The role of the synaptic vesicle priming factors CAPS-1 and CAPS-2 in synaptic vesicle docking

Next, I decided to focus on CAPS-1/2 proteins, which are thought to regulate synaptic vesicle priming in concert with Munc13s. Autaptic hippocampal cultures from CAPS1/2 DKO neurons revealed a complex physiological phenotype: similar to Munc13-1/2 DKO neurons, 39% of all CAPS-1/2 DKO neurons lack any presynaptic activity and are deficient of fusion-competent vesicles (Jockusch et al., 2007). However, in the remaining 61% cells, synaptic responses are not completely abolished, but EPSC sizes and the RRP

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Figure 3.4. Three-dimensional electron tomographic analysis of synaptic vesicle docking in Munc13-1/2 DKO neurons

Tomographically reconstructed subvolumes of control (A) and Munc13-1/2 DKO (B) synapses from 200 nm-thick sections. Three-dimensional models of tomographically reconstructed control (C) and Munc13-1/2 DKO (D) synaptic profiles. Ultrastructural features reconstructed in the models include the active zone plasma membrane (white), synaptic vesicles (membrane-attached, green;

non-attached, grey), and large dense core vesicles (LDCVs; beige). Orthogonal views of control (E) and Munc13-1/2 DKO (F) tomographic models displaying the spatial arrangement of membrane-attached synaptic vesicles within the reconstructed active zone area. Mean number of SVs within 100 nm of the AZ normalized to AZ area (G). Mean number of SVs within 40 nm of the AZ normalized to AZ area (H). Mean number of membrane-attached SVs (within 0-2 nm of the AZ) normalized to AZ area (I). Mean number of membrane-attached and closely-tethered SVs (within 0-4 nm of the AZ) normalized to AZ area (J). Mean outer SV diameter (K). Mean SV volume including the membrane bilayer (L). Spatial distribution of SVs within a 100 nm distance of the AZ membrane (M). Distribution of synaptic vesicle diameters of vesicles within 100 nm of the AZ (N).

G-L: Control: N=2, n=16; Munc13-1/2 DKO: N=3, n=15 (Mean + SEM), P<0.001: ***; P<0.01: **;

P<0.05: *. M, N: (511 SVs in control and 402 SVs in Munc13-1/2 DKO synaptic profiles). Scale bar: B, 100 nm.

As previously shown, homozygous CAPS-2 KO neurons are phenotypically indistinguishable from wild-type neurons (Jockusch et al., 2007). For the two-dimensional analysis of electron micrographs from ultrathin sections, four CAPS-1/2 DKO animals (N=4) were compared with three littermate control animals (N=3) from three cultures. The control animals were either wild-type or heterozygous for the CAPS-1 KO allele in a homozygous CAPS-2 KO background.

3.1.3.2.1. 2D-EM analysis of synaptic morphology in CAPS-1/2 DKO neurons

Morphologically, no major differences in the gross synaptic ultrastructure of neurons lacking both CAPS isoforms were observed in electron micrographs from ultrathin (60 nm) sections (Fig. 3.5 A, B). The total number of synaptic vesicles per synaptic profile did not differ in CAPS-1/2 deficient neurons in comparison to control samples (control: 63.15 ± 3.641, n=109; CAPS-1/2 DKO: 60.74 ± 2.907, n=115 / P=0.9228, n.s.; Fig. 3.5 C).

Moreover, the synaptic vesicle terminal density was not significantly altered (control: 1.454

± 0.059, n=109; CAPS-1/2 DKO: 1.535 ± 0.057, n=115 / P=0.2858, n.s.; Fig. 3.5 D), with only a slight decrease in the synaptic vesicle cluster density (control: 3.436 ± 0.061, n=109; CAPS-1/2 DKO: 3.285 ± 0.056, n=115 / P=0.0308, *; Fig 3.5 E) and no changes in the mean PSD length (control: 330.5 ± 11.06, n=109; CAPS-1/2 DKO: 315.2 ± 8.849, n=115 / P=0.5539, n.s.; Fig. 3.5 F). In contrast to synapses lacking Munc13s, I did not observe an increase in the number of recycling endosomes per synaptic profile in neurons deficient for CAPS-1 and -2 in comparison to control neurons (control: 1.248 ± 0.170, n=109; CAPS-1/2 DKO: 1.270 ± 0.142, n=115 / P=0.5173, n.s.; Fig.3.5 G). Also, no change in the number of LDCVs in synapses lacking all CAPS isoforms was detected in my experimental setting (control: 0.147 ± 0.043, n=109; CAPS-1/2 DKO: 0.191 ± 0.046,

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3.1.3.2.2. 3D-ET analysis of synaptic vesicle docking in CAPS-1/2 DKO neurons

In the electron tomographic three-dimensional analysis, I analyzed samples from three 1/2 DKO (N=3) and three control animals (N=3) (Fig. 3.6 A-F). I noticed that CAPS-1/2 DKO synaptic profiles analyzed by electron tomography exhibited a drastic reduction in the number of membrane-associated vesicles (0-2 nm) in comparison to control synaptic profiles (control: 1.202 ± 0.143, n=22; CAPS-1/2 DKO: 0.083 ± 0.026, n=19 / P<0.001, ***; Fig. 3.6 I), and also a strong decrease in the number of closely tethered (0-4 nm) synaptic vesicles (control: 1.598 ± 0.158, n=22; CAPS-1/2 DKO: 0.180 ± 0.063, n=19 / P<0.001, ***; Fig. 3.6 J). However, the number of vesicles within 40 nm of the presynaptic plasma membrane (control: 2.660 ± 0.196, n=22; CAPS-1/2 DKO: 2.393 ± 0.191, n=19 / P=0.3400, n.s.; Fig. 3.6 H) and the number of vesicles within 100 nm of the presynaptic release sites were unaltered (control: 7.121 ± 0.413, n=22; CAPS-1/2 DKO:

6.015 ± 0.391, n=19 / P=0.0614, n.s.; Fig. 3.6 G). In contrast to synapses lacking Munc13s, synaptic vesicles in synapses lacking both CAPS isoforms appeared to be randomly distributed within the first 100 nm from the active zone membrane, rather than accumulating at an 8-10 nm distance (Fig. 3.6 M). Moreover, neither the mean outer synaptic vesicle diameter (control: 45.19 ± 0.560, n=22; CAPS-1/2 DKO: 45.54 ± 0.649, n=19 / P=0.6434, n.s.; Fig. 3.6 K, N) nor the mean outer synaptic vesicle volume were significantly different from controls (control: 49181 ± 1896, n=22; CAPS-1/2 DKO: 50355 ± 2022, n=19 / P=0.6743, n.s.; Fig. 3.6 L) for CAPS-1/2 DKO neurons.

In my analysis, 63% of all sampled CAPS-1/2 DKO synaptic profiles (12 out of 19 tomograms) were completely devoid of membrane-attached vesicles (0-2 nm) and 58% of all CAPS-1/2 DKO synaptic profiles (11 out of 19 tomograms) also lacked vesicles closely tethered to the active zone membrane (0-4 nm). In comparison to control synaptic profiles, the number of membrane-attached (0-2 nm) and closely tethered (0-4 nm) synaptic vesicles in CAPS-1/2 DKO neurons was reduced to 7% and 12%, respectively.

Tomograms from CAPS-1/2 DKO synapses that still harbored membrane-attached synaptic vesicles, docking (0-2 nm) and close tethering (0-4 nm) was reduced to 19% and 28% compared to controls, respectively.

Figure 3.5. Two-dimensional ultrastructural analysis of synaptic morphology in CAPS-1/2 DKO neurons

Electron micrographs of control (A) and CAPS-1/2 DKO (B) synaptic profiles acquired from 60 nm-thick ultrathin sections. Mean number of synaptic vesicles (SVs) per synaptic profile (C). Mean number of SVs normalized to synaptic terminal area (SV terminal density; D). Mean number of SVs normalized to SV cluster area (SV cluster density; E). Mean postsynaptic density (PSD) length (F) Mean number of endosomes per synaptic profile (G). Mean number of large dense-core vesicles (LDCVs) per synaptic profile (H). C-H: Control: N=3, n=109; CAPS-1/2 DKO: N=4, n=115 (Mean + SEM), P<0. 001: ***; P<0.01: **; P<0.05: *. Scale bar: B, 500 nm.

These ultrastructural findings indicate that CAPS proteins perform not only to produce functionally prime synaptic vesicles, but, similar to Munc13s, have an important role in docking synaptic vesicles to the presynaptic active zone membrane. I interpret these findings as further support for the hypothesis that the RRP of functionally primed vesicles is comprised of membrane-attached synaptic vesicles capable of securing fast synaptic release (Murthy et al., 2001; Schikorski and Stevens, 2001). This pool of readily-releasable and docked vesicles has been shown here to be either completely depleted or strongly diminished in neurons lacking proteins from the Munc13 or CAPS family.

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Figure 3.6. Three-dimensional electron tomographic analysis of synaptic vesicle docking in CAPS-1/2 DKO neurons

Tomographically reconstructed subvolumes of control (A) and CAPS-1/2 DKO (B) synapses from 200 nm-thick sections. Three-dimensional models of tomographically reconstructed control (C) and CAPS-1/2 DKO (D) synaptic profiles. Ultrastructural features reconstructed in the models include the active zone plasma membrane (white) and synaptic vesicles (membrane-attached, green;

non-attached, grey). Orthogonal views of control (E) and CAPS-1/2 DKO (F) tomographic models displaying the spatial arrangement of membrane-attached synaptic vesicles within the reconstructed active zone area. Mean number of SVs within 100 nm of the AZ normalized to AZ area (G). Mean number of SVs within 40 nm of the AZ normalized to AZ area (H). Mean number of membrane-attached SVs (within 0-2 nm of the AZ) normalized to AZ area (I). Mean number of membrane-attached and closely-tethered SVs (within 0-4 nm of the AZ) normalized to AZ area (J).

Mean outer SV diameter (K). Mean SV volume including the membrane bilayer (L). Spatial distribution of SVs within a 100 nm distance of the AZ membrane (M). Distribution of synaptic vesicle diameters of vesicles within 100 nm of the AZ (N). G-L: Control: N=3, n=22; CAPS-1/2 DKO: N=3, n=19 (Mean + SEM), P<0.001: ***; P<0.01: **; P<0.05: *. M, N: (716 SVs in control and 560 SVs in CAPS-1/2 DKO synaptic profiles). Scale bar: B,100 nm.