Data_Sheet_1_Cerebrospinal fluid level of proNGF as potential diagnostic biomarker in patients with frontotemporal dementia.docx

IntroductionFrontotemporal dementia (FTD) is an extremely heterogeneous and complex neurodegenerative disease, exhibiting different phenotypes, genetic backgrounds, and pathological states. Due to these characteristics, and to the fact that clinical symptoms overlap with those of other neurodegenerative diseases or psychiatric disorders, the diagnosis based only on the clinical evaluation is very difficult. The currently used biomarkers help in the clinical diagnosis, but are insufficient and do not cover all the clinical needs.MethodsBy the means of a new immunoassay, we have measured and analyzed the proNGF levels in 43 cerebrospinal fluids (CSF) from FTD patients, and compared the results to those obtained in CSF from 84 Alzheimer’s disease (AD), 15 subjective memory complaints (SMC) and 13 control subjects.ResultsA statistically significant difference between proNGF levels in FTD compared to AD, SMC and controls subjects was found. The statistical models reveal that proNGF determination increases the accuracy of FTD diagnosis, if added to the clinically validated CSF biomarkers.DiscussionThese results suggest that proNGF could be included in a panel of biomarkers to improve the FTD diagnosis.</p

Functional Characterization of Human ProNGF and NGF Mutants: Identification of NGF P61SR100E as a “Painless” Lead Investigational Candidate for Therapeutic Applications

<div><p>Background</p><p>Nerve Growth Factor (NGF) holds a great therapeutic promise for Alzheimer's disease, diabetic neuropathies, ophthalmic diseases, dermatological ulcers. However, the necessity for systemic delivery has hampered the clinical applications of NGF due to its potent pro-nociceptive action. A “painless” human NGF (hNGF R100E) mutant has been engineered. It has equal neurotrophic potency to hNGF but a lower nociceptive activity. We previously described and characterized the neurotrophic and nociceptive properties also of the hNGF P61S and P61SR100E mutants, selectively detectable against wild type hNGF. However, the reduced pain-sensitizing potency of the “painless” hNGF mutants has not been quantified.</p><p>Objectives and Results</p><p>Aiming at the therapeutic application of the “painless” hNGF mutants, we report on the comparative functional characterization of the precursor and mature forms of the mutants hNGF R100E and hNGF P61SR100E as therapeutic candidates, also in comparison to wild type hNGF and to hNGF P61S. The mutants were assessed by a number of biochemical, biophysical methods and assayed by cellular assays. Moreover, a highly sensitive ELISA for the detection of the P61S-tagged mutants in biological samples has been developed. Finally, we explored the pro-nociceptive effects elicited by hNGF mutants <i>in vivo</i>, demonstrating an expanded therapeutic window with a ten-fold increase in potency.</p><p>Conclusions</p><p>This structure-activity relationship study has led to validate the concept of developing painless NGF as a therapeutic, targeting the NGF receptor system and supporting the choice of hNGF P61S R100E as the best candidate to advance in clinical development. Moreover, this study contributes to the identification of the molecular determinants modulating the properties of the hNGF “painless” mutants.</p></div

Painful effect induced by hNGF WT or mutants.

<p>Pooled data of the mechanical allodynic response (A) and the thermal (hot) hyperalgesic response (B) evoked by intraplantar (i.pl.) injection (20 μl) of hNGF WT, hNGF R100E, hNGF P61S, hNGF P61S R100E or their vehicle (Veh, isotonic saline), measured 5 hours post-treatment. Data are mean ± sem of at least n = 4 mice per group; *P<0.05 vs. Veh or hNGF WT 0.1 μg or hNGF P61S 0.1 μg; #P<0.05 vs. hNGF WT 1 μg or hNGF P61S 0.1 μg; §P<0.05 vs. hNGF WT 10 μg or hNGF P61S 10 μg. One-way ANOVA followed by Bonferroni post-test.</p

Chemical denaturation.

<p>Guanidinium Chloride (Gdm-Cl) denaturation profile of WT (blue), P61S (green), P61SR100E (red), R100E (purple) hNGF (panel A), hproNGF (Panel B). The fraction of folded protein is plotted as a function of Gdm-Cl concentration. The inset in Panel A shows an alternative normalization, obtained to consider the anomalous behaviour of the mutants that show an increase in the emission fluorescence intensity at low Gdm-Cl concentrations (0–1.5M). The data were normalized assuming that the mutants, unlike hNGF WT, are in a fully folded state (Fraction folded = 1) at 0.5M Gdm-Cl (instead of that at 0M Gdm-Cl) and allow to better compare all the proteins at a glance.</p

ELISA assay to measure hNGF P61S tagged mutants.

<p>Panel A. Standard calibration curve, obtained with hNGF P61S (red diamonds) or hNGF P61SR100E (orange squares). Panel B. Calibration curves carried out with hNGF P61S (red diamonds), rat NGF (green squares), mouse NGF (yellow circles) and hNGF WT (blue triangles). The experimental points are the mean values of the different experiments, the error bars represent the standard deviations.</p

A comparison of the ΔC50 values concerning the in vitro stability test of human NGF Wild type and the mutants.

<p>In order to measure the differences in stability between hNGF and the mutants after the treatments (4°C and 22°C incubation, freeze-thaw cycles, lyophilization) a comparison of the ΔC50 values was evaluated. The reference curve, corresponding to the hNGF or the mutant untreated, exhibits a ΔC50 value equal to 0, so that ΔC50 values higher than 0 indicate that the stability of the NGF sample tested was affected. The ΔC50 for the different treatments were calculated using the formula indicated in the Materials and Methods section. The errors were calculated based on the error propagation formulae. The hNGF R100E mutant exhibits in two treatments high error values (in bold). This is due to the fact that the experimental points corresponding to those treatments did not fit with the theoretical curve, causing high errors. This behavior indicates a strong destabilization of the protein (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136425#pone.0136425.s002" target="_blank">S2 Fig</a>).</p><p>A comparison of the ΔC50 values concerning the in vitro stability test of human NGF Wild type and the mutants.</p

Summary of the binding affinities of human proNGF wild type and the mutants for the MAb anti-NGF: R&D (MAB 256) and αD11 [29] and the MAb anti-proNGF Millipore (clone EP1318Y) in Surface Plasmon Resonance binding experiments (proNGF concentration range: 0.1–100 nM).

<p>Summary of the binding affinities of human proNGF wild type and the mutants for the MAb anti-NGF: R&D (MAB 256) and αD11 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136425#pone.0136425.ref029" target="_blank">29</a>] and the MAb anti-proNGF Millipore (clone EP1318Y) in Surface Plasmon Resonance binding experiments (proNGF concentration range: 0.1–100 nM).</p

NGF crystallographic structure showing the mutation.

<p>NGF crystallographic structure (PDB ID: 1www, in blue) in which the mutated Arginine (R100) and the neighbouring Aspartic Acid (D93), engaged in a ionic bridge, are highlighted in red. Fig 12 has obtained using Pymol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136425#pone.0136425.ref049" target="_blank">49</a>].</p

Kinetics of proteolytic cleavage of proNGF WT and mutants.

<p>Representative SDS-PAGE of hproNGF WT (panel A), hproNGF P61S (panel B), hproNGF P61SR100E (panel C), hproNGF R100E (panel D) digested by trypsin. The lanes correspond to aliquots of the reaction mixtures, taken at time 0 (immediately after trypsin addition) and after 0.5, 1, 1.5, 2, 3, 4, 6, 20 hrs. The loading position of the molecular weight marker is indicated by M.</p

Inflection points extrapolated by the chemical denaturation profiles of human NGF and proNGF Wild Type and mutants.

<p>Inflection points extrapolated by the chemical denaturation profiles of human NGF and proNGF Wild Type and mutants.</p