Pharmacotherapy for Neuropathic Pain: A Review

<p><b>Article full text</b></p> <p><br></p> <p>The full text of this article can be found here<b>.</b> <a href="https://link.springer.com/article/10.1007/s40122-017-0091-4">https://link.springer.com/article/10.1007/s40122-017-0091-4</a></p><p></p> <p><br></p> <p>The full text of all of the articles in this Supplement can be found here. <a href="https://link.springer.com/journal/40122/6/1/suppl/page/1">https://link.springer.com/journal/40122/6/1/suppl/page/1</a></p><p></p> <p><br></p> <p><b>Provide enhanced content for this article</b></p> <p><br></p> <p>If you are an author of this publication and would like to provide additional enhanced content for your article then please contact <a href="http://www.medengine.com/Redeem/”mailto:[email protected]”"><b>[email protected]</b></a>.</p> <p><br></p> <p>The journal offers a range of additional features designed to increase visibility and readership. All features will be thoroughly peer reviewed to ensure the content is of the highest scientific standard and all features are marked as ‘peer reviewed’ to ensure readers are aware that the content has been reviewed to the same level as the articles they are being presented alongside. Moreover, all sponsorship and disclosure information is included to provide complete transparency and adherence to good publication practices. This ensures that however the content is reached the reader has a full understanding of its origin. No fees are charged for hosting additional open access content.</p> <p><br></p> <p>Other enhanced features include, but are not limited to:</p> <p><br></p> <p>• Slide decks</p> <p>• Videos and animations</p> <p>• Audio abstracts</p> <p>• Audio slides</p

SOD1 stimulates lamellipodial protrusions in Neuro 2A cell lines

<p>We here investigated the effects of overexpressed superoxide dismutase (SOD)1 and amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants G93A and G147S in Neuro 2A (N2A) cell lines, and found a three-fold increase in lamellipodia either in cells cultured under differentiated or undifferentiated growth conditions. In undifferentiated N2A cells, SOD1 constructs promoted lamellipodial protrusions to similar extent as the overexpression of Rac1, and SOD1-mediated lamellipodia were prevented by coexpression of the N17 dominant-negative form of Rac1, or shRNA for a downstream effector of Rac1, the insulin receptor tyrosine kinase substrate p53 (IRSp53) or its binding partner LIN7. Moreover, no additive effect was measured by coexpression of the SOD1 constructs with Rac1, IRSp53 or LIN7. Collectively these data support a role for SOD1 in the regulation of Rac1-mediated lamellipodia pathway, a property fully retained by the two SOD1 mutants.</p

Immunofluorescence staining on primary neuronal cultures.

<p>Immunofluorescence staining with GHS-R of either cortical (CN) or hippocampal (HN) neurons at different stages of development. Both neuronal cultures show correct maturation in vitro, as assayed by positive staining of vesicular marker for excitatory neurotransmitter transporter Vglut which shows punctuated expression along neuritis at mature stages of development.</p

Comparison of GHS-R levels in cortical and hippocampal neurons.

<p>A. GHS-R mRNA expression levels in cortical (red) and hippocampal (blue) primary neurons normalized on expression levels at 4 div. GHS-R show a significant increase in expression levels at 9 and 16 div, with a significant reduction at later stages (21 div) in both neuronal populations. B. Relative mRNA expression of GHS-R at different developmental stages of hippocampal (red) and cortical (blue) neuronal cells normalized on GAPDH. GHS-R is significantly more expressed in hippocampal rather than cortical neurons at 9 and 16 div.</p

Evaluation of anti-GHS-R Mab specificity.

<p>A. Western blot of analysis of a selected brain region (hypothalamus) and a non-brain GHS-R bearing tissue (heart) showing a single band of the predicted molecular weight for GHSR at 48 kDa B. Immunoprecipitation of rat cortical (C) and hippocampal (H) primary neurons lysates with Mab 1D8B2 and commercial polyclonal antibody. Lysates from rat hippocampal and cortical neurons after nine days in vitro were immunoprecipitated with Mab (8 µg) or polyclonal antibody (CTRL), resolved and transferred to nitrocellulose membranes. The western blot analysis were performed with the polyclonal antibody when the monoclonal was used for immunoprecipitation and the other way around, both immunoprecipitation analysis clearly showed a 48 kD band corresponding to the predicted size of GHS-R. β-actin was used as a loading control. Sizes (kD) of molecular mass markers are indicated on the right.</p

Production and characterization of Mab anti-GHS-R.

<p>A. Immunoprecipitation of 22RV1 cell lysates with Mab 1D8B2 and commercial polyclonal antibody (CTRL). Lysates from cells were immunoprecipitated with Mab (8 µg) or polyclonal antibody, resolved and transferred to nitrocellulose membranes. The western blot analysis were performed with the polyclonal antibody when the monoclonal was used for immunoprecipitation and the other way around, both immunoprecipitation analysis clearly showed a 48 kD band corresponding to the predicted size of GHS-R. Sizes (kD) of molecular mass markers are indicated on the left. These experiments were performed independently at least twice with similar results. B. Binding analysis of Mab 1D8B2 to 22RV1 cells by flow cytometry. Purified monoclonal antibody (6 µg) were analyzed with a flow cytofluorimetric analysis to assess the binding of antibodies to the GHSR on the surface of the cells. The control consisted of an anti-GHSR purified polyclonal antibody. Experiments were performed four times with reproducible results C. Representative image of 22VR1 cells cotransfected with GHS-R siRNA and eGFP. eGFP (green) expressing cells do not show positivity for GHSR (red). D. Relative mRNA expression of GHS-R in transfected 22VR1 normalized on GAPDH. GHS-R expression is significantly lower in transfected cells(siRNA GHSR) than in non-transfected cells (GHSR UT).</p

Gene expression analysis in NB and HeLa cells and correlation analysis.

<p>(A) Relative gene expression analysis of the <i>ALK</i>, <i>PHOX2B</i> and <i>PHOX2A</i> genes, carried out in a panel of NB and in HeLa cell lines by real-time RT-qPCR using a pool of normal tissue RNAs as reference sample (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013108#s4" target="_blank">Methods</a>), shows over-expression of the three genes in all but two NB cell lines tested (GI-ME-N and ACN). (B) X-Y Plots showing a significant correlation between the expression level of <i>PHOX2B</i> and <i>PHOX2A vs.ALK</i> (left) and <i>PHOX2Avs. PHOX2B</i> (right) genes in the analyzed cell lines. Pearson's correlation coefficient indicates a very significant correlation of the three transcription levels <i>vs.</i> each other. Values are the mean ± s.d. of N = 3 independent RT-qPCR analyses performed in triplicate.</p

<i>In vitro</i> interaction of PHOX2B with the <i>ALK</i> promoter.

<p>A) EMSAs were performed using probes containing one of the ATTA sites of the region under analysis (ATTA 1, ATTA 2, ATTA 3 and the complex ATTA 4/5). Each labeled probe was incubated in the absence of nuclear extracts (lane 1), with IMR-32 nuclear extracts (lanes 2–4) or the <i>in vitro</i> expressed PHOX2B-Myc fusion protein (lanes 5–7). As negative control the oligonucleotides were also incubated with the <i>in vitro</i> reaction performed using the empty vector pcDNA3.1 M/H (lane 8). The competition experiments were performed in the presence of a molar excess of the unlabeled oligonucleotides (lanes 3 and 6). The anti-PHOX2B or the anti-c-Myc antibodies were added to the samples run in lanes 4 and 7, respectively. On the left, the arrows indicate the specific retarded bands detected; on the right, one or two asterisks indicate the supershifted complexes containing PHOX2B obtained by incubation of IMR-32 nuclear extracts with the anti-PHOX2B antibody (*) or the <i>in vitro</i> expressed protein with the anti-cMyc antibody (**), respectively. The free probes are shown at the bottom of the gels. B) ChIP assay. Chromatin extracted from IMR-32 cells was immunoprecipitated using the antibody against PHOX2B; pre-immune chicken IgY and the anti-acetylated histone H4 antibodies were used as negative and positive controls, respectively. The input represent 0,5% of the total chromatin extract. The precipitated DNA has undergone PCR amplification by using primers bordering the ATTA 3 and the ATTA 4/5 boxes in the <i>ALK</i> promoter.</p

PHOX2B effect on the <i>ALK</i> promoter sequentially deleted plasmids.

<p>A) Schematic representation of deleted plasmid inserts, progressively shorter from the entire wt <i>ALK</i> promoter region considered (−671 bp), down to the so called deletion 2 (del2; −351 bp), and to the so called deletion 3 (del3; −31 bp) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013108#pone-0013108-g004" target="_blank">figure 4A</a>). The promoter (grey bar), the 5′UTR (black bar) and the ATTA boxes (black circles) are shown. B) Activity of the <i>ALK</i> promoter fragments, expressed as percentage of the activity of the wt construct. Values are the mean ± s.d. of N = 3 independent experiments performed in HeLa cells. C) PHOX2B-mediated induction of the <i>ALK</i> promoter deleted plasmids, expressed as fold increase of the Luciferase activity obtained with respect to the use of the empty vector (pcDNA3.1) on the wt promoter. Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate in HeLa cells.</p

siRNA-mediated silencing of <i>ALK, PHOX2B</i> and <i>PHOX2A</i> in NB cells.

<p>Effects on the transcription level of the <i>ALK</i> (left side graphs), <i>PHOX2B</i> (middle graphs) and <i>PHOX2A</i> (right side graphs) genes after knock-down of the same genes in SHSY-5Y (A), IMR-32 (B) and HTLA-230 (C) cells. Gene-specific knock-down, evaluated 48 hours post-transfection by real-time RT-qPCR analysis, is very effective but also <i>PHOX2</i>-directed siRNAs are able to downregulate <i>ALK</i> at a similar extent (**: <i>P</i><0.01; ***: <i>P</i><0.001). Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate. (D) Gene silencing was confirmed at 72 hours post-transfection by Western blot.</p