Indeed, the binding affinity of hNGFR100 mutants to p75NTR is strongly reduced, while that for TrkA remains unchanged. Despite the identical TrkA binding affinity, hNGFR100 mutants differ from hNGF in their ability to activate downstream TrkAdependent signalling pathways, with a notable selective reduction in the ability to activate PLC�C1c and Erks. Mechlorethamine hydrochloride Notably, hNGFR100 mutants do not sensitize nociceptors and fail to induce pain in vivo. Thus, hNGFR100 mutants may share some properties of other NGF mutants described to be selective TrkA agonists with respect to p75NTR, but have the additional property of selectively modulating a subset of the signaling pathways downstream of TrkA, and to be proven as less potent nociception activators. On the basis of these properties, the hNGFP61S/R100E therapeutic candidate was designed, aimed at optimizing the therapeutic window for NGF. The characterization of the hNGFP61S/R100E double mutant is provided in this paper. Moreover, until recently no study addressed the issue whether administration of NGF, or of NGF-related molecules, might affect the neurodegeneration in 
FAD related APP mouse models. We show that hNGFP61S/R100E is characterized by a reduced ability to activate PLC�C1c, Erks and c-jun, providing a molecular explanation for the reduced pro-nociceptive activity, observed in a mechanical allodynia pain model. hNGFP61S/R100E showed no difference with respect the hNGF counterparts in a number of different survival, differentiation and proliferation cellular assays. Thus, hNGFR100P61S/R100E maintains the neurotrophic signaling stream unchanged, while showing a reduced signaling involved in nociceptor sensitization and pain perception. Notably, hNGFP61S/R100E is very effective to rescue or prevent neurodegeneration and learning and memory deficits in mouse models, including an APP-based model, in which the neurodegeneration is not directly caused by NGF inhibition. We observed a higher effectiveness of hNGFP61S/R100E, with respect to hNGFR100E, in rescuing the neurodegeneration in the AD11 model, as well as a slight difference between the R100E and the P61S/R100E NGF proteins in nociceptive sensitization assays, most likely involving a structural cross-talk between S61 and R100 residues. Thus, the P61S substitution, which per se does not determine any measurable influence, Cinoxacin compared to wild type NGF,, could exert a long distance influence on the residue at position 100. In all the available 3D structures of hNGF complexes loop 58�C68 is only partially resolved. Indeed the region 61�C66, including the residue P61, is structurally not defined. On the contrary, in the crystal structures of mouse NGF the homologous loop, including S61, is structurally well defined. Thus, the 58�C68 loop in hNGF has likely a higher intrinsic conformational flexibility compared to mNGF. This loop is structurally positioned over the C58�CC108 and C68�CC110 cysteine knot, and it may pose structural constraints on the possible conformations taken by the disulphide bridges of the cysteine knot. The C58�CC108 and C68�CC110 disulphide bridges pair b-strand-2 to b-strand-4. Residue R100 is located at the N-term of b-strand-4. The P61S substitution in hNGF, by rendering the 58�C68 loop structurally similar to that observed in mNGF, might cause a local conformational change in the proximity of the interacting cysteine knot, thereby exerting a long-distance effect on R100, which could explain the observed subtle modifi