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Research Article
Aberrant function and structure of retinal ribbon synapses in the absence of complexin 3 and complexin 4 Kerstin Reim1, Hanna Regus-Leidig2, Josef Ammermüller3, Ahmed El-Kordi4, Konstantin Radyushkin4, Hannelore Ehrenreich4, Johann Helmut Brandstätter2,* and Nils Brose1,* 1
Department of Molecular Neurobiology and Center for the Molecular Physiology of the Brain, Max Planck Institute of Experimental Medicine, D-37075 Göttingen, Germany 2 Department of Biology, Animal Physiology, University of Erlangen-Nuremberg, D-91058 Erlangen, Germany 3 AG Neurobiology, Carl von Ossietzky University of Oldenburg, D-26111 Oldenburg, Germany 4 Division of Clinical Neuroscience and Center for the Molecular Physiology of the Brain, Max Planck Institute of Experimental Medicine, D-37075 Göttingen, Germany *Authors for correspondence (e-mails:
[email protected];
[email protected])
Journal of Cell Science
Accepted 10 January 2009 Journal of Cell Science 122, 1352-1361 Published by The Company of Biologists 2009 doi:10.1242/jcs.045401
Summary Complexins regulate the speed and Ca2+ sensitivity of SNAREmediated synaptic vesicle fusion at conventional synapses. Two of the vertebrate complexins, Cplx3 and Cplx4, are specifically localized to retinal ribbon synapses. To test whether Cplx3 and Cplx4 contribute to the highly efficient transmitter release at ribbon synapses, we studied retina function and structure in Cplx3 and Cplx4 single- and double-knockout mice. Electroretinographic recordings from single and double mutants revealed a cooperative perturbing effect of Cplx3 and Cplx4 deletion on the b-wave amplitude, whereas most other detected effects in both plexiform synaptic layers were additive. Light and electron microscopic analyses uncovered a disorganized outer plexiform layer in the retinae of mice lacking Cplx3 and Cplx4, with a significant proportion of photoreceptor terminals
Key words: Complexin, Exocytosis, SNARE, Ribbon synapse, ERG, Photoreceptors, Bipolar cells
Introduction Ca2+-triggered fusion of synaptic vesicles is one of the most tightly regulated membrane fusion reactions. It is executed by SNARE complexes, which in most neurons contain the vesicle protein synaptobrevin 2 (also known as Vamp2), and the plasma membrane proteins SNAP25 and syntaxin 1 (Jahn and Scheller, 2006). SNARE complex function at neuronal synapses is controlled by regulatory proteins, which determine the extreme speed and accuracy of synaptic excitation-secretion coupling (Wojcik and Brose, 2007). Complexins are prominent representatives of this group of SNARE regulators (Brose, 2008). Complexins bind and stabilize assembled SNARE complexes (Bracher at al., 2002; Chen et al., 2002). Their deletion in mice causes profound deficits in release probability, evoked transmitter release and reduced spontaneous release at neuronal synapses (Reim et al., 2001; Xue et al., 2007; Xue et al., 2008), as well as reduced secretory granule fusion in adrenal chromaffin cells (Cai et al., 2008), which indicates that complexins act as positive regulators at or following the Ca2+-triggering step of synaptic vesicle fusion. The notion that complexins act as facilitators of SNARE-mediated secretory vesicle fusion is also supported by the observation that overexpression of Cplx2 in wild-type chromaffin cells increases chromaffin granule secretion (Cai et al., 2008). However, data obtained from in vitro fusion assays and the phenotype of
complexin-deficient Drosophila mutants are more compatible with a role of complexins as inhibitory pre-fusion clamps that maintain SNARE complexes in a highly fusogenic state but prevent them from executing fusion until triggered by Ca2+ (Giraudo et al., 2006; Schaub et al., 2006; Tang et al., 2006; Huntwork and Littleton, 2007). It is therefore possible that complexins exert both positive and negative regulatory effects on SNARE function, the relative contribution of which may differ between organisms and cell types (Xue et al., 2007; Brose, 2008). Mice have four complexin genes (Cplx1-Cplx4), three of which (Cplx1-Cplx3) are expressed in brain (Reim et al., 2005). Cplx3 expression in mouse brain is very low and does not contribute significantly to synaptic transmission in the brain regions tested so far (Xue et al., 2008). All four complexin isoforms are expressed in the retina, where Cplx1 and Cplx2 are found in conventional synapses of amacrine cells, whereas Cplx3 and Cplx4 are predominantly expressed in ribbon synapses of photoreceptors and bipolar cells (Reim et al., 2005). All complexins bind SNARE complexes and their overexpression rescues the phenotypic deficits of hippocampal neurons lacking Cplx1 and Cplx2, indicating that the basic functional properties of complexins are similar in conventional synapses (Reim et al., 2005). Ribbon synapses are a specialized subclass of chemical synapses characterized by a presynaptic plate-like organelle, the ribbon, which
containing spherical free-floating ribbons. These structural and functional aberrations were accompanied by behavioural deficits indicative of a vision deficit. Our results show that Cplx3 and Cplx4 are essential regulators of transmitter release at retinal ribbon synapses. Their loss leads to aberrant adjustment and fine-tuning of transmitter release at the photoreceptor ribbon synapse, alterations in transmission at bipolar cell terminals, changes in the temporal structure of synaptic processing in the inner plexiform layer of the retina and perturbed vision.
Journal of Cell Science
Role of Cplx3 and Cplx4 in the retina is anchored to the plasma membrane in close vicinity to voltage-gated Ca2+-channels (tom Dieck et al., 2005). Ribbons are typically surrounded by a large number of synaptic vesicles, which can be morphologically divided into docked vesicles that contact the active zone, tethered vesicles that cluster on the surface of the ribbon but do not touch the plasma membrane, and free vesicles that are distributed in the synaptic cytoplasm. Such large vesicle pools are obviously necessary to support the high rates of synaptic vesicle fusion that enable ribbon synapses to respond precisely to graded changes in membrane potentials (Parsons and Sterling, 2003; Heidelberger et al., 2005; Sterling and Matthews, 2005), but little is known about the molecular composition and the functional properties of the presynaptic fusion machinery that determine the unique characteristics of retinal ribbon synapses. To test whether Cplx3 and Cplx4 contribute to the unique efficacy of transmitter release at retinal ribbon synapses, we studied retina function in mice lacking Cplx3 and/or Cplx4. Results Generation of Cplx4 single-knockout and Cplx3/4 double-knockout mice
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Fig. 1. Mutation of the murine Cplx4 gene. (A) Deletion of Cplx4 in the mouse. Maps of the wildtype Cplx4 gene, the respective targeting vector, and the resulting mutant gene. Exons 1 and 2 (black boxes, E1,E2), the position of the outside probe (OP) used to identify the mutant allele (open bar), and diagnostic restriction enzyme sites are indicated. Neo, neomycin resistance gene; TK, thymidine kinase gene. (B) Southern blot analysis of Cplx4 deletion in mice. Tail DNA from an adult wild-type mouse (+/+), and mice heterozygous (+/–) or homozygous (–/–) for the Cplx4 mutation were analyzed as described in Materials and Methods. Positions of wild-type (WT) and knockout (KO) bands are indicated. (C) Analysis of Cplx4 expression in wild-type, heterozygous and homozygous mice. Brain and retina homogenates (10 μg protein per lane) from adult wild-type (+/+), heterozygous (+/–), and homozygous (–/–) mice were analyzed by SDS-PAGE and immunoblotting using an anti-Cplx4 antibody. The position of Cplx4 is indicated. (D) Analysis of Cplx3 expression in retina of wild-type, heterozygous and homozygous Cplx3 mutant mice. Retina homogenates (10 μg protein per lane) from adult wild-type (+/+), heterozygous (+/–), and homozygous (–/–) mice were analyzed by SDS-PAGE and immunoblotting using an anti-Cplx3 antibody. The position of Cplx3 is indicated. (E) Expression of Cplx3 and Cplx4 in retina of Cplx3/4 double deletion mutant mice generated from two double heterozygous parents. Retina extracts (10 μg protein per lane) of adult wild-type (+/+, +/+), Cplx3 knockout (–/–, +/+), Cplx4 knockout (+/+, –/–), and double deletion mutant mice (–/–, –/–) were analyzed by SDS-PAGE and immunoblotting using anti-Cplx3 and anti-Cplx4 antibodies, respectively. Syntaxin 3 served as a loading control.
Cplx4 single-knockout mice were generated by homologous recombination (Fig. 1A). To confirm the mutation of the Cplx4 gene, Southern blot analyses of offspring resulting from interbreeding of mice heterozygous for the Cplx4 mutation were performed (Fig. 1B). The results demonstrated that the respective genotypes (wild-type +/+, heterozygous +/–, homozygous –/–) appeared in the expected mendelian frequency. Western blot analysis using extracts of adult retinae showed that Cplx4 expression is completely abolished in homozygous mutants whereas protein levels in heterozygous animals were reduced (Fig. 1C). As expected, Cplx4 protein was not detectable in wild-type or mutant brains (Fig. 1C) (Reim et al., 2005). As we described previously, deletion of the Cplx3 gene leads to complete loss of Cplx3 protein in brain (Xue et al., 2008). Western blot analyses using extracts from adult retinae showed that expression of Cplx3 is abolished in homozygous Cplx3 knockouts (Fig. 1D). Double mutants lacking Cplx3 and Cplx4 were generated by interbreeding of parents double heterozygous for the Cplx3 and Cplx4 mutations. Again, standard western blot analyses of retinae from adult animals confirmed that expression of Cplx3 and Cplx4 is abolished in homozygous double deletion mutants (Fig. 1E). Mice lacking Cplx3 or Cplx4 and double-knockout animals showed no obvious phenotypic changes and bred normally. To assess changes in protein composition of mutant synapses, we compared the levels of several synaptic proteins (Cplx1-Cplx4, syntaxin 1, syntaxin 2, syntaxin 3, SNAP25, synaptobrevin 2, Bassoon, Ribeye, synaptophysin, synaptotagmin 1, VGLUT1, VIAAT, RIM, Munc131–Munc13-3) in mutant and wild-type retinae by western blotting (Fig. 2). Except for the ribbon-specific structural protein Ribeye, for which we observed lower levels in the Cplx3/4 double-knockout
mutants, the amounts of tested proteins were comparable between the three different types of homozygous knockouts and their respective wild-type controls. To study the altered Ribeye expression levels in more detail and to test whether the deletion of Cplx3 and Cplx4 causes subtle alterations of Cplx1 and Cplx2 expression that are not detectable by standard western blotting, we analyzed the amounts of Cplx1 and Cplx2 in retina extracts of Cplx3/4 double mutants by quantitative western blot analysis. Because Cplx3 and Cplx4 are ribbon-synapsespecific SNARE-regulating proteins, we also examined whether their lack influences the expression of key retinal SNARE complex components (syntaxin 1, syntaxin 3, SNAP25, synaptobrevin 2) or structural proteins such as the active zone cytomatrix protein Bassoon. The results of these quantitative analyses, expressed as a percentage of wild-type levels and summarized in Table 1, showed that only the Ribeye expression levels in Cplx3/4 double mutants were altered significantly (62.5±9.1% of wild-type levels, n=3, P
