Neuroprotective and Neuroregenerative Effects of Nimodipine in a Model System of Neuronal Differentiation and Neurite Outgrowth

Nimodipine is a Ca2+-channel antagonist mainly used for the management of aneurysmal subarachnoid hemorrhage (aSAH) to prevent cerebral vasospasms. However, it is not clear if the better outcome of nimodipine-treated patients is mainly due to vasodilatation or whether other cellular neuroprotective or neuregenerative effects of nimodipine are involved. We analysed PC12 cells after different stress stimuli with or without nimodipine pretreatment. Cytotoxicity of 200 mM EtOH and osmotic stress (450 mosmol/L) was significantly reduced with nimodipine pretreatment, while nimodipine has no influence on the hypoxia-induced cytotoxicity in PC12 cells. The presence of nimodipine also increased the NGF-induced neurite outgrowth in PC12 cells. However, nimodipine alone was not able to induce neurite outgrowth in PC12 cells. These results support the idea that nimodipine has general neuroprotective or neuregenerative effect beside its role in vasodilatation and is maybe useful also in other clinical applications beside aSAH

Advanced glycation endproducts interfere with adhesion and neurite outgrowth.

Advanced glycation endproducts (AGEs) represent a non-enzymatic posttranslational protein modification. AGEs are generated by a series of chemical reactions of free reducing monosaccharides, such as glucose, fructose or metabolites of the monosaccharide metabolism with amino groups of proteins. After oxidation, dehydration and condensation, stable AGE-modifications are formed. AGE-modified proteins accumulate in all cells and tissues as a normal feature of ageing and correlate with the glucose concentration in the blood. AGEs are increased in diabetic patients and play a significant role in the pathogenesis of most age-related neural disorders, such as Alzheimer's disease. We examined the role of AGEs on neurite outgrowth of PC12 cells. We induced the formation of AGEs using the reactive carbonyl compound methylglyoxal (MGO) as a physiological metabolite of glucose. We found that AGE-modification of laminin or collagen interfered with adhesion but not with neurite outgrowth of PC12 cells. Furthermore, the AGE-modification of PC12 cell proteins reduced NGF-induced neurite outgrowth. In conclusion, our data show that AGEs negatively influence neural plasticity

Cell adhesion of PC12 cells to AGE-modified ECM proteins.

<p>AGE-modified and non-modified collagen IV and laminin (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112115#pone-0112115-g002" target="_blank">Fig. 2</a>) were coated on E-plates. Cell adhesion of 5×10⁵ PC12 cells was quantified by RTCA real time analysis as described. Adhesion to non-modified ( = control) substrates was set to 100% and adhesion to AGE-modified substrates was calculated in % of control. Each bar represents values of three independent experiments carried out in triplicates (*p≤0.0001).</p

Cell viability after MGO treatment.

<p>PC12 cells were incubated with PBS, 0.1 mM MGO, 0.3 mM MGO or 1 mM MGO for 4 hours. A. Micrographs of typical PC12 cells. B. Tryphan blue staining of PC12 cells. Bars represent three independent experiments carried out in quadruplicates. Cell viability of cells cultured in the presence of PBS ( = control) was set to 1 and cell viability expressed in relation to the control. C. FACS analysis of PC12 cells stained with annexin V and propidium iodide.</p

AGE-modification of PC12 cells using MGO.

<p>PC12 cells were incubated with PBS, 0.1 mM MGO, 0.3 mM MGO or 1.0 mM MGO for 4 hours. A. Washed cells were solubilized and subjected to SDS-gel electrophoresis. Proteins were blotted and detected using monoclonal CML26 antibody B&C. Permeabilized (B) and non-permeabilized (C) were analyzed by flow cytometry using monoclonal CML26 antibody.</p

Real-time analysis of neurite outgrowth of AGE-modified PC12 cells.

<p>PC12 cells were AGE-modified using 1 mM MGO for 4 h as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112115#pone-0112115-g004" target="_blank">Figs. 4</a> &<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112115#pone-0112115-g005" target="_blank">5</a>. Cells were cultured on LN-coated E-plates and neurite outgrowth was induced by application of 100 ng/ml NGF. Neurite outgrowth was continuously quantified over 48 hours by RTCA as described in Pollscheit et al. (2012). Total neurite outgrowth of non-modified control cells during 48 h was set to 100% and neurite outgrowth of AGE-modified cells was expressed in % of control. Bars represent two independent experiments carried out in quadruplicates.</p

Formation of advanced glycation endproducts (AGE).

<p>Formation of advanced glycation endproducts (AGE).</p

AGE modification of ECM proteins using MGO.

<p>20 µg collagen IV (Col. IV) or laminin (LN) were spotted on a nitrocellulose membrane. One half of the membrane was incubated with 1 mM MGO for 4 hours. AGE formation was detected by dot blot analysis using the monoclonal CML26 antibody.</p