Astrocytes, Recycling, and the Brain-Derived Neurotrophic Factor

What can quantum dot technology reveal about how astrocytes recycle?

By Alomone Team • June 2023

Astrocytes keep making scientific waves as we discover more and more about their nature – we recently we talked about their friend or foe nature in epilepsy. It now seems astrocytes are also recycling: a paper from the Korea Brain Research Institute (KBRI) has illuminated a hidden part of astrocyte function in the regulation of brain functions. By tracking the journey of brain-derived neurotrophic factor (BDNF), the researchers revealed a fascinating mechanism that involves the uptake, transport, and secretion of BDNF by astrocytes.

BDNF performs a diverse set of actions when it comes to regulating brain functions, acting through a receptor called TrkB. We’ve known for some time that astrocytes express TrkB receptors and can internalize extracellular BDNF through receptor-mediated endocytosis. However, the exact mechanism behind the re-secretion of internalization BDNF has been elusive. But now, the authors of this fascinating new study used quantum dot (QD)-conjugated mature BDNF (QD-BDNF) as a proxy for extracellular BDNF, which allowed them to monitor the movement of BDNF in live cortical astrocytes.

Illuminating Astrocytes

In the course of their research, the team found that vesicle-associated membrane protein 3 (Vamp3), known to be involved in endosomal recycling, is pivotal when it comes to the release of endocytic BDNF from astrocytes. Immunocytochemistry and QD imaging, combined with Alomone’s biotin-labeled human BDNF, revealed that endocytic BDNF particles were enriched in vesicles containing Vamp3 in astrocytes.

Stimulating the astrocytes with ATP triggered antero- or retrograde transport and exocytosis of the BDNF-containing vesicles, effectively recycling the BDNF back into the extracellular space. To help shed more light on the role of Vamp3, the researchers knocked down Vamp3 expression, which disrupted the release of BDNF from astrocytes without affecting its uptake or transport. Although the researchers were unsure exactly how ATP stimulation increased this antero- or retrograde transport of endocytic BDNF-containing vesicles, they suspected that Ca²⁺ or lipid signaling could be involved.

Imaging BDNF in Astrocytes

**Figure 1. QD-BDNF as a tool for monitoring endocytic BDNF in astrocytes.** (A) Schematic diagram of biotinylated mBDNF conjugated with streptavidin-QD655 (QD-BDNF). (B) Left: Representative fluorescence image of purified QD-BDNFs (2 nM). Scale bar = 10 µm, inset scale bar = 5 µm. Right: distribution of the 2D sizes of QD-BDNF particles (1073 particles from 40 cells). (C) Representative images of EGFP-expressing astrocytes treated with QD-BSA or QD-BDNF. Scale bar = 10 µm. Below: magnified views of the indicated locations (numbers). Scale bar = 10 µm. (D) Representative images of endocytic QD-BDNFs in astrocytes expressing scrambled shRNA (Control), TrkB-shRNA #1 (shTrkB #1), or #2 (shTrkB #2). Scale bar = 10 µm. Inset: magnified view of the indicated location (white box). Scale bar = 5 µm. Bar graphs: average QD-BDNF densities under each condition. **P < 0.01. N = 10 cells for each group. E. Average shape indices of QD-BSA- and QD-BDNF-treated astrocytes. *P < 0.05. N = 5 or 16 cells. (F) Average QD-BDNF densities at each incubation time treated with 2 nM of QD-BDNF. *P < 0.05. N = 10 cells in each condition. G. Representative images of astrocytes treated with 0.5, 1, 2, or 5 nM QD-BDNF. Scale bar = 10 µm, inset scale bar = 5 µm. (H) Average QD-BDNF densities with minimum (0.3 µm2) or larger sizes (> 0.3 µm2). *P < 0.05, **P < 0.01. (I)Average fractions of QD-BDNF with minimum or larger sizes among total intracellular QD-BDNF. *P < 0.05. N = 9–10 cells in each condition. The quantification of QD-BDNF particle numbers and the shape index of astrocytes were determined by using ImageJ/FIJI software (Ver. 2.1.0/1.53c, NIH). Image from J, Yoon et al. Sci Rep 11, 21203 (2021).

Astrocytes and SNARE

Astrocytes have long been recognized for their essential roles in supporting neuronal function, but their involvement in BDNF recycling and secretion adds another skill to their repertoire. Here, the researchers observed that the direct internalization and recycling of BDNF in astrocytes were mediated by the TrkB.T1 receptor.

As mentioned, astrocytes were also treated with ATP. The cells responded to ATP stimulation by releasing endocytic BDNF through a soluble NSF attachment protein receptor (SNARE)-dependent mechanism. This implies that astrocytes actively participate in modulating extracellular BDNF concentrations, contributing to the intricate regulation of brain functions alongside TrkB.T1.

Vamp3 the Regulator

The researchers identified Vamp3 as a key regulator of ATP-triggered endocytic BDNF secretion. Although Vamp3-containing vesicles, including early endosomes and secretory granules, contained the majority of endocytic QD-BDNF particles, there were also other vesicular fractions that showed significant colocalization with QD-BDNF. This suggests that other vesicles, such as Rab7- or Rab11-positive endosomes, are involved in the recycling process.

Since Vamp3 is an enriched vSNARE in astrocytes, the researchers hypothesized that recycling endocytic BDNF-containing vesicles in astrocytes could be under the control of Vamp3. They found that Vamp3-EGFP-containing vesicles often secreted QD-BDNF secretion upon ATP stimulation, supporting their hypothesis. And in their Vamp3 knockdown experiments, they saw ATP-induced QD-BDNF exocytosis reduced by 76%. However, the flip side of this is that Vamp3 wasn’t required for endocytosis or transport of QD-BDNF, which they demonstrated after observing no changes in QD-BDNF uptake and transport in astrocytic Vamp3 knockdown models. Together, this suggests that Vamp3 is a main but selective regulator of endocytic BDNF secretion.

Calcium and Complexity

The researchers delved deeper into the molecular mechanisms underlying endocytic BDNF secretion from astrocytes and discovered that the chelation of ATP-induced Ca²⁺ elevation partially reduced QD-BDNF exocytosis. Interestingly, directly increasing intracellular Ca²⁺ concentration wasn’t enough to cause QD-BDNF exocytosis, which suggests there are likely additional signaling pathways involved.

One potential pathway involves the modification of cyclic adenosine monophosphate (cAMP) levels through P2 receptor activation or A2 receptors. These pathways might activate cAMP-dependent signaling pathways, which are needed for vesicle docking or exocytosis, thus influencing endocytic BDNF release. Finally, Vamp3-independent mechanisms likely play a role in BDNF secretion, as the team observed QD-BDNFs in Vamp3-negative vesicles, as well as ATP-triggered QD-BDNF release from these vesicles.

Multiple Pathways at Play

The findings of this study provide evidence for the selective role of Vamp3 in regulating endocytic BDNF secretion from astrocytes during BDNF recycling. But that wasn’t the whole story: the involvement of Ca²⁺ and ATP, changes in cAMP concentrations, and Vamp3-independent mechanisms picked up through the presence of QD-BDNFs in Vamp3-negative vesicles suggest that BDNF recycling is not a simple affair. And while Vamp3 is a selective regular of this process, there must be multiple signaling pathways contributing to the astrocytic recycling of BDNF.

Understanding the molecular events governing BDNF recycling in astrocytes could have significant implications for synaptic plasticity and cognitive functions. Consequently, more work is needed to unravel additional aspects of BDNF recycling. Researchers will likely focus on other molecular players and signaling pathways involved in this process. Additionally, studying the impact of altered BDNF recycling on synaptic plasticity, learning, and memory will provide valuable insights into the broader implications of this intricate mechanism.

BDNF’s Recycling Journey

Thanks to QD technology, scientists have been able to shed a little more light on BDNF’s journey and how it’s recycled within astrocytes. By employing QD-BDNF as a proxy for extracellular BDNF, the researchers could track its movements within cultured cortical astrocytes and start to untangle the multiple pathways involved in this seemingly simple process. This research has added to our understanding of the dynamic interplay between different cell types in the brain and emphasizes the significance of astrocytes in maintaining brain health.

Bolstering your Brain Research

If you need reagents to help study BDNF, we’ve got you covered:

Neurotrophins/Growth Factors

Antibodies

Blockers/Antagonists

ANA-12 (#A-215) – An antagonist of TrkB receptor
AZD-1332 (#A-495) – An inhibitor of TrkA, TrkB, TrkC receptors
GNF 5837 (#G-145) – A pan-Trk (neurotrophin receptor) Inhibitor

Explorer kits

Neurotrophic Factor Explorer Kit (#EK-500) – contains neurotrophic factor proteins, ideal for screening purposes

Research packs

Research Packs contains all you need for BDNF research: Antibodies to BDNF and its receptors, recombinant BDNF and specific pharmacological tools all in one economical package: