In vivo knockdown of Piccolino disrupts presynaptic ribbon morphology in mouse photoreceptor synapses

Regus-Leidig, Hanna; Fuchs, Michaela; Löhner, Martina; Leist, Sarah R.; Leal-Ortiz, Sergio; Chiodo, Vince A.; Hauswirth, William W.; Garner, Craig C.; Brandstätter, Johann Helmut

doi:10.3389/fncel.2014.00259

Cited by 50 publications

(60 citation statements)

References 47 publications

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“…Alternatively, numerous structural proteins have been shown to localize to the ribbon complex (Brandstatter et al, 1999), including Piccolo, Bassoon, Cast/ERC, Rim1, Rim2, but each of these proteins are also found at conventional synapses, which lack the ribbon structure. Of note, a ribbon-specific isoform of Piccolo has been described in retina (Regus-Leidig et al, 2014b, Regus-Leidig et al, 2013), suggesting that ribbon-specific splicing may underlie some of the diversity in ribbon shape and size.…”

Section: Discussionmentioning

confidence: 99%

Synaptic Ribbons Require Ribeye for Electron Density, Proper Synaptic Localization, and Recruitment of Calcium Channels

et al. 2016

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Summary Synaptic ribbons are structures made largely of the protein Ribeye that hold synaptic vesicles near release sites in non-spiking cells in some sensory systems. Here we introduce frame-shift mutations in the two zebrafish genes encoding for Ribeye and thus remove Ribeye protein from neuromast hair cells. Despite Ribeye depletion, vesicles collect around ribbon-like structures that lack electron density, which we term “ghost-ribbons”. Ghost-ribbons are smaller in size, but possess a similar number of smaller vesicles and are poorly localized to synapses and calcium channels. These hair cells exhibit enhanced exocytosis as measured by capacitance, and recordings from afferent neurons post-synaptic to hair cells show no significant difference in spike rates. Our results suggest that Ribeye makes up most of the synaptic ribbon density in neuromast hair cells, and is necessary for proper localization of calcium channels and synaptic ribbons.

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Section: Discussionmentioning

confidence: 99%

Synaptic Ribbons Require Ribeye for Electron Density, Proper Synaptic Localization, and Recruitment of Calcium Channels

et al. 2016

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“…Recent studies have begun to unravel the structural and molecular underpinnings of the photoreceptor ribbon synapse (39, 40). In addition to Ribeye (41) and Piccolino (a splice variant of Piccolo) (42), a few molecular components have been associated with precise photoreceptor presynapse morphology and wiring with bipolar cells; these include scaffold protein 4.1G (also called Epb4.1l2), cell adhesion protein ELFN1 (43), and extracellular proteins Pikachurin (44) and auxillary calcium channel subunit α2δ4 (45). Here, we define a set of 26 new molecular determinants that limit the size and/or axonal positioning of the spherule in the appropriate sublamina, two essential features associated with functional specificity of the rod photoreceptor synapse.…”

Section: Discussionmentioning

confidence: 99%

NRL- and CRX-guided gene network modulates photoreceptor presynapse size and positioning during retinal development

Whitaker

Mondal

Fann

et al. 2019

Preprint

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29 Unique morphologies of rod and cone photoreceptor presynaptic terminals permit the formation 30 of synapses onto interneurons during retina development. We integrated multiple "omics" 31 datasets of developing rod and S-cone-like photoreceptors and identified 719 genes that are 32 regulated by NRL and CRX, critical transcriptional regulators of rod differentiation, as 33 candidates for controlling presynapse morphology. In vivo knockdown of 72 candidate genes in 34 the developing retina uncovered 26 genes that alter size and/or positioning of rod spherules in 35 the outer plexiform layer. Co-expression of seven cDNAs with their cognate shRNAs rescued 36 the rod presynapse phenotype. Loss of function of four genes in germline or by an AAV-37 mediated CRISPR/Cas9 strategy validated RNAi screen findings. A protein interaction network 38 analysis of the 26 positive effectors revealed additional candidates in the NRL/CRX-regulated 39 presynapse morphology-associated gene network. Follow-up knockdowns of two novel 40 candidates support the proposed network. Our studies demonstrate a requirement of multiple 41 components in a modular network for rod presynapse morphogenesis and provide a functional 42 genomic framework for deciphering genetic determinants of morphological specification during 43 development. 443 45 Author Summary 46 The relationship between neuronal morphology and function has been recognized for over 100 47 years. However, we still have poor understanding of genes and proteins that control 48 morphogenesis of a specific neuron. In the current study, we address this connection between 49 gene expression and neural morphology by identifying and knocking down a subset of 50 expressed genes in rod photoreceptors. We ascertained a number of candidate genes 51 controlling photoreceptor pre-synaptic terminal morphology, which is necessary for its 52 connection with second-order neurons in the retinal circuit. Furthermore, we have curated a 53 more plausible network of genes, either identified in our study or predicted, that are enriched for 54 processes underlying photoreceptor morphogenesis. We suggest that our work will provide a 55 framework for dissecting genetic basis of neuronal architecture and assist in better design of cell 56 replacement therapies for retinal degeneration. 57 4 58 Introduction 59 The human central nervous system contains approximately 86 billion neurons that form 15060 trillion synapses (1, 2). Each neuronal cell type possesses a stereotyped morphological 61 standard, creating precise synaptic connections. Over 100 years ago, Santiago Ramón y Cajal 62 predicted intimate structure-function relationships within neural circuits based on elaborate 63 observations in the retina and the brain (3). The developmental trajectory of a neuron constricts 64 its essential features towards that of its class to ensure a typical morphology and function, which 65 are stringently and largely controlled by transcriptional control mechanisms (4-8). Genetic 66 determinants that define neuronal size, ...

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“…For example, Piccolo appears at emerging synapses formed between mossy fiber boutons and cerebellar granule cells as well as between parallel fiber boutons and Purkinje cell dendrites during the earliest stages of cerebellar development (Zhai et al, 2001). The large size of Piccolo and the complexity of the Pclo gene has thwarted most efforts to elucidate its function, though critical roles in retinal ribbon synapse formation and visual function (Regus-Leidig et al, 2014; Muller et al, 2019) as well as the integrity of hippocampal synapses has been identified (Waites et al, 2013). What remains unclear is how Piccolo contributes to cerebellar development and whether, as suggested by genetic studies, it has a primary role in the etiology of PCH3.…”

Section: Introductionmentioning

confidence: 99%

Loss of Piccolo function in rats induces Pontocerebellar Hypoplasia type 3-like phenotypes

Falck

Bruns

Hoffmann

et al. 2019

Preprint

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Piccolo, a presynaptic active zone protein, is best known for its role in the regulated assembly and function of vertebrate synapses. Genetic studies suggest a further link to several psychiatric disorders as well as Pontocerebellar Hypoplasia type 3 (PCH3), although a causal relationship is lacking. We have characterized recently generated knockout (Pclogt/gt) rats. Analysis revealed a dramatic reduction in brain size compared to wildtype (Pclowt/wt) animals, attributed to a decrease in the size of the cerebral cortical, cerebellar and pontine regions. Analysis of the cerebellum and brainstem revealed a reduced granule cell (GC) layer and a reduction in size of pontine nuclei. Moreover, the maturation of mossy fiber (MF) afferents from pontine neurons and the expression of the α6 GABAA receptor subunit at the MF-GC synapse are perturbed, as well as the innervation of Purkinje cells by cerebellar climbing fibers (CFs). Ultrastructural and functional studies revealed a reduced size of MF boutons, with fewer synaptic vesicles and altered synaptic transmission. These data imply that Piccolo is required for the normal development, maturation and function of neuronal networks formed between the brainstem and cerebellum. Consistently, behavioral studies demonstrated that adult Pclogt/gt rats display impaired motor coordination, despite adequate performance in tasks that reflect muscle strength and locomotion. Together these data suggest that loss of Piccolo function in patients with PCH3 could be causal for many of the observed anatomical and behavioral symptoms, and that the further analysis of these animals could provide fundamental mechanistic insights into this devastating disorder.Significance StatementPontocerebellar Hypoplasia type 3 is a devastating developmental disorder associated with severe developmental delay, progressive microcephaly with brachycephaly, optic atrophy, seizures and hypertonia with hyperreflexia. Recent genetic studies have identified non-sense mutations in the coding region of the Piccolo gene, suggesting a functional link between this disorder and the presynaptic active zone. Our analysis of Piccolo knockout rats supports this hypothesis, formally demonstrating that anatomical and behavioral phenotypes seen in patients with PCH3 are also exhibited by these Piccolo deficient animals.

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In vivo knockdown of Piccolino disrupts presynaptic ribbon morphology in mouse photoreceptor synapses

Cited by 50 publications

References 47 publications

Synaptic Ribbons Require Ribeye for Electron Density, Proper Synaptic Localization, and Recruitment of Calcium Channels

Synaptic Ribbons Require Ribeye for Electron Density, Proper Synaptic Localization, and Recruitment of Calcium Channels

NRL- and CRX-guided gene network modulates photoreceptor presynapse size and positioning during retinal development

Loss of Piccolo function in rats induces Pontocerebellar Hypoplasia type 3-like phenotypes

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