Activity-induced synaptic capture and exocytosis of the neuronal serine protease neurotrypsin

Abstract

Extracellular proteolysis plays an essential role in synaptic remodeling that is indispensable for cognitive function. The extracellular serine protease neurotrypsin was implicated in cognitive function, because humans lacking a functional form of neurotrypsin suffer from severe mental retardation. By immunoelectron microscopy, neurotrypsin has been localized to presynaptic terminals, suggesting a local proteolytic function after its synaptic release. Here, we studied axonal trafficking and synaptic exocytosis of neurotrypsin by live imaging of hippocampal neurons expressing neurotrypsin fused with enhanced green fluorescent protein or its pH-sensitive variant, superecliptic pHluorin. In differentiated neurons, we identified neurotrypsin in mobile transport vesicles along axons and in both an intracellular and an extracellular pool at synapses. Short depolarization triggered rapid synaptic exocytosis of neurotrypsin. Once externalized, neurotrypsin lingered at its synaptic release site for several minutes before it disappeared. Cell depolarization also enhanced synaptic capture of intracellular neurotrypsin transport vesicles, and elevated synaptic activity increased both number and motility of mobile axonal neurotrypsin vesicles. We further observed trading of neurotrypsin vesicles between adjacent synapses. These activities may support the replenishment of neurotrypsin after activity-induced synaptic exocytosis. Together, the activity-dependent recruitment of neurotrypsin to synapses and its exocytosis and transient persistence at its synaptic release site argue for a spatially and temporally restricted proteolytic action at the synapse. Thereby, neurotrypsin may play a role in activity-dependent remodeling of the synaptic circuitry that is key to adaptive synaptic changes in the context of cognitive functions, such as learning and memory.

Figure 1.

Confocal microscopy confirms localization of neurotrypsin to axons and presynaptic boutons. A, Costaining of neurotrypsin–EGFP-expressing hippocampal neurons (A1) with the PSD95 (A2) and the presynaptic protein synapsin (A3). The overlay is shown in A4. Pairwise colocalizations of neurotrypsin–EGFP puncta with either PSD95 or synapsin are indicated by arrowheads in the high-magnification images. Examples of extrasynaptic neurotrypsin–EGFP, most likely representing vesicular transport packages, are indicated by arrows in the high-magnification images of A1–A4. B, Neurites positive for neurotrypsin–EGFP are not costained with the dendritic protein MAP2 (B2), suggesting axonal localization of neurotrypsin (arrows in B1–B4). Many neurotrypsin–EGFP puncta are colocalized with vGlut, a presynaptic marker of excitatory synapses (arrowheads in high-magnification images). Faint extrasynaptic neurotrypsin–EGFP puncta are indicated by arrows in the high-magnification images. The regions of the high-magnification images are boxed in A1–A4 and B1–B4, respectively. Scale bars: B4, 15 μm; high-magnification images, 2 μm.

Figure 2.

Synaptic neurotrypsin is found in intracellular and extracellular pools by live imaging of cells infected with an adenovirus expressing neurotrypsin–pHluorin. A, Labeling of active synapses with FM 4-64. B, In normal buffer (pH 7.4), only extracellular neurotrypsin–pHluorin is visible (B1). Most extracellular neurotrypsin colocalizes with synapses labeled with FM 4-64 (B2). C, Addition of NH₄Cl unmasks intracellular pools of neurotrypsin (C1). Note the additional neurotrypsin–pHluorin dots within FM 4-64-positive areas (synapses) in the presence of NH₄Cl (C2) compared with normal imaging buffer (B2). Boxed areas in B2 and C2 are shown at higher magnification. In addition, nonsynaptic dots representing transport vesicles appear in NH₄Cl-containing buffer (arrowheads in C1 and C2). Scale bars: overviews, 5 μm; high-magnification images, 1 μm.

Figure 3.

Chronic disinhibition of hippocampal cultures with PTX enhances the synaptic localization of neurotrypsin. Hippocampal neurons were cotransfected with neurotrypsin and VAMP2–EGFP and stained with anti-neurotrypsin antibody. Cells were treated with APV/CNQX (A) or PTX (B) 24 h before fixation. In APV/CNQX-treated cells, only a small fraction of the VAMP2–EGFP puncta contained neurotrypsin (arrows in A3). Chronic neuronal activity led to a higher occupation of synapses by neurotrypsin, as indicated by the high degree of colocalization of neurotrypsin with VAMP2–EGFP in PTX-treated cells (arrows in B3). Scale bar, 5 μm.

Figure 4.

K⁺-induced depolarization of hippocampal neurons results in translocation of neurotrypsin to synapses. A, Image sequence from a time-lapse experiment of hippocampal neurons expressing neurotrypsin–EGFP. Neurotrypsin–EGFP signals were recorded for a period of 1 min before and 12:20 min after a 90 s period of K⁺-induced depolarization (A1–A10). Synapses were stained with FM 4-64 at the end of the experiment (A11). The image of the FM 4-64-labeled synapses was used to generate overlays with selected images of neurotrypsin–pHluorin (A12–A14). Before depolarization, no synaptic neurotrypsin was observed in this particular segment (A1, A2). At the end of the depolarization period, we observed enhanced neurotrypsin trafficking (arrows in A3). Later, transport vesicles started to be captured at active synapses (arrowheads in A4/A12, A5/A13). Neurotrypsin was often redistributed in small transport vesicles after lingering for several minutes at a particular synapse (arrow in A6), remaining at the synapse in only small amounts (right arrowhead in A6). At other synapses, neurotrypsin was continuously accumulated (left arrowhead in A5–A10 and overlays A13, A14). Between neighboring synapses, an exchange of neurotrypsin transport packages could be observed (arrow in A6–A12). Scale bar, 1 μm. B, Synaptic neurotrypsin was quantified in 20 μm axonal segments before and after KCl depolarization. The number of synaptic puncta before depolarization was used for normalization. At 100 s after stimulation, significant increase of synaptic neurotrypsin was observed. During the following 300 s, the number of synaptic neurotrypsin increased gradually. At ∼400 s after KCl treatment, the occupation of the synapses with neurotrypsin was twofold compared with the control situation and did not increase further (***p < 0.001, two-way ANOVA, Bonferroni's post hoc test, n = 12 cells).

Figure 5.

Vesicular transport of neurotrypsin–EGFP is enhanced after K⁺-induced depolarization and PTX-mediated disinhibition. A, Image series of an axonal segment before (0–8 s) and after (220–228 s) KCl application. Arrows indicate transport vesicles. Note the faster movement of the indicated vesicle after KCl. Also note the higher density of vesicles after stimulation. B, Frequency distribution of neurotrypsin–EGFP transport vesicle velocities from hippocampal neurons at DIV 18 treated with PTX or APV/CNQX for 24 h. Note the shift to higher velocities in PTX-treated cultures. C, Vesicles were counted in live neurons as they passed an axonal segment of 20 μm in 100 s time windows. Data were normalized to the number of vesicles counted during the first 100 s. If cells were not depolarized, the trafficking remained unchanged during the 800 s of observation (open circles). If cells were treated with PTX before imaging, depolarization had no effect on the trafficking (open triangles). However, if cells were kept in APV/CNQX, depolarization led to an increased trafficking within 200 s after KCl treatment (filled triangles). The increase in trafficking was up to 2.5-fold 600 s after onset of depolarization. **p < 0.01; ***p < 0.001.

Figure 6.

The synaptic extracellular pool of neurotrypsin is increased in PTX-disinhibited neurons. A, B, Axonal segment of neurons transfected with neurotrypsin–pHluorin. Synapses were labeled using FM 4-64 (A3, B3). In the cultures supplemented with APV/CNQX, there was only little synaptic extracellular neurotrypsin (arrow in A1). Addition of NH₄Cl revealed intracellular pools of neurotrypsin. The intracellular pools located at a synapse are indicated by an arrow, an arrowhead, and an open arrow in A2 and A4. At numerous synaptic intracellular pools (visualized by NH₄Cl), no extracellular neurotrypsin was observed (arrowhead and open arrow in A1). If cells were cultured in the presence of PTX, an overall increased fluorescence of both intracellular and extracellular pools was observed (B1–B4). At synapses, both extracellular and intracellular neurotrypsin were more prominent (arrows in B1, B2, B4). Scale bar, 2 μm.

Figure 7.

Depolarization leads to synaptic exocytosis of neurotrypsin and buildup of intracellular neurotrypsin stores at synapses. A, Live imaging of hippocampal neurons at DIV 18 infected with an adenovirus expressing neurotrypsin–pHluorin. Synapses were stained at the end of the experiment using FM 4-64. The image of the FM 4-64-stained synapses was used to generate overlays with selected images of neurotrypsin–pHluorin. At the beginning of the experiment, little synaptic extracellular neurotrypsin was found (arrow in −15:54). Short perfusion with pH 5.5 imaging buffer abolished all observed fluorescence (−13:39). Perfusion with NH₄Cl-containing buffer revealed the presence of intracellular pools of neurotrypsin–pHluorin at synaptic (arrow at −03:42 and overlay at −03:42) and extrasynaptic sites. Starting from 0:00 min, cells were depolarized for 90 s with KCl. Shortly after depolarization, increased extracellular fluorescence could be observed at synapses in which intracellular neurotrypsin was present before depolarization (arrow in 00:27) and also at sites in which previously no neurotrypsin fluorescence was found (arrowhead at 01:18 and overlay at 01:18). At a number of synapses, externalized neurotrypsin could be observed for awhile after the end of depolarization (arrow and arrowhead in 01:51). In the minutes after KCl depolarization, new intracellular pools were formed at synapses that previously did not contain neurotrypsin (open arrow in 02:57, 12:27). Most intracellular pools were replenished after neurotrypsin–pHluorin exocytosis (arrowhead in 12:27), although a few were not (arrow in 02:57 and 12:27). Scale bar, 2 μm. B, Normalized fluorescence intensity of individual synaptic sites during a live-imaging experiment. B1 shows the course of fluorescence of a synaptic spot with an intracellular pool (gray bar at 100 s and 700 s) and little extracellular fluorescence (red bar) before depolarization. There was a strong increase in fluorescence when KCl was added (open bar, 1000 s). Note that the intracellular pool was diminished and not restored (gray bar, 1100 s and 1700 s). In B2, intracellular pools were present before depolarization, but only little was released during KCl depolarization (open bar at 1000 s in B2). Immediately after depolarization, the intracellular pool remained unaltered (gray bar, 1100 s). However, several minutes after KCl application, the intracellular fluorescence was more than doubled (gray column, 1700 s). Other synapses contained neither intracellular nor extracellular neurotrypsin before depolarization (B3). Also, there was no immediate increase of fluorescence when KCl was added (open bar at 1000 s in B3). Only several minutes after depolarization a large intrasynaptic pool was found (1700 s, gray bar). C, Time course of the change of fluorescence during and after depolarization at individual synapses containing neurotrypsin. The increase in fluorescence varied between individual synapses, ranging from 1.1-fold (blue line) to 1.65-fold (black line). Note that also the velocity of the signal change varied between individual synapses, in both the increasing and decreasing phase. D, The number of synapses containing intracellular or external neurotrypsin was increased 2.06 ± 0.20 times 135 s after onset of depolarization and stayed stable during the time of imaging. E, We observed a 3.07 ± 0.58-fold increase in the number of synapses with extracellular neurotrypsin 30 and 120 s after onset of depolarization. The number of synapses with extracellular neurotrypsin was back to basal level after 7 min (mean ± SEM, n = 6 cells).