Vamp3-dependent secretion of endocytic BDNF from astrocytes

Brain-derived neurotrophic factor (BDNF) regulates diverse brain functions via TrkB receptor signaling. Due to the expression of TrkB receptors, astrocytes can internalize extracellular BDNF proteins via receptor-mediated endocytosis. Endocytosed BDNF can be re-secreted upon stimulation, but the molecular mechanism underlying this phenomenon remains unrecognized. Our study reveals that vesicle-associated membrane protein 3 (Vamp3) selectively regulates the endocytic release of BDNF from astrocytes. By using quantum dot (QD)-conjugated mature BDNF (QD-BDNF) as a proxy for the extracellular BDNF protein, we monitored the uptake, transport, and secretion of BDNF from cultured cortical astrocytes. Our data showed that endocytic QD-BDNF particles were enriched in Vamp3-containing vesicles in astrocytes and that ATP treatment suciently triggered either the antero- or retrograde transport and exocytosis of QD-BDNF-containing vesicles. Downregulation of Vamp3 expression disrupted endocytic BDNF secretion from astrocytes but did not affect uptake or transport. Collectively, these results provide evidence of the selective ability of astrocytic Vamp3 to control endocytic BDNF secretion during BDNF recycling. utilizing recombinant mBDNF proteins linked to quantum dots (QDs) revealed that Vamp3 was selectively involved in the exocytosis of endocytic mBDNF. Our QD-linked mBDNF sensor was sucient for examining the transport and activity-dependent secretion of endosomes, as reported previously 7,16,17 , due to the excellent photostability and high signal-to-noise ratio of QDs in live cells. These results support the notion that mBDNF recycling in astrocytes serves as an additional source of extracellular BDNF, which is crucial for activity-dependent synaptic plasticity.


Introduction
Brain-derived neurotrophic factor (BDNF) regulates diverse brain functions, including cell survival, differentiation, synaptic connectivity, and cognitive processes [1][2][3] . Secretion of either the pro-form of BDNF (proBDNF) or the mature form of BDNF (mBDNF) from dense-core vesicles depends on the Ca 2+mediated actions of vesicular exocytosis machineries such as Soluble NSF Attachment protein Receptor (SNARE) proteins 4,5 . Extracellular pro-BDNF and mBDNF bind to pan-neurotrophin receptor p75 (p75NTR) and tropomyosin-related kinase B (TrkB), respectively 2 and can reside in endosomal compartments in nearby target cells after receptor-mediated endocytosis. While the BDNF-TrkB complex in neuronal endosomes can be retrogradely transported or remain active in the form of a "signaling endosome", extracellular BDNF can also be recycled by re-secretion in response to neuronal activity 6-8 .
Astrocytes are also thought to recycle extracellular BDNF proteins. ProBDNF was shown to be internalized through p75NTR-dependent endocytosis, and this endocytosed neuronal proBDNF appeared to be re-secreted as mBDNF 9,10 . The maintenance of long-term potentiation (LTP) and memory acquisition requires the astrocytic secretion of endocytic BDNF 9,10 . On the other hand, mBDNF seems to be absorbed by astrocytes due to their strong expression of TrkB 11,12 ; however, the re-secretion of endocytic mBDNF has not yet been directly assessed. Neurons require complexin-1/2 and synaptotagmin 6 for the activity-dependent re-secretion of endocytic mBDNF 7 , but the molecular mechanisms underlying the recycling of mBDNF in astrocytes are unknown.
Astrocytes respond to neurotransmitters or active substances, such as glutamate and ATP, displaying the increase in the intracellular Ca 2+ concentration by the activation of corresponding receptors 13,14 . Because vesicular exocytosis is dependent on Ca 2+ -dependent SNARE proteins, the astrocytic Ca 2+dependent actions of SNARE machinery could feasibly be involved in the release of mBDNF from astrocytes. Diverse SNARE machinery proteins, such as vesicle-associated membrane proteins 2, 3, and 7 (Vamp2, Vamp3, and Vamp7, respectively) 15 , are expressed in astrocytes, but which astrocytic SNARE proteins are implicated in endocytic BDNF secretion remains unknown. In this study, we provide direct evidence that Vamp3 is a key SNARE protein controlling endocytic mBDNF release from astrocytes. Monitoring the direct uptake, transport and activity-dependent exocytosis of endocytic mBDNF in astrocytes utilizing recombinant mBDNF proteins linked to quantum dots (QDs) revealed that Vamp3 was selectively involved in the exocytosis of endocytic mBDNF. Our QD-linked mBDNF sensor was su cient for examining the transport and activity-dependent secretion of endosomes, as reported previously 7,16,17 , due to the excellent photostability and high signal-to-noise ratio of QDs in live cells. These results support the notion that mBDNF recycling in astrocytes serves as an additional source of extracellular BDNF, which is crucial for activity-dependent synaptic plasticity.

Methods
All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Brain Research Institute (IACUC-2017-0047). All experiments were carried out in accordance with the approved guidelines and regulations.

Primary astrocyte culture
We utilized an AWESAM astrocyte culture protocol as reported previously 21 with minor modi cations to acquire cultured astrocytes that had an in vivo-like morphology. Cortical astrocytes were prepared from embryos from wild-type C57BL/6 mice on days E17-18. Cortices were dissected in dissection medium (10 mM HEPES in HBSS) at 4°C and then incubated in 0.25% trypsin-EDTA in a 37°C water bath for 20 min with gentle inversion every 5 min. After trypsinization, the tissue was washed in dissection medium at 4°C ve times and then triturated with 1 ml of NB + medium (2% B27 supplement, 2 mM GlutaMax, 5,000 U/ml penicillin and 5,000 µg/ml streptomycin in neurobasal medium). Dissociated cells were ltered through a cell strainer and plated on 0.04% polyethylenimine (PEI)-coated cell culture dishes (4 x 10 6 cells/60 mm dish) in culture media (10% FBS, 5,000 U/ml penicillin and 5,000 µg/ml streptomycin in DMEM). Seven days after plating the dissociated cells, the dishes were shaken at 110 rpm for 6 hours. The cells were then washed with 1x PBS three times, treated with 0.25% trypsin, and plated on 0.04% PEI-coated glass-bottom dishes (3 x 10 4 cells/dish) or 18 mm coverslips in a 12-well plate (1 x 10 4 cells/well) in NB + medium containing HBEGF (50 µg/ml).
To screen Vamp3 siRNA, C8-D1A (mouse type 1 astrocyte cell line) cells were cultured in DMEM supplemented with 10% FBS at 37°C under 5% CO 2 . Each siRNA (100 nM) was transfected into C8-D1A cells using RNAi Max according to the manufacturer's protocol. Two days after transfection, samples were analyzed by western blotting with an anti-Vamp3 primary antibody or β-actin-HRP and HRP-conjugated anti-rabbit secondary antibody. The screening of Vamp3 siRNAs revealed that siVamp3 #1 effectively diminished the level of endogenous Vamp3 (Fig. S2). Therefore, only siVamp3 #1 was employed in the experiments.
TrkB-targeting shRNA lentiviral particles were purchased from Sigma (shRNA-pLKO.1-hPGK-puro-CMV-tGFP). The shRNA target sequences are described in the Key Resources Table. To assess the knockdown e ciency of TrkB shRNA, cortical neurons from E17-18 C57BL/6 mouse embryos were cultured. Each Lenti-shTrkB particle was transduced into cortical neurons at 5 DIV. Three days after transduction, total RNA was extracted using TRIzol reagent. Each RNA sample (0.3 µg) was reverse transcribed into cDNA by using SuperScript III reverse transcriptase. To determine the reduction in TrkB RNA levels, PCR was performed using TrkB and β-actin primers. Because shTrkB #1 reduced the level of endogenous TrkB more effectively than shTrkB #2 (Fig. S1), only shTrkB #1 was used in the experiments.

Immunocytochemistry
To determine the localization of QD-BDNF, cultured astrocytes were incubated with 2 nM QD-BDNF for 20 min and then xed with 4% paraformaldehyde (PFA). For immunostaining, the cells were permeabilized with 0.1% Triton X-100 for 10 min and then blocked with 5% normal goat serum for 1 hour at room temperature. After blocking, the cells were incubated with anti-Rab5, anti-Rab7, anti-Rab11, anti-Lamp1, anti-Vamp3, or anti-chromograninB for 1 hour and then incubated with an anti-Alexa 488 secondary antibody for 1 hour at room temperature.

QD imaging
For monitoring endocytic BDNF, 50 nM biotinylated mature BDNF (bt-BDNF) or 50 nM biotinylated bovine serum albumin (bt-BSA) was incubated with 50 nM streptavidin-conjugated quantum dot 655 (st-QD655) at 4°C overnight at a ratio of 2:1. QD-BDNF or QD-BSA was then ltered with a 100 kDa Amicon lter to remove unconjugated mBDNF, BSA, or QDs, and 1% BSA containing PBS was added to the ltrates. Astrocytes were incubated with QD-BDNF or QD-BSA on 12-13 DIV, and the medium was then replaced with an extracellular solution (in mM; 119 NaCl, 2.5 KCl, 20 HEPES, 2 CaCl 2 , 30 glucose, and 2 MgCl 2 , pH 7.4) containing 4 µM QSY21. Time-lapse images were taken by using a confocal laser scanning microscope (TCS SP8, Leica) at a 1 Hz rate using a 63 X oil objective. ATP (100 µM) or ionomycin (1 µM)-containing extracellular solution was added to stimulate the astrocytes. QD655 uorescence was excited with a 561 nm laser and assessed with a HyD (hybrid) detector in the range of 650-695 nm.

Image and statistical analyses
Image processing and analysis were performed using ImageJ/FIJI software (NIH, USA). To analyze the kinetics or secretion of BDNF particles, regions of interest (ROIs) of astrocytic processes were manually selected and linearized. The linearized time-lapse images were transformed into kymographs using the KymographBuilder plugin in ImageJ/FIJI. After extracting the X and Y coordinate data for each particle from the kymograph, the direction, distance, and velocity were determined.
Colocalization of QD-BDNF and endosomal markers was analyzed by using the Colocalization Threshold plugin in ImageJ/FIJI. To analyze the complexity of astrocytes induced by BDNF, the shape index (perimeter 2 /area -4π) was utilized as described previously 12,22 .
Statistical analyses were performed using Prism 8.0 software (GraphPad). Statistically signi cant differences between two groups were determined using Student's unpaired t-test, and three or more groups were compared using one-way ANOVA with Dunnett's multiple comparisons test. The Kolmogorov-Smirnov test was used to examine the statistical signi cance of the percentages of cumulative distribution between the two groups. All data were from three independent batches of cultured astrocytes and are indicated as the mean ± standard error of the mean (SEM).

Results
Monitoring endocytic BDNF in cultured astrocytes using QD-BDNF To directly monitor endocytic BDNF in astrocytes, we utilized biotinylated recombinant mature BDNF directly associated with streptavidin-QDs as described previously (ref. 7 ; see Methods for detailed information). With this method, the uorescence of the extracellular QD-conjugated mature BDNF complex (QD-BDNF; Fig. 1A) could be cancelled by a hydrophilic uorescence quencher, QSY21 (4 µM), in the extracellular media, but QD-BDNF uorescence was recovered after endocytosis (Fig. 1A, C). Under our imaging conditions, the smallest and most observable two-dimensional size of puri ed QD-BDNF was approximately 0.3 µm 2 , indicating a single QD-BDNF particle (Fig. 1B). The intracellular uptake of QD-BDNF particles into astrocytes was mediated by receptor-mediated endocytosis, as (1) QD-BSA treatment resulted in no intracellular QD particles (Fig. 1C), and (2) the number of intracellular QD-BDNF particles (Fig. 1D) from astrocytes was signi cantly reduced by shRNA-mediated genetic knockdown (KD) of TrkB expression (Fig. S1). Moreover, our QD-BDNF particles were bioactive, because cultured astrocytes showed more complex morphology after QD-BDNF treatment (Fig. 1E), consistent with a previous report 12 . Since astrocytic TrkB.T1-dependent structural complexity is important for the structural and functional maturation of astrocytes 12 , QD-BDNF uptake under our conditions appeared to be mediated by TrkB.T1.
We next explored the ideal concentration and incubation time for the QD-BDNF treatment of cultured astrocytes to track single QD particles. QD-BDNF (0.5 5 nM) was applied to cultured astrocytes for 5 minutes (min) up to 4 hours. Treatment with 2 nM QD-BDNF for 20 min resulted in the most intracellular single QD-BDNF particles (Fig. 1F-I), and all QD-BDNF tracking and secretion experiments were therefore carried out under this condition.

ATP triggers the transport and secretion of endocytic BDNF in astrocytes
We next monitored intracellular QD-BDNF particles in astrocytes to investigate the transport and secretion of endocytic mBDNF. Since astrocytes can be stimulated by extracellular ATP due to the expression of diverse P2 receptors 23 , 100 µM ATP was added to QD-BDNF-containing astrocytes expressing EGFP ( Fig. 2A, B) to induce the activity-dependent transport and secretion of QD-BDNF. Most QD-BDNF particles remained immobile (stationary mode) before ATP treatment (Fig. 2C). However, ATP stimulation triggered either the anterograde or retrograde transport of QD-BDNF (Fig. 2C), leading to an increase in the distance of QD-BDNF tra cking (Fig. 2D) despite the ATP-insensitive speeds of QD-BDNF transport (Fig. 2E). These results suggest that the transport of endocytic BDNF is activity-dependent.
We next assessed whether ATP stimulation evokes endocytic BDNF release in astrocytes. The exocytosis of endocytic QD-BDNF could be detected by the disappearance of QD-BDNF uorescence due to the exposure of QD-BDNF to the QSY21 quencher via opened vesicle pores 7 . Despite a few spontaneous QD-BDNF exocytosis events (5.28 ± 1.76 %), QD-BDNF exocytosis was signi cantly increased (19.37 ± 4.75 %; Fig. 2F) after the ATP treatment, consistent with another study 24 . This ATP-induced QD-BDNF secretion was abolished by the expression of the tetanus toxin light chain (TLC) in astrocytes (Fig. 2F), supporting the idea that endocytic BDNF release is SNARE-dependent. Preincubation with BAPTA-AM also largely reduced the ATP-induced QD-BDNF secretion, indicating that ATP-induced Ca 2+ elevation is required for endocytic BDNF secretion (Fig. 2F). However, direct Ca 2+ elevation by ionomycin treatment did not trigger QD-BDNF secretion (Fig. 2F). These results indicate that intracellular Ca 2+ is necessary for QD-BDNF secretion but suggest that the cooperative actions of Ca 2+ signaling with other signaling pathways are critical for the exocytosis of endocytic BDNF-containing vesicles. Finally, as reported in neurons 7 , BDNF secretion events were frequently observed in immobile vesicles before ATP treatment (Fig. 2G), suggesting that the arrival of endocytic BDNF vesicles at secretion sites is a prerequisite for exocytosis events.
QD-BDNF particles were widely detected in all the tested vesicular fractions (Fig. 3A, B). Of note, colocalization of QD-BDNF with Vamp3 was prominent ( Fig. 3B), suggesting that internalized BDNF molecules were preferentially sorted into Vamp3-positive vesicles. To further characterize the Vamp3-positive QD-BDNF-containing vesicles, additional immunocytochemistry analyses of astrocytes with both QD-BDNF particles and Vamp3-EGFP were performed with vesicular marker antibodies (Fig. 3C). Regardless of whether QD-BDNF particles were detected, Vamp3-positive vesicles were enriched in vesicles containing Rab5, Rab7, or ChgB (Fig. 3C, D). However, Vamp3-positive vesicles with QD-BDNF were more colocalized with Rab5-or Lamp1-positive vesicles than those without QD-BDNF (Fig. 3C, D). Given that astrocytic Vamp3-containing vesicles are implicated in the exo-and endocytotic cycling of endosomes 25 , our results suggest that Vamp3 participates in endocytic BDNF recycling in astrocytes.
Vamp3 is required for ATP-induced endocytic BDNF secretion from astrocytes Since our results showed that endocytic BDNFs were enriched in Vamp3-containing astrocytic vesicles (Fig. 3), ATP-induced BDNF secretion may frequently occur at Vamp3-positive vesicles. We thus compared the fraction of QD-BDNF particles displaying the exocytosis event from Vamp3 (+) vesicles to that from Vamp3-negative (-) vesicles (Fig. 4A). Few very spontaneous QD-BDNF secretion events were observed regardless of the presence of Vamp3 in QD-BDNF-containing vesicles (Fig. 4B), indicating that spontaneous endocytic BDNF release does not involve Vamp3. However, ATP-induced QD-BDNF secretion events was observed from both Vamp3-positive and Vamp3-negative vesicles (Fig. 4B). Moreover, QD-BDNFs in Vamp3-positive vesicles were secreted more frequently than those in Vamp3-negative vesicles (Fig. 4C). These results indicate that activity-induced endocytic BDNF secretion primarily occurs via Vamp3-positive vesicles.
Next, we tested whether Vamp3 directly participates in endocytic BDNF exocytosis by using the siRNA mediated KD method (Fig. S2C). We rst assessed whether the endocytosis or transport of QD-BDNF was affected by Vamp3 KD (Fig. 5A). Vamp3 KD failed to alter the endocytosis (Fig. 5B) or ATP-induced antero-or retrograde transport of QD-BDNF ( Fig. 5C-E). Although why ATP stimulation resulted increased endocytic BDNF transport remains unclear ( Fig. 2C, D), the modi cation of vesicle tra cking or sorting by P2 receptor-mediated Ca 2+ or lipid signaling may be implicated 26-28 . By contrast, astrocytes with Vamp3 KD showed signi cantly reduced ATP-triggered QD-BDNF secretion (Fig. 5F). This reduced QD-BDNF exocytosis was successfully restored by the delivery of the siRNA-insensitive Vamp3 construct together with Vamp3 siRNAs (Fig. 5F). Together, these results indicate that Vamp3 selectively controls endocytic BDNF exocytosis in astrocytes.

Discussion
In this work, we showed the direct uptake and recycling of mBDNF in astrocytes by utilizing QD-BDNF as a proxy for the extracellular BDNF protein. After secreted from source cells, neurotrophin proteins seem to be internalized by binding to corresponding Trk receptors on nearby target cells, but direct monitoring of endogenous neurotrophin has been hampered due to their relatively low concentration in live cells. Because QD is a uorescent nanoparticle with an excellent photostability and could stably tracked in live cells with a high signal-to-noise ratio, the QD-linked neurotrophin sensor has been widely used to examine the transport and activity-dependent secretion of neurotrophin-containing endosomes in live cells 7,16,17 . Using QD-linked mBDNF, a previous study founds TrkB-dependent mBDNF internalization, as well as complexin 1/2 (Cpx1/2) / synaptotagmin 6 (Syt6)-dependent re-secretion of endocytic mBDNF 7 . However, it has not examined whether mBDNF is directly internalized and recycled in astrocytes and what molecular mechanisms handle endocytic mBDNF secretion from astrocytes, although astrocytic p75NTR-dependent endocytosis of neuronal proBDNF and its re-secretion were reported 10 .
When treated with puri ed QD-BDNF particles, there was an increase in the complexity of astrocytic morphology (Fig. 1E), as found from other studies showing TrkB.T1-dependent structural complexity and maturation of astrocytes 11,12 . Given that TrkB-shRNA expression diminished QD-BDNF internalization (Fig. 1D), QD-linked mBDNF endocytosis and morphological changes seem to be mediated by TrkB.T1. Because ATP stimulation of astrocytes was su cient for triggering Ca 2+ and SNARE-dependent release of endocytic QD-BDNF (Fig. 2F), our study proposes that neuronal mBDNF directly takes part in the process of astrocytic modulation of extracellular BDNF concentration, in addition to TrkB.T1-dependent regulation of astrocyte functions.
We revealed a key molecular mechanism, Vamp3-dependent exocytosis, that controls activity-dependent endocytic mBDNF secretion from astrocytes. Among all tested vesicular pools, Vamp3-positive vesicles in the early endosomes or lysosome fraction contained most endocytic QD-BDNFs (Fig. 2).
However, other vesicular fractions such as Rab7 or Rab11-positive endosomes or ChgB-positive secretory granules appear to contain a portion of endocytic BDNF, because we found signi cant colocalization of QD-BDNF in both Vamp3-positive and -negative vesicles with corresponding vesicular markers but no signi cant colocalization with MitoTrackers (Fig. 2D). Because Vamp3 is an enriched vSNARE in astrocytes 29 and involved in endosome recycling 25 , it is possible that recycling of endocytic BDNF-containing vesicles in astrocytes requires the role of Vamp3. Indeed, our ndings support this notion; we observed the secretion of QD-BDNF by ATP stimulation frequently from Vamp3-EGFP-containing vesicles (Fig. 4). Vamp3 KD was successful in diminishing ATP-induced QD-BDNF exocytosis (Fig. 5), supporting the idea that Vamp3 mediates exocytosis of endocytic BDNF-containing vesicles. However, neither endocytosis nor transports of QD-BDNF requires Vamp3, as shown by no changes in QD-BDNF uptake and transports by astrocytic Vamp3 KD (Fig. 5). These results indicate Vamp3 selectively controls exocytosis of endocytic BDNF-containing vesicles. It is unclear how ATP stimulation of astrocytes caused increased the antero-or retrograde transport of endocytic BDNF-containing vesicles, but modi cation of vesicle tra cking or sorting by P2 receptor-mediated Ca 2+ or lipid signaling 26-28 may be implicated.
Our work also uncovered the complex molecular nature underlying endocytic BDNF secretion from astrocytes. We discovered that chelation of ATP-induced Ca 2+ elevation signi cantly reduces QD-BDNF exocytosis, whereas a direct increase in intracellular Ca 2+ concentration cannot evoke QD-BDNF exocytosis (Fig. 3F). These ndings imply the requirement of additional signaling pathway for full exocytosis of endocytic BDNF-containing vesicles. For example, modi cation of cAMP concentration through P2 receptor activation 30,31 or A2 receptors 32 , may in uence endocytic BDNF release by activating cAMPdependent signaling pathways important for vesicle docking or exocytosis 29,33 . Moreover, Vamp3-independent mechanisms may also be implicated in regulating endocytic BDNF release, because we observed a signi cant number of ATP-triggered QD-BDNF release events from Vamp3 (-) vesicles (Fig. 4B).
These ndings support the notion that astrocytic mBDNF recycling involves multiple but differential signaling pathways. Additional studies will further explore the other aspects of molecular events regulating BDNF recycling in astrocytes and their physiological functions in synaptic plasticity and cognitive functions.

Declarations
Data availability All materials, data, and associated protocols in this study will be available upon requests. Please contact the corresponding author (phj2@kbri.re.kr).

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