Journal: Frontiers in Cellular Neuroscience

Article Title: ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses

doi: 10.3389/fncel.2022.866802

Figure Lengend Snippet: BDNF prodomain in low concentration (1 nM) pre-synaptically increases ACh quantal size and simultaneously induces oppositely directed presynaptic effects affecting the evoked ACh release at newly formed NMJs of reinnervated mouse m. EDL. (A) Representative recordings of MEPPs (left above) and mean MEPP amplitude and cumulative probability plots (right above), frequency and time-course parameters (left to right below) in control ( n = 20) and upon application of BDNF prodomain ( n = 22). (B) Mean MEPP amplitude in control ( n = 16) and during inhibition of vesicular ACh transporter by vesamicol (1 μM, n = 17) (left) and mean MEPP amplitude and cumulative probability plots in control ( n = 15) and upon application of BDNF prodomain in the presence of vesamicol ( n = 16). (C) Changes in the EPP amplitude (left) and their quantal content (right) in control ( n = 31) and in the presence of BDNF prodomain ( n = 41). Inset shows MEPP amplitudes.

Article Snippet: We used recombinant human proBDNF (its cleavable form) and BDNF prodomain (purchased from Alomone Labs, Jerusalem, Israel); tertiapin-Q as a selective blocker of inward-rectifier K + channels, iberiotoxin as a selective blocker of the big conductance Ca 2+ -activated K + channels, nitrendipine as a L-type calcium channel blocker, Y-27632 dihydrochloride as a selective inhibitor of ROCK, and (±)-Vesamicol hydrochloride as a direct inhibitor of vesicular ACh transport (all purchased from Tocris, Bio-Techne, Minneapolis, MN, United States); TAT-Pep5 as a p75 receptor signaling inhibitor (purchased from Sigma-Aldrich, United States).

Techniques: Concentration Assay, Inhibition

Journal: Frontiers in Cellular Neuroscience

Article Title: ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses

doi: 10.3389/fncel.2022.866802

Figure Lengend Snippet: BDNF prodomain (1 nM) but not proBDNF (1 nM), induces strong inhibition of spontaneous end evoked ACh release at mature NMJs. (A) Mean MEPP amplitude, cumulative probability plots, frequency, and time-course parameters (left to right) in control ( n = 19) and upon application of proBDNF ( n = 26). (B) Representative recordings of MEPPs (top left) and mean MEPP amplitude, cumulative probability plots, frequency, (top right) and their time-course parameters (bottom) in control ( n = 23) and upon application of BDNF prodomain ( n = 33). (C) Representative recordings of EPPs during a short (1 s) high-frequency (50 Hz) train in control (above) and upon application of BDNF prodomain (below). (D) Changes in the EPP amplitude (above) and in the quantal content of EPPs (below) in control ( n = 22) and in the presence of proBDNF ( n = 21). Inset shows MEPP amplitudes.

Article Snippet: We used recombinant human proBDNF (its cleavable form) and BDNF prodomain (purchased from Alomone Labs, Jerusalem, Israel); tertiapin-Q as a selective blocker of inward-rectifier K + channels, iberiotoxin as a selective blocker of the big conductance Ca 2+ -activated K + channels, nitrendipine as a L-type calcium channel blocker, Y-27632 dihydrochloride as a selective inhibitor of ROCK, and (±)-Vesamicol hydrochloride as a direct inhibitor of vesicular ACh transport (all purchased from Tocris, Bio-Techne, Minneapolis, MN, United States); TAT-Pep5 as a p75 receptor signaling inhibitor (purchased from Sigma-Aldrich, United States).

Techniques: Inhibition

Journal: Frontiers in Cellular Neuroscience

Article Title: ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses

doi: 10.3389/fncel.2022.866802

Figure Lengend Snippet: BK channels do not but GIRK channels mediate BDNF prodomain-induced inhibition of evoked ACh release at mature NMJs. (A) Changes in the EPP amplitude (left) and in the quantal content of EPPs (right) in control ( n = 15) and upon BDNF prodomain (1 nM) in the presence of BK-blocker iberiotoxin (ITx, 100 nM) with L-type Ca 2+ -channel blocker nitrendipine (Nitr., 1 μM) ( n = 21). (B) Changes in the EPP amplitude (left) and in the quantal content of EPPs (right) in control ( n = 15) and in the presence of GIRK blocker tertiapin-Q (100 nM, n = 17). (C) Changes in the EPP amplitude (left) and in the quantal content of EPPs (right) in control ( n = 16) and upon BDNF prodomain (1 nM) in the presence of tertiapin-Q ( n = 19). Insets show MEPP amplitudes.

Article Snippet: We used recombinant human proBDNF (its cleavable form) and BDNF prodomain (purchased from Alomone Labs, Jerusalem, Israel); tertiapin-Q as a selective blocker of inward-rectifier K + channels, iberiotoxin as a selective blocker of the big conductance Ca 2+ -activated K + channels, nitrendipine as a L-type calcium channel blocker, Y-27632 dihydrochloride as a selective inhibitor of ROCK, and (±)-Vesamicol hydrochloride as a direct inhibitor of vesicular ACh transport (all purchased from Tocris, Bio-Techne, Minneapolis, MN, United States); TAT-Pep5 as a p75 receptor signaling inhibitor (purchased from Sigma-Aldrich, United States).

Techniques: Inhibition

Journal: Frontiers in Cellular Neuroscience

Article Title: ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses

doi: 10.3389/fncel.2022.866802

Figure Lengend Snippet: p75 receptors and Rho-signaling pathway underlie BDNF prodomain-triggered inhibition of evoked ACh release at mature NMJs. Moreover, the inhibitory effect of the BDNF prodomain (1 nM) on the evoked neuromuscular transmission depends on the endogenous activity of synaptic purinoreceptors at mature NMJs. (A) Changes in the EPP amplitude (left) and in the quantal content of EPPs (right) in control ( n = 20) and upon BDNF prodomain (1 nM) in the presence of Rho-GDI-associated p75 signaling inhibitor TAT-Pep5 (1 μM, n = 21). (B) Changes in the EPP amplitude (left) and in the quantal content of EPPs (right) in control ( n = 17) and in the presence of ROCK inhibitor Y-27632 (3 μM, n = 21). (C) Changes in the EPP amplitude (left) and in the quantal content of EPPs (right) in control ( n = 15) and upon BDNF prodomain (1 nM) in the presence of Y-27632 ( n = 21). (D) Changes in the EPP amplitude (left) and in the quantal content of EPPs (right), registered from NMJs of Panx1 –/– mice in control ( n = 24) and in the presence of BDNF prodomain ( n = 22). Insets show MEPP amplitudes.

Article Snippet: We used recombinant human proBDNF (its cleavable form) and BDNF prodomain (purchased from Alomone Labs, Jerusalem, Israel); tertiapin-Q as a selective blocker of inward-rectifier K + channels, iberiotoxin as a selective blocker of the big conductance Ca 2+ -activated K + channels, nitrendipine as a L-type calcium channel blocker, Y-27632 dihydrochloride as a selective inhibitor of ROCK, and (±)-Vesamicol hydrochloride as a direct inhibitor of vesicular ACh transport (all purchased from Tocris, Bio-Techne, Minneapolis, MN, United States); TAT-Pep5 as a p75 receptor signaling inhibitor (purchased from Sigma-Aldrich, United States).

Techniques: Inhibition, Transmission Assay, Activity Assay

Journal: Biophysical Reports

Article Title: Multimodal single-molecule microscopy with continuously controlled spectral resolution

doi: 10.1016/j.bpr.2021.100013

Figure Lengend Snippet: Multicolor single-particle tracking of axonal transport in live neurons. ( A ) Schematic illustration descripting distal axonal uptake of the different neurotrophic factors and their transportation to the proximal axon and cell body through endosomes. The three neurotrophic factors used in this experiment, proBDNF, BDNF, and NGF, were labeled with Qdot565 ( green ), Qdot625 ( red ), and Qdot800 ( magenta ), respectively. ( B ) False-colored overlay of time-averaged localization (RPA = 180°) full FOV time lapse, processed with background removal. In each frame, four consecutive acquisitions with emission filter switching were taken ( white , no filter; cyan , 575/15; yellow , 605/15; and magenta , 809/81) and 405 nm excitation. The FOV shows the signaling activity in the proximal axons near neuronal cell bodies. Orange rectangle marks the axon presented in ( E ). Scale bars, 30 μ m. ( C ) PSF examples of an endosome transporting all three neurotrophic factors through the axon marked in ( B ). Left: three sequential localization (RPA = 180°) acquisitions with emission filters switching (same color code as in ( B )). Middle: single localization (RPA = 180°) acquisition with no emission filter. Right: CoCoS color-detection mode with no emission filter and optimal dispersion (RPA = 172°); color bar on the right illustrates the spectral-dispersion map (nonaccurate). ( D ) Theoretical spectra of the three Qdots with PSF visual representation for illustration purposes (nonaccurate. For a theoretically calculated dispersed PSF, see Fig. S13 ). ( E ) Example time-lapse frames followed by a kymograph of the marked axon in ( B ), showing the retrograde transport of the endosome presented in ( C ). Left: three consecutive acquisitions per frame are shown with RPA = 180°, 405 nm excitation, and 575/15 ( cyan ), 605/15 ( yellow ), and 809/81 ( magenta ) emission filters. A spatiotemporal mismatch between the acquisitions is clearly visible when the transport velocity is high (0, 3.2, and 6.4 s frames). Right: a single acquisition per frame is shown with no emission filter and with RPA = 172°. At the bottom of each time lapse, a kymograph of the full time lapse is presented, showing a halt in transportation at cell death after 30 s of imaging. Horizontal scale bars, 3 μ m; vertical scale bars, 30 s. ( F ) Example of CoCoS PSFs for each of the protein combinations inside the transporting endosomes as detected in the FOV of ( B ). Left: single acquisition with RPA = 172°. Right: false-color overlay of three consecutive acquisitions with RPA = 172° and emission filters switching.

Article Snippet: Meanwhile, human BDNF-biotin (B-250-B; Alomone Labs), human pro-BDNF-biotin (B-256-B; Alomone Labs), and mouse NGF-biotin (N-240-B; Alomone Labs) were mixed separately in a molar ratio of 3:1 with Qdot-625 streptavidin, Qdot-565 streptavidin, and Qdot-800 streptavidin, respectively (Q10131MP; Q10143MP; and Q10173MP; Molecular Probes, Eugene, OR).

Techniques: Single-particle Tracking, Labeling, Activity Assay, Imaging

Journal: Human Molecular Genetics

Article Title: The pro-domains of neurotrophins, including BDNF, are linked to Alzheimer's disease through a toxic synergy with Aβ

doi: 10.1093/hmg/ddv130

Figure Lengend Snippet: The pro-domain of BDNF and Aβ 1–42 exhibits synergistic toxicity in SH-SY5Y human neuroblastoma cell cultures. ( A ) Addition of 25, 100 and 1000 n m oligomeric Aβ 1–42 to the culture medium of SH-SY5Y human neuroblastoma cells for 48–60 h resulted in significant toxicity when compared with vehicle. Each bar represents mean ± SEM ( n = 3). Statistical comparisons were made by one-way analysis variance test. * P < 0.05. ( B ) Cells were treated with 200 ng/ml recombinant pro-domain of BDNF for 48–60 h. Met66 BDNF pro-domain alone did not have an effect on cell viability in control cells. ( C ) Cells were treated with Met66 pro-domain of BDNF (200 ng/ml) in combination with 25 n m Aβ 1–42 for 48–60 h. In the presence of Aβ 1–42 cell death was increased upon addition of Met66 BDNF pro-domain ( n = 9) performed. Each bar represents mean ± SEM ( n = 9). Statistical comparisons were made by paired t test. ** P < 0.01. ( D ) Twenty-six paired experiments indicated that cell death was higher when a particular preparation of Aβ 1–42 (25 n m , n = 9 preparations of Aβ 1–42 ) was supplemented with Met66 BDNF pro-domain ( y -axis) than for that preparation of Aβ 1–42 alone ( x -axis, dashed line indicates the null hypothesis of no effect of the Met66 BDNF pro-domain). ( E ) Similar results were seen when non-differentiated and ( F ) differentiated cells were treated with the Met66 BDNF pro-domain (200 ng/ml) in combination with 1 μ m Aβ 1–42 for 48 h. ( G ) Cells were treated with the Met66 BDNF pro-domain (200 ng/ml) in combination with 25 n m Aβ 1–42 oligomers for 48–60 h before SDS–PAGE and western blotting with antibodies against p75 NTR and sortilin. β-Actin expression was used as a loading control. Western blots indicated that levels of p75 NTR but not sortilin were higher following Aβ 1–42 oligomer treatment in cultures of human neuroblastoma cells. Representative blots are shown ( n = 3). ( H ) The p75 NTR and ( I ) sortilin intensities from the western blots were normalized against the level of β-actin. Each bar represents mean ± SEM ( n = 3). Statistical comparisons were made by one-way analysis variance test. * P < 0.05.

Article Snippet: Twenty four hours after plating, cells were washed and maintained in 0.5% (v/v) FBS-containing medium for 18 h. After that cells were replaced with 0.2% (v/v) FBS-containing medium and then incubated further 48–72 h in the presence of Aβ 1–42 oligomers with or without recombinant human wild type (Val66) or variant (Met66) BDNF pro-domain (Alomone labs).

Techniques: Recombinant, SDS Page, Western Blot, Expressing