Precursor and mature NGF live tracking: one versus many at a time in the axons

Nadai, Teresa De; Marchetti, Laura; Rienzo, Carmine Di; Calvello, Mariantonietta; Signore, Giovanni; Matteo, Pierluigi Di; Gobbo, Francesco; Turturro, Sabrina; Meucci, Sandro; Viegi, Alessandro; Beltram, Fabio; Luin, Stefano; Cattaneo, Antonino

doi:10.1038/srep20272

Cited by 22 publications

(48 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The medium was changed every 1-2 days to prevent salt concentrations increasing due to medium evaporation. Moreover, after 24 hours from the seeding, 50 ng/ml of NGF were added to the soma compartment and 100 ng/ml to the axon compartment to generate a gradient of NGF and induce DRG axons to reach the 25 axonal side by crossing the microgrooves 26 . A volume difference between the two compartments (∼50 μl, higher volume on the soma side) was applied to fluidically isolate them 58 .…”

Section: Neuron Seedingmentioning

confidence: 99%

“…Tracking of single vesicles containing NGF was performed using homemade scripts in MatLab (The MathWorks), starting as described in De Nadai et al 26 . Briefly, we selected manually the channels present in each field of view, and used functions distributed by Raghuveer Parthasarathy 61 for sub-pixel localization and tracking (https://pages.uoregon.edu/raghu/particle_tracking.html).…”

Section: Single Particle Tracking Analysismentioning

confidence: 99%

See 1 more Smart Citation

Graphene Promotes Axon Elongation through Local Stall of Nerve Growth Factor Signaling Endosomes

et al. 2020

Self Cite

View full text Add to dashboard Cite

Several works reported increased differentiation of neuronal cells grown on graphene; however, the molecular mechanism driving axon elongation on this material has remained elusive. Here, we study the axonal transport of nerve growth factor (NGF), the neurotrophin supporting development of peripheral neurons, as a key player in the time course of axonal elongation of dorsal root ganglion neurons on graphene. We find that graphene drastically reduces the number of retrogradely transported NGF vesicles in favor of a stalled population in the first two days of culture, in which the boost of axon elongation is observed. This correlates with a mutual charge redistribution, observed via Raman spectroscopy and electrophysiological recordings. Furthermore, ultrastructural analysis indicates a reduced microtubule distance and an elongated axonal topology. Thus, both electrophysiological and structural effects can account for graphene action on neuron development. Unraveling the molecular players underneath this interplay may open new avenues for axon regeneration applications.

show abstract

Section: Neuron Seedingmentioning

confidence: 99%

Section: Single Particle Tracking Analysismentioning

confidence: 99%

Graphene Promotes Axon Elongation through Local Stall of Nerve Growth Factor Signaling Endosomes

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…The sortilin/p75NTR complex binds to the prodomain of neurotrophins and has a high affinity for proNGF, but not NGF [13]. Consistently, NGF and proNGF are retrogradely transported in neurons with comparable kinetics; however, the majority of NGF-positive vesicles contain TrkA alone, while proNGF-positive vesicles contain both TrkA and p75NTR [18]. …”

Section: Introductionmentioning

confidence: 99%

ProNGF, but Not NGF, Switches from Neurotrophic to Apoptotic Activity in Response to Reductions in TrkA Receptor Levels

Ioannou

Fahnestock

2017

IJMS

View full text Add to dashboard Cite

Nerve growth factor (NGF) promotes the survival and differentiation of neurons. NGF is initially synthesized as a precursor, proNGF, which is the predominant form in the central nervous system. NGF and proNGF bind to TrkA/p75NTR to mediate cell survival and to sortilin/p75NTR to promote apoptosis. The ratio of TrkA to p75NTR affects whether proNGF and mature NGF signal cell survival or apoptosis. The purpose of this study was to determine whether the loss of TrkA influences p75NTR or sortilin expression levels, and to establish whether proNGF and mature NGF have a similar ability to switch between cell survival and cell death. We systematically altered TrkA receptor levels by priming cells with NGF, using small interfering RNA, and using the mutagenized PC12nnr5 cell line. We found that both NGF and proNGF can support cell survival in cells expressing TrkA, even in the presence of p75NTR and sortilin. However, when TrkA is reduced, proNGF signals cell death, while NGF exhibits no activity. In the absence of TrkA, proNGF-induced cell death occurs, even when p75NTR and sortilin levels are reduced. These results show that proNGF can switch between neurotrophic and apoptotic activity in response to changes in TrkA receptor levels, whereas mature NGF cannot. These results also support the model that proNGF is neurotrophic under normal circumstances, but that a loss in TrkA in the presence of p75NTR and sortilin, as occurs in neurodegenerative disease or injury, shifts proNGF, but not NGF, signalling from cell survival to cell death.

show abstract

Section: Introductionmentioning

confidence: 99%

“…Building on work by Sung et al ( 2011 ), our group has recently developed a new site-directed and stoichiometry-controlled labeling of NGF and proNGF (Marchetti et al, 2014 ; De Nadai et al, 2016 ). Accordingly, the coding sequence of human proNGF can be modified by the C-terminal insertion of the YBBR tag coding sequence (De Nadai et al, 2016 ). This 11-amino-acid long peptide (DSLEFIASKLA) was identified from a series of truncated proteins derived from the ybbR ORF gene of Bacillus subtilis and shown to be an efficient substrate for the enzymatic activity of the Sfp synthase enzyme (Yin et al, 2005 ).…”

Section: Introductionmentioning

confidence: 99%

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

Matteo

Calvello

Luin

et al. 2017

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Precursor and mature NGF live tracking: one versus many at a time in the axons

Cited by 22 publications

References 41 publications

Graphene Promotes Axon Elongation through Local Stall of Nerve Growth Factor Signaling Endosomes

Graphene Promotes Axon Elongation through Local Stall of Nerve Growth Factor Signaling Endosomes

ProNGF, but Not NGF, Switches from Neurotrophic to Apoptotic Activity in Response to Reductions in TrkA Receptor Levels

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

Contact Info

Product

Resources

About