Synaptic Effects of Munc18-1 Alternative Splicing in Excitatory Hippocampal Neurons.

The munc18-1 gene encodes two splice-variants that vary at the C-terminus of the protein and are expressed at different levels in different regions of the adult mammalian brain. Here, we investigated the expression pattern of these splice variants within the brainstem and tested whether they are functionally different. Munc18-1a is expressed in specific nuclei of the brainstem including the LRN, VII and SOC, while Munc18-1b expression is relatively low/absent in these regions. Furthermore, Munc18-1a is the major splice variant in the Calyx of Held. Synaptic transmission was analyzed in autaptic hippocampal munc18-1 KO neurons re-expressing either Munc18-1a or Munc18-1b. The two splice variants supported synaptic transmission to a similar extent, but Munc18-1b was slightly more potent in sustaining synchronous release during high frequency stimulation. Our data suggest that alternative splicing of Munc18-1 support synaptic transmission to a similar extent, but could modulate presynaptic short-term plasticity

Munc18-1a is expressed in the auditory brainstem of mice.

<p>(A) Munc18-1a immunostaining shows specific Munc18-1a expression in the Superior olivary complex (SOC), Facial motor nucleus (VII), and Lateral reticular nucleus (LRN) of the brainstem of wild-type (WT) mice. Scale bar 200 μm. (B) Munc18-1b-Venus is not expressed in nuclei of the brainstem of homozygous Munc18-1-Venus knock-in (M18V KI) mice. (C and D) Higher magnification of indicated regions in A and B, respectively, shows that Munc18-1a is highly expressed in the SOC, which lacks Munc18-1b-Venus expression. Scale bar 100 μm. (E) Bright field (bf) image of a coronal section of P21 wild-type mouse brain (images E-J all from same section), showing part of the brainstem including MNTB (white oval) and LSO. (F) Fluorescence image of immunostaining against Munc18-1a (M18-1a), showing relative high expression in the MNTB and LSO. Scale bar 100 μm. (G) Fluorescence image of immunostaining against Munc18-1b (M18-1b), showing relative low expression in the MNTB and LSO. (H) Composite image of E, F, G showing non-complementary expression of Munc18-1a (red) and Munc18-1b (green). (I and J) Confocal zoom from image in F and G of the MNTB region showing (I) Munc18-1a expression in presynaptic Calyces (arrowheads) and post-synaptic principal cells (*). In contrast, expression of Munc18-1b is punctate but unspecific for Calyces (J). Scale bar 10 μm.</p

Morphology of hippocampal neurons expressing a single splice variant of Munc18-1.

<p>(A) Amino acid sequences of the C-terminus of mouse Munc18-1a and 1b starting at site 562 (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138950#pone.0138950.ref010" target="_blank">10</a>]). Phosphorylation sites identified in high-throughput screens are highlighted (Fig 1b, M18-1a: S590, S594; M18-1b: T581, T588, S593, S594) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138950#pone.0138950.ref016" target="_blank">16</a>]. Arrow points to a predicted CaMKII phosphorylation site in Munc18-1a identified using NetPhos 2.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138950#pone.0138950.ref040" target="_blank">40</a>] and Phosida [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138950#pone.0138950.ref038" target="_blank">38</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138950#pone.0138950.ref039" target="_blank">39</a>]. (B) Representative immunoblot of Munc18-1 protein levels in rescued <i>munc18-1</i> null neurons. Neurons were analysed at DIV13 with western blot analysis using double immuno-fluorescent labelling for Munc18-1 (red) and α-Tubulin (green). (C-E) Munc18-1a or -1b was reintroduced using lentivirus in hippocampal <i>munc18-1</i> null neurons cultured in the absence of glia. (C) Examples of MAP2 (dendritic marker) and VAMP2 (synaptic marker) immunostainings to visualize neuronal morphology. Scale bar = 100 μm. (D) Quantification of neuronal morphology. Number of synapses (M18-1b: 212.1 ± 8.9 synapses, n = 79; M18-1a: 241.3 ± 10.0 synapses, n = 78, Unpaired t test, p = 0.0309). Synapse density (M18-1b: 0.248 ± 0.009 synapses/μm dendrite, n = 79; M18-1a: 0.233 ± 0.006 synapses/μm dendrite, n = 78, Mann-Whitney Test, p = 0.1466). Total dendritic length (M18-1b: 0.895 ± 0.040 mm, n = 79; M18-1a: 1.057 ± 0.041 mm, n = 78, Mann-Whitney Test, p = 0.009). Number of branches (M18-1b: 127.2 ± 5.6 branches, n = 79; M18-1a: 147.0 ± 6.0 branches, n = 78, Mann-Whitney Test, p = 0.0043). (E) Sholl analysis suggests that dendritic branching is more elaborate in Munc18-1a expressing neurons. Shown are of the number of dendrite crossings per ring. Rings are placed with increasing radius around the soma (increments of 5 μm).</p

Alternative splicing of Munc18-1 does not affect spontaneous release.

<p>Munc18-1a or -1b was reintroduced using lentivirus in autaptic hippocampal <i>munc18-1</i> null neurons cultured on glia islands. (A) Example traces of spontaneous release of synaptic vesicles in the absence of stimulation. (B) Quantification of the mEPSC size and decay kinetics. mEPSC size (M18-1b: 18.11 ± 0.88 pA, n = 46; M18-1a: 16.29 ± 0.71 pA, n = 39, Unpaired t test with Welch correction, p = 0.11). mEPSC decay (M18-1b: 3.18 ± 0.15, n = 46; M18-1a: 2.84 ± 0.12, n = 39, Unpaired t test with Welch correction, p = 0.08). (C) Both splice variants support spontaneous release at similar rate. mEPSC frequency (M18-1b: 5.60 ± 1.29 Hz, n = 46; M18-1a: 6.21 ± 1.74 Hz, n = 39, Mann-Whitney Test, p = 0.5665.</p

Alternative splicing of Munc18-1 does not affect basal evoked release.

<p>Munc18-1a or -1b was reintroduced using lentivirus in autaptic hippocampal <i>munc18-1</i> null neurons cultured on glia islands. (A) Typical examples of paired pulse traces, traces from different intervals are plotted superimposed. (B) Both splice variants support evoked release to the same extent. Initial EPSC size (M18-1b: 3.21 ± 0.31 nA, n = 45; M18-1a: 3.50 ± 0.35 nA, n = 38, Unpaired t test, p = 0.54). (C) Quantification of the RRP estimate derived from back-extrapolation of the cumulative charge released during a RRP depleting train (100 pulses at 40 Hz) (M18-1b: 377.8 ± 52.7 pC, n = 35; M18-1a: 287.3 ± 33.3, n = 29, Unpaired t test with Welch correction, p = 0.15). (D) Quantification of the paired pulse ratio for different intervals (20 ms interval: M18-1b, 0.89 ± 0.04 EPSC^2nd/EPSC^1st; M18-1a, 0.79 ± 0.03 EPSC^2nd/EPSC^1st, Unpaired t test, p = 0.060).</p

Rate of EPSC depression during STP protocols depends on the splice variant expressed.

<p>Munc18-1a or -1b was reintroduced using lentivirus in autaptic hippocampal <i>munc18-1</i> null neurons cultured on glia islands. (A) Quantification of EPSC depression during 2.5 Hz stimulation normalized to the first EPSC amplitude (inset shows absolute value of the first EPSC). (B) Quantification of EPSC depression during 5 Hz stimulation normalized to the first EPSC amplitude (inset shows absolute value of the first EPSC). (C) Quantification of EPSC depression during 10 Hz stimulation normalized to the first EPSC amplitude. Left inset shows absolute value of the first EPSC. Right inset shows the relative depression at the end of the train normalized to the first EPSC (M18-1b, 0.26 ± 0.03 EPSC^#100/EPSC^#1; M18-1a, 0.20 ± 0.02 EPSC^#100/EPSC^#1, Unpaired t test with Welch correction, p = 0.042). (D) Quantification of the build-up of asynchronous charge during 10 Hz stimulation. (E-F) EPSC depression during two stimulation trains (100 pulses at 40Hz) given with a 2 seconds inter-train interval. (E) Shown are the responses to the first five pulses in the train, normalized to the first pulse. Inset shows the recovery of EPSC amplitude after two seconds (1^st EPSC of 2^nd train / 1^st EPSC of 1^st train). (F) Quantification of charge transferred within 25ms after each pulse relative to the first pulse in a train. Left inset shows the absolute EPSC charge at the start of the first train, right inset shows the first 5 pulses.</p

Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins

Synapses are specialized neuronal cell-cell contacts that underlie network communication in the mammalian brain. Across neuronal populations and circuits, a diverse set of synapses is utilized, and they differ in their molecular composition to enable heterogenous connectivity patterns and functions. In addition to pre- and post-synaptic specializations, the synaptic cleft is now understood to be an integral compartment of synapses that contributes to their structural and functional organization. Aiming to map the cleft proteome, this study applied a peroxidase-mediated proximity labeling approach and used the excitatory synaptic cell adhesion protein SynCAM 1 fused to horseradish peroxidase (HRP) as a reporter in cultured cortical neurons. This reporter marked excitatory synapses as measured by confocal microcopy and was targeted to the edge zone of the synaptic cleft as determined using 3D dSTORM super-resolution imaging. Proximity labeling with a membrane-impermeant biotin-phenol compound restricted labeling to the cell surface, and Label-Free Quantitation (LFQ) mass spectrometry combined with ratiometric HRP tagging of membrane vs. synaptic surface proteins was used to identify the proteomic content of excitatory clefts. Novel cleft candidates were identified, and Receptor-type tyrosine-protein phosphatase zeta was selected and successfully validated. This study supports the robust applicability of peroxidase-mediated proximity labeling for synaptic cleft proteomics and its potential for understanding synapse heterogeneity in health and changes in diseases such as psychiatric disorders and addiction

Southern blots and J-immunoblots of genomic DNA of and (bloodstream form) digested with a variety of frequently cutting restriction enzymes

<p><b>Copyright information:</b></p><p>Taken from "Telomeric localization of the modified DNA base J in the genome of the protozoan parasite "</p><p></p><p>Nucleic Acids Research 2007;35(7):2116-2124.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874636.</p><p>© 2007 The Author(s)</p> The DNA was treated as in . The blot was incubated with the J-antisera (α-J) followed by hybridization with a telomeric probe (telo). () Southern blot of genomic DNA of . () Southern blot of genomic DNA of (bloodstream form). The bands migrating at the top of the lanes 1, 2 and 4 that strongly react with the J-antisera are probably due to the 50 bp repeats as these are digested by RsaI (which was used in the lanes 3, 5, 6 and 7). Lanes 1. AluI, HpaII, BsrGI, HaeII; 2. AluI, HpaII, Sau3AI, TaqI; 3. AluI, HpaII, CfoI, RsaI; 4. BsrGI, HaeII, Sau3AI, Taq I; 5. BsrGI, HaeII, CfoI, RsaI; 6. Sau3AI, RsaI, CfoI, TaqI; 7. AluI, HpaII, BsrGI, HaeII, Sau3AI, RsaI, CfoI, TaqI. ND stands for not digested

Southern blot and J-immunoblot of genomic DNA of various kinetoplastid parasites digested with frequently cutting restriction enzymes

<p><b>Copyright information:</b></p><p>Taken from "Telomeric localization of the modified DNA base J in the genome of the protozoan parasite "</p><p></p><p>Nucleic Acids Research 2007;35(7):2116-2124.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874636.</p><p>© 2007 The Author(s)</p> DNA was digested with the restriction enzymes AluI, AvaII, CfoI, HinfI, RsaI, SspI, size-fractionated and blotted as described in . The left panel shows the result after incubation with the J-antiserum (α-J). The right panel shows the result of the hybridization with the telomeric probe (telo)

Southern blot and J-immunoblot of genomic DNA of Friedlin, and digested with frequently cutting restriction enzymes

<p><b>Copyright information:</b></p><p>Taken from "Telomeric localization of the modified DNA base J in the genome of the protozoan parasite "</p><p></p><p>Nucleic Acids Research 2007;35(7):2116-2124.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874636.</p><p>© 2007 The Author(s)</p> () Genomic DNA of Friedlin was digested with the enzymes AluI, BsrGI, BstUI, CfoI, HaeII, HpaII, Sau3AI and TaqI and size-fractionated by electrophoresis in an agarose gel and blotted on a nylon membrane. The blot was incubated with the J-antiserum (α-J) and after analysis, hybridized with telomeric (telo) and LST-RA radioactively labeled DNA oligonucleotide probes. () Southern blot and J-immunoblot of genomic DNA of , , and . DNA of was digested with AluI, AvaII, CfoI, HinfI, RsaI, SspI. DNA of and was digested with the enzymes listed in A. The combination of restriction enzymes was optimized to digest the greatest variety of DNA repetitive sequences. The left panel shows the result after incubation with the J-antiserum (α-J). The right panel shows the result of the hybridization with the telomeric probe (telo)