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International journal of molecular sciences 23(12), 6596 (2022). doi:10.3390/ijms23126596, The human genetic variant BDNF (V66M) represents the first example of neurotrophin family member that has been linked to psychiatric disorders. In order to elucidate structural differences that account for the effects in cognitive function, this hproBDNF polymorph was expressed, refolded, purified, and compared directly to the WT variant for the first time for differences in their 3D structures by DSF, limited proteolysis, FT-IR, and SAXS measurements in solution. Our complementary studies revealed a deep impact of V66M polymorphism on hproBDNF conformations in solution. Although the mean conformation in solution appears to be more compact in the V66M variant, overall, we demonstrated a large increase in flexibility in solution upon V66M mutation. Thus, considering that plasticity in IDR is crucial for protein function, the observed alterations may be related to the functional alterations in hproBDNF binding to its receptors p75NTR, sortilin, HAP1, and SorCS2. These effects can provoke altered intracellular neuronal trafficking and/or affect proBDNF physiological functions, leading to many brain-associated diseases and conditions such as cognitive impairment and anxiety. The structural alterations highlighted in the present study may pave the way to the development of drug discovery strategies to provide greater therapeutic responses and of novel pharmacologic strategy in human populations with this common polymorphism, ultimately guiding personalized medicine for neuropsychiatric disorders., Published by Molecular Diversity Preservation International, Basel

Dystroglycan (DG), composed of and subunits, belongs to the dystrophin-associated glycoprotein complex. alpha-DG is an extracellular matrix protein that undergoes a complex post-translational glycosylation process. The bifunctional glycosyltransferase like-acetylglucosaminyltransferase (LARGE) plays a crucial role in the maturation of alpha-DG, enabling its binding to laminin. We have already structurally analyzed the N-terminal region of murine alpha-DG (alpha-DG-Nt) and of a pathological single point mutant that may affect recognition of LARGE, although the structural features of the potential interaction between LARGE and DG remain elusive. We now report on the crystal structure of the wild-type human alpha-DG-Nt that has allowed us to assess the reliability of our murine crystallographic structure as alpha-DG-Nt general model. Moreover, we address for the first time both structures in solution. Interestingly, small-angle X-ray scattering (SAXS) reveals the existence of two main protein conformations ensembles. The predominant species is reminiscent of the crystal structure, while the less populated one assumes a more extended fold. A comparative analysis of the human and murine alpha-DG-Nt solution structures reveals that the two proteins share a common interdomain flexibility and population distribution of the two conformers. This is confirmed by the very similar stability displayed by the two orthologs as assessed by biochemical and biophysical experiments. These results highlight the need to take into account the molecular plasticity of alpha-DG-Nt in solution, as it can play an important role in the functional interactions with other binding partners.

The immobilization of biomolecules at screen printed electrodes for biosensing applications is still an open challenge. To enrich the toolbox of bioelectrochemists, graphite screen printed electrodes (G-SPE) were modified with an electropolymerized film of pyrrole-2-carboxilic acid (Py-2-COOH), a pyrrole derivative rich in carboxylic acid functional groups. These functionalities are suitable for the covalent immobilization of biomolecular recognition layers. The electropolymerization was first optimized to obtain stable and conductive polymeric films, comparing two different electrolytes: sodium dodecyl sulphate (SDS) and sodium perchlorate. The G-SPE modified with Py-2-COOH in 0.1 M SDS solution showed the required properties and were further tested. A proof-of-concept study for the development of an impedimetric sensor for perfluorooctanoic acid (PFOA) was carried out using the delipidated human serum albumin (hSA) as bioreceptor. The data interpretation was supported by size exclusion chromatography and small-angle X-ray scattering (SEC-SAXS) analysis of the bioreceptor-target complex and the preliminary results suggest the possibility to further develop this biosensing strategy for toxicological and analytical studies.

The dystroglycan (DG) adhesion complex has a crucial role for muscle stability and is involved in a number of autosomal recessive neuromuscular disorders ranging from severe congenital (Muscle-Eye-Brain and Walker-Warburg syndrome) to milder limb-girdle muscular dystrophies (LGMD2P). The complex undergoes a still poorly understood molecular maturation/targeting pathway, in which a precursor molecule undergoes an early proteolytic event in the endoplasmic reticulum and is cleaved into two subunits, alpha (extracellular) and beta (transmembrane); this is followed by a multistep glycosylation process which mainly takes place within the Golgi apparatus. Most of the currently identified dystroglycanopathies depend on an impaired sugar decoration of the alpha-dystroglycan subunit that, when hypoglycosylated, shows a reduced binding affinity towards laminin-2 (dubbed as secondary dystroglycanopathies). However, lately a novel subgroup of primary dystroglycanopathies, depending on missense mutations of the dystroglycan core protein, is emerging. We have established an array of diverse tools for the analysis of primary dystroglycanopathies. Namely, the production of novel mice lines, the analysis of transfected cellular lines and the recombinant expression of isolated domains for biochemical and crystallization analysis, as well as computational biochemistry. Our cellular, microscopic and biochemical analysis of the murine counterpart (C667F) of the recently identified missense mutation C669F (inducing a severe muscle-eye-brain phenotype) located within the ectodomain of human beta-DG has allowed us to show that i) the processing of the alpha-beta DG precursor is altered; ii) the variant protein is mainly stuck in the ER and iii) there is some degree of oligomerization and/or structural organization within these C667F aggregates. Following the award of an AFM grant to study primary dystroglycanopathies, we have strongly invested into the generation of a first mouse model. The company GenOway is currently taking care of preparing the knocked-in gametes which will be employed at Bonn University for generating the desired heterozygous and homozygous mouse colony that will be used for morphologic and histologic analysis in the brain and muscle.

Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix and it is composed of two subunits: the extracellular ?-DG and the transmembrane ?-DG. ?-DG is a highly glycosylated protein and its hypoglycosylation weakens its interaction with the extracellular matrix proteins, such as laminins, resulting in a functionally compromised protein. The DG complex undergoes a still poorly understood molecular maturation/targeting pathway, where a precursor is cleaved into ? and ? subunits. Furthermore, the ?-DG subunit is decorated with complex glycans in a multistep glycosylation process. Most of the dystroglycanopathies currently identified are due to an impaired functional state of the enzymes involved in ?-DG maturation (secondary dystroglycanopathies). More recently, missense mutations of the dystroglycan core protein has been identified as the cause of distroglycanopathies (primary dystroglycanopathies) of diverse severity. Indeed, a set of point mutations on the N-terminal region of ?-DG (a.a. 50-313 in mouse) determine the hypoglycosylation of the DG complex, due to the impairment of a key step in ?-DG glycosylation operated by the bifunctional glycosyltransferase LARGE1. Moreover, a point mutation affecting ?-DG, namely the mutation of cysteine 667 to phenylalanine results in a pathological retention of ?-DG at the endoplasmic reticulum level leading to a severe pathological state. With the aim of elucidating the structural implications of the pathological mutations leading to primary distroglycanopathies, we have undertaken a multi-technique study, involving biochemical and biophysical methods as well. Indeed, synchrotron radiation (SR) provides an invaluable tool for the elucidation of the molecular structure of biological macromolecules in both crystals and solution. We have used SR-based X-ray crystallography in order to determine the molecular structure, at the atomic resolution, of ?-DG point mutants. Moreover, SR-based Small Angle X-ray Scattering (SAXS) has been used as a low-resolution probe for the structural organization of ? and ? DG mutants at near-physiological conditions. The results of the SR-based experiments, combined with biochemical and microscopic analysis allowed to shed light on the molecular and structural basis of primary dystroglycanopathies.

A better understanding of the interactions between perfluroocatanoic acid (PFOA) and human serum albumin (HSA) can improve the design of protein-based electrochemical biosensors for fluorinated environmental contaminants, namely per- and poly-fluoroalkyl substances (PFAS). In the last decade, the affinity of PFOA for fatted and defatted serum albumin was confirmed by numerous toxicological studies 1-3 . The results showed that PFOA can bind strongly to serum albumins, such as HAS, mimicking fatty acids binding and affecting the functions of these transport proteins. To transpose these findings in the design of bioreceptors, a multi-analytical study was carried out comparing directly the performances of fatted and defatted HSA in terms of stability, number of binding sites and affinity towards PFOA and other PFAS . Isothermal titration calorimetry (ITC) measurements allowed to define the stoichiometry and the affinity constants, while native nano-electronspray ionization MS (nESI-MS) provided additional information about the stability of the proteins and the protein-target complex. To identify the binding site and discriminate between fatted and defatted HSA, vapor diffusion crystallization was performed. All analysis confirmed the higher affinity of PFOA for the defatted HSA even though the fatted HSA showed an higher stability. Both types of HSA were immobilized on the graphite-screen printed electrodes modified with graphene oxide (GO) or electropolymerized o-phenylene diamine (oPD). Impedance electrochemical spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the immobilization strategies performances. Also direct detection by EIS and indirect detection using a redox mediator were compared to evaluate the binding event between PFOA and HAS. The combination of toxicological data and electrochemistry allowed to devise a new detection strategy for environmental contaminants, mimicking a biological process happening in vivo for biosensing purposes.

Nerve Growth Factor (NGF) is the prototype of the neurotrophins family and induces cell growth and differentiation in neuronal cell types, both in the central and peripheral nervous system. Even though it was discovered almost 70 years ago, and its tertiary structure is known since 1991, many molecular and functional properties remain elusive. At the same time, its pharmaceutical interest is still high, since the protein is involved in many disease mechanisms, like chronic pain and neurodegenerative disorders. Small endogenous ligands that interact with NGF are of increasing interest, due to their likely capability of modulating its biological activity. Among these molecules, ATP was shown to mediate NGF neurotrophic activity through it receptors, TrkA and p75NTR. However, no structural information on the binding sites nor on the mechanism of the interaction is available so far. Aiming at gaining new information on this aspect, we undertook a biophysical study on NGF/ATP binding, by means of solution NMR. We have focused our studies on the recombinant human NGF (rhNGF), which is the molecule of medical interest, more that the better structural characterized mouse protein (mNGF). At first, we have obtained 15N- and 13C15N-labeled rhNGF, suitable for the NMR studies. We have optimized the protocols set up in our previous work on the mouse protein, to better adapt the expression conditions to the human protein. The 2D HSQC NMR collected spectra allowed us to identify structural features of the rhNGF in comparison to mNGF in solution. We know from our previously published data that the hNGF and mNGF do not overlap in their biochemical, biophysical and in vitro functional properties, reflected in their 3D structure in solution and highlighted by their available respective crystal structures. We therefore proceeded to the assignment of both the backbone and side chains of hNGF, to fully characterize the protein, by means of 3D NMR experiments (15N and 13C NOESYs). We investigated the binding effects of ATP and of a set of different divalent ions by means of Differential Scanning Fluorimetry and identified the suitable conditions for the NMR studies. We then moved to the investigation of the NGF/ATP binding, using a protein-based solution NMR approach. We recorded 2D HSQC spectra following a titration with increasing amounts of ATP or the more stable analogue, ATP-PCP. We could identify the binding site of ATP and the results will be described.

Plant cell walls are complex macromolecular composites and their degradation by microorganisms requires a large and diverse panel of hydrolytic enzymes. Moreover, the degradation and fermentation of complex polysaccharides by microbiota is essential for many ecosystem-processes including nutrient cycling and herbivores nutrition. Among the different structural polysaccharides composing the plant cell walls, xylan is the second most abundant one and its degradation in rumen involves the removal of O-acetyl esters decorating the O-2 and O-3 positions of the D-xylanopyranosyl moiety. A transcriptomic analysis of a gene cluster, present in the ruminal anaerobe Prevotella ruminicola 23, revealed a repertoire of xylanolytic enzymes with different functions and cellular localization [1]. Among them Axe7A, an acetylxylan esterase belonging to the CAZy CE7 carbohydrate esterase family, has been established [2]. Interestingly Axe7A amino acidic sequence includes, besides the characteristic catalytic domain, an additional 100 residues long N-terminal domain no previously found in the any other member of the CE7 family members, showing low similarity (around 10% identity) with any other structurally characterized protein annotated in the PDB. In order to gain insights into the functional role of its N-terminal domain, the crystal structure of Axe7XE7A has been solved by SAD phasing and refined at 2.6 Å resolution. Axe7A shares the 32 hexameric doughnut-shape commonly found in with all the CE7 deacetylases [3], while the unique protomer architecture has been disclosed in the CE7 family, for the first time. The N-terminal domain, characterized by a ?-sandwich fold, is actively engaged in the oligomer formation. We are further investigating, by SAXS, the behaviour of Axe7A in solution aiming at validate the crystallographic oligomeric assembly in nearly-physiological conditions.

Nerve Growth Factor (NGF), the prototype of the neurotrophin family, is essential for maintenance and growth of different neuronal populations. The X-ray crystal structure of NGF has been known since the early ‘90s and shows a β-sandwich fold with extensive loops that are involved in the interaction with its binding partners. Understanding the dynamical properties of these loops is thus important for molecular recognition. We present here a combined solution NMR/molecular dynamics study which addresses the question of whether and how much the long loops of NGF are flexible and describes the N-terminal intrinsic conformational tendency of the unbound NGF molecule. NMR titration experiments allowed identification of a previously undetected epitope of the anti-NGF antagonist antibody aD11 which will be of crucial importance for future drug lead discovery. The present study thus recapitulates all the available structural information and unveils the conformational versatility of the relatively rigid NGF loops upon functional ligand binding.

Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix. DG is composed of two subunits: alpha-DG and beta-DG. alpha-DG is a highly glycosylated extracellular protein which is essential for high-affinity binding of extracellular matrix proteins such as laminins. The hypoglycosylation of alpha-DG weakens this interaction affinity resulting in severe pathological states. The N-terminal region (aa 50-313 in mouse) of alpha-DG plays a crucial role in the glycosylation of the ?-DG mucin-like domain, being required by the glycosyltransferase LARGE during the extension of the O-glycans implicated in laminin binding. Furthermore, pathological missense mutations, observed at the N-terminal region of alpha-DG, are responsible of alpha-DG hypoglycosylation states resulting in primary distroglycanopathies1. We have been investigating the structures of the three point mutants V72I (V72I-ma-DG), D109N (D109N-ma-DG) and T190M2 (T190M-ma-DG) of mouse alpha-DG (ma-DG), combining X-ray crystallography and in solution Small Angle X-ray Scattering (SAXS). The purpose of this study is to gain evidences about the structural determinants of N-terminal ?-DG that are functionally relevant for its glycosylation pathway. The crystal structures of the three mutants display the same overall structure of WT-ma-DG. In contrast, the solution structural models obtained by SAXS analysis depict a different scenario, where the WT-a-DG is quite flexible in solution, assuming more than one conformation. The comparison of SAXS data from WT-ma-DG and its mutants demonstrates the presence of a perturbation, in both the conformations as well as in the partition among different populations, which is mediated by the flexible peptide linking the Ig-like and S6 small-ribosomal domains, embodied in the alpha-DG N-terminal region. SAXS data thus suggest a more complex and dynamical situation with respect to the crystallographic evidences that may have functional implications for DG post-translational processing and for the interactions with its binding partners.

Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix. DG is composed of two subunits: alpha-DG and beta-DG. alpha-DG is a highly glycosylated extracellular protein, whereas beta-DG is an integral membrane protein that also interacts with dystrophin in the cytoplasm. alpha-DG's glycosylation is essential for high-affinity binding of extracellular matrix proteins such as laminins. The hypoglycosylation of alpha-DG weakens this interaction affinity resulting in severe pathological states. The N-terminal region (a.a 50-313 in mouse) of alpha-DG plays a crucial role in the glycosylation of the alpha-DG mucin-like domain, being required by the glycosyltransferase LARGE during the extension of the O-glycans implicated in laminin binding. Furthermore, pathological missense mutations, observed at the N-terminal region of alpha-DG, are responsible of alpha-DG hypoglycosylation state. We have been investigating the structures of N-terminal region of the WT human alpha-DG (WT-ha-DG) and of the two point mutants V72I (V72I-ma-DG) and D109N (D109N-ma-DG) of mouse alpha-DG by X-ray crystallography, and in solution by Small Angle X-ray Scattering (SAXS). The purpose of this study is to gain evidences about the structural determinants of N-terminal alpha-DG that are functionally relevant for its glycosylation pathway. The crystal structure of the ha-DG does not significantly deviate from the already determined crystal structure of WT mouse alpha-DG (WT-ma-DG). The overall fold is conserved and differences are restricted to the most flexible part of the protein, i.e. the loop encompassing residues 159-179, which is only partially visible after crystal structure refinement. In addition, the crystal structures of the two mutants V72I-ma-DG and D109N-ma-DG display the same overall structure of WT-ma-DG, suggesting for negligible effects of the point mutations on the overall fold of alpha-DG. The two mutants show limited and local structural dissimilarities with respect to WT-ma-DG, probably influencing the interaction with potential binding partners. In contrast, the solution structural models obtained by SAXS analysis depict a different scenario, where the WT-a-DG is quite flexible in solution, assuming more than one conformation, with the most populated close to the crystal conformation and a second less populated one, which is more extended with respect to the principal conformation. The comparison of SAXS data from WT-ma-DG and its mutants demonstrates the presence of a perturbation, in both the conformations as well as in the partition among different populations. SAXS data thus suggest a more complex and dynamical situation with respect to the crystallographic evidences that may have functional implications for DG post-translational processing and for the interactions with its binding partners.

A sub-group of LuxR family of proteins that plays important roles in quorum sensing, a process of cell-cell communication, is widespread in proteobacteria. These proteins have a typical modular structure consisting of N-ter autoinducer binding and C-ter helix-turn-helix (HTH) DNA binding domains. The autoinducer binding domain recognizes signaling molecules which are most often N-acyl homoserine lactones (AHLs) but could also be other novel and yet unidentified molecules. In this study we carried out a series of specific domain swapping and promoter activation experiments as a first step to engineer synthetic signaling modules, taking advantage of the modularity and the versatile/diverse signal specificities of LuxR proteins. In our experiments the N-ter domains from different LuxR homologs were either interchanged or placed in tandem followed by a C-ter domain. The rational design of the hybrid proteins was supported by a structure-based homology modeling studies of three members of the LuxR family (i.e., LasR, RhlR, and OryR being chosen for their unique ligand binding specificities) and of selected chimeras. Our results reveal that these LuxR homologs were able to activate promoter elements that were not their usual targets; we also show that hybrid LuxR proteins retained the ability to recognize the signal specific for their N- ter autoinducer binding domain. However, the activity of hybrid LuxR proteins containing two AHL binding domains in tandem appears to depend on the organization and nature of the introduced domains. This study represents advances in the understanding of the modularity of LuxR proteins and provides additional possibilities to use hybrid proteins in both basic and applied synthetic biology based research.

The homodimer NGF (nerve growth factor) exerts its neuronal activity upon binding to either or both distinct transmembrane receptors TrkA and $p75^{NTR}$. Functionally relevant interactions between NGF and these receptors have been proposed, on the basis of binding and signaling experiments. Namely, a ternary $TrkA/NGF/p75^{NTR}$ complex is assumed to be crucial for the formation of the so-called high-affinity NGF binding sites. However, the existence, on the cell surface, of direct extracellular interactions is still a matter of controversy. Here, supported by a small-angle x-ray scattering solution study of human NGF, we propose that it is the oligomerization state of the secreted NGF that may drive the formation of the ternary heterocomplex. Our data demonstrate the occurrence in solution of a concentration-dependent distribution of dimers and dimer of dimers. A head-to-head molecular assembly configuration of the NGF dimer of dimers has been validated. Overall, these findings prompted us to suggest a new, to our knowledge, model for the transient ternary heterocomplex, i.e., a $TrkA/NGF/p75^{NTR}$ ligand/receptors molecular assembly with a (2:4:2) stoichiometry. This model would neatly solve the problem posed by the unconventional orientation of $p75^{NTR}$ with respect to TrkA, as being found in the crystal structures of the TrkA/NGF and $p75^{NTR}/NGF$ complexes.

Recent studies have identified a novel interkingdom signaling circuit, via plant signaling molecules, and a bacterial sub-family of LuxR proteins, bridging eukaryotes and prokaryotes. Indeed pivotal plant-bacteria interactions are regulated by the so called Plant Associated Bacteria (PAB) LuxR solo regulators that, although closely related to the quorum sensing (QS) LuxR family, do not bind or respond to canonical quorum sensing N-acyl homoserine lactones (AHLs), but only to specific host plant signal molecules. The large body of structural data available for several members of the QS LuxR family complexed with different classes of ligands (AHLs and other compounds), has been exploited to dissect the cartography of their regulatory domains through structure-based multiple sequence alignments, structural superimposition and a comparative analysis of the contact residues involved in ligand binding. In the absence of experimentally determined structures of members of the PAB LuxR solos subfamily, an homology model of its prototype OryR is presented, aiming to elucidate the architecture of its ligand-binding site. The obtained model, in combination with the cartography of the regulatory domains of the homologous QS LuxRs, provides novel insights into the 3D structure of its ligand-binding site and unveils the probable molecular determinants responsible for differences in selectivity towards specific host plant signal molecules, rather than to canonical QS compounds.

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability. Fil: Corbalan, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Runti, Giulia. Università degli Studi di Trieste; Italia Fil: Adler, Conrado. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Covaceuszach, Sonia. Consiglio Nazionale delle Ricerche; Italia Fil: Ford, Robert C.. University of Manchester; Reino Unido Fil: Lamba, Doriano. Consiglio Nazionale delle Ricerche; Italia Fil: Beis, Konstantinos. Imperial College London; Reino Unido Fil: Scocchi, Marco. Università degli Studi di Trieste; Italia Fil: Vincent, Paula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

Nerve growth factor (NGF) deficits are linked to Alzheimer's Disease (AD), due to the role of NGF on basal forebrain cholinergic neurons (BFCN). We have further established that a disequilibrium in NGF signaling and/or processing from its precursor proNGF is also directly and causally related to the aberrant activation of an amyloidogenic route to neurodegeneration. The therapeutic potential of using human NGF to provide a long-lasting cholinergic trophic support, thereby preventing or slowing cognitive decline in AD patients, has therefore a strong rationale. However, a simple and practical means of delivering NGF to the brain in a safe and long-term manner, limiting the undesired adverse effects of NGF in activating nociceptive responses, has represented a significant challenge. For this reason, pilot clinical studies have been performed so far with invasive approaches requiring neurosurgery. We obtained a proof of principle, in neurodegeneration animal models, of an alternative, non-invasive delivery of NGF through an intranasal route, which facilitates access of NGF to the central nervous system (CNS), while minimizing the biodistribution of NGF to compartments where it activates undesired effects, such as pain. The ideal NGF product for a non invasive NGF-based therapy would be a recombinant NGF that, while exhibiting an identical biological activity to that of human NGF, can be traced, against the endogenous NGF, in order to optimize the therapeutical dose range and meet the required therapeutic window. We describe an engineered mutein of hNGF, hNGF-61, that is selectively recognized, against endogenous NGF, by a specific antibody. hNGF-61 mutein has an identical potency and bioactivity profile as hNGF, in vitro and in vivo. Moreover, hNGF-61 and hNGF are equally effective in rescuing the behavioral and neurodegenerative phenotype in adult and aged AD11 anti-NGF mice. Finally, we demonstrated that intranasally delivered hNGF-61 is significantly more effective than ocularly applied hNGF-61, to determine phenotypic rescue in AD11 mice. The development of hNGF-61 towards clinical applications in AD patients is under way.

The anti-nerve growth factor (NGF) monoclonal antibody alphaD11 is a potent antagonist that neutralizes the biological functions of its antigen in vivo. NGF antagonism is expected to be a highly effective and safe therapeutic approach in many pain states. A comprehensive functional and structural analysis of alphaD11 monoclonal antibody was carried out, showing its ability to neutralize NGF binding to either tropomyosine receptor kinase A (TrkA) or p75 receptors. The 3-D structure of the alphaD11 Fab fragment was solved at 1.7 A resolution. A computational docking model of the alphaD11 Fab-NGF complex, based on epitope mapping using a pool of 44 NGF mutants and experimentally validated by small-angle X-ray scattering, provided the structural basis for identifying the residues involved in alphaD11 Fab binding. The present study pinpoints loop II of NGF to be an important structural determinant for NGF biological activity mediated by TrkA receptor.

Nerve growth factor (NGF) is involved in pain transduction mechanisms and plays a key role in many persistent pain states, notably those associated with inflammation. On this basis, both the NGF ligand and its receptor TrkA (tyrosine kinase A) represent an eligible target for pain therapy. Although the direct involvement of NGF in pain modulation is well established, the effect of a direct functional block of the TrkA receptor is still unknown. In this study, we have demonstrated that MNAC13, the only anti-TrkA monoclonal antibody for which function neutralizing properties have been clearly shown both in vitro and in vivo , induces analgesia in both inflammatory and neuropathic pain models, with a surprisingly long-lasting effect in the latter. The formalin-evoked pain licking responses are significantly reduced by the MNAC13 antibody in CD1 mice. Remarkably, treatment with the anti-TrkA antibody also produces a significant antiallodynic effect on neuropathic pain: repeated i.p. injections of MNAC13 induce significant functional recovery in mice subjected to sciatic nerve ligation, with effects persisting after administration. Furthermore, a clear synergistic effect is observed when MNAC13 is administered in combination with opioids, at doses that are not efficacious per se . This study represents a direct demonstration that neutralizing antibodies directed against the TrkA receptor may display potent analgesic effects in inflammatory and chronic pain.

Infection by HIV-1 causes persistent, long-term high virus production in macrophages. Major evidence, both in humans and in primate models, shows the crucial role of macrophages in sustaining virus production and in mediating a cytopathic effect on bystander CD4+T lymphocytes and neuronal cells. In the present study, we used severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID mice) to investigate thein vivoeffect of HIV-1-infected macrophages on virus spread and CD4+T lymphocyte depletion, and the ability of a mAb against nerve growth factor (NGF, a neurokine essential for the survival of HIV-1-infected macrophages) to suppress the pathogenetic events mediated by infected macrophages. Injection of mice with as few as 500 HIV-exposed macrophages causes (i) complete depletion of several millions of autologous CD4+T lymphocytes, (ii) sustained HIV viremia, and (iii) spreading of HIV-1 DNA in mouse lymphoid organs. In contrast,in vivotreatment with an anti-NGF Ab completely abrogates all effects mediated by HIV-infected macrophages. Taken together, the results demonstrate the remarkable power of macrophages in sustainingin vivoHIV-1 infection, and that such a phenomenon can be specifically abrogated by an anti-NGF Ab. This may open new perspectives of experimental approaches aimed at selectively eliminating persistently infected macrophages from the bodies of HIV-infected patients.