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. 2010 Apr 6;107(14):6504-9.
doi: 10.1073/pnas.1002307107. Epub 2010 Mar 23.

Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis

Konark Mukherjee  1 , Xiaofei Yang, Stefan H Gerber, Hyung-Bae Kwon, Angela Ho, Pablo E Castillo, Xinran Liu, Thomas C Südhof
Affiliations

Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis

Konark Mukherjee et al. Proc Natl Acad Sci U S A. .

Abstract

Piccolo and bassoon are highly homologous multidomain proteins of the presynaptic cytomatrix whose function is unclear. Here, we generated piccolo knockin/knockout mice that either contain wild-type levels of mutant piccolo unable to bind Ca(2+) (knockin), approximately 60% decreased levels of piccolo that is C-terminally truncated (partial knockout), or <5% levels of piccolo (knockout). All piccolo mutant mice were viable and fertile, but piccolo knockout mice exhibited increased postnatal mortality. Unexpectedly, electrophysiology and electron microscopy of piccolo-deficient synapses failed to uncover a major phenotype either in acute hippocampal slices or in cultured cortical neurons. To unmask potentially redundant functions of piccolo and bassoon, we thus acutely knocked down expression of bassoon in wild-type and piccolo knockout neurons. Despite a nearly complete loss of piccolo and bassoon, however, we still did not detect an electrophysiological phenotype in cultured piccolo- and bassoon-deficient neurons in either GABAergic or glutamatergic synaptic transmission. In contrast, electron microscopy revealed a significant reduction in synaptic vesicle clustering in double bassoon/piccolo-deficient synapses. Thus, we propose that piccolo and bassoon play a redundant role in synaptic vesicle clustering in nerve terminals without directly participating in neurotransmitter release.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structures of piccolo and bassoon, and generation of piccolo KO and KI mice. (A) Comparison of piccolo and bassoon domain structures. Homologous regions are indicated as % identity between piccolo and bassoon (Zn, zinc-finger domains; CC, predicted coiled-coil domains). Epitope locations for the two piccolo antibodies used are shown above the piccolo structure, and the C-terminal region in the piccolo C2A-domain that is encoded by exon 14, and targeted in our genetic experiments, is shown below the diagram, as is the even more C-terminal alternatively spliced area. For bassoon, the central region that is deleted in the bassoon KO (20) is boxed. (B) Generation of piccolo KO and KI mice. Genomic clones of piccolo containing exon 14 (plmPic1 and plmPic4) were used to generate a targeting vector that includes a neomycin gene resistance cassette for positive selection (NEO, flanked by flp recombination sites) and a diphtheria gene cassette for negative selection (DT). Exon 14 was flanked by loxP recombination sites and mutated to substitute the two Ca2+-binding aspartate residues in loop 1 for alanines [14* (9)]. (C) Mice containing the mutant piccolo gene were generated by homologous recombination (#1). Cre-recombinase-mediated excision of exon 14 causes suppression of piccolo expression (referred as piccolo KO mice; #2). Flp-recombinase-mediated excision of the neomycin resistance cassette reactivates piccolo expression, resulting in piccolo KI mice in which wild-type levels of piccolo with a mutant Ca2+-binding site in the C2A-domain are expressed (#3). Finally, combined flp- and cre-recombinase mediated excision of both the neomycin resistance cassette and exon 14 results in the partial expression of truncated piccolo lacking the C2A- and C2B-domains (#4). (D) Immunoblot analysis of the indicated active zone proteins in brain homogenates from wild-type (WT), homozygous KI (PicKI/KI), heterozygous KO (PicWT/KO), homozygous KO (PicKO/KO), and homozygous partial KO (PicPKO/PKO) mice. Blots were probed with 125I-labeled secondary antibodies, and signals were visualized with a phosphorimager. (E) Quantitation of active zone protein levels, as shown in D, in three independent experiments (mean ± SEM; n = 3; *, P < 0.05 by Student's t test; n.s., not significant).
Fig. 2.
Fig. 2.
Survival and weight of piccolo KO mice. (A and B) Survival of the offspring of crosses between heterozygous piccolo KO mice (PicWT/KO), plotted as a function of genotype and analyzed at P21 (A) or P60 (B). The actual (black bars) and expected (gray bars, based on Mendelian inheritance) distribution of genotypes for wild-type (WT), heterozygous (PicWT/KO), and homozygous KO (PicKO/KO) mice are shown. The indicated P values were calculated using a χ-test comparing observed and expected distributions (n = 133). (C) Plot of the observed mortality of littermate wild-type (WT), heterozygous (PicWT/KO), and homozygous piccolo KO (PicKO/KO) mice at P60. Indicated P value was calculated using a χ-test (n = 133). (D) Representative image of littermate male wild-type (WT) and piccolo KO (PicKO/KO) mice. (E and F) Weight curves of littermate female (E) and male (F) piccolo wild-type (WT), heterozygous (PicWT/KO), and homozygous piccolo KO (PicKO/KO) mice. Data shown are mean ± SEM; indicated P values were calculated using a two-tailed paired t test (n, number of mice in each group).
Fig. 3.
Fig. 3.
Inhibitory synaptic transmission in piccolo- and bassoon-deficient neurons. All experiments were performed on cortical neurons cultured from littermate wild-type and piccolo KO mice, and infected with lentiviruses. WT and BasKD, cortical neurons cultured from wild-type mice and infected with control lentiviruses or lentivirus expressing the bassoon shRNA, respectively; PicKO and PicKO+BasKD, cortical neurons cultured from piccolo KO mice and infected with control lentiviruses or lentivirus expressing the bassoon shRNA, respectively. Neurons were infected twice (at DIV2 and at DIV8) and analyzed at DIV14–DIV16 by voltage-clamp whole-cell recordings. (A) Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) monitored in the presence of 1 μM tetrodotoxin, 10 μM CNQX, and 50 μM APV. Representative traces are shown on the left, and summary graphs of the mIPSC frequency and amplitudes are shown on the right. (B) Inhibitory postsynaptic currents (IPSCs) evoked by isolated action potentials elicited by a local electrode in 20 μM CNQX and 50 μM APV (27). Representative traces are shown on the left, and summary graphs of the IPSC amplitudes are shown on the right. (C) IPSCs evoked by a 10-Hz, 1-s stimulus train in the presence of 10 μM CNQX and 50 μM APV. Representative traces are shown on the left, summary graphs of the total charge transfer induced by the train are shown in the center, and the degree of synaptic depression as a function of the action potential number is plotted on the right. (D) IPSCs evoked by a 30-s application of 0.5 M sucrose in the presence of 1 μM tetrodotoxin, 20 μM CNQX, and 50 μM APV to measure the size of the readily releasable pool (RRP). Representative traces are depicted on the left, and summary graphs of the total synaptic charge transfer during the first 10 s (which measures the RRP) are shown on the right. All data shown are mean ± SEM; numbers in bars list the total number of neurons recorded in at least three independent experiments. P-value calculations using Student's t test revealed that none of the manipulations induced a statistically significant change in synaptic responses (n.s., nonsignificant).
Fig. 4.
Fig. 4.
Ultrastructure of synapses lacking piccolo and bassoon. (A–F) Representative electron micrographs of synapses in cortical neurons that were cultured from wild-type mice and infected with control lentiviruses (Wild-type; A, C, and D), and from littermate piccolo KO mice and infected with lentivirus expressing bassoon shRNA (PicKO + BasKD; B, E, and F). Neurons were infected twice (at DIV2 and at DIV8) and analyzed at DIV14. [Scale bars: C (for A–C), 600 nm; F (for E and F), 400 nm.] (G–O) Quantifications of synaptic parameters in anonymized electron micrographs of cultured cortical neurons. Two sets of neurons were analyzed separately: neurons from littermate wild-type and piccolo KO mice that were not infected with lentiviruses (WT, wild-type neurons; PicKO, piccolo KO neurons), and neurons from wild-type mice that were infected with control lentivirus (WT + control) or from littermate piccolo KO mice that were infected with lentivirus expressing the bassoon shRNA (PicKO + BasKD). (G) Average number of synaptic vesicles per bouton. (H) Average density of synaptic vesicles in the terminal. (I) Average bouton area. (J) Average size of synaptic vesicles. (K) Average number of synaptic vesicles within 150 nm of the active zone. (L) Average number of docked vesicles per active zone. (M) Average PSD length. (N) Average number of docked vesicles per PSD length. (O) Percentage of presynaptic terminals containing a vesicle cluster. Parameters are shown as mean ± SEM (n = 75 synapses) from a single experiment that was independently repeated once for the double-deficient comparison with comparable results (Fig. S5). Statistical significance was assessed by Student's t test (n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001).
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