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In Vivo Time-Lapse Imaging Reveals Differential Activity-Induced Regulation of Proteasome Activity in Subcellular Regions of the Optic Tectum in Xenopus laevis Tadpoles.
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The proteasome is a major organelle responsible for protein degradation in neurons and has been implicated in the regulation of signal transduction and activity-dependent plasticity mechanisms that are essential for normal neuronal function. However, our understanding of the regulation of proteasome activity in the brain is limited by the currently available assays and tools. Here, we used a fluorogenic substrate-based probe, TAS1, to directly monitor proteasome activity in the brain of Xenopus laevis tadpoles with time-lapse two-photon microscopy. With the spatial resolution enabled by in vivo imaging, our data revealed a significant difference in proteasome activity between brain regions enriched in neuronal soma versus neuropil under both basal and pharmacologically stimulated conditions, suggesting differential activity-induced regulation of proteasome activity across neuronal subcellular compartments. These results demonstrate the feasibility of using TAS1 to track proteasome activity in vivo and provide new evidence for the differential regulation of proteasome activity in different subcellular compartments of neurons in the intact neural circuit.
Figure 1. Experimental setup and analysis methods. (A) Chemical structure of the TAS1 probe and its cleaved products. (B) Schematic of the experimental setup. Awake tadpole was immobilized in the imaging chamber, and TAS1 was injected intraventricularly. The animal was then imaged on a 2-photon microscope. Z-stacks of one tectal lobe were acquired at different time points with the same acquisition parameters. Regions of interest (ROIs) were drawn for quantification, and average intensity in each ROI was determined at every time point. V: ventricle, N: neuropil, CBL: cell body layer; NPL: neural progenitor layer. ROIs were drawn across the cell body layer (ROIs 1–3) and neuropil (ROIs 4–6) regions.
Figure 2. TAS1 fluorescence signal can be detected in live tadpole brains. (A, B) Representative image of TAS1 fluorescent signal in the optic tectum 10 min (A) and 45 min (B) following intraventricular injection. Tectal lobes shown 120–140 μm from the dorsal surface. (C) Zoomed in image of TAS1 signal in one tectal lobe 30 min post-injection. (D) Dorsal surface of the brain where blood vessels can be seen. (E) TAS1 puncta colocalize with LysoTracker Red puncta in vivo. Representative images are shown for animals injected with TAS1 only, LysoTracker Red (LT) only, or coinjected with both TAS1 and LT. Histogram on the right shows the average percentage of TAS1 puncta that colocalized with LT puncta across all images (mean ± SEM, n = 6). Scale bar: A, B, D, 80 μm; C, E, 10 μm. All images in this and the following figures are single optical sections.
Figure 3. TAS1 signal increases over time in live tadpole brains. (A) Representative time-lapse images of TAS1 and Atto590 signals in the optic tectum. (B) Quantification of average fluorescence intensity over time in the cell body layer (CBL) and neuropil layer (N) in the representative animals. The linear range of each curve was used to calculate the slope (dashed line). (C) Average slopes in CBL and N across animals injected with TAS1 or Atto590. TAS1: n = 9, Atto590: n = 4; **: p < 0.01, ****: p < 0.0001, n.s.: not significant, two-way ANOVA with Holm–Šídák correction for multiple comparisons. Scale bar: 80 μm.
Figure 4. Epoxomicin (EP) and MG-132 effectively inhibit TAS1 measured proteolytic activity. (A) Representative time-lapse images of TAS1 fluorescent intensity across animals injected with TAS1 alone or coinjected with EP or MG-132. (B) Quantification of average fluorescence intensity over time in the cell body layer (CBL) and neuropil layer (N) in representative animals. The linear range of each curve was used to calculate the slope (dashed line). (C) Slope in CBL and N in control animals and those coinjected with proteasome inhibitors. Lines connect animals from the same batch imaged side by side. TAS1: n = 9, EP: n = 9, MG-132: n = 4; *: p < 0.05, **: p < 0.01, n.s.: not significant; two-way ANOVA with Holm–Šídák correction for multiple comparisons. Scale bar: 80 μm.
Figure 5. Bicuculline (Bic) increases proteasome activity in the tadpole brain. (A) Evaluation of proteasome activity in tadpole brain lysates using in vitro Suc-LLVY-AMC assay. (B). Representative time-lapse images of TAS1 fluorescent intensity in the optic tectum of control and Bic-treated animals. (C) Quantification of average fluorescent intensity over time in the cell body layer (CBL) and neuropil layer (N) in representative animals. The linear range of each curve was used to calculate the slope. (D) Slope in CBL and N in control animals and those exposed to Bic. Lines connect animals from the same batch imaged side by side. TAS1: n = 9, Bic: n = 9; *: p < 0.05, **: p < 0.01; two-way ANOVA with Holm–Šídák correction for multiple comparisons. (E) Average slope in CBL and N in the presence of Bic, normalized to batch-matched TAS1 only control. n = 9, **: p < 0.01, Wilcoxon matched-pairs signed rank test. Scale bar: 80 μm.
