Tracking BDNF’s Long-Distance Signal

New insights from compartmentalized neurons

Brain-derived neurotrophic factor (BDNF) has an impressive number of roles to play – it’s needed for synaptic plasticity, dendritic growth, and neuronal survival. But how does it send long-distance signals from axons to the cell body? A recent study tackled this question by taking a closer look at the molecular mechanisms that drive BDNF-TrkB signaling endosome transport, with a focus on the role of phospholipase C-gamma (PLC-γ) and calcium.(1)

The Challenge of Long-Distance BDNF Signaling

BDNF binds to the TrkB receptor and activates downstream signaling cascades, including the ERK, PLC-γ and PI3K-mTOR pathways, which are involved in neuronal development and function. Although the researchers has previously found that BDNF induces dendritic arborization,(2) it wasn’t clear how these signals travel efficiently from axons to the cell body over long distances. So here they tested whether PLC-γ activation in axons was necessary for TrkB endocytosis and retrograde transport – an important step for propagating neurotrophic signals.

Compartmentalized Neurons and Fluorescent BDNF Tracking

To explore this, the researchers used compartmentalized microfluidic cultures of mouse cortical neurons to isolate axonal and somatic compartments. They tracked BDNF signaling endosomes using a biotinylated BDNF from Alomone Labs (human BDNF-Biotin (#B-250-B)), labeled with DyLight 488-conjugated streptavidin for fluorescent imaging​.

Neurons were pre-treated with different inhibitors, including U73122 (a PLC-γ inhibitor) and BAPTA-AM (a calcium chelator), before adding the fluorescently labeled BDNF to the axonal compartment. They quantified the movement of BDNF-containing vesicles in both the microgrooves (axonal region) and soma​ (Figure 1).

Figure 1. Axonal PLC-γ activity and intracellular Ca2+ are required for axonal BDNF-containing signaling endosome generation and retrograde transport to cell bodies in mouse cortical neurons. (A) DIV 7 compartmentalized cortical neurons were retrograde labeled with Ctb555 (red) overnight. At DIV 8, the cell body compartment was treated with TrkB-Fc, and the AC was treated with DyLight 488-labeled streptavidin (f-streptavidin, green) alone or with biotinylated BDNF conjugated to DyLight 488-labelled streptavidin (f-BDNF, green), as indicated in the methodology section, for 6 h in the absence or presence of vehicle or 5 μM U73122 (U73122) or 20 μM BAPTA-AM (BAPTA). Then, the cells were fixed, mounted in Mowiol containing Hoechst (blue) and prepared for confocal microscopy. Scale bar, 5 μm (B) Quantification of green-labelled vesicles in cell bodies of neurons containing Ctb555. Only vesicles larger than 200 nm2 were considered for the analysis. Forty-five neurons from three independent compartmentalized cultures were considered. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni correction for multiple comparisons. ****p < 0.0001. (C) Left panels, representative images of f-streptavidin or f-BDNF (green) associated fluorescence. White lines are selecting the region of the microgroove label with Ctb555 (red) shown in the right panels. White arrows indicate green-labelled vesicles. Right panels, Representative images of axons (labeled with Ctb555, red) in microgrooves of neurons treated as described in (A). Scale bar, 5 μm. (D) Quantification of green-labelled vesicles in microgrooves of axons containing Ctb555. Forty-five microgrooves from three independent compartmentalized cultures were considered for the analysis. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni correction for multiple comparisons. ****p < 0.0001. [full image figure 5, here: https://pmc.ncbi.nlm.nih.gov/articles/PMC11040097/]

Key Findings: PLC-γ and Calcium Control BDNF Transport

What they found was that PLC-γ and calcium signaling are essential for the proper transport of BDNF-containing signaling endosomes. Without them, BDNF struggles to reach the cell body, and key processes like TrkB endocytosis, CREB activation, and dendritic growth are disrupted. Here’s how these mechanisms shape long-distance BDNF signaling

1. PLC-γ is essential for BDNF-TrkB endocytosis
   • Blocking PLC-γ with U73122 reduced the number of BDNF-containing signaling endosomes in the axon.
   • This inhibition also significantly reduced TrkB internalization, suggesting that PLC-γ activity is crucial for forming signaling endosomes​.
2. Calcium regulates endosome formation and transport
   • Chelating intracellular calcium with BAPTA-AM disrupted BDNF endosome transport to the soma – this reinforces the role of Ca²⁺ in retrograde transport​.
3. BDNF signaling endosomes require PLC-γ and calcium for efficient transport
   • Quantification of fluorescent vesicles in cell bodies showed a significant decrease in transported BDNF when PLC-γ or calcium signaling was blocked​.
   • Without PLC-γ activity, CREB phosphorylation and dendritic arborization were also reduced, confirming that long-distance BDNF signaling relies on these molecular events​.

Why This Matters

This study provides clear evidence that PLC-γ and calcium signaling regulate TrkB endocytosis and the retrograde transport of BDNF-containing signaling endosomes. Without these key molecular events, neurons fail to transmit BDNF signals from axons to the soma and limit dendritic branching and neuronal plasticity.

Understanding these processes could have implications for neurodevelopmental disorders and neurodegenerative diseases, where defective BDNF signaling has been implicated.(3) Future work could explore how modulating PLC-γ activity or calcium levels might enhance BDNF transport and improve neuronal health.

The Power of High-Quality Reagents in Neuroscience

Research like this depends on access to reliable reagents. With help from biotinylated BDNF, conjugated to DyLight 488, the researchers here we able to track BDNF-containing endosomes and visualize neurotrophic signaling in live neurons​.

At Alomone Labs we design, make, and test a variety of high-purity biotinylated proteins so that researchers can accurately track and study important molecules like NGF (mouse NGF 2.5S-Biotin (#N-240-B)) as well as BDNF. Beyond neurotrophic factors, we have curated a collection of directly conjugated antibodies that specifically target the extracellular portion of a protein, designed for live-cell imaging, immunofluorescence, and flow cytometry. These fluorophore-conjugated extracellular antibodies give researchers the chance to examine metabolite transporters in live cells without the need for secondary antibodies​.

And everything is made in-house. We have developed neurotrophic factors, conjugated antibodies and toxins, and a host of live-cell compatible reagents to help you track molecular dynamics in real-time and uncover new mechanisms in neuroscience.

 

Reference

1. Moya-Alvarado, X. Valero-Peña, A. Aguirre-Soto, F. J. Bustos, O. M. Lazo, F. C. Bronfman, PLC-γ-Ca2+ pathway regulates axonal TrkB endocytosis and is required for long-distance propagation of BDNF signaling. Front Mol Neurosci 17, 1009404 (2024). DOI: https://doi.org/10.3389/fnmol.2024.1009404.
2. G. Moya-Alvarado, R. Tiburcio-Felix, M. R. Ibáñez, A. A. Aguirre-Soto, M. V. Guerra, C. Wu, W. C. Mobley, E. Perlson, F. C. Bronfman, BDNF/TrkB signaling endosomes in axons coordinate CREB/mTOR activation and protein synthesis in the cell body to induce dendritic growth in cortical neurons. eLife 12, e77455. DOI: https://doi.org/10.7554/eLife.77455.
3. G. Moya-Alvarado, M. V. Guerra, R. Tiburcio, E. Bravo, F. C. Bronfman, The Rab11-regulated endocytic pathway and BDNF/TrkB signaling: Roles in plasticity changes and neurodegenerative diseases. Neurobiol Dis 171, 105796 (2022). DOI: https://doi.org/10.1016/j.nbd.2022.105796.