• depolarization
• fluorophores
• neurons
• neurotrophins
• NGF

Being the first discovered neurotrophic factor, Nerve Growth Factor (NGF) is well characterized to be crucial during the growth, differentiation and sustenance of several neuronal populations of a living organism. Relevance of NGF in nervous system physiology is proved by the observation that the missense mutation in the 100th aminoacid of NGF sequence (R100W) results in HSAN V disease, a grave hereditary neuropathy developing loss of pain perception. NGF exists and is biologically active also in its precursor form, the proNGF. This, triggers distinct genes activation and biological responses with respect to the mature form, is the predominant form of NGF in the Central Nervous System and imbalances in its levels have been causally correlated to Alzheimer Disease.
Since axonal transport was discovered to be one of the most important mechanisms governing the neurotrophin function, trafficking of NGF in the living neuron was extensively studied. However, the same for NGF variants has not been widely investigated yet and, for this reason, the aim of my thesis was to implement a site-specific labelling method for neurotrophins to observe the pathway of immature and mutated forms of NGF. Furthermore, attention was paid to the influence of electrical activity on the axonal transport of NGF, an important aspect of neuron physiology which has not yet been investigated.
To label neurotrophins, a peptide tag named ybbR, is inserted in the C-terminus of the protein coding sequence through a method already developed by my group. The tag was attached in the plasmid containing the chimeric proBDNF–NGFR100W construct, which fuses the NGF R100W mature sequence along with proBDNF prodomain part, in order to overcome the low production efficiency previously found by my group for the proNGF R100W construct. This hybrid construct was used as template to insert the ybbR tag immediately upstream the stop codon. Wild type version of this chimera underwent the same protocol, which consists in a site-directed mutagenesis, operated with a two-step PCR in order to introduce subsequently the splitted coding sequence of the 11 aminoacids-long tag. Purified PCR product was used to transform Escherichia coli XL-10 Gold Ultracompetent cells and seeded in agar plates. Clones were screened by DNA sequencing, and confirmed 7 and 9 positive clone for wild type and R100W mutant respectively.
Furthermore, I produced two other neurotrophins: proNGFybbR and proNGFybbR RSAA, a cleavage-resistant version of proNGF, which is not proteolytically processed by the cells. Their coding plasmids, already available in the lab, were transformed in Escherichia coli BL21 (DE3) strain. The expression product was purified from inclusion bodies, and in vitro refolding was performed. Fast Protein Liquid Cromatography (FPLC) was used to obtain the pure protein. Only proNGFybbR underwent trypsin digestion and subsequent FPLC purification followed to get the mature NGFybbR form. Final yield of protein production was 10.8 mg per liter of bacteria for proNGFybbR RSAA and 3 mg per liter for NGFybbR.
Once the proteins were obtained, we performed a labelling reaction using a Coenzyme A (CoA) and fluorophore-maleimide conjugate, which can be covalently transferred to the ybbR tag fused to the neurotrophins, by a reaction catalyzed by the Sfp synthase enzyme. To understand which fluorophore shows better performance to label the obtained neurotrophins, Alexa Fluor 647-CoA and Abberior STAR 635P-CoA adducts, which I previously synthesized and purified by reversed phase High Performance Liquid Cromatography (HPLC), along with six other fluorophores adducts already available (Alexa Fluor 488, Abberior 488, Atto 488, Alexa Fluor 568, Atto 550, Atto 633 conjugated to CoA) were tested to evaluate their performance in labelling of NGF and a cleavage-resistant version. The different reactions were analyzed by SDS-PAGE combined to a detection in fluorescence by a gel imager equipped for fluorescence excitation.
Quantitative analysis of the obtained bands of labelled proteins led to select Alexa Fluor 647 as the best fluorophores among the far-red fluorescent dyes to label NGF.
NGFybbR labelled with Alexa Fluor 647-CoA was finally purified in HPLC. Purified fluoNGF was quantified by spectrofluorimetry and then administrated to Dorsal Root Ganglion (DRG) to detect its path inside axons. DRG cells were extracted and cultured in microfluidic compartmentalized chambers to allow separation of axons from cell somas and, after elongation of the axons, their tips were supplied with fluoNGF. Axonal transport was observed in an epifluorescence microscopy equipped for live cell imaging in the absence or presence of a chemical depolarization induced by addition of 45mM KCl in the cell medium. Results enhanced that the NGF vesicles already internalized mobilize immediately after the depolarization trigger and then return to a resting condition dependently to the time progression from the initial KCl stimulus. Hence, we supposed that, rather than an alteration in NGF internalization levels, is the neurotrophin axonal flux to be influenced by KCl-induced neural activity.