GlyT1 determines the glycinergic phenotype of amacrine cells in the mouse retina.

The amino acid glycine acts as a neurotransmitter at both inhibitory glycinergic and excitatory glutamatergic synapses predominantly in caudal regions of the central nervous system but also in frontal brain regions and the retina. After its presynaptic release and binding to postsynaptic receptors at caudal glycinergic synapses, two high-affinity glycine transporters GlyT1 and GlyT2 remove glycine from the extracellular space. Glycinergic neurons express GlyT2, which is essential for the presynaptic replenishment of the transmitter, while glial-expressed GlyT1 was shown to control the extracellular glycine concentration. Here we show that GlyT1 expressed by glycinergic amacrine cells of the retina does not only contribute to the control of the extracellular glycine concentration in the retina but is also essential for the maintenance of the glycinergic transmitter phenotype of this cell population. Specifically, loss of GlyT1 from the glycinergic AII amacrine cells impairs AII-mediated glycinergic neurotransmission and alters regulation of the extracellular glycine concentration, without changes in the overall distribution and/or size of glycinergic synapses. Taken together, our results suggest that GlyT1 expressed by amacrine cells in the retina combines functions covered by neuronal GlyT2 and glial GlyT1 at caudal glycinergic synapses.

Identification and immunocytochemical characterization of Piccolino, a novel Piccolo splice variant selectively expressed at sensory ribbon synapses of the eye and ear.

Piccolo is one of the largest cytomatrix proteins present at active zones of chemical synapses, where it is suggested to play a role in recruiting and integrating molecules relevant for both synaptic vesicle exo- and endocytosis. Here we examined the retina of a Piccolo-mutant mouse with a targeted deletion of exon 14 in the Pclo gene. Piccolo deficiency resulted in its profound loss at conventional chemical amacrine cell synapses but retinal ribbon synapses were structurally and functionally unaffected. This led to the identification of a shorter, ribbon-specific Piccolo variant, Piccolino, present in retinal photoreceptor cells, bipolar cells, as well as in inner hair cells of the inner ear. By RT-PCR analysis and the generation of a Piccolino-specific antibody we show that non-splicing of intron 5/6 leads to premature translation termination and generation of the C-terminally truncated protein specifically expressed at active zones of ribbon synapse containing cell types. With in situ proximity ligation assays we provide evidence that this truncation leads to the absence of interaction sites for Bassoon, Munc13, and presumably also ELKS/CAST, RIM2, and the L-type Ca(2) (+) channel which exist in the full-length Piccolo at active zones of conventional chemical synapses. The putative lack of interactions with proteins of the active zone suggests a function of Piccolino at ribbon synapses of sensory neurons different from Piccolo's function at conventional chemical synapses.

Nxnl2 splicing results in dual functions in neuronal cell survival and maintenance of cell integrity.

The rod-derived cone viability factors, RdCVF and RdCVF2, have potential therapeutical interests for the treatment of inherited photoreceptor degenerations. In the mouse lacking Nxnl2, the gene encoding RdCVF2, the progressive decline of the visual performance of the cones in parallel with their degeneration, arises due to the loss of trophic support from RdCVF2. In contrary, the progressive loss of rod visual function of the Nxnl2-/- mouse results from a decrease in outer segment length, mediated by a cell autonomous mechanism involving the putative thioredoxin protein RdCVF2L, the second spliced product of the Nxnl2 gene. This novel signaling mechanism extends to olfaction as shown by the progressive impairment of olfaction in aged Nxnl2-/- mice and the protection of olfactory neurons by RdCVF2. This study shows that Nxnl2 is a bi-functional gene involved in the maintenance of both the function and the viability of sensory neurons.

Intraflagellar transport proteins in ciliogenesis of photoreceptor cells.

BACKGROUND INFORMATION: The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian photoreceptor cilia. RESULTS: Electron microscopy revealed that ciliogenesis in mouse photoreceptor cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron-dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light-sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of photoreceptor cell development. CONCLUSIONS: Our data conclusively provide evidence for the participation of IFT proteins in photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well-established function for IFT in mature cilia and flagella.

TOPORS, implicated in retinal degeneration, is a cilia-centrosomal protein.

We recently reported that mutations in the widely expressed nuclear protein TOPORS (topoisomerase I-binding arginine/serine rich) are associated with autosomal dominant retinal degeneration. However, the precise localization and a functional role of TOPORS in the retina remain unknown. Here, we demonstrate that TOPORS is a novel component of the photoreceptor sensory cilium, which is a modified primary cilium involved with polarized trafficking of proteins. In photoreceptors, TOPORS localizes primarily to the basal bodies of connecting cilium and in the centrosomes of cultured cells. Morpholino-mediated silencing of topors in zebrafish embryos demonstrates in another species a comparable retinal problem as seen in humans, resulting in defective retinal development and failure to form outer segments. These defects can be rescued by mRNA encoding human TOPORS. Taken together, our data suggest that TOPORS may play a key role in regulating primary cilia-dependent photoreceptor development and function. Additionally, it is well known that mutations in other ciliary proteins cause retinal degeneration, which may explain why mutations in TOPORS result in the same phenotype.

Optimized recombinant dense bodies of human cytomegalovirus efficiently prime virus specific lymphocytes and neutralizing antibodies without the addition of adjuvant.

Control of human cytomegalovirus (HCMV) infection correlates with the reconstitution of antiviral T lymphocytes in haematopoietic stem cell transplant recipients. A vaccine to foster this reconstitution and to ameliorate the severe consequences of HCMV reactivation is yet unavailable. This work focused on providing a rationale for the amendment of the yields and the antigenic composition of a vaccine, based on subviral dense bodies (DB) of HCMV. Modified DB were generated that contained the HLA-A2 presented IE1 model peptide TMYGGISLL, integrated at different positions in the major DB protein pp65. Insertion at position W175 of pp65 allowed efficient formation of recDB in the cytoplasm of infected cells and resulted in considerable yields of these particles. Even in the absence of adjuvant, these particles proved to be highly immunogenic with respect to CD8 and CD4 T cell and neutralizing antibody responses.

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells.

The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies. Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65-VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus. The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function.

Intraflagellar transport molecules in ciliary and nonciliary cells of the retina.

The assembly and maintenance of cilia require intraflagellar transport (IFT), a process mediated by molecular motors and IFT particles. Although IFT is a focus of current intense research, the spatial distribution of individual IFT proteins remains elusive. In this study, we analyzed the subcellular localization of IFT proteins in retinal cells by high resolution immunofluorescence and immunoelectron microscopy. We report that IFT proteins are differentially localized in subcompartments of photoreceptor cilia and in defined periciliary target domains for cytoplasmic transport, where they are associated with transport vesicles. IFT20 is not in the IFT core complex in photoreceptor cilia but accompanies Golgi-based sorting and vesicle trafficking of ciliary cargo. Moreover, we identify a nonciliary IFT system containing a subset of IFT proteins in dendrites of retinal neurons. Collectively, we provide evidence to implicate the differential composition of IFT systems in cells with and without primary cilia, thereby supporting new functions for IFT beyond its well-established role in cilia.

Different roles for KIF17 and kinesin II in photoreceptor development and maintenance.

Kinesin 2 family members are involved in transport along ciliary microtubules. In Caenorhabditis elegans channel cilia, kinesin II and OSM-3 cooperate along microtubule doublets of the axoneme middle segment, whereas OSM-3 alone works on microtubule singlets to elongate the distal segment. Among sensory cilia, vertebrate photoreceptors share a similar axonemal structure with C. elegans channel cilia, and deficiency in either kinesin II or KIF17, the homologue of OSM-3, results in disruption of photoreceptor organization. However, direct comparison of the two effects is confounded by the use of different species and knockdown strategies in prior studies. Here, we directly compare the effects of dominant-negative kinesin II and KIF17 expression in zebrafish cone photoreceptors. Our data indicate that dominant-negative kinesin II disrupts function at the level of the inner segment and synaptic terminal and results in cell death. In contrast, dominant-negative KIF17 has no obvious effect on inner segment or synaptic organization but has an immediate impact on outer segment assembly.

Immunoelectron microscopy of vesicle transport to the primary cilium of photoreceptor cells.

Cilia are organelles of high structural complexity. Since the biosynthetic machinery is absent from cilia all their molecular components must be synthesized in organelles of the cytoplasm and subsequently transported to the cilium. Ciliary cargos are thought to be translocated in the membrane of transport vesicles or association with these vesicles to the base of the cilium where the vesicles fuse with the periciliary target membrane for further delivery of their cargo into the ciliary compartment by the intraflagellar transport (IFT). Here we describe a modified preembedding labeling method as an alternative technique to conventional postembedding methods eligible for analyses of ciliary cargo vesicles and the distribution of ciliary molecules in subciliary compartments for immunoelectron microscopy. The preembedding labeling method preserves the antigenicity of ciliary antigens and its application reveals differential localization of individual IFT proteins in vertebrate photoreceptor cilia. Since membrane vesicles are conserved, the preembedding protocol additionally allows the identification of ciliary cargo vesicles by immunolabeling of individual IFT proteins and ciliary targeting molecules in ciliary photoreceptor cells. These results do not only confirm the central function of IFT molecules in ciliary transport, but further strengthen their role in transport processes in the cytoplasm. Furthermore, evidence for different alternative transport routes of cargo vesicles directed to different target membranes is gathered.

Mutations in LCA5, encoding the ciliary protein lebercilin, cause Leber congenital amaurosis.

Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We detected homozygous nonsense and frameshift mutations in LCA5 in five families affected with LCA. In a sixth family, the LCA5 transcript was completely absent. LCA5 is expressed widely throughout development, although the phenotype in affected individuals is limited to the eye. Lebercilin localizes to the connecting cilia of photoreceptors and to the microtubules, centrioles and primary cilia of cultured mammalian cells. Using tandem affinity purification, we identified 24 proteins that link lebercilin to centrosomal and ciliary functions. Members of this interactome represent candidate genes for LCA and other ciliopathies. Our findings emphasize the emerging role of disrupted ciliary processes in the molecular pathogenesis of LCA.