDNF at high-affinity sites (up to 80 pM [125I]BDNF) were unchanged upon PSA removal. In contrast, PSA removal produced a three fold increase in the total number of [125I]BDNF binding sites, derived from [125I]BDNF concentrations reaching 3.2 nM. In control conditions (without endoN), the apparent Kd values of [125I]BDNF at high- and low-affinity sites in primary septal neurons were comparable to that established in chick dorsal root ganglia neurons (Rodriguez-Tebar and Barde 1988). The apparent Kd of [125I]BDNF at low-affinity sites in the presence of endoN may be overestimated since it was difficult to attain the saturation of these sites upon PSA removal. To evaluate the contribution of the p75NTR sites in the modulation of [125I]BDNF binding by PSA, we used an excess of unlabelled NGF (3 nM) to saturate the p75NTR sites and render them unavailable for [125I]BDNF binding. We found that preventing the binding of [125I]BDNF to p75NTR reduced the impact of PSA removal on the Bmax and apparent Kd of [125I]BDNF. Taken together, our results revealed that the p75NTR sites significantly contribute to the increases in [125I]BDNF binding, at lower affinity sites, observed in absence of PSA and using a range of [125I]BDNF concentrations up to 3.2 nM.

In conclusion, we showed that PSA clearly limits BDNF-induced ChAT activity in septal cholinergic neurons. The removal of PSA increased the binding of BDNF to its receptors, including p75NTR, which are required with TrkB to stimulate BDNF-induced ChAT. We propose that upon PSA removal, the stimulation of additional p75NTR, along with TrkB receptors, would trigger maximal ChAT activity (Fig. 7).


Figure 7.  Proposed mechanism of PSA's action on BDNF-induced ChAT activity. In embryonic septal neurons: (a) Basal ChAT activity occurs in the absence of trophic factors, and (b) BDNF-induced ChAT activity requires the stimulation of both TrkB and p75NTR receptors. (c) Our current results indicate that PSA removal, following a treatment with EndoN, significantly increases the binding of BDNF to its receptors, especially p75NTR, which together with TrkB, causes an increase in ChAT activity. It is proposed that PSA limits ligand and receptor interactions involved in BDNF-induced ChAT activity. The stoichiometry of TrkB, BDNF, p75NTR complexes and the nature of TrkB and p75NTR interactions activating ChAT are unknown in this model.

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It is possible that the role of PSA in modulating BDNF actions depends on the type of receptors present on the cells, i.e. TrkB and p75NTR. Septal cholinergic neurons express TrkB and p75NTR, whereas previous studies suggesting that PSA facilitate BDNF's actions have focused on cells expressing TrkB only (Müller et al. 2000; Kiss et al. 2001; Vutskits et al. 2001).

Recently, an elegant study demonstrated that PSA is crucial to proper brain development (Weinhold et al. 2005). Its timely expression in the brain is likely to coordinate important events such as neuronal differentiation and maturation. PSA's expression in the rodent brain is drastically reduced during the second and third week postnatal (Seki and Arai 1993; Wood et al. 1997). This corresponds to the age when ChAT activity is up-regulated (Auburger et al. 1987; Cavicchioli et al. 1991), and it is usually attributed to concomitant increases in the levels of neurotrophins and their receptors. Our results suggest that the ontogenetic loss of PSA on septal neurons can allow a greater number of p75NTR to bind BDNF and, along with activated TrkB, contribute to the increase in ChAT activity to assure the appropriate maturation of cholinergic neurons.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was funded by the Canadian Institutes of Health Research, Canadian Neurotrauma Research Program, Canada Innovation Foundation (New Opportunities) and the Ontario Innovation Trust (I.A). Alison Burgess was partially funded by University of Toronto Open Fellowship, and the Peterborough K.M. Hunter Graduate Scholarship. The authors would like to thank Drs Freda Miller, Geneviève Rougon and Anurag Tandon for their insightful comments on this manuscript. Ms. Shawna Rideout-Gros and Dr Isabelle Frappé provided technical assistance with the isolation of primary neurons, and Dr Xuezhi Cui helped in the Western blotting experiments. We appreciated the expert assistance of Ms. Gisele Knowles in confocal microscopy. The Centre for Cytometry and Scanning Microscopy at Sunnybrook Research Institute was supported by a Canadian Institutes of Health Research Multi-User Equipment & Maintenance Grant. Dr Eric Yang at the Proteomics Core Facility of the Toronto Angiogenesis Research Centre, Sunnybrook Research Institute, provided technical assistance for Western blot quantification. We are grateful to Dr Louis Reichardt for his generous gift of the REX p75NTR antibody and to Dr Geneviève Rougon for her contribution in supplying endoneuraminidase N.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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