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An Optimized Procedure for the Site-Directed Labeling of NGF and proNGF for Imaging Purposes.

ABSTRACT: Neurotrophins are growth factors of fundamental importance for the development, survival and maintenance of different neuronal and non-neuronal populations. Over the years, the use of labeled neurotrophins has helped in the study of their biological functions, leading to a better understanding of the processes that regulate their transport, traffic, and signaling. However, the diverse and heterogeneous neurotrophin labeling strategies adopted so far have often led to poorly reproducible protocols and sometimes conflicting conclusions. Here we present a robust, reliable, and fast method to obtain homogeneous preparations of fluorescent proNGF and NGF with 1:1 labeling stoichiometry. This strategy is well suited for several applications, ranging from advanced imaging techniques such as single particle tracking, to analyses that require large amounts of neurotrophins such as in vivo monitoring of protein biodistribution. As a proof of the quality of the labeled NGF and proNGF preparations, we provide a quantitative analysis of their colocalization with proteins involved in the signaling endosome function and sorting. This new analysis allowed demonstrating that proNGF localizes at a sub-population of endosomes not completely overlapped to the one hosting NGF.

SUBMITTER: Di Matteo P

PROVIDER: S-EPMC5288393 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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An Optimized Procedure for the Site-Directed Labeling of NGF and proNGF for Imaging Purposes.

Di Matteo Pierluigi P Calvello Mariantonietta M Luin Stefano S Marchetti Laura L Cattaneo Antonino A

Frontiers in molecular biosciences 20170202

Neurotrophins are growth factors of fundamental importance for the development, survival and maintenance of different neuronal and non-neuronal populations. Over the years, the use of labeled neurotrophins has helped in the study of their biological functions, leading to a better understanding of the processes that regulate their transport, traffic, and signaling. However, the diverse and heterogeneous neurotrophin labeling strategies adopted so far have often led to poorly reproducible protocol ...[more]

PMID: 28210618

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Project description:The homeostasis between mature neurotrophin NGF and its precursor proNGF is thought to be crucial in physiology and in pathological states. Therefore, the measurement of the relative amounts of NGF and proNGF could serve as a footprint for the identification of disease states, for diagnostic purposes. Since NGF is part of proNGF, their selective identification with anti-NGF antibodies is not straightforward. Currently, many immunoassays for NGF measurement are available, while the proNGF assays are few and not validated by published information. The question arises, as to whether the commercially available assays are able to distinguish between the two forms. Also, since in biological samples the two forms coexist, are the measurements of one species affected by the presence of the other? We describe experiments addressing these questions. For the first time, NGF and proNGF were measured together and tested in different immunoassays. Unexpectedly, NGF and proNGF were found to reciprocally interfere with the experimental outcome. The interference also calls into question the widely used NGF ELISA methods, applied to biological samples where NGF and proNGF coexist. Therefore, an immunoassay, able to distinguish between the two forms is needed. We propose possible ways forward, toward the development of a selective assay. In particular, the use of the well validated anti-NGF ?D11 antibody in an alphaLISA assay with optimized incubation times would be a solution to avoid the interference in the measurement of a mixed sample containing NGF and proNGF. Furthermore, we explored the possibility of measuring proNGF in a biological sample. But the available commercial kit for the detection of proNGF does not allow the measurement of proNGF in mouse brain tissues. Therefore, we validated an SPR approach for the measurement of proNGF in a biological sample. Our experiments help in understanding the technical limits in the measurement of the NGF/proNGF ratio in biological samples, and propose concrete solutions toward the solution of this problem.

Project description:BACKGROUND: Growing evidence shows that, in vivo, the precursor of Nerve Growth Factor (NGF), proNGF, displays biological activities different from those of its mature NGF counterpart, mediated by distinct, and somewhat complementary, receptor binding properties. NGF and proNGF induce distinct transcriptional signatures in target cells, highlighting their different bioactivities. In vivo, proNGF and mature NGF coexist. It was proposed that the relative proNGF/NGF ratio is important for their biological outcomes, especially in pathological conditions, since proNGF, the principal form of NGF in Central Nervous System (CNS), is increased in Alzheimer's disease brains. These observations raise a relevant question: does proNGF, in the presence of NGF, influence the NGF transcriptional response and viceversa? In order to understand the specific proNGF effect on NGF activity, depending on the relative proNGF/NGF concentration, we investigated whether proNGF affects the pattern of well-known NGF-regulated mRNAs. RESULTS: To test any influence of proNGF on pure NGF expression fingerprinting, the expression level of a set of candidate genes was analysed by qReal-Time PCR in rat adrenal pheochromocytoma cell line PC12, treated with a mixture of NGF and proNGF recombinant proteins, in different stoichiometric ratios. These candidates were selected amongst a set of genes well-known as being rapidly induced by NGF treatment. We found that, when PC12 cells are treated with proNGF/NGF mixtures, a unique pattern of gene expression, which does not overlap with that deriving from treatment with either proNGF or NGF alone, is induced. The specific effect is also dependent on the stoichiometric composition of the mixture. The proNGF/NGF equimolar mixture seems to partially neutralize the specific effects of the proNGF or NGF individual treatments, showing a weaker overall response, compared to the individual contributions of NGF and proNGF alone. CONCLUSIONS: Using gene expression as a functional read-out, our data demonstrate that the relative availability of NGF and proNGF in vivo might modulate the biological outcome of these ligands.

Dataset Information

An Optimized Procedure for the Site-Directed Labeling of NGF and proNGF for Imaging Purposes.

Publications

An Optimized Procedure for the Site-Directed Labeling of NGF and proNGF for Imaging Purposes.

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