An important part of neuronal differentiation is the outgrowth of axons and dendrites, collectively known as neurites. The primary morphological event that facilitates synaptic connections by neurons is characterized by a process that extends from the cell soma and is led by a growth cone. The neurite outgrowth assay is thus an assay of neuronal differentiation, and is often used to test the neuritogenic potential of a given substance. The neurons are grown at low density, in order to prevent the exchange of growth factors and other substances, through which they would otherwise be able to affect the neurite outgrowth in neighboring neurons. In essence, neurons are grown for a certain period of time in the presence of the molecule of interest, after which the outgrowth of the neurites can be quantified.
The neuritogenic potential of NGF and the six NGF-derived peptides in CGN cultures were evaluated by plating primary CGN at low density on permanox chamber slides. The cells were left for 24 hours in the presence of medium alone (untreated control), medium supplemented with 5µg/ml p2d peptide (positive control), and various concentrations of the six different peptides or the native NGF protein. Neurons were stained for GAP-43 to visualize neurites and the neurite outgrowth was quantified as described in section 4.2.3. The p2d peptide derived from a homophilic binding site in the neural cell adhesion molecule (NCAM), has previously been shown to induce neurite outgrowth (Pedersen et al 2004) and was used as a positive control. The effects of the control media versus the p2d peptide upon neurite outgrowth are shown in figure 5.1.1a-c.
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Figure 5.1.1. CGN were plated at low density and were left to differentiate for 24 hours in the presence of a) medium alone (untreated control), b) medium supplemented with 5µg/ml p2d peptide (positive control), various concentrations of the six different peptides or the native NGF protein. c) presents the graphical difference between untreated controls and positive controls. The overall neuritogenic effect of the positive control compared to the untreated controls was tested in a paired t-test with *** p<0.001.
The NGF protein was tested for its ability to induce neurite outgrowth in primary CGN at two different concentrations. Figure 5.1.2 shows that NGF was not able to induce neurite outgrowth compared to the untreated control. The maximal stimulation was found to be 103 ± 17.3% at a concentration of 100ng/ml.
Figure 5.1.2. Primary CGN were plated at low density and left to differentiate for 24 hours in the presence of medium alone (untreated control), medium suplemented with the p2d peptide (positive control), or two concentrations of the NGF protein. Approximately 200 cells were counted for each condition tested, and the effect of the protein was compared to the untreated control. Results are expressed as the percentage of untreated control and presented as ± SEM, n = 5. The overall
neuritogenic effect of the NGF protein compared to its untreated control was tested in a one-way ANOVA for repeated measurements, and no effect was observed.
33 The neuritogenic effects of the six different NGF-derived peptides appear from figure 5.1.3a-f.
Figure 5.1.3. Primary CGN were plated at low density and left to differentiate for 24 hours in the presence of medium alone (untreated control), medium suplemented with the p2d peptide (positive control), or various concentrations of the NGF-derived peptides. Approximately 200 cells were counted for each condition tested, and the effect of the peptides was compared to the untreated control. Results are expressed as the percentage of untreated control and presented as ± SEM, n = 5. The overall
neuritogenic effect of the NGF-derived peptides compared to their untreated controls was tested in a one-way ANOVA for repeated measurements. The results were as follows: hNgf_C1 p<0.05, hNgf_C2 p<0.01, hNgf_D not significant, hNgf_E p<0.0001, hNgf_EE p<0.0001 and hNgf_N p<0.01. The results of the following Dunnett's Multiple Comparison Test was * = p<0.05 and ** = p<0.01.
34 The neuritogenic effect of the hNgf_EE was found to be very pronounced, since this peptide significantly enhanced neurite outgrowth with up to 1509 ± 229% at a concentration of 9µg/ml and 1498 ± 186% at a concentration of 3µg/ml as compared to the untreated control. In figure 5.1.4 an example of the neuritogenic effect of the hNgf_EE peptide can be seen.
Figure 5.1.4. Primary CGN were plated at low density and left to differentiate for 24 hours in the presence of a) medium alone (untreated control), b) medium suplemented with the p2d peptide (positive control), or c) 9µg/ml hNgf_EE.
The effects of the hNgf_C2 and hNgf_E peptides were not as pronounced as the effects of hNgf_EE, but still they showed a strong neuritogenic effect. The hNgf_E peptide showed a maximal stimulation of 473 ± 91.86% at a concentration of 27µg/ml, compared to the untreated control. The maximal stimulation by hNgf_C2 was found to be 396 ± 147% at a concentration of 9µg/ml compared to the untreated control. The hNgf_C2, hNgf_E and hNgf_EE peptides seem to stimulate neurite outgrowth in a dose-response dependent relationship.
The hNgf_C1 peptide was found not to promote neurite outgrowth very well, it had its maximal stimulation at a concentration of 9µg/ml at 150 ± 77%, but was not significantly different from the untreated control.
hNgf_D and hNgf_N did not show any neuritogenic effects, since their maximal stimulation were at 68 ± 13% and 75 ± 17% at the concentrations 9µg/ml and 0.3µg/ml, respectively, when compared to the untreated control. Thus, CGN cultures treated with the NGF-derived peptides hNgf_EE, hNgf_E and hNgf_C2 exhibited a pronounced dose-dependent neuritogenic response. For the hNgf_D and hNgf_N peptides, no stimulation of the neurite outgrowth was observed.
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