Cilia - The sensory antennae in the eye

Accepted Manuscript Cilia - The sensory antennae in the eye Helen May-Simera, Kerstin Nagel-Wolfrum, Uwe Wolfrum PII:

S1350-9462(16)30088-X

DOI:

10.1016/j.preteyeres.2017.05.001

Reference:

JPRR 670

To appear in:

Progress in Retinal and Eye Research

Received Date: 4 November 2016 Revised Date:

4 May 2017

Accepted Date: 8 May 2017

Please cite this article as: May-Simera, H., Nagel-Wolfrum, K., Wolfrum, U., Cilia - The sensory antennae in the eye, Progress in Retinal and Eye Research (2017), doi: 10.1016/ j.preteyeres.2017.05.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Helen May-Simera1, Kerstin Nagel-Wolfrum2, Uwe Wolfrum2* Institute of Molecular Physiology, 1Cilia Biology, 2Molecular Cell Biology, Johannes Gutenberg-Universität Mainz, 55099 Mainz, Germany

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* Corresponding author: Tel.: + 49 (0) 6131 3925148; fax: + 49 (0) 6131 3923815 e-mail address: [email protected] (U. Wolfrum)

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Ocular cilia

Cilia - the sensory antennae in the eye

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Abstract 1. Introduction 1.1 Revival of an ancient organelle - Emerging novel functions of cilia 1.2 Cilia architecture and evolutionarily conserved functional modules 2. Ocular cilia – Location and function 2.1 Cilia in the cornea 2.2 Cilia in the ciliary body 2.3 Cilia in the iris 2.4 Cilia in the trabecular meshwork 2.5 Cilia in the lens 2.6 Cilia in the retinal pigment epithelium 2.7 Cilia in the retina 2.7.1 Cilia in retinal non-photoreceptor cells 2.7.2 Ciliary proteins in non-ciliated retinal cells 3. Cilia in photoreceptor cells 3.1 Photoreceptor cell structure, molecular architecture, and function 3.1.1 The outer segment - the photoreceptor cilium 3.1.1.1 The outer segment axoneme 3.1.1.2 Partitioning incisures 3.1.2 Calyceal processes structurally support rod and cone outer segment of non-rodent vertebrates 3.1.3 The photoreceptor transition zone 3.1.4 The basal body and the periciliary region in photoreceptor cells 3.1.5 The striated ciliary rootlets 3.2 Functional modules in photoreceptor cilia 3.2.1 The basal body a major microtubule organization center 3.2.2 The periciliary region 3.2.3 Endocytosis at the ciliary base 3.2.4 Ciliogenesis in photoreceptor cells 3.2.5 The phototransduction module 3.2.6 Outer segment renewal/disk neogenesis module 3.3 Ciliary transport in photoreceptor cells 3.3.1 Unidirectional transport of outer segment compounds 3.3.2 Rhodopsin transport via a secretory pathway 3.3.3 Rhodopsin transport in the ciliary membrane 3.3.4 Intraflagellar transport (IFT) 3.3.4.1 IFT20 in ciliary transport 3.3.4.2 Retrograde transport 1

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3.3.5 Alternative ciliary transport pathways 3.3.6 Co-transport, trafficking chaperones and transport of complexes 3.3.7 Transport and targeting of soluble molecules 3.3.8 Bidirectional movement of signalling molecules 3.4 Novel unexpected functional modules in photoreceptor cilia 3.5 Ciliary photoreceptor evolution 4. Ocular pathogenesis in ciliopathies 4.1 Non-syndromic ocular pathogenesis 4.2 Syndromic ocular pathogenesis 5. Treatment approaches for ocular phenotype in ciliopathies 5.1 Advancements in gene addition 5.2 Read-through of nonsense mutations 5.3 Exon skipping 5.4 Stem cell therapy 5.5. Retinal implants and optogenetics 6. Future directions Acknowledgements Funding sources References Figure legends

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Figure 1) Schematic representation of a prototypic cilium.

Figure 2) Schematic representation of different regions in the vertebrate eye that contain cilia.

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Figure 3) Examples of cilia in ocular tissues. Figure 4) Compartmentalization of ciliated photoreceptors.

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Figure 5) Transmission electron microscopy of photoreceptor cells. Figure 6) Ciliogenesis in the photoreceptor cells. Figure 7) Schematic representation of transport modules in photoreceptor cells. Figure 8) Subciliary localization of IFT molecules and BBS components in mouse photoreceptor cells. Figure 9) Gene-based therapeutic approaches for the ocular phenotype of ciliopathies. 2

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Abstract Cilia are hair-like projections found on almost all cells in the human body. Originally believed to function merely in motility, the function of solitary non-motile (primary) cilia was long overlooked. Recent research has demonstrated that primary cilia function as signalling hubs that sense environmental cues and are pivotal for organ development and function, tissue hoemoestasis, and maintenance of human health. Cilia share a common anatomy and their diverse functional features are achieved by evolutionarily conserved functional modules, organized into sub-compartments. Defects in these functional modules are responsible for a rapidly growing list of human diseases collectively termed ciliopathies. Ocular pathogenesis is common in virtually all classes of syndromic ciliopathies, and disruptions in cilia genes have been found to be causative in a growing number of non-syndromic retinal dystrophies. This review will address what is currently known about cilia contribution to visual function. We will focus on the molecular and cellular functions of ciliary proteins and their role in the photoreceptor sensory cilia and their visual phenotypes. We also highlight other ciliated cell types in tissues of the eye (e.g. lens, RPE and Müller glia cells) discussing their possible contribution to disease progression. Progress in basic research on the cilia function in the eye is paving the way for therapeutic options for retinal ciliopathies. In the final section we describe the latest advancements in gene therapy, read-through of non-sense mutations and stem cell therapy, all being adopted to treat cilia dysfunction in the retina.

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Keywords: Cilia, Ciliopathy, Eye, Retina, Degeneration, Therapy

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1. Introduction 1.1 Revival of an ancient organelle - Emerging novel functions of cilia Anthony van Leeuwenhoek was one of the pioneers of microbiology. Handcrafting his own microscopes, he was able to achieve far higher magnification than had been previously available. As early as 1675 he described observing ‘incredibly thin feet, or little legs, which moved ‘very nimbly’ when viewing protozoa through his microscope (Dobell, 1932). What he was observing were cilia, microtubule based appendages extending from the cell membrane. The term cilia (or cilium in singular), Latin for eyelash, was probably first coined in 1786 by Otto Muller (Muller, 1786). For many decades, cilia were thought to function in cell motility, similar to eukaryotic flagella. Then in the late 1800’s scientists began to observe another type of cilium on eukaryotic cells, namely a solitary, non-motile cilium (Kovalevskij, 1867; Langerhans, 1876; Zimmermann, 1898). At the time Zimmerman proposed a sensory role for these nonmotile cilia (Zimmermann, 1898), yet this was quickly forgotten and these organelles were largely ignored in the following decades. In the 1960’s the advent of electron microscopy renewed interest in cilia biology. Cilia are found on only a few specific cell types in multicellular invertebrates (Mencarelli et al., 2008). In contrast to this, virtually all vertebrate cell types are ciliated (Gerdes et al., 2009). A distinction between primary vs. motile cilia was made, based on the initial appearance of a singular ‘primary’ cilium, which precedes multiple ‘motile’ cilia in the central nervous system (Sorokin, 1968). Motile cilia, comprised of nine outer microtubule doublets with a central pair of microtubules (9+2), were thought to be required for generation of movement (Figure 1). In contrast, primary cilia, which lack the central microtubule pair (9+0), were long thought to be a redundant, vestigial organelle (Federman and Nichols, 1974). It was not until the start of the new millennia, that the significance of primary cilia became apparent. With the identification that primary cilia have a sensory role, which underlies many human diseases, research into this once long forgotten organelle has exploded (Beales and Jackson, 2012). Motile cilia predominantly serve to move fluid across membrane surfaces, for example mucus-flow on epithelial cells of the lungs and cerebrospinal fluid in the ventricles of the brain (Lee, 2011). In contrast, primary cilia transduce a multitude of sensory stimuli, including chemical concentrations of growth factors, hormones, odorants, and developmental morphogens, as well as osmolarity, light intensity and fluid flow (Goetz and Anderson, 2010; Satir et al., 2010). Furthermore the cilia-associated basal body complex serves as a gatekeeper for the regulation of down stream intracellular signalling events (May-Simera et al., 2012). Several properties of cilia facilitate their role as sensory transducers. They project away from the cell body, and so serve as good probes of the external milieu; their overlying membrane and cytoplasmic contents are relatively well isolated from the cell body; the machinery for their assembly makes possible rapid, regulated transport to and from the cell body; and the assembly machinery seems exploitable for use directly in signalling pathways (Snell et al., 2004). In effect, the 4

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reliance on the cilium for multiple signal transduction pathways means that the cilium is essential for the proper development of almost all organs in the body. The eye is no exception to this. With so many different cell types and components, many of them maintain primary cilia even into adulthood. And, as will be described in section three, the sensory photoreceptor cells of the retina, harbor one of the most specialized primary cilia in the mammalian body.

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1.2 Cilia architecture and evolutionarily conserved functional modules The wide divergence in ciliary function may suggest that this organelle differs depending on cell type. However, all cilia share a common architecture and structural composition. Cilia are composed of a long microtubule axoneme surrounded by an external membrane that is continuous with the plasma membrane of the cell (Figure 1). In standard motile cilia, the internal structure is comprised of a ‘9+2’ axoneme that contains nine outer doublet microtubules and two central microtubules. The outer doublet microtubules are comprised of a complete A-microtubule to which an incomplete B-microtubule is attached. The axoneme also contains inner and outer dynein arms that generate the force for motility (Kamiya, 1995), and radial spokes which contribute to regulation of motility (Pigino and Ishikawa, 2012). Primary cilia (interchangeably called sensory cilia) lack the central pair of microtubules (9+0), dynein arms and radial spokes. This basic division is overly simplified, as sensory perception has been shown to be an attribute of both motile and primary cilia (Bloodgood, 2010). Additionally a 9+2 microtubule configuration, does not automatically make a cilium motile, as can be seen in the non-motile olfactory sensory cilia or the kinocilium of mechanosensitve hair cells which have a 9+2 structure (Eley et al., 2005; Lidow and Menco, 1984). At the proximal end of all cilia, the nine outer microtubule doublets are continuous with the basal body, consisting of a ring of three triplet microtubules, which anchors the cilium to the cell (Rosenbaum and Witman, 2002) (Figure 1). The basal body is a modified mother centriole that serves as the foundation upon which the cilium is constructed. It also serves as a microtubule-organizing centre (MTOC) for cytoplasmic microtubules. As such the basal body is an important platform for protein interactions and serves as a docking station for proteins en route to the cilium (Marshall, 2007; Nigg, 2007). One of the key components of a cilium is the transition zone, which is emerging as one of the critical sites for regulation of ciliary function. The transition zone connects the ciliary axoneme with the basal body complex, and is surrounded by a specialized periciliary membrane. The transition zone starts where the triplet microtubules of the basal body end, and extends to the basal plate, upon which the central pair of microtubules (in motile cilia) are nucleated. The region of the transition zone is defined by Y-linkers, which connect the outer microtubule doublets to the plasma membrane (Figure 1) (O’Toole et al., 2007).

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The ciliary tip is another specialized region of the cilium, although little is known about its protein composition and ultrastructure. Studies have shown that ciliary length is controlled by addition or removal of microtubule subunits at the tip (Marshall and Rosenbaum, 2001; Pedersen et al., 2006) and that many signalling components localize there (Goetz and Anderson, 2010). Cilia not only share common morphologic features but also functional properties, which are reflected in evolutionarily conserved functional modules. These functional modules encompass ciliary trafficking, signalling, regulation and motility. Although there is significant overlap and crosstalk between the individual modules, they serve as classifications for understanding and organising ciliary mechanisms. One of these functional modules encompasses ciliary trafficking. Cilia lack the machinery for protein synthesis so all proteins required for growth, maintenance and function of the diverse types of cilia must be transported from the site of synthesis in the cell body to the ciliary compartment. Trafficking of proteins from the Golgi and cytosol to the base of the cilium occurs via polarized vesicle trafficking. A more specialised form of vesicle trafficking is required for protein trafficking along the ciliary axoneme. This process has been termed intraflagellar transport (IFT) and is integral to the cilium’s structure and function (Cole, 2003; Cole et al., 1998). IFT is covered in more detail in chapter two and three. A further functional module encompasses signalling. A defining feature of the cilium is the presence of a ciliary membrane that is continuous with the plasma membrane and surrounds the organelle. This is in sharp contrast to the prokaryotic flagellum (which is evolutionarily unrelated) and allows the cilium to function in signal transduction pathways through the accumulation of receptors in the ciliary membrane (Satir et al., 2010). The precise composition of ciliary membrane components and inventory of signalling modules differs between cell type and cell stage, but primary cilia have been shown to coordinate signalling via several receptors including, tyrosine kinases (RTKs), Hedgehog (Hh), Wingless (Wnt), transforming growth factor-β (TGF-β), Ca2+,, neuronal and purinergic receptors, as well as through communication with the extracellular matrix (Christensen et al., 2012; May-Simera et al., 2012; Schou et al., 2015; Wong and Reiter, 2008; Wood and Rosenbaum, 2015). Surprisingly, an association between primary cilia and the DNA damage repair pathway has been identified (Attanasio, 2015; Chaki et al., 2012). Moreover, ciliogenesis per se is highly regulated by extracellular signals and intracellular signalling cascades (Kim and Dynlacht, 2013). As many different receptors or channels can be present in the same cilium at the same time, there is an enormous amount of complexity and potential crosstalk between various signalling pathways. The diversity and dynamics of important ciliary membrane proteins and their effector molecules in the cilium and at the basal body has only begun to be catalogued. Many of these signalling pathways are active in different stages of ocular development and in different cell types. As will be discussed below, we are only starting

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to identify cilia in various ocular tissues and as yet very little is known about how the cilium contributes to signalling pathways in ocular development or function.

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2. Ocular cilia – Location and function Almost every eukaryotic cell has the potential to generate a cilium. Therefore, it is not surprising that primary cilia are widely distributed in the different tissues found in the vertebrate eye (Figure 2). Although current research focuses predominantly on cilia in photoreceptor cells, there are many other ciliated cell types in the vertebrate eye including the cornea, ciliary body, iris, lens, trabecular meshwork, and the retinal pigment epithelium (RPE). Although the functions of these cilia are barley understood, it is highly likely that these cilia contribute to organogenesis and tissue homeostasis. Disruption of ciliary function may also contribute to visual impairment, not only in syndromic ciliopathies but also in non-syndromic ocular disorders. Not only will we review novel insights into photoreceptor cilia, but also the current knowledge and understanding of cilia found on other cells types in the vertebrate eye.

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2.1 Cilia in the cornea The cornea is the outermost layer of the vertebrate eye (Figure 2B). It is a see-through, dome shaped structure, which covers the front of the eye and is comprised of five layers (Eghrari et al., 2015). As the cornea has no blood supply, it relies on tears and fluid from the aqueous humor, lying behind the cornea, for nourishment. The outermost epithelial layer is separated from the innermost endothelium layer via two membranes and a collagen matrix, the stroma. Cornea endothelial cells are vital in regulating the fluid content of the cornea. Early electron microscopy studies identified primary cilia on rabbit and human cornea endothelial cells, although the extent of ciliation varied (Doughty, 2004; Gallagher, 1980). Later work suggested that the appearance of primary cilia on cornea endothelial cells might be linked to fluid flow (Doughty, 1998), which could explain the discrepancies in the literature. Further studies also found ciliated cornea endothelial cells in other vertebrate species (Collin and Barry Collin, 2004; Collin and Collin, 2000). More recently it has been shown that primary cilia are required for the development of the corneal endothelium in mouse, orchestrating coordinated morphogenesis of the tissue (Blitzer et al., 2011). In adult these cilia are disassembled, yet are able to reassembly quickly in response to injury. Primary cilia have also been found in the corneal epithelium during development and also in adult, where they are predominantly present on basal cells (Grisanti et al., 2016). Conditional ablation of cilia in these cells resulted in an increase in proliferation and cell density, which caused a thickening of the cornea epithelium. Disruption of the Notch signalling pathway preceded these events, leading the authors to propose that in cornea epithelial cells, the primary cilium maintains tissues homeostasis via modulation of Notch signalling (Grisanti et al., 2016).

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2.2 Cilia in the ciliary body Although termed the ‘ciliary body’ the presence of cilia in the ciliary body has only briefly been described and their precise function has not been assigned. The ciliary body, described as a ring shaped thickening, separates the posterior chamber from the vitreous chamber (Lang, 2007) (Figure 2B). This structure is comprised of so-called ciliary muscle, ciliary stroma, and both pigmented and non-pigmented epithelial cells. Inward folding of the pigmented and non-pigmented epithelium forms the ciliary processes. The ciliary muscle controls the shape of the lens, whilst the ciliary epithelium regulates aqueous humor production. Primary cilia were commonly observed via transmission electron microscopy in both pigmented and non-pigmented epithelial cells of the ciliary body in rabbit and monkey (Ohnishi and Tanaka, 1980; Tenkova and Chaldakov, 1988). Although this work was done prior to the ‘rediscovery’ of the critical role of primary cilia in cell signalling and tissue homeostasis, the authors propose that these cilia may have a sensory role, which would allow a local response to the altered composition of the intraocular fluid and changes in the vascular pressure (Ohnishi and Tanaka, 1980). The authors also found cilia in the various cell types of the ciliary stroma, again suggesting a function of cilia in maintaining aqueous humor dynamics (Tenkova and Chaldakov, 1988), which they speculate may be functioning via regulation of cyclic AMP.

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2.3 Cilia in the iris Very little is known about cilia in the iris. One report documented the appearance of primary cilia in the iris stroma in rabbit and human (Vrabec, 1971). The authors noted differences in cilia length and shape among differing cell types of the iris stroma, yet some confusion between primary and motile cilia might have arisen. The age of the donor tissue was not reported, which might also have accounted for differences in cilia length.

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2.4 Cilia in the trabecular meshwork The trabecular meshwork is a spongy tissue in the anterior chamber of the eye that maintains the intraocular pressure by regulating drainage of the aqueous fluid (Llobet et al., 2003) (Figure 2B). Early reports of the presence of primary cilia in the trabecular meshwork in monkey eye went largely unnoticed (Vrabec, 1971). Trabecular meshwork cells are responsible for maintaining intraocular pressure. An imbalance in ocular pressure has severe deleterious consequences. Low ocular pressure can cause structural changes; elevated ocular pressure can result in optic nerve damage, a hallmark of glaucoma. Defects in the ciliary protein OCRL, an inositol polyphosphate 5-phosphatase, causes Lowes Syndrome, patients of which also suffer from glaucoma (Attree et al., 1992; Luo et al., 2012). Luo et al., were able to show that primary cilia are responsible for the trabecular meshwork’s ability to respond to changes in intraocular pressure (Luo et al., 2014) (Figure 3A). They also showed that primary cilia on trabecular meshwork cells shorten in response to fluid flow and elevated hydrostatic pressure, and also promote the transcription of tumor necrosis factor alpha (TNF-α), transforming growth factor beta 8

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(TGF-β), and glioma-associated oncogene 1 (GLI1). Increased expression of TNF-α and TGF-β is thought to play a role in the progression of glaucoma (Ozcan et al., 2004; Yan et al., 2000). On a more functional level, it has been suggested that OCRL interactions with transient receptor potential vanilloid 4 (TRPV4) channels in trabecular meshwork cilia regulate osmosis and Ca2+ influx in the eye, thereby regulating ocular pressure (Luo et al., 2014).

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2.5 Cilia in the lens The lens of the vertebrate eye is an ectodermally-derived tissue comprised of two major cell types; highly ordered fiber cells covered by monolayer epithelial cells. Precise alignment of lens fiber cells is critical for lens function, and any disturbance to their organization impairs light transmission (McAvoy et al., 1999). Coordinated alignment of lens fiber cells is orchestrated via non-canonical Wnt (planar cell polarity) signalling (Chen et al., 2008). Many components of the planar cell polarity pathway are asymmetrically localized in lens fiber cells and regulate fiber cell alignment (Sugiyama et al., 2011, 2010; Sugiyama and McAvoy, 2012). Primary cilia are localized on virtually all lens cells (Figure 3B). Asymmetric positioning of cilia in lens fiber cells is coupled with asymmetric localization of planar cell polarity proteins (Sugiyama et al., 2010). Surprisingly, despite the fact that primary cilia have been shown to affect planar cell polarity in other tissues (May-Simera et al., 2015), they do not appear to be important for lens fiber cell alignment (Sugiyama et al., 2016). Even complete loss of primary cilia had no effect on tissue morphogenesis, although this might be due to the fact the the basal body, which likely co-ordinates the ciliary response to Wnt signalling, was still retained in these tissues.

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2.6 Cilia in the retinal pigment epithelium The retinal pigment epithelial (RPE) is a monolayer epithelium that sits at the back of the eye and is tighly associated with the outer segments of the retinal photoreceptors (Figure 2C). RPE cell lines have been extensively used by the cilia community to study many aspects of ciliogenesis, the most common being ARPE-19 and hTERT RPE-1. However, as with other cell lines, their properties are far removed from native tissue (Alge et al., 2006). Electron micrographs showed ciliary structures (axoneme, basal body, rootlet) in adult cat, monkey and human RPE (Fisher and Steinberg, 1982), although the orientation and origin of these was unclear. Nor was it possible to determine the percentage of cells in which a ciliary structure was observed. An earlier electron microscopy study had also identified primary cilia in human RPE cells (Allen, 1965), in which it was observed that these cilia extended into an intracellular pocket. These could be remnants of retracted cilia. In 2002, a study described cilia in the rat RPE (Nishiyama et al., 2002). The authors identified primary cilia on flat mount RPE preparations via immunohistochemistry against the ciliary marker acetylated α-tubulin. The percentage of ciliated cells declined postnatally to less than a few percent in adult. Intriguingly, tight junction marker, claudin, 9

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was also shown to strongly decorate the ciliary axoneme in RPE cells, strengthening the association between ciliary and cell junction mechanisms. It is highly likely that differences in RPE cell ciliation may depend on stages of development and species. A more recent study examined the role of the primary cilium during development and differentiation of the RPE in mouse and human induced pluripotent stem cell (iPSC) derived RPE cells (May-Simera et al., submitted). In mouse RPE they found that primary cilia appear as early as E14.5 and are likely required to downregulate canonical Wnt signalling during maturation of the RPE (Figure 3C). RPE defects in cilia mutants preceeded retinal degeneration of the adjacent photreceptors and was accompanied by upregulation of canonical Wnt genes. Experimentally enhanced ciliation improved human iPSC-derived RPE differentiation also via manipulation of the Wnt signalling pathway.

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2.7 Cilia in the retina Retina photoreceptors contain a highly modified primary cilium, the connecting cilium and outer segment. The importance of these ciliary structures has been extensively studied and will be described in detail in section three. Surprisingly, other retinal cell types have also been described harboring primary cilia, though any physiological function is currently only speculative.

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2.7.1 Cilia in retinal non-photoreceptor cells Primary cilia are found on almost all cell types of the vertebrate retina (Figure 2A). Electron micrographs clearly demonstrated the presence of short primary cilia in the ganglion cell layer and in the inner nuclear layer in both guinea pig and human retinas (Allen, 1965). These primary cilia have the classic 9+0 microtubule doublet configuration and range in length from 1.2-1.8 µm. The origin of these primary cilia is not clear and is almost impossible to distinguish via electron microscopy. The author noted that these primary cilia were more commonly found in neurons along the ‘outer edges’ of these cell layers (at the junction to the plexiform layers), yet acknowledged that this may be because cells in these regions were more often scrutinized. Using a specific type of silver stain, ‘Richardson’s’, that exclusively stains cilia, Boycott and Hopkins (Boycott and Hopkins, 1984) also showed the presence of primary cilia in an adult cat retina. Using this technique they concluded that in addition to photoreceptors, amacrine and ganglion cells each have a primary cilium in the cat retina. They also showed changes in ciliary morphology with increasing age (Boycott and Hopkins, 1984). Limitations in terms of resolution, staining and imaging methods might have precluded the identification of primary cilia in other cell types. Müller glia cells are arguably the most intriguing cell types in the retina (Figure 2A), being the only cell type that possesses regeneration capacity, not only in teleost but also in mammals. Upon retinal injury, Müller glia are able to re-entre the cell cycle, proliferate and differentiate into neuronal lineages (Das et al., 2006; Ooto et al., 2004), therefore acting as dormant retinal stem cells. In zebrafish Müller glia are able to 10

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regenerate all major retinal cell types and restore vision (Goldman, 2014). Primary cilia where observed in Müller glia close to the outer limiting membrane in both juvenile and adult teleost retina (Ennis and Kunz, 1986) but are also present on Müller glia in the mammalian retina (Figure 3D). Mature Müller glia primary cultures isolated from rat retina where shown to have primary cilia, which regulate Sonic hedgehog-induced Müller glia cell proliferation and dedifferentiation (Ferraro et al., 2014). Combined, these findings highlight a possible role for Müller glia primary cilia in regulation of retinal regeneration.

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2.7.2 Ciliary proteins in non-ciliated retinal cells Surprisingly, bone fide ciliary proteins are also present in non-ciliary compartments of retinal cells. IFT20 was found as an authentic Golgi resident (see also 3.3.4.1), anchored in the cis-Golgi as a specific effector, independent of canonical IFT machinery (Keady et al., 2011). IFT20 functions as a molecular mediator between the sorting machinery of the Golgi apparatus and targeted vesicular transport of membrane proteins destined for the cilium (Follit et al., 2008, 2006; Sedmak and Wolfrum, 2010). Non-ciliary dendritic IFT complexes, comprising IFT-B particle molecules and the anterograde IFT motor KIF17, were identified at post-synaptic terminals and dendritic processes of secondary retinal neurons, namely bipolar and horizontal cells (Sedmark and Wolfrum, 2010). In addition, BBS molecules associated with the BBSome are not only restricted to ciliary transport, but appear to be involved in microtubule-based transport of membrane components in general (May-Simera et al., 2015, 2009; Novas et al., 2015). This is supported by the prominent localization of several BBS molecules in the post-synaptic terminals and dendrites of retinal neurons (Smith et al., 2013; Wolfrum et al., 2012; Spitzbarth et al., manuscript in preparation). The non-ciliary IFT/BBS processes in retinal neurons possibly involve exocytosis related to glutamate receptor delivery and positioning in the post-synaptic membrane. A non-ciliary IFT system is also associated with exocytosis at the immune synapse required for T-cell interaction with antigen-presenting cells (Finetti et al., 2009; Finetti and Baldari, 2013). However, non-ciliary IFT complexes at the immune synapse seem to differ in composition from those found at dendritic processes and synapses of retinal neurons (Sedmak and Wolfrum, 2010). There is growing evidence that the IFT/BBS complex evolved from the exocytotic clathrin/COP1 coatamer system of early ancestral non-ciliated cells (Jékely and Arendt, 2006; Jin et al., 2010; van Dam et al., 2013), and that the exocytotic delivery systems in ciliary and post-synaptic compartments coevolved from this ancestor (Sedmark and Wolfrum, 2010). 3. Cilia in photoreceptor cells Most researchers working on retinal photoreceptors mistakenly assume that the connecting cilium is the only ciliary component of a photoreceptor cell. However, the connecting cilium corresponds to the transition zone region of a primary cilium (Figure 11

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4A,B). Therfore, the entire outer segment can be considered a highly specialised primary sensory cilium. Considering that the first step in seeing is the translation of light into electrical signals, which occurs in the photoreceptor outer segments, its important to view these processes in terms of specialised ciliary functions.

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3.1 Photoreceptor cell structure, molecular architecture, and function Vertebrate photoreceptor cells are the most abundant cells of the neuronal retina and are arranged parallel to one another in the photoreceptor layer (Figure 2A). Two principle types of photoreceptor cells exist, rods and cones. Rod photoreceptors are committed to high-sensitive photon capture at low light intensities, the backbone of the scotopic visual system. There are usually two or more different types of cone photoreceptors. Expressing different opsins, cone photoreceptors allow for colour discrimination at high-resolution in the photopic visual system, but are up to 100-fold less sensitive than rods. The human retina has three types of cones, namely M-, L-, and S-cones, which differ in their spectral sensitivity. The percentage of cones vs rods varies between species and roughly correlates with the pattern of daily activity (Peichl, 2005). Nocturnal mammals have 0.5% to 3% cones, crepuscular and arrhythmic species have 2% to 10% cones, and diurnal mammals have a wide range, from 8% to 95%, of cones among their photoreceptors. Rod and cone photoreceptors are not evenly distributed throughout the retina. In most species, cones are concentrated in distinct regions of the retina. In human and other old world primates, cones are mostly concentrated in the macula region of the retina. In the fovea centralis, each cone is connected to one bipolar cell, thus this region is responsible for the high visual resolution. Both photoreceptor cell types are highly specialized, polarized neurons, which consist of morphological and functional distinct cellular compartments (Figure 4) (Roepman and Wolfrum, 2007). The photosensitive outer segment is a specialized primary cilium. It is bridged via a narrow so-called connecting cilium - the transition zone of a prototypic cilium - to the inner segment. The inner segment compartment contains all organelles for biosynthesis and metabolism of the cell. Due to the extraordinary high-energy demand of photoreceptor cells (Okawa et al., 2008), the inner segments contain numerous mitochondria in the so-called ellipsoid region. The inner segment continues proximally into the cell body, with its nucleus (“pericaryon”) densely packed into the outer nuclear layer, and the synaptic region. At the synaptic region the generated electrical signals are transmitted to the horizontal and bipolar cells, the first neurons in the post-receptoral retinal network of the inner neuronal retina. The specialized synaptic terminals of photoreceptors, namely the rod spherules and cone pedicles, are characterized by synaptic ribbons known for their complex chemical synapses, which continually release neurotransmitters (Regus-Leidig and Brandstätter, 2012). 3.1.1 The outer segment - the photoreceptor cilium

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The outer segment of both cone and rod photoreceptor cells is characterized by hundreds of membrane disks, which are arranged into coin-like stacks (Sjöstrand, 1953) (Figure 5A, B). In rods, flattened membranous disks are arranged separately from the plasma membrane and enclose an intradiskal space of about 6 nm. The cytoplasmic interdiskal distance between the disks measures between 12-15 nm (Gilliam et al., 2012). In contrast to the rods, the outer segment disk membranes in cones are mostly described as a continuum of the plasma membrane. However, this “text book” rule is only true for lower vertebrates; cone outer segments in mammals can also contain stacks of membrane disks separated from the plasma membrane (Anderson et al., 1978; Cohen, 1970; Pearring et al., 2013). Oligomerization of transmembrane glycoprotein peripherin 2 (PRPH2), also known as RDS (retinal degeneration slow), and ROM-1 is required for the formation of the membrane boundaries of the flattened disks (Goldberg et al., 2016; Stuck et al., 2015). Higher order PRPH2/RDS tetramers tethered by disulfate-bridges in the intradiskal space facilitate bending of the membrane into the characteristic “hairpinshape” of the closed disks. PRPH2/RDS forms part of a rim complex together with the ABCA4 and glutamic acid rich proteins (GARPs) which are alternative splice variants of the rod specific CNGB1. The interaction of PRPH2/RDS with the GARP domain of the transmembrane β-subunit of the cGMP gated channel (CNGB1) mediates fibril linkages between the rim of the disks and the plasma membrane of the outer segment (Körschen et al., 1999; Poetsch et al., 2001). Homophilic interactions of cytoplasmic GARP2 molecules of the rim complex is suggested to contribute to disk stacking (Kaupp and Seifert, 2002). Mutations in PRPH2/RDS cause a variety of inherited retinal dystrophies such as autosomal recessive and dominant retinitis pigmentosa (arRP/adRP) as well as macular degeneration (Stuck et al., 2016). In the open disk region of cones, PRPH2/RDS localization is restricted to the region of the outer segment adjacent to the axoneme. Another rim protein, prominin-1, is found at the edge of the “open” disks, (Han et al., 2012; Stuck et al., 2016, 2015). Mutant prominin-1 disrupts disk morphogenesis, causing autosomal dominant macular degeneration (Yang et al., 2008). The two types of photoreceptors differ in their shape: in comparison to rods, the outer segment of cones is more conical shaped (Pearring et al., 2013). In most vertebrate species, cone outer segments are shorter (~20 µm) and wider than rod outer segments (20-60 µm). In addition, there are dramatic interspecies differences in the dimensions of photoreceptor outer segments. For example, rod outer segments measure ∼1.4 µm in diameter in rodents and ~1.7 µm in diameter in human, respectively (own unpublished data). Rod outer segments of amphibians and fish are wider and can measure more than 15 µm in diameter in salamanders such as the axolotls (Ambystoma mexicanum), or closer to 20 µm as in the Australian lungfish (Neoceratodus forsteri) (Bailes et al., 2006). In all vertebrate retinas with the exception of placental mammals, multiple photoreceptor cells, termed paired or double photoreceptor cells, can be identified. These are usually double cones (Rowe, 2000). Double cones share large parts of the inner segment cell membrane but always have two nuclei, two synaptic process and separated 13

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outer segments with identical or unidentical opsins. Paired or double cones can either be equal (e.g. amphibians) or unequal (e.g. sauropsides/reptiles and birds) in shape, the latter consisting of a principle and an accessory photoreceptor. In either case, all cones are ensheathed by a specific extracellular matrix sheath composed of proteoglycans which bind peanut agglutinin (PNA). This feature is commonly utilised for histochemical detection of cones in the retina via application of fluorescently tagged PNA (Long and Aguirre, 1991; Reiners et al., 2005)

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3.1.1.1 The outer segment axoneme Most of the space in the outer segments are taken up by the stacked disks. The cytoplasm is restricted to the interdiskal space, the axonemal compartment, and the cytoplasmic region of the incisures in the outer segment disks. The axonemal microtubules extend from the microtubule doublets of the connecting cilium. In contrast to the axoneme of a prototypic primary cilium, characterized by microtubule doublets, the outer segment axoneme contains doublet and singlet microtubules (Figure 4A, B) (Cohen, 1965; Roepman and Wolfrum, 2007; Roof et al., 1991). The microtubule-binding protein, RP1, is a prominent component of the cytoskeleton of the outer segment axoneme (Liu et al., 2004a) and is often used as a molecular marker of the axonemal compartment (Karlstetter et al., 2014). The RP1 protein is also essential for correct stacking of outer segment disks (Liu et al., 2003, 2002) and defects in the RP1 gene cause autosomal dominant retinitis pigmentosa (adRP) (Liu et al., 2002). In contrast to the axoneme of a proteotypic primary cilia, only a subset of IFT or BBSome proteins localize to the rod outer segment axoneme (see below (Sedmak and Wolfrum, 2010; Smith et al., 2013; Wolfrum et al., 2012). Although in most species, the axoneme penetrates only one third of the outer segment, in the large rod outer segments of lower vertebrates such as amphibians, the axoneme can reach the tip of outer segment. Intriguingly in the cones of teleost fish, the outer segment axoneme can be replaced by an accessory outer segment (Hodel et al., 2014; Nagle et al., 1986). Similar to the conventional axoneme, the accessory outer segment originates from the connecting cilium, contains microtubules, and projects alongside the principle outer segment of the cone (Figure 5F-H). A thin cytoplasmic bridge connects both outer segment compartments. So far, the function of the accessory outer segment is speculative. A role in transport and exchange of metabolites between the cone inner segment and outer segment as well as with the surrounding extensions of the RPE cells has been suggested. Recently, myosin VIIa an actin filament-based molecular motor related to the human Usher syndrome (USH1B) was found to be concentrated in the accessory outer segment of cones in the zebrafish retina (Hodel et al., 2014). Although actin filaments are not obvious in the accessory outer segment (Nagle et al., 1986), the high concentration of myosin VIIa supports a role in ciliary transport as previously hypothesized. 3.1.1.2 Partitioning incisures 14

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The rim of the membranous disks of rod outer segments invaginate to form incisures, thereby partitioning the outer segment disks (Eckmiller, 2000; Papermaster et al., 1978) (Figure 5B, C). The number of incisures per rod outer segment differs between vertebrate species and appears to be related to the diameter and circumference of the outer segment. In wide rod outer segments of fish and amphibians up to 30 incisures create a characteristic flower-like shape in cross sections; the narrow outer segments of many mammalian species only have one incisura (e.g. rodents and bovine) (Brown et al., 1963; Cohen, 1965; Eckmiller, 2000.; Körschen et al., 1999; Steinberg and Wood, 1975). Nevertheless, multiple incisures are also found in the narrow outer segments of primates (up to 30) and human (up to 12). The depth of the incisures also varies between different vertebrate species. The incisures of amphibians are deeper than the shallow incisures found in human. The prominent amphibian incisures are characterized by microtubules, which extend from the base of the outer segment longitudinally towards the tip (Eckmiller, 2000; Roof et al., 1991). In addition, proteins of the PRPH2/RDS rim complex, described above, are also found in incisures (Körschen et al., 1999). GFP tagged PRPH2/RDS disrupts the incisures in the outer segments of transgenic Xenopi, indicating a structural role of these rim molecules for the formation of incisures (Tam et al., 2004). Viewed laterally, the numerous incisures of the amphibian appear as longitudinal parallel striations without interruptions (Eckmiller, 2000). In contrast, the single incisures in rodents are not aligned in a continuum; they can transversally shift along the outer segment length, making it look as though the stacking of the outer segment disks were twisted. Although incisures are conserved prominent structures in the otherwise well studied system of the rod outer segment, their function is still not clear. It is thought that they facilitate diffusion of cytoplasmic nutrients and signalling molecules in a longitudinal direction as well as along the outer segment axis (Makino et al., 2012) Conversely, the analysis of GFP constructs in Xenopus rods revealed that incisures can form a barrier for diffusion in the separated compartments of the outer segment (Najafi et al., 2012).

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3.1.2 Calyceal processes structurally support rod and cone outer segment of nonrodent vertebrates Calyceal processes are microvillus-like extensions of the inner segment which cup the basal fifth of the outer segments of rod and cone photoreceptor cells (Figure 5C, D) (Nagle et al., 1986). They were first described in the early 60’s (Brown et al., 1963; Cohen, 1965). Calyceal processes are nearly ubiquitous in vertebrate photoreceptor cells. As a rule, cones possess more calyceal processes than rods, e.g. macaque cones have around 14-16 as opposed to rods which have around ten (Sahly et al., 2012). There are also interspecies differences in the number of calyceal processes per photoreceptor cell: the voluminous outer segments of amphibian rods are encased by 25 or more calyceal processes, primates and pigs possess about a dozen, and surprisingly calyceal processes are virtually absent in rodents (Lin-Jones et al., 2004; Nagle et al., 1986; Sahly et al., 15

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2012). The absence of calyceal processes in rodents might be due to the extremely tight packing of the photoreceptor outer segments in their very compact eye. The function of calyceal processes is far from understood. Nevertheless, their spatial arrangement and molecular architecture strongly suggests that they serve the structural stabilization of the bottom 1/5th of the outer segment, e.g. against lateral mechanical forces. Like other microvilli, calyceal processes are characterized by an F-actin core in which actin filaments are bundled by F-actin cross-linking proteins, such as fimbrins/plastins, fascin 2 and espin (Höfer and Drenckhahn, 1993; Lin-Jones and Burnside, 2007; Sahly et al., 2012). At the base of the calyceal processes F-actin roots protrude from the F-actin core into the distal half of the inner segment, forming a basket of submembranous actin bundles (Burnside, 1978; Sahly et al., 2012). In addition, the myosin class III Myo3A associates with the actin filaments in calyceal processes and the distal inner segment (Dosé et al., 2003). More recently, proteins related to the Usher syndrome (USH) have been identified in the calyceal processes (Sahly et al., 2012; Wolfrum et al., 2010). USH proteins are known to form protein networks which associate with membrane adhesion complexes in the stereocilia of inner ear hair cells and in the periciliary region of photoreceptor cells (see section 3.1.4). In the calyceal processes, USH proteins (described below) are thought to form a protein complex required for the adhesion between microvilli membrane to the the outer segment membrane. This theory is strengthen by the observation that both sensory systems, the inner ear and the eye, are affected in patients with USH syndrome. Both of these sensory systems harbor microvilli and microvillus-ciliary structures interconnected by USH protein networks. Rodent models for USH1 gene defects do not develop retinal degeneration displayed in USH patients (Williams, 2008). The absence of calyceal processes in rodent photoreceptor cells may be the cause for this discrepancy between mouse and man. Further examination into the role of calyceal processes should illuminate their role not only in the cell biology of photoreceptor cells but also into the pathmechanisms underlying the retinal degeneration in USH.

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3.1.3 The photoreceptor transition zone In the literature, the connecting cilium is often misleadingly referred to as the ciliary ”axoneme”. The connecting cilium correlates with the transition zone, the short evolutionarily conserved ciliary subdomain at the junction between the basal body and the true axoneme of a prototypic primary and motile cilium (Figure 4B) (Horst et al., 1990; Reiter et al., 2012; Roepman and Wolfrum, 2007; Röhlich, 1975; Sang et al., 2011). The diameter of the connecting cilium (~ 250-300 nm) correlates with the diameter of a primary cilium. However, the length of the connecting cilium can be much longer than the length of a transition zone in a typical primary cilium, which measures ~ 200-300 nm. The length of the photoreceptor connecting cilium is extremely variable in vertebrates, from 1.2 µm in rodent rods and 0.6 µm in human rods (Overlack et al., 2011) to 250 nm in rods of lower vertebrates, amphibians or fish. This length seems negatively 16

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correlate with the diameter of the inner and outer segments; photoreceptor cells with wider inner and outer segments have shorter connecting cilia and vice versa. Furthermore, various disease states can influence this length, causing retinal denegation: for example in a mouse model for Huntington's disease, expression of the mutant Huntingtin protein with an expanded polyglutamine at the N-terminus, causes abnormal elongation of the connecting cilium (Karam et al., 2015). In contrast, in a gene-trap Fam161aGT/GT mouse, a model for autosomal-recessive retinitis pigmentosa 28 (RP28), the connecting cilium is significantly shorter (Karlstetter et al., 2014). In a classical study, Röhlich (1975) analyzed the connecting cilium of rat rods by freeze-fracture and thin-sectioning electron microscopy, revealing the striking morphology of the transition zone. Transverse sections through the connecting cilium revealed radial Y-shaped structures (Y-linkers) that connected each ciliary microtubule doublet to the plasma membrane (Figure 5E, E`). These correspond to transverse strands of intramembranous particles which were previously described in the ciliary necklace of motile cilia (Gilula and Satir, 1972). In the eighties, Basharse and co-workers (summarized in Horst et al., 1990) identified high molecular weight glycoconjugates as surface components of the Y-linkers in crude “axoneme” preparations from bovine retinas (Fleischman et al., 1980). In these studies, a K26 monoclonal antibody was generated which recognized only a single 425 kD component in the “axoneme” fraction and stained the photoreceptor connecting cilium and motile cilium transition zone in motile cilia of oviduct epithelia cells via immunohistochemistry (Horst et al., 1990). The nature of the K26 immunoreactive glycoprotein has not been described so far. Since then numerous proteins localising specifically to the connecting cilium and transition zone have been identified as good candidates for molecular components of the Y-linker and ciliary necklace structures (Khanna, 2015). However, so far only the CEP290/NPHP6/BBS14 protein could be specifically assigned to the Y-linkers in the flagella of the green alga Chlamydomonas or the connecting cilium in mouse (Craige et al., 2010; R. A. Rachel et al., 2015). Nevertheless, the components of several multiprotein complexes which are related to syndromic ciliopathies with a prominent retinal phenotype, including Meckel syndrome (MKS), Joubert syndrome (JBTS), nephronophthisis (NPHP), and Senior–Loken syndrome (SLSN) as well as the severe retinal dystrophy Leber congenital amaurosis (LCA) have been shown to localize to the transition zone of primary cilia (Sang et al., 2011). Based on their prominent localization, it is hypothesized that they participate in the composition of the Y-linkers. The detailed molecular signature and composition of the primary cilia transition zone has recently been summarized (Garcia-Gonzalo and Reiter, 2017). Super-resolution microscopy, namely stimulated emission depletion (STED), has aided the generation of an architectural map of key molecules in the transition zone in primary cilia of human hTERT RPE-1 cells (Yang et al., 2015). Although such a comprehensive, high-resolution analysis of the connecting cilium is lacking, based on their homology the current map should be transferrable to the connecting cilium of vertebrate photoreceptor cells. 17

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However, comparisons of staining patterns for some molecules does reveal differences between the transition zone and the connecting cilium. For example, centrins, small Ca2+EF-proteins, which are regularly present in the pericentriolar matrix and in centrioles of centrosomes and basal bodies (Trojan et al., 2008a) have also been identified as prominent, evolutionary conserved components of the connecting cilium of vertebrate photoreceptor cells (Giessl et al., 2004; Schmitt and Wolfrum, 2001; Wolfrum, 1995; Wolfrum and Salisbury, 1998) (Figure 4D). Therefore, they are commonly used as a molecular marker for the connecting cilium (Karlstetter et al., 2014; Ronquillo et al., 2016; Trojan et al., 2008a). In contrast, Yang and coworkers (2015) only mapped centrin2 to the basal body, but not to the transition zone in hTERT RPE-1 cells. This discrepancy may indicate differences in epitope accessibility during immunostaining procedures between primary cilia in RPE-1 cells vs photoreceptor cilia. Besides common components shared with the transition zone of prototypic primary cilia, the photoreceptor connecting cilium harbors unique components due to its specialization. Two prominent examples are alternative splice variants in the retinitis pigmentosa GTPase regulator (RPGR) gene, RPGR_Orf15 which includes exon 15 and a large isoform of RPGRIP1, RPGRIP1α1, which is specifically expressed in photoreceptor cells (Brunner et al., 2010; Castagnet et al., 2003; Ferreira, 2005; Roepman et al., 2000). In photoreceptor cells, RPGR localizes to the connecting cilium and basal body (Hong et al., 2003; Khanna et al., 2005; Rao et al., 2016). In the last years numerous binding proteins for RPGR have been reported, which probably assemble in supramolecular complexes associated with connecting cilium function (Khanna, 2015). For example RPGR binds to the trafficking chaperon PDEδ (Linari et al., 1999), and to the C-terminus of RPGRIP1, which physically links this complex to nephrocystin-4. This interaction suggests that RPGR is part of distinct a RPGR-NPHP complex (Linari et al., 1999; Patil et al., 2012; Roepman et al., 2005) More recently, a molecular connection between RPGR_Orf15 and the USH protein network via a direct interaction with the USH2D scaffold protein whirlin has been demonstrated (Wright et al., 2012). There is evidence for the enrollment of these RPGR_Orf15 related protein complexes in the control of ciliary transport. A recent study indicated that RPGR regulates entry or retention of soluble proteins in photoreceptor cilia but spares the trafficking of structural and signalling proteins to the outer segment (Rao et al., 2015). Mutations in components of this network lead to severe retinal dystrophies, such as retinitis pigmentosa (RPGR, RP3). RP3 is mutated in up to 20% of all RP patients; Considering the high conservation of transition zone and connecting cilium components, its not surprising that molecular defects in these molecules are associated with syndromic retinal dystrophies, such as the Usher syndrome (USH), nephronophthisis, a combination of nephronophthisis and retinitis pigmentosa, termed Senior-Løken syndrome (SLSN), Bardet-Biedl syndrome, Joubert syndrome type and Meckel syndrome type (discussed in section four).

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3.1.4 The basal body and the periciliary region in photoreceptor cells During ciliogenesis, the microtubule doublets of the connecting cilium extend from the A and B microtubules of the microtubule triplets found in the basal body. Basal bodies are evolutionarily conserved structures, which differentiate from the mother centriole of the centrosome and attach to the plasma membrane via distal transition fibers. The transition fibers serve as docking sites for cargo and IFT particles, which transport proteins within the ciliary compartment. In mature photoreceptor sensory cilia, the daughter centriole, also named the adjacent centriole, remains associated with the basal body embedded in the pericentriolar matrix of the periciliary region of the inner segment. This is consistent with other primary cilia but different from motile multi-ciliated cells.

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3.1.5 The striated ciliary rootlets Photoreceptor cells are characterized by an exceptionally large sensory primary cilium, therefore it has to be anchored to the inner segment via the basal body complex and the ciliary rootlet. These confer structural integrity and long-term survival. Both centrioles of the basal body complex are linked by rootletin fibers, which are also the major components of the prominent striated ciliary rootlets of vertebrate photoreceptor cells (Figure 4B, C) (Yang et al., 2006). The striated ciliary rootlets extend from the basal body and adjacent centriole and project into the inner segment (Wolfrum, 1992). The length of the ciliary rootlet varies between vertebrate species. In some mammals, including human, photoreceptor cell ciliary rootlets are extremely long and project through the inner segment, encompassing the nucleus and terminating at the synaptic terminal (Cohen, 1960; Sjöstrand, 1953; Spira and Milman, 1979). The fibers in the ciliary rootlets are composed of rootletin, a 220 kD protein encoded by the CROCC gene (Yang et al., 2002). Rootletin is capable of forming homodimers arranged in parallel which elongate to form higher order polymers similar to intermediate filaments (Yang et al., 2002). Rootletin polymers are anchored at the proximal portion of both centrioles of the basal body complex by physical interactions with the C-Nap1 (Yang et al., 2006) also known as CEP250. CEP250 has recently been associated with human Usher syndrome (Khateb et al., 2014). Rootletin was serendipitously discovered by evaluating the crossreactivity of antibodies against unrelated proteins with the ciliary rootlet in photoreceptor cells (Yang et al., 2002). In immunohistochemical experiments, diverse antibodies and even preimmune sera can strongly react with the ciliary rootlet in tissue sections. This obvious cross-reactivity with the rootlet is critical and has to be taken into account in the interpretation of immunostainings of the ciliary rootlet. Despite rootletin being essential for forming ciliary rootlets (Yang et al., 2005), rootletin knockout mice exhibit no obvious functional deficits in the retina. Although their photoreceptor cells lack striated rootlets, photoreceptors only degenerate slowly over time. Application of mechanical stress revealed a striking fragility at the ciliary base (Yang et al., 2005). Thus, it seems that similar to intermediate filaments, rootletin fibers support the ciliary rootlet against mechanical forces. In addition to conferring structural 19

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integrity, the ciliary rootlet is thought to organize organelles such as mitochondria or the ER, and possibly serves as a platform for proteins, such as the kinesin light chain (KLC3) which has been found to interact with the globular head domain of rootletin (Yang et al., 2002). Interestingly, the ciliary rootlets in primary cilia differ from the flagellar rootlets found in green algae, such as Tetraselmis or Chlamydomonas (Salisbury, 1995). In contrast to ciliary rootlets of photoreceptor and other primary cilia, flagellar rootlets of green algae and the homologue spasmoneme of some sessile ciliates contract in response to an increase in Ca2+-concentration. These contractions are associated with conformational changes in centrins, which are a major component of the contractile rootlets of protists, but absent from primary ciliary rootlets of primary cilia (Trojan et al., 2008a). Early reports on the presence of centrins in the rootlets of arthropod primary cilia (Wolfrum, 1991) were based on false-positive cross-reactivity of antibodies.

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3.2 Functional modules in photoreceptor cilia Photoreceptor cells have morphologically defined compartments in which specific molecular, biochemical and biological processes are performed (Hofmann et al., 2006). These functional modules are required for signal-transduction, transport of cargo to the outer segment, or disk neogenesis module at the outer segment base. We will describe the molecular machinery required for photoreceptor cilia function on the modular, submodular and molecular level below.

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3.2.1 The basal body a major microtubule organization center In addition to facilitating ciliogenesis, the basal body complex contributes to microtubule organizing center (MTOC) activity in photoreceptor cells (Muresan et al., 1993; Troutt et al., 1990). In photoreceptor cells, microtubules project into the inner segment and cell body, providing transport tracks for microtubule motor complexes. Cytoplasmic microtubules are anchored with their minus ends at the subdistal appendages, but nucleate predominantly at the γ-tubulin ring complexes (γTuRC) in the pericentriolar matrix encompassing the basal body apparatus (Petry and Vale, 2015). Recently, defects in a component of the γ-TuRC have been associated with a chorioretinopathy in humans (Scheidecker et al., 2015). In addition, mutations in TUBGCP4, which encodes γ-tubulin complex protein 4, known to regulate the nucleation and organization of microtubules, cause a spectrum of anomalies in eye development, particularly photoreceptor anomalies (Scheidecker et al., 2015). Proteomic and genomic screens identified many basal body components and suggested additional candidate components, yet to be validated, which associate with basal body function. Initial mass spectrometric analyses of photoreceptor cilia suggested almost 1,200 outer segment protein components and almost 2,000 proteins which were supposed to be associated with the entire ciliary apparatus (Liu et al. 2007). Proteomics of cleaner photoreceptor outer segment preparations from murine retina revealed a list of 511 20

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putative outer segment protein components which, as expected, still contains several impurities (Kwok et al., 2008). More recently a genome-wide knockdown screen targeting proteins related to ciliogenesis revealed further novel proteins associated with the basal body complex in primary cilia of photoreceptor cells (Wheway et al., 2015). These proteins were assigned to an organelle-specific protein landscape and revealed novel molecular modules associated with ciliary function (Boldt et al., 2016).

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3.2.2 The periciliary region Microtubules anchored to the basal body serve as tracks for cytoplasmic dynein-mediated transport of vesicles, bearing ciliary cargo, towards their minus ends (Tai et al., 1999). In the periciliary region surrounding the basal body, inner segment transport molecules target specific sites for cargo loading onto transport complexes, which deliver ciliary compounds to their destination. In frog photoreceptor cells opsin-transport vesicles are targeted to the periciliary ridge complex surrounding the base of the connecting cilium in the periciliary compartment of the inner segment (Papermaster, 2002; Papermaster et al., 1986; Peters et al., 1983). Proteins related to the human Usher syndrome (USH) were identified as molecular components of the periciliary ridge complex (Liu et al., 2007; Maerker et al., 2008). Several studies of the periciliary USH protein complex demonstrated the molecular and structural homology of the periciliary ridge complex in amphibian cells to a ciliary-periciliary membrane adhesion complex (PMC) of mammalian photoreceptor cells (Maerker et al., 2008; Mathur and Yang, 2015; Wolfrum, 2011). The PMC is an evolutionary conserved protein network found in all photoreceptor cells of vertebrates, thus far (Wolfrum, 2011). In this complex, the transmembrane components VLGR1 (very large G-protein coupled receptor, GPR98, ADGRV1, USH2C) and USH2A bridge the adjacent membranes of the inner segment and connecting cilium with their long extracellular domains, forming fibrous links visible by electron microscopy (Figure 4B) (Maerker et al., 2008). The open extracellular space between the membranes resembles the ciliary pocket of non-photoreceptor cilia (Figure 5A, D). Homolog fibers composed of the extracellar domains of VLGR1 and USH2A are found as so-called ankle-links in the differentiating hair bundle of mechanosensitive hair cells linking neighboring stereovilli (Mathur and Yang, 2015; McGee et al., 2006). VLGR1 dificient mice lack these fibrous links between the adjacent membranes in both, hair cells and photoreceptor cells (Maerker et al., 2008; McGee et al., 2006). Both these transmembrane USH2 proteins are anchored to the cytoplasm by binding to the USH2D scaffold protein whirlin via their intracellular domains (Maerker et al., 2008; Sorusch et al., 2017; van Wijk et al., 2006). In the cytoplasm of the inner segment, whirlin additionally interacts with the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) (Maerker et al., 2008). By its association with cytoplasmic dynein, SANS is linked to transport machinery (Papal et al., 2013), which transports rhodopsin vesicles along microtubule tracks to the periciliary inner segment. It 21

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is thought that cargo vesicles are targeted to the membrane by the USH membrane adhesion complex via interaction of whirlin and SANS (Maerker et al., 2008; Wolfrum, 2011). After fusion of the transport vesicle, cargo, such as rhodopsin, is incorporated into the periciliary target membrane for trafficking across the connecting cilium.

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3.2.3 Endocytosis at the ciliary base More recently, the MAGUK protein Magi2 (membrane-associated guanylate kinase inverted-2) was found as an additional component associated with the periciliary USH protein network by interacting with the USH1G protein SANS (Bauß et al., 2014). It had been demonstrated that Magi2 is enrolled in the endocytosis of neurotransmitter receptors (Danielson et al., 2012). More recent studies revealed that Magi2 also mediates endocytosis at the base of primary cilia in cultured cells and photoreceptor cells (Bauß et al., 2014; Wolfrum et al., 2014). These findings affirm the periciliary region as a hot spot for endocytosis from the ciliary pocket of primary cilia (Clement et al., 2013; Ghossoub et al., 2013, 2011). Bauß et al. (2014) demonstrated for the first time that endocytosis at the ciliary pocket is essential for ciliogenesis and/or maintenance of primary cilia. In photoreceptor cells, the high abundance of the transferrin receptor in the membrane surrounding the ciliary pocket suggests that the iron carrier transferrin is endocytosed at this ciliary compartment. Controlled iron uptake is essential for the homeostasis of photoreceptor cells (Picard et al., 2011). Interestingly, there is additional evidence that Magi2 mediated endocytosis is negatively regulated by the binding of phosphorylated SANS associated with vesicular transport (see above) (Bauß et al., 2014).

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3.2.4 Ciliogenesis in photoreceptor cells Photoreceptor cells differentiate at the apical side of the postnatal neuroblastic retina layer (Sedmak and Wolfrum, 2010). Studies in rodents show that ciliogenesis in the differentiating photoreceptor cells is not completely synchronized. The first stages are similar to the steps required for ciliogenesis of primary cilia (Pedersen et al., 2008; Sedmak and Wolfrum, 2010; Sorokin, 1962, 1968) (Figure 6). An intracellular primary ciliary vesicle appears at the distal end of the mother centriole, which maturates into the basal body. During this process, two sets of accessory structures, the distal and sub-distal appendages, appear at the basal body barrel. While the distal appendages protrude towards the membrane of the primary vesicle, the sub-distal appendages project into the pericentriolar cytoplasm. The ciliary vesicle grows by fusion of post-Golgi vesicles. Simultaneously microtubules doublets extend from the basal body (namely the A and B doublet of the microtubule triplet from the mother centriole) and invaginate the vesicle. During the course of differentiation, ciliary components e.g. IFT molecules and BBS proteins associate with post-Golgi vesicles and are thereby trafficked to and incorporated into the growing ciliary vesicle (Sedmak and Wolfrum, 2010). Recently details of the molecular mechanism underlying the ciliary vesicle formation and extension in primary cilia have been identified (Lu et al., 2015). Two Eps15-homology-domain (EHD)22

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containing proteins, namely EHD1 and EHD3 recruit the SNARE protein SNAP29 and thereby regulate the conversion of small distal appendage vesicles into larger ciliary vesicles. During this process, the extending ciliary vesicle encloses the growing ciliary bud until the ciliary vesicle fuses with the plasma membrane exposing the cilium to the extracellular milieu. In photoreceptor cilia, the distal part of the evolving cilium swells and elongated tube-like membranous vesicles appear, which fuse to disk-like membranes. Although some of these membranous disks are already shaped as in mature outer segments, they still lack the characteristic over all organization. For final maturation of the outer segment structures, expression of structural proteins, e.g. disk-rim proteins and rhodopsin is essential. In the absence of rhodopsin, such as in the retina of rhodopsin knockout mice, rods form cilia with only a rudimentary outer segment which do not contain any stacked disks (Connell et al., 1991; Lee et al., 2006). It is important to note that during retinal morphogenesis the photoreceptor ciliogenesis proceeds in close association with maturation of the retinal pigment epithelium (RPE). This close association may be critical for differentiation. Although both epithelia are separated by the subretinal space, there is physical interaction via the abundant microvilli of the RPE cells with the maturing photoreceptor cells. Exchange of signalling molecules cannot be excluded.

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3.2.5 The phototransduction module The outer segment of rod and cone photoreceptor cells are specialized to capture photons. They harbor the entire phototransduction machinery, for propagation of the incoming light signal, termination of receptor signalling and its adaption. The visual transduction cascade is one of the best-studied G protein coupled signalling cascades, which has been discussed in-depth in several comprehensive reviews (Arshavsky and Burns, 2012; Koch and Dell’Orco, 2015). In brief, photons cause the isomerization of 11-cis retinal, a chromophore covalently bound to visual pigments (rhodopsin in rods or opsins in cones). Photoexcited pigments activate the heterotrimeric G protein, transducin, whose dissociated α-subunit then activates cGMP-phosphodiesterase (PDE6). The resulting decrease in cytoplasmic cGMP leads to the closure of cGMP-gated cation channels (CNG) located in the photoreceptor plasma membrane.The heterotetrameric CNG-gated channels are composed of three α-subunits and one β-subunit which are cell specific (Zheng et al., 2002). Closure of the CNG hyperpolarizes the photoreceptor cell. In consequence, less glutamate is released at the synaptic terminals connected to the second order neurons for conduction and processing of the electrical signal. Termination of receptor signalling is induced by the phosphorylation of rhodopsin by G-protein coupled receptor kinase (GRK1) which allows binding of arrestin (ARR1) (Gurevich et al., 2011). To return to the dark state, the other molecules of the signalling cascade are deactivated in several additional biochemical reactions (Lamb and Pugh, 2006). CNG channels open due to the rise of cGMP levels, which is facilitated by the 23

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activation of the retinal guanylyl cyclase (RetGC1) through Ca2+-sensing guanylatecyclase-activating proteins (GCAPs) (Baehr and Palczewski, 2007; Koch and Dell’Orco, 2015). This leads to a partially depolarized state of photoreceptor cells and an increase of glutamate release at the synapses in the dark. Finally all-trans retinal is converted back to 11-cis retinal to regenerate rhodopsin (Saari, 2012, 2000). The enzyme pathway of the visual cycle is carried out in RPE cells in close interaction with photoreceptor outer segment compartments. For the cone system, a non-canonical visual cycle has been recently identified in Müller glia cells (Kaylor et al., 2014). Mutations within most of the genes encoding phototransduction proteins have been associated with retinal dystrophies. A comprehensive list of retinal disease genes is regularly updated on https://sph.uth.edu/retnet/home.htm. Mutations in the genes encoding rhodopsin, CNGA1, CNGB1, PDE6A or PD6B cause retinitis pigmentosa (RP), mutations in the genes encoding arrestin, rhodopsin kinase or NCKX1 cause congenital stationary night blindness (CSNB). Mutations in the genes encoding cone CNG channel subunits CNGA3 and CNGB3, cone PDEH or cone transducin (GNAT2) give rise to Achromatopsia, and mutations in the GUCY2D gene encoding in GC-1 have been linked to Leber congenital amaurosis (LCA) and cone-rod dystrophy (CRD). Defects in genes encoding for components of the visual cycle, e.g. ABCA4, RDH5, RDH12, LRAT, RPE65) also lead to retinal diseases (see section four).

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3.2.6 Outer segment renewal/disk neogenesis module Photoreceptor cells continually regenerate outer segments throughout their lifetime. This phenomenon serves as a preventive mechanism for molecular aging and accumulation of damaged molecular components, such as damage by photo-oxidative stress related to constant photon bombardment. Rod photoreceptor cells regularly shed disks from the distal outer segment on a daily basis (10%/day), which are than phagocytosed by the adjacent RPE cells. The phagocytosis of outer segment disk packages requires coordinated activities of numerous cell surface and cytosolic proteins (Mazzoni et al., 2014). For example, phosphatidylserin found on the plasma membrane of outer segment tips is recognized by a lactadherin-αvβ-integrin-focal adhesion kinase (FAK) complex on the RPE cell as a “digest-me-signal”. New membrane disks are continually replaced at the base of the outer segment (see below). Outer segment renewal is probably the most extensive turnover of the ciliary compartment in cilia biology. Richard Young first described outer segment renewal in an elegant pulse-chase study in several vertebrate species in the sixties (Young, 1967). For this, he monitored the appearance of radioactively tagged proteins in the retina after feeding of radioactive amino acids. Autoradiogram analyses of retinal sections demonstrated that tagged proteins first appear at the site of protein biosynthesis in the inner segment, but subsequently locate to discrete bands at the base of the outer segment. The band of labeled proteins move apically as new unlabeled disk proteins are incorporated at the base of the outer segment. In rodents or Xenopus, phagocytosed radioactive proteins were 24

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found in RPE cells after ~ 10 days (Besharse et al., 1977; Young, 1967). In contrast, the renewal of the rod outer segments was nearly five-times slower in the frog Rana pipiens (Young and Droz, 1968). The renewal of cone outer segments was similarly proven in species with cone-dominant retinas, such as the cat or the ground squirrel (Anderson et al., 1978; Anderson and Fisher, 1975). More recently analyses of outer segment renewal using fluorescently-tagged rhodopsin and peripherin-2, indicated that light activation regulates ciliary protein transport and outer segment disk renewal in photoreceptor cells (Hsu et al., 2015). This study suggested that rhodopsin is preferentially incorporated into newly formed disks in the dark while the disk rim protein peripherin-2 is integrated under light conditions, resulting in alternating stacks of separate rhodopsin-rich and peripherin-2-rich disks. However, these findings may be over-interpreted since a previous study had shown that the lightdependent axial variation in rhodopsin observed by these authors is only a property of transgenically expressed eGFP-rhodopsin. Endogenous rhodopsin does not follow this banding pattern, as measured by microspectrophotometry (Haeri et al., 2013). Although the renewal of outer segment disks was identified five decades ago, the mechanism by which new disks are added at the base of the outer segment long remained an enigma and two contrary hypotheses have been proposed. Initial studies suggested that disks are formed by the evagination and subsequent expansion of the plasma membrane (Steinberg et al., 1980). In the endosome model, disk membranes are formed by multiple vesicles fusion to grow into larger disks (Obata and Usukura, 1992; Chuang et al., 2007). The latter hypothesis was supported by electron microscopy and the identification of molecular components which have been associated with vesicular trafficking and membrane fusion in other systems. However, a systematic analysis of nascent rod outer segment disk membranes by electron microscope comparing different fixation methods (Ding et al., 2015) combined with recent 3D tomography studies in diverse mammalian species strongly support the evagination hypothesis (Volland et al., 2015; Burgoyne et al., 2015). Further supporting the evagination hypothesis, Wensel and colleagues did not observe any vesicles in the connecting cilium or at the base of the outer segment upon cryo-EM (Gilliam et al., 2012). For an comprensive overview please see Goldberg et al., 2016. In any case, the process of disk neogenesis is thought to be facilitated by actin filaments (Chaitin et al., 1984). These actin filaments are found to be anchored to the axonemal microtubules. They project into the disk neogenesis compartment at the base of the outer segment and attach to vesicle membranes or the plasma membrane via their fast growing barbed-ends (Chaitin and Burnside, 1989; Obata and Usukura, 1992). Nonmuscle myosin II associates with these actin filaments, assembling a potential contractile system (Chaitin and Coelho, 1992). Interestingly, actin filaments are not only essential for the coordination of the formation of the disk membrane region, but also for trafficking of outer segment plasma membrane proteins such as CNGA1 to the outer segment (Nemet et al., 2014). 25

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The pentaspan transmembrane glycoprotein prominin-1 (PROM-1) is found at the far rim of the nascent disks (Han et al., 2012; Stuck et al., 2016). Prominin-1, also known as CD133, associates with lipid rafts by interacting with plasma membrane cholesterol and exhibits a profound preference for membrane curvature (Corbeil et al., 2001). Defects in prominin-1 disrupts disk morphogenesis and results in progressive degeneration of photoreceptor cells, associated with autosomal-dominant macular degeneration, Stargardt disease or cone-rod dystrophies (Yang et al., 2008). At the outer segment base, prominin1 associates with actin filaments and interacts with the photoreceptor-specific protocadherin, PCDH21 (pr-CAD) (Yang et al., 2008) which was also found at the rim of the nascent disk membranes (Rattner et al., 2001). PCDH21 was identified as a component of fibers which link the fragile nascent disk membranes of the outer segment base with the apical membrane of the inner segment of rod photoreceptor cells (connecting junctions in Figure 4B) (Burgoyne et al., 2015). Interestingly, another protocadherin, PCDH15, is localized vis-à-vis in the apical inner segment (Overlack et al., 2010) and may act as a counterpart from the inner segment membrane. PCDH15 is known to interact with cadherin 23 (CDH23) to form the tip-link filaments linking neighboring stereocilia of mechanosensitve hair cells (Kazmierczak et al., 2007). Molecular modeling and mutation analyses have indicated that binding of PCDH15 and CDH23 is mediated via polar amino acids in their N terminal ectodomains. This differs from the strand-swap binding mode of classical cadherins, but is similar to other protocadherins, especially PCDH21 (Elledge et al., 2010). The spatial proximity, together with the shared molecular binding mode, suggests that PCDH21 may interact with PCDH15 to form the fibrous junctions between the membranes of inner and outer segments. During disk maturation, the fibrous junctions are dispersed by a single proteolytic cleavage of PCDH21 resulting in the release of a soluble ectodomain and a Cterminal fragment including the transmembrane domain, which remains within the outer segment plasma membrane (Rattner et al., 2004).

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3.3 Ciliary transport in photoreceptor cells Similar to prototypic cilia, the photoreceptor primary cilium lacks biosynthesis machinery for proteins and lipids. All components required for outer segment morphogenesis, maintenance and sensory function must be transported from the site of synthesis in the inner segment to the outer segment (Figure 7). It is possible to discriminate between the unidirectional transport of membrane bound components of the outer segment disks and the bidirectional translocation of soluble signalling molecules into and out of the outer segments (Karan et al., 2008; Pearring et al., 2013). 3.3.1 Unidirectional transport of outer segment compounds In rods, outer segment proteins associated with disk membranes are incorporated in nascent disks at the base of the outer segment. Outer segment proteins remain within

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disks during maturation and aging, until they are phagocytosed by RPE cells and then degraded.

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3.3.2 Rhodopsin transport via a secretory pathway Most insights into the mechanisms of membrane protein trafficking and targeting to the outer segment come from studies on rhodopsin transport (see reviews by Nemet et al., 2015; Wang and Deretic, 2014). Similar to other transmembrane proteins, rhodopsin is synthesized at the rough ER and sequentially transported towards its final destination via submodules, following a conventional secretory pathway regulated by small GTPases acting as molecular switches. Molecular interactions and the precise sequence of events required during rhodopsin trafficking has been systematically analyzed for decades. Similar to other ciliary membrane proteins, such as polycystin-1 and polycystin-2 (PKD2) (Geng et al., 2006; Ward et al., 2011) or the nucleotide-gated olfactory channel CNGB1b subunit (Jenkins et al., 2006), rhodopsin processes a VxPx motif in its Cterminus (Chuang and Sung 1998). Rhodopsin´s VxPx motif is sufficient to target rhodopsin’s C terminus to the apical membrane of cultured cells (Tai et al., 2001) and to the outer segment of frogs and zebrafish (Tam et al., 2000; Perkins et al., 2002). The Cterminus of rhodopsin is highly conserved among vertebrate species and is a hot spot for mutations in RP patients (Sung and Tai, 2000). In addition, rhodopsin contains a targeting motif in the cytoplasmic helix 8, comprised of amino acids phenylalanine and arginine (FR), necessary for recruiting further ciliary targeting complex components. Deretic and coworkers studied rhodopsin transport in the frog retina, taking advantage of increased membrane trafficking due to the huge outer segments of amphibian rod photoreceptors (summarized in: Papermaster, 2002). Applying a combination of biochemical tools, such as pulse chase studies with radiolabeled compounds, subcellular fractionation and co-immunoprecipitation they identified key players in formation and targeting of rhodopsin transport to the plasma membrane (reviewed in Wang and Deretic, 2014). After maturation of rhodopsin in the Golgi apparatus the small GTPases Arf4 and Rab11, the Arf GAP (GTPase activating protein) ASAP1 and the Rab11-Arf interacting protein FIP3, combine to initiate the assembly of a ciliary targeting complex. First, the activated small GTPase Arf4 binds to the VxPx motif in the C-terminus of rhodopsin and ASAP1 recognizes the FR motif. Following this, FIP3 and Rab11 are recruited to the complex. ASAP1 promotes membrane curvature through its BAR domain and thereby vesicle budding from the trans Golgi network (TGN) (Mazelova et al. 2009). Following GTP hydrolysis of Arf4 and its release, the remaining components recruit Rab8 and Rabin8. Both, Rab8 and its effector Rabin8 are proposed to regulate the fusion of the cargo vesicle with the target membrane by association with the BBSome (Nachury et al. 2007). A direct role for Rab8a or Rab11a in rhodopsin transport has been verified by overexpression of dominant-negative proteins or shRNA knockdown in Xenopus (Moritz et al., 2001; Reish et al., 2014), but could not be confirmed in retina-specific Rab11a and Rab8a single or double knockout mice (Ying et al., 2016). These observations may reflect 27

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significant interspecies variation in rhodopsin trafficking or may be based on molecular redundancy of Rab components. Further validation and clarification of this fundamental process in photoreceptor cells is necessary. The outlined working model describes the formation of the ciliary targeting complex at the TGN and can partly explain targeting to the ciliary base. However, it does not cover any description of the machinery required for the vectorial transport of rhodopsin after budding from the TGN through the inner segment to the ciliary base. Yeast-2-hybrid screens revealed that the VxPx motif does not only bind Arf4, but also the dynein light chain Tctex-1 (DYNLT1) (Tai et al., 1999). After release of Arf4 from the targeting complex, the VxPx motif is free to bind Tctex-1, which links the transport vesicle to cytoplasmic dynein. In vitro assays revealed that Tctex-1 and cytoplasmic dynein are essential for vectorial transport of rhodopsin bearing vesicles along microtubules. Electron microscopy and super resolution light microscopy demonstrated that microtubules were tracks for the transport of rhodopsin bearing vesicles (Overlack et al., 2011; Tai et al., 1999). More recently a group of proteins related to Usher syndrome have been identified on transport vesicles, together with spectrin βV, further linking rhodopsin to the cytoplasmic dynein motor complex (Papal et al., 2013). In conclusion, there is strong evidence for cytoplasmic dynein-mediated transport of rhodopsin bearing cargo vesicles from the TGN along microtubules towards their minusend located at the base of the photoreceptor cilium. As discussed above transport vesicles containing rhodopsin fuse with the apical inner segment membrane at a well defined membrane domain of the periciliary membrane complex (PMC) (Maerker et al., 2008; Yang et al., 2015). At the PMC and the basal body, ciliary cargo is transferred to ciliary transport machinery for delivery to the outer segment destination (Sedmak and Wolfrum, 2010; Wolfrum and Schmitt, 2000).

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3.3.3 Rhodopsin transport in the ciliary membrane For a long time the transport of opsin across the connecting cilium to the outer segment was an enigma. Failed and none-reproducible early immunoelectron microscopy labeling of opsin in the ciliary membrane gave rise to hypotheses favoring vesicular transport of rhodopsin (Horst et al., 1990) which are still being discussed (Chuang et al., 2015). However, since the turn of the millennia improved immunoelectron microscopy evidently revealed that rhodopsin molecules are in fact transported within the ciliary membrane to the outer segment (Liu et al., 1999; Wolfrum and Schmitt, 2000). More recently, an independent study nicely reproduced these data (Burgoyne et al., 2015). Immunoelectron microscopy analyses of opsin in photoreceptor cells of shaker-1 mice, deficient for myosin VIIa, revealed the accumulation of opsin in the ciliary membrane (Liu et al., 1999; Wolfrum and Schmitt, 2000). Based on these findings, myosin VIIa was hypothesed to participate in the transport of opsin across the connecting cilium to the outer segment base. Immunoelectron microscopy also provided evidence for submembranous actin filaments in the connecting cilium along which the myosin motor may 28

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move (Wolfrum and Schmitt, 2000). However, since myosin VIIa is highly concentrated in the RPE (Liu et al., 1997) and is essential for the phagocytosis of the outer segment tips by RPE cells (Liu et al., 1998), phagocytosis defects may affect the cargo transport to the outer segment. Therefore, the backup of opsin molecules in the ciliary membrane could be a secondary consequence due to disruption of phagocytosis of outer segment tips by myosin VIIa deficient RPE cells and not a consequence of defective trafficking in the connecting cilium.

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3.3.4 Intraflagellar transport (IFT) As in all cilia, the transport machinery in the photoreceptor cilium is based on intraflagaellar transport (IFT) particles (Cole et al., 1998; Pazour et al., 2002b; Rosenbaum and Witman, 2002; Sedmak and Wolfrum, 2010). Rhodopsin is probably the most often-cited cargo of the intraflagellar transport (IFT) system in photoreceptor cells. A crucial role of IFT for the delivery of outer segment cargo was proposed in 1999 (Rosenbaum et al., 1999). The first supporting evidence came from mislocalization of rhodopsin in mouse models with disrupted anterograde IFT motor molecules (Marszalek et al., 2000). However, recent studies on rod specific kif3a and kif17 single knockout and double knockout mice indicated that both kinesin-IIs are probably not essential for the transport of rhodopsin or other phototransduction membrane proteins in rod photoreceptor cells (Jiang et al., 2015a, 2015b). These findings, coupled with the observation that loss of IFT88 also does not have an effect on rhodopsin trafficking (Jiang et al., 2015b), either suggest alternative non-IFT related mechanisms required for rhodopsin trafficking across the connecting cilium, or imply a high degree of functional redundancy between IFT transport motors. Nevertheless, these studies showed that both kinesin-IIs are required for the early stages of ciliogenesis, the correct formation of the connecting cilium and formation of the outer segment axoneme during photoreceptor cell differentiation. Ciliogenesis defects in kif3a deficient cells may also explain the observed reduction in fluorescence recovery after photobleaching (FRAP) of RHO-EGFP in connecting cilia of Kif3a−/− mouse rod photoreceptor cells in vivo (Trivedi et al., 2012). The essential role of IFT molecules for correct development and maintenance of the photoreceptor outer segments was first demonstrated in the Tg737orpk mouse, which contains a mutation in the IFT-B protein IFT88 (Pazour et al., 2002a, 2000). Further studies in mouse and zebrafish showed that mutations and knock outs of multiple IFT components prevent outer segment formation in photoreceptor cells (Crouse et al., 2014; Hudak et al., 2010; Keady et al., 2011; Krock and Perkins, 2008; Omori et al., 2008; Sukumaran and Perkins, 2009; Tsujikawa and Malicki, 2004). Immunostaining revealed that IFT complex A and B proteins are concentrated in the periciliary region near the basal body of the photoreceptor cilium (Figure 8) (Pazour et al., 2002a; Sedmak and Wolfrum, 2010). There, the IFT molecules are found to be associated with transport vesicles and centriolar satellites (Sedmak and Wolfrum, 2011, 2010). They may participate in the transfer of ciliary cargo from these transport complexes to the IFT trains 29

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which are thought to be assembled in the periciliary region at the ciliary base (Pigino et al., 2009; Sedmak and Wolfrum, 2010). In the periciliary compartment, IFT molecules may act in concert with components of the BBSome, especially for sorting and delivery of ciliary membrane proteins (Nachury et al., 2010). This is consistent with the localization of all eight BBSome components at the base of the photoreceptor cilium (Figure 8) (Smith et al., 2013; Spitzbarth et al., manuscript in preparation; Wolfrum et al., 2012). In addition to their high concentration at the ciliary base, prominent localization of IFT molecules has been consistently found at the outer segment base at the disk neogenesis module (Pazour et al., 2002a; Sedmak and Wolfrum, 2010). In this compartment, the IFT trains may release cargo molecules required for the formation of novel disks (Pazour et al., 2002a; Sedmak and Wolfrum, 2010). Although guanylate cyclase (GC-1) has been identified in complexes with IFT components by biochemical experiments there is no direct evidence for IFT cargos in photoreceptor cells so far (Bhowmick et al., 2009). Interestingly, the BBSome components are not present in the disk neogenesis module at base of the outer segment (Smith et al., 2013; Spitzbarth et al., manuscript in preparation; Wolfrum et al., 2012) suggesting that BBS molecules are not involved in release of disk cargo. Less abundant IFT and BBS molecules are detected in the connecting cilium of photoreceptor cells (Pazour et al., 2002a; Sedmak and Wolfrum, 2010; Wolfrum et al., 2012). This may reflect the high velocity of the IFT particle and attached BBS molecules passing through the connecting cilium. A subset of IFT molecules, namely IFT88 and IFT140 associate with the axoneme longitudinally, projecting through the outer segment past the connecting cilium (Sedmak and Wolfrum, 2010). Only a few BBS molecules of the BBSome complex are found in the axoneme of photoreceptor cells (Smith et al., 2013; Spitzbarth et al., manuscript in preparation; Wolfrum et al., 2012). This may suggest that only a subset of IFT proteins or BBS proteins participate in IFT or are transported along these axonemal microtubules. As in other primary cilia, structural components of the axoneme such as microtubule tubulin subunits are relevant IFT cargo and are transported along microtubule tracks towards the tip where tubulin subunits are incorporated into microtubules at their plus end (Hao et al., 2011). 3.3.4.1 IFT20 in ciliary transport In contrast to other IFT molecules, IFT20 is the only molecule found in the Golgi apparatus in addition to the periciliary region (Follit et al., 2009, 2006; Sedmak and Wolfrum, 2010). IFT20 is anchored in the cis-Golgi by the golgin GMAP210 (Follit et al., 2008). GMAP210 and IFT20 can interact with rhodopsin independently from other IFT B molecules and an acute deletion of IFT20 results in cone opsin accumulation at the Golgi complex (Keady et al., 2011). Mutations in the kinase regulatory subunit VPS15 (PIK3R4) affects IFT20 release from the Golgi and causes a ciliopathy (Stoetzel et al., 2016). In conclusion, these data supports a model that IFT20 participates in the formation 30

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of transport carriers in the Golgi complex and in the transport of membrane proteins from the Golgi complex to the base of cilium (Sedmark and Wolfrum, 2010; Keady et al 2011). The association of IFT20 with transport vesicles at docking zone the cargo vesicles in periciliary region of photoreceptor cells further support this hypothesis (Sedmark and Wolfrum, 2010, 2011). Although there is no doubt that IFT20 is essential for the correct formation and maintenance of photoreceptor cilium, the role of IFT20 in the canonical IFT transport machinery through the photoreceptor cilia is still controversy discussed (Sedmark and Wolfrum, 2010; Keady et al 2011).

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3.3.4.2 Retrograde transport IFT-A components are predominantly thought to be required for retrograde trafficking of IFT trains transported by the cytoplasmic dynein 2 complex (heavy chain: DYNC2H1) (Pedersen and Rosenbaum, 2008). A canonical retrograde dynein-2 motor complex was identified in the photoreceptor cilium (Mikami et al., 2002) and morpholino knockdowns in zebrafish revealed an essential role for the dynein-2 complex for outer segment maintenance (Krock et al., 2009). Interestingly, the dynein-1 heavy chain, which is the characteristic dynein in the cell body, was additionally found in the axoneme of outer segments of zebrafish photoreceptor cells and may act as a second retrograde IFT motor (Insinna et al., 2010). Immuoelectron microscopy on murine and human photoreceptor cells revealed that IFT-A components co-localize with all IFT-B proteins and BBSome proteins at the ciliary base (assembly of IFT trains), along the connecting cilium, and with a subset of IFT-B proteins (e.g. IFT88) and BBSome components in the outer segment axoneme (Sedmak and Wolfrum, 2010; Smith et al., 2013; Wolfrum et al., 2012). Since the outer segments are continually renewed (see section 3.2.6), the retrograde facet of IFT in photoreceptor cilia might be considered less critical than in other cilia. Nevertheless, IFT particles including the associated molecular motor complexes need to be recovered. In contrast to earlier work in zebrafish (Tsujikawa and Malicki, 2004) a recent study show that the IFT complex A protein IFT140, is required for development and maintenance of outer segments in the murine retina (Crouse et al., 2014), which is confirmed by findings of IFT-A components in other primary cilia. Recent data indicated that BBS molecules play a critical role in retrograde IFT in primary cilia and in particular in retinal photoreceptor cells (Datta et al., 2015; Liew et al., 2014). Proteomic analysis of the outer segment fraction of photoreceptor cells from BBS (Lztfl1/Bbs17) mutant mice revealed an enrichment of non-outer segment proteins, which was confirmed in situ by immunohistochemistry showing accumulation of such proteins in the photoreceptor outer segment. These findings suggest that a major function of BBS proteins in photoreceptors cells is to facilitate the retrograde transport of proteins from the outer segment back to the inner segment or to prevent entry of non-resident proteins into the outer segment compartment (Datta et al., 2015). Further studies are needed to validate this mechanism in primary cilia and photoreceptor cilia. 31

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3.3.5 Alternative ciliary transport pathways It has previously been suggested that the VxPx motif is a general outer segment/ciliary targeting signal for both, integral and peripheral membrane proteins in photoreceptor cells (Luo et al., 2004). However, so far besides rhodopsin it has only be found as a functional targeting signal in retinol dehydrogenase (prRDH) (Luo et al., 2004) which catalyzes the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment (Maeda et al., 2005). The ciliary delivery of other outer segment proteins appear to rely on altherantive targeting motifs and transport mechanisms. In contrast to the CNGB1b channel subunit of CNG channels in olfactory sensory cells (Jenkins et al., 2006), the visual CNGB1a subunit does not possess a VxPx motif. Its ciliary delivery is driven by the membrane adaptor ankyrin-G, which directly binds to the C-terminus of CNGB1a (Kizhatil et al., 2009). Ankyrin-G is required for both the postGolgi transport of CNGB1a and its anchoring to the plasma membrane of the rod outer segment. A more recent study demonstrated that the actin cytoskeleton at the base of the outer segment is essential for deivery of CNGA1 into the outer segment plasma membrane (Nemet et al., 2014). Another abundant outer segment protein, the disk rim protein peripherin-2 (PRPH2/RDS), is transported separately (Fariss et al., 1997). Although PRPH2 and rhodopsin might assemble into a functional complex once inside rod outer segments (Becirovic et al., 2014), the majority of PRPH2 traffics via an unconventional secretory pathway bypassing the TGN (Abd-El-Barr et al., 2007; Tian et al., 2014). However, the rhodopsin-PRPH2 complex could not be reproduced in a more recent publication (Pearring et al., 2015). Similar to rhodopsin, the C-terminal tail of PRPH2 is important for ciliary targeting, yet it lacks any well-defined targeting motif and no known proteins which recognize its C-terminus have been identified (Salinas et al., 2013; Tam et al., 2004; Tian et al., 2014).

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3.3.6 Co-transport, trafficking chaperones and transport of complexes Several outer segment proteins do not possess major ciliary targeting sequences and therefore require cofactors for their transport to the outer segment. The progressive rodcone degeneration (PRCD) protein, a protein of unknown function is guided together with rhodopsin to the outer segment (Spencer et al., 2016). Similarly guanylate cyclase-1 (GC-1) has also been shown to be trafficked in a complex with rhodopsin (Pearring et al., 2015). Further studies show that GC-1 is also co-transported with retinal degeneration 3 (RD3) protein to the outer segment (Azadi et al., 2010; Zulliger et al., 2015b). Impaired RD3-mediated GC-1 expression and trafficking leads to severe retinal dystrophy. During this transport, RD3 negatively regulates GC-1 activity, which is activated by RD3 release (Peshenko et al., 2011). Cone S- and M/L-opsins need be regenerated with the chromophore 11-cis retinal to permit transport to the outer segments (Zhang et al., 2008). Furthermore, the presence of 11-cis retinal is also essential for proper transport of several

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membrane-associated molecules of the cone phototransduction pathway, namely GC-1, cone transducin, cone PDE6α', and the opsin kinase GRK1. Trafficking chaperones assist the transport and delivery of lipidated cargo proteins to the outer segment. The PrBP/δ protein also known as the δ-subunit of the cGMP phosphodiesterase PDE binds to prenylated proteins like PDE or farnesylated cargo such as the GRK1 for their delivery to the outer segment (Zhang et al., 2007). Another lipid binding chaperone is UNC119, which binds acylated protein cargo such as the α-subunit of transducing Gtα to assist its trafficking to photoreceptor outer segments (Wright et al., 2011; Zhang et al., 2011). Both trafficking chaperones PrBP/δ and UNC119 release their cargo upon binding of GTP-activated ADP-ribosylation factor (Arf)-like small GTPases Arl2 and Arl3 (Ismail, 2016). In the cilium release of the cargo from the carrier is triggered by guanyl exchange factor (GEF) Arl13B which switches Arl3 into Arl3_GTP (Gotthardt et al., 2015). At the ciliary base the retinitis pigmentosa 2 (RP2) protein stimulates the hydrolysis of Arl3_GTP which leads to the dissociation of the carriers from Arl3_GDP (Evans et al., 2010). Recently, an additional role of ARL3 in the regulation of IFT in photoreceptor ciliogenesis has been identified (Hanke-Gogokhia et al., 2016) in addition to its function as a release factor of trafficking chaperones in mature photoreceptor cells. Another example of a co-transport submodule is the trafficking of the RGS9-Gβ5 GTPase activating complex to the ciliary compartment of photoreceptor cells. In rods, the RGS9-Gβ5 GTPase activating complex, required for the activation of transducin, is anchored to the surface of the disk membrane by the R9AP protein (Arshavsky and Wensel, 2013). Interestingly, RGS9-Gβ5 contains a targeting signal motif which completely excludes the protein from the outer segment (Gospe et al., 2011). Binding of R9AP erases this signal and traffics the multi-protein complex to its destination in the outer segment.

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3.3.7 Transport and targeting of soluble molecules Early studies suggested that soluble proteins diffuse freely between the different compartments of the photoreceptor cell (Nair et al., 2005; Rosenzweig et al., 2007). Specific estimates of protein diffusion rates into and out of the ouster segment have been calculated (Calvert et al., 2006). More recently a ciliary pore complex has been proposed to act as a size dependent diffusion barrier at the ciliary base (Kee et al., 2012). However, theoretical analyses and in vivo experiments have not yet provided any evidence for a diffusion barrier for soluble proteins, either at the transition zone of primary cilia (Breslow et al., 2013) or at the connecting cilium of photoreceptor cells (Najafi et al., 2012). Fluorescent proteins of the same size as outer segment soluble proteins (between 27 kDa to 81 kDa) cross the connecting cilium at the same rate (Najafi et al., 2012). Nevertheless, the narrowness of cytoplasmic space between tightly packed disks significantly hinders the penetration of soluble proteins. This limited accessibility of

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outer segment cytoplasmic volume markedly reduces the fraction of soluble proteins present in this (Najafi et al., 2012).

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3.3.8 Bidirectional movement of signalling molecules Light-induced translocation of several phototransduction proteins occurs between the inner and outer segment. In dark-adapted rod photoreceptor cells, the Ca2+-binding protein recoverin is present in all subcellular compartments. Upon illumination it translocates form outer segments to synaptic terminals (Strissel et al., 2005). This allows recoverin to facilitate its dual functions. On one hand, it regulates the activity of rhodopsin kinase in the outer segment (Chen et al., 2012; Klenchin et al., 1995) and on the other hand it modulates synaptic output (Sampath et al., 2005). The light dependent reciprocal movements of arrestin and the heterotrimeric visual G protein, transducin, has been intensely studied over the last 15 years. Pearring et al. (2013) recently reviewed a detailed examination of this topic. The translocation of arrestin and transducin is thought to contribute to the adaptation of photoreceptor cells enabling them to perform at a wide range of ambient illumination. In response to light, arrestin moves to the rod outer segment whereas transducin translocates into the inner segment (Whelan and McGinnis, 1988). The kinetics of arrestin and transducin movements are comparable with the diffusion rate of soluble GFP (Calvert et al., 2010; Nair et al., 2005), each occurring on the timescale of minutes. In the dark, lipid modifications on each subunit allows the transducin heterotrimer GαtGβ1γ1 to bind to the disk membranes with a high affinity. After illumination, binding of transducin to activated rhodopsin causes a separation of the GTP-bound Gαt from the non-dissociable Gβ1γ1. This lowers the membrane affinity of the subunits followed by subsequent dissociation of Gαt and Gβ1γ1 from the disk membrane. The trafficking chaperone UNC119 assists translocation of transducin to the outer segment in the dark. Phosducin and PrBP/δ are required for the retranslocation of Gβ1γ1. The diffusion of transducin is linked to saturation of the membrane bound phosphodiesterase PDE effector, and because cones cannot be saturated by bright light, transducin translocation does not occur in cones (Lobanova et al., 2010). It is noteworthy that the interaction and co-localization of centrins with transducin led to the hypothesis that the assembly of centrin-transducin complexes may regulate the transition of transducin through the connecting cilium of photoreceptor cells (Pulvermüller et al., 2002; Trojan et al., 2008). However, recent studies in Cetn1 and Cetn2 knockout mice did not provide any evidence for this hypothesis (Avasthi et al., 2013; Ying et al., 2014). The ciliary function of centrins and their interaction with Gβ1γ1 has yet to be determined. In the dark arrestin (Arr1) is found predominately in the inner segment and only at a low concentration in the outer segment, mainly localized in the cytoplasmic space of the axoneme (Strissel et al., 2006; Smith et al., 2013). Although Arr1 is thought to freely diffuse throughout all compartments of the photoreceptor cells in the dark, the high concentration of Arr1 in the inner segment can be explained by the fact that the inner 34

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segment represents a significantly larger available cytoplasmic volume than other compartments of the rod photoreceptor cell. In addition, Arr1 may be removed from the diffusion equilibrium/gradient by binding to microtubules or other structural partners present in the inner segment (Nair et al., 2005). Furthermore, the low concentration of Arr1 in the outer segment can be also explained by steric volume exclusion of Arr1 oligomers from the space between the outer segment disks (Najafi et al., 2012). The first hypothesis for light-induced Arr1 translocation to the outer segment was based upon its high affinity binding to phosphorylated rhodopsin R* (Pearring et al., 2013). However, rhodopsin phosphorylation is not required for light-induced Arr1 movement to the outer segment (Mendez et al., 2003). Because Arr1 translocation is dependent on activation levels of phototransduction, an alternative model has been suggested in which a light-induced intracellular signal triggers the release of Arr1 from its low affinity binding sites in the inner segment and leads to subsequent diffusion along the concentration gradient (Pearring et al., 2013). A good candidate for the initiation of Arr1 translocation is the activation of phospholipase C and PKC downstream of rhodopsin. A study showed that pharmacological activation of PLC and PKC could initiate Arr1 translocation, even in the absence of light (Orisme et al., 2010). More recently, BBS5 was identified as a substrate for PKC (Smith et al., 2013). Interestingly, BBS5 directly interacts with Arr1 and light-dependent PKC phosphorylation of BBS5 leads to the release of Arr1 (Smith et al., 2013). How this light-induced release of Arr1 from phosphorylated BBS5 contributes to the light-dependent delivery of Arr1 from the axoneme into the outer segment disk compartment has not yet been clarified. Although all current hypotheses favor the bidirectional translocation of Arr1 and transducin based on diffusion, there are reports which indicate cytoskeletal involvement (Peterson et al., 2003; Reidel et al., 2008). During dark adaptation, depolymerization of microtubules as well as actin filaments disrupt the translocation of both arrestin and transducin in rod photoreceptor cells of the murine retina (Reidel et al., 2008). During light-adaptation, only the translocation of Arr1 within the outer segment was impaired after destabilization of microtubules. These differences suggest that the two-way trafficking of Arr1 and transducin into and out of the outer segment may depend on different mechanisms. The disruption of cytoskeletal elements may physically modify the diffusion channels, which are used by diffusing molecules or actively support the translocation. 3.4 Novel unexpected functional modules in photoreceptor cilia Although most proteins involved in phototransduction have already been identified and characterized, little is known about the proteins that are responsible for outer segment structure, maintenance and renewal. Omics approaches and new technologies in molecular genetics (e.g. whole exome sequencing) have identified numerous putative molecules and retinal disease causing mutations in genes, which have not been previously associated with the ophthalmologic system. Proteomics on photoreceptor outer segment 35

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preparations of the murine retina identified 41 proteins that function in rod and cone phototransduction and the visual cycle (Kwok et al., 2008). However, 475 proteins not related to the latter “visual” processes were also found. These included proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously associated with outer segments and/or other primary cilia including a subset of Rab and SNARE proteins, which are known to participate in vesicle trafficking and membrane fusion (Kwok et al., 2008). In the outer segment, SNARE proteins have been implicated in disk morphogenesis. A genome-wide knockdown screen targeting ciliogenesis genes revealed a large number of putative ciliary proteins related to unexpected novel functional modules (Wheway et al., 2015). These proteins were subsequently assigned to the organelle/ciliaspecific protein landscape on the basis of affinity proteomics data sets demonstrating functional networks of such novel protein communities and modules associated with cilia function (Boldt et al., 2016). Selective validation demonstrated proteins and networks related to biological functions such as mRNA processing, protein translation, DNA damage repair, folding and degradation processes that have not previously been associated with photoreceptor proteins and function in the photoreceptor cilium. For example, pre-mRNA processing factors (PRPFs), well known components of spliceosomes present in the nucleus, were identified to be required for ciliogenesis of primary cilia (Wheway et al., 2015). Furthermore, immunohistochemistry by light and electron microscopy revealed the localization of PRPF proteins at the ciliary base and the connecting cilium of mouse and human photoreceptor cells and may fulfil an additional function independent of their nuclear role in splicing. Since mutations in PRPF6, PRPF8 and PRPF31 lead to autosomal dominant retinitis pigmentosa (RP types 60, 13 and 11, respectively) pathogenic mechanism underlying retinal degeneration may be linked to photoreceptor cilium functions. Such findings opened new perspectives for the comprehension of the pathogenesis of these disease subtypes.

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3.5 Ciliary photoreceptor evolution Photoreceptor cells evolved very early in the phylogeny of metazoans. The photoreceptor cells of animals can be classified into two types, rhabdomeric and ciliary (Lamb et al., 2007). In rhabdomeric photoreceptor cells, the light sensitive membranes are organized in microvilli whereas ciliary photoreceptor cells extend membranes from a modified primary cilium. As a rule in both types, different opsins are found coupled to distinct downstream signalling pathways: the rhabdomeric r-opsins with the transduction cascade based on phospholipase C (PLC) and the ciliary c-opsins, with transduction based on phosphodiesterase (PDE) (Nilsson and Arendt, 2008). Nevertheless, a growing number of similarities, for example in the expression of transcription factors, strongly indicates a common origin of photoreceptor cells in all animals. Although most deuterostomes including chordates have exclusively ciliated photoreceptor cells, there is evidence that 36

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the bilateral common ancestor of protostomes and deuterostomes possessed both rhabdomeric and ciliary photoreceptors (Arendt, 2003). In the early chordate amphioxus, rhabdomeric and ciliary receptors coexist in separate organs (Lacalli, 2004) and it is not clear when in the vertebrate evolution the rhabdomeric photoreceptors were lost (Lamb et al., 2007). Remarkably the photoreceptor cells of cnidarian jellyfish and vertebrate rods and cones share c-opsin, together with the PDE transduction and ciliary outer segments (Kozmik et al., 2008). The disk structure of the vertebrate outer segment membrane evolved in several stages of the chordate phylogeny: In photoreceptor cells of ascidian larvae, the membranes extend from a ciliary stick approximately longitudinally with respect to the cell axis. In jawless hagfish, the membranes radiate more laterally, from a central cilium. Already in the retinal photoreceptors of the jawless lampreys (Cyclostomata) the membranes are arranged as neatly stacked and highly ordered disks with a lateral connecting cilium, similar to higher vertebrates.

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4. Ocular pathogenesis in ciliopathies Primary cilia dysfunction is known to lead to ocular pathogenesis. Mutations in over 250 different genes have been found to be causative for a wide range of retinal dystrophies (https://sph.uth.edu/Retnet/). A large proportion of these genes encode cilia proteins. Distinctions can be made between retinal disease genes that code for proteins required for ciliary signalling, photoreceptor assembly, photoreceptor structure and photoreceptor function (Wheway et al., 2014; Yildiz and Khanna, 2012). Retinal dystrophies can be accompanied with a range of other phenotypes, as is common in virtually all classes of so- called ‘syndromic ciliopathies’. However, disruptions in cilia genes have also been identified in a growing number of non-syndromic retinal dystrophies. Different mutations in the same gene can cause a variety of phenotypes, ranging from distinctly different ciliopathies to non-syndromic retinal dystrophies. Examples for this include the transition zone protein CEP290 (Centrosomal protein 290) (Coppieters et al., 2010) and centriole protein OFD1 (Orofaciodigital syndrome 1) (Coene et al., 2009; Webb et al., 2012). For a comprehensive and up to date list of genes and loci related to retinal diseases, please refer to https://sph.uth.edu/Retnet/. Here we highlight some of the most prominent examples. 4.1 Non-syndromic ocular pathogenesis Disruptions in cilia genes have been found to be causative in non-syndromic retinal dystrophies which can been classified as non-syndromic retinal ciliopathies (EstradaCuzcano et al., 2012). This class of retinal dystrophies can be categorized depending on the type of photoreceptors that are principally affected. The most well-known nonsyndromic retinal ciliopathies include retinitis pigmentosa, Cone dystrophy, and Leber Congenital Amaurosis. Distinguishing between different non-syndromic retinal ciliopathies is not clear cut, not only because of overlapping clinical presentation, but also due to the fact that they often have overlapping genetic causes.

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Certain mutations in cilia genes seem to only affect photoreceptor cells and cause nonsyndromic retinal dystrophies (e.g. USH2A, BBS8, RP1). One reason for this could be that the highly specialized primary cilium in photoreceptor cells is particularly sensitive to minor disturbances (see section 3). Alternative splicing, resulting in cell type specific isoforms, may also contribute to these phenomena.

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Retinitis pigmentosa (RP) is the most common inherited retinal degeneration and affects up to 1 in 3,000 individuals (Veltel et al., 2008). Initially characterized by rod photoreceptor loss, patients typically begin to develop night blindness, which develops into tunnel vision and decreased central vision. Symptoms tend to begin at around 20 year of age and progress slowly. In advanced stages of the disease cone photoreceptors are also affected. Fundus examinations of RP patients show dark pigmentary clumps, which give the syndrome its name. Surprisingly tritanopic dyschromatopsia (blue-yellow color blindness) is also associated with RP. Other ocular symptoms include cataracts, myopia, astigmatism and keratoconus (conical shaped cornea). Onset of RP in childhood is termed juvenile retinitis pigmentosa (Gu et al., 1997). The major types of nonsyndromic RP are usually distinguished by their pattern of inheritance: autosomal dominant (adRP), autosomal recessive (arRP), or X-linked (XLRP). For the nomenclature of RP genes the prefix “RP” is added and the chronical number of identification: RP1 to RP77. Numerous of these RP genes are cilia genes. By some estimates cilia genes represent almost 40% of the genetic causes of RP (Estrada-Cuzcano et al., 2012). An example of a cilia gene in which mutations have been found to cause adRP is RP1, which was shown to localize to the photoreceptor axoneme (Liu et al., 2004b) and is required for correct stacking of outer segment disks (Liu et al., 2003) (see section 3.2.6). Mutations in numerous cilia genes have been found to be causative for arRP. These include ARL6, BBS1, BBS9, C2orf71, C8orf7, CLRN1, FAM161A, MAK, OFD1, RP1, RP2, RPGR, TOPORS, TTC8 (BBS8), TULP1 and USH2A (Estrada-Cuzcano et al., 2012). Many of these genes also cause syndromic ciliopathies (discussed below) depending on the mutation. Alternative splicing of ciliopathy gene BBS8 (described below), ‘only’ causes RP. The IVS1-2A>G mutation in exon 2A produces a frame shift in the reading frame thereby abolishing the mature protein (Murphy et al., 2015). Because this exon is only expressed in photoreceptor cells, other cell types are unaffected by this mutation. USH2A mutations are one of the most common causes of RP and also causes Ushers syndrome type 2 (Lenassi et al., 2015). The following cilia genes are associated with XLRP: OFD1 (RP23) codes for a centrosomal protein that interacts with other ciliopathy-assosiated proteins (Webb et al., 2012), RPGR (RP3) a GTPase which loclaizes to the conecting cilium and outer segments (Megaw et al., 2015) and RP2 the GTPase-activating protein (GAP) for the small GTPase ARL3 is enrolled in ciliary trafficking (see section 3.3.6) (Zhang et al., 2015). Both Cone dystrophy (CD) and Cone-rod dystrophy (CRD) are less common than RP. CD patients typically present with a disturbance in colour vision and associated 38

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visual loss in the first or second decade of life (Michaelides et al., 2006). Cone function is lost progressively. Rod function is initially unaffected, but can also deteriorate as the disease progresses. This leads to considerable overlap between progressive CD and CRD. CRD is likewise characterized by an initial loss of cone photoreceptors, which is closely followed or simultaneous with loss of rod photoreceptors (Hamel, 2007). Patients present with photo aversion, decreased visual acuity, colour vision defects and decreased sensitivity of the central visual field. Night blindness and loss of peripheral vision are also common as a consequence of the loss of rods. Distinguishing CD from CRD is predominately done biased on electroretinogram (ERG) recordings. Mutations in RPGR, the protein of which localizes to the connecting cilium, have been identified in both CD and CRD patients. Mutations in four other cilia genes (RPGRIP1, C8orf37, RAB28, TTLL5) most of which localize to the base of the cilium have also been found in CRD patients (Estrada-Cuzcano et al., 2012; Roosing et al., 2013; Sergouniotis et al., 2014). Leber Congenital Amaurosis (LCA) is characterized by severe retinal degeneration and nystagmus (Chung and Traboulsi, 2009). Patients are usually born with profound vision loss with no recordable ERG responses. Similar to RP, other ocular symptoms can include keratoconus, cataracts and abnormal fundus. Additional ocular symptoms associated with LCA are photophobia (increased sensitivity to light) and hyperopia (extreme farsightedness). Characteristic of LCA is a specific behavior termed ‘Franceschetti’s oculo-digital sign’. This is observed by patients pressing, rubbing and poking their eyes with a knuckle or finger (Franceschetti, 1947). Thirteen different types of LCA are distinguishable based on genetics and ocular phenotypes. Mutations in cilia genes account for about a quarter of the genetic loci associated with the disease (Chung and Traboulsi, 2009), although one has to be careful not to confuse LCA with other syndromic disorders in which ocular symptoms mimic LCA. Mutations in CEP290 are some of the most common causes of LCA (den Hollander et al., 2006; Estrada-Cuzcano et al., 2012), which is not surprising considering that CEP290 is one of the most critical cilia proteins. It is thought to be a component of the Y-linkers in the transition zone/connecting cilium and is required for ciliary trafficking (Coppieters et al., 2010; R. Rachel et al., 2015). Another important ciliary gene causative for LCA is Lebercilin (den Hollander et al., 2007). Lebercilin uniquely localizes to the connecting cilium in photoreceptor cells and interacts with IFT molecules (Boldt et al., 2011). LCA mutations in lebercilin disrupt this interaction thereby disturbing IFT trafficking. Mutations in two cilia genes have been found to be causative for Macular dystrophy (MD). MD affects the photoreceptors in the macular and results in loss of colour vision and visual acuity. Defects in RPGR and RP1L1 which localize to the connecting cilium were identified in MD patients (Akahori et al., 2010; Bassuk et al., 2014; Fujinami et al., 2016; Shu et al., 2007). 4.2 Syndromic ocular pathogenesis

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Primary cilia dysfunction leads to an overlapping group of human diseases collectively termed ciliopathies. The most common features of ciliopathy patients include visual dysfunction, polydactyly, obesity, learning disabilities, renal dysfunction, infertility and auditory dysfunction (Beales et al., 1999; Braun and Hildebrandt, 2017; Lee and Gleeson, 2011). An ever-increasing list of secondary features can also arise. Historically, ciliopathies were categorized based on combinations of associated phenotypes. As gene sequencing becomes more readily available, diagnosis can now be confirmed upon the presence of disease causing mutations in ciliopathy genes. So far, over 20 different ciliopathies have been identified. Although the incidence of individual ciliopathies is rare, it is estimated that 1 in 1,000 people are affected by a cilia associated disease gene (ww.ciliopathyalliance.org). As primary cilia are absolutely required for regulation of various signalling pathways during embryonic development, complete loss of cilia is incompatible with life. Consequently, mutations in key cilia genes are not observed in the patient population. Ciliopathy syndromes are mostly heterogeneous with a large degree of inter and intra familial variation. Mutations in the same gene can cause varying syndromes and there is little genotype to phenotype correlation. Some of the complexity can be explained by the fact that ciliary genes may exert additional cilia-unrelated functions some mutations may only affect a specific subset of primary cilia in certain tissues or at specific developmental time points, whilst other genetic defects may influence different ciliary modules influencing a subset of ciliary functions. Ocular pathogenesis is common in virtually all classes of syndromic ciliopathies. Most early reports simply described the retinal dystrophy accompanying ciliopathy symptoms as an atypical retinitis pigmentosa. Prior to the advent of genetic testing, onset of retinal degeneration was one of the first symptoms that could lead to a diagnosis, thus skewing literature values for age of onset and penetrance. These days, age of diagnosis has been dramatically reduced, so we are able to learn more about the early asymptomatic stages of ocular disturbances. One overriding feature amongst virtually all the ciliopathies is the clinical variation in retinal degeneration, which cannot always be attributed to the causative mutation. Siblings with identical mutations often exhibit different clinical features (Badano et al., 2003; Makino and Tampo, 2014). This could be explained by the presence of potential modifier alleles (Badano et al., 2006; Khanna et al., 2009). In recent years there have been numerous reviews addressing the retinal aspect of ciliopathies (Bujakowska et al., 2017; Patnaik et al., 2015; Rachel et al., 2012; Sorusch et al., 2014; Werdich et al., 2014; Wheway et al., 2014). Below we highlight the ocular pathogenesis of the more common ciliopathy syndromes. Alström syndrome is caused by mutations in which ALMS1, which encodes a basal body protein. Even though there is just one gene causative for Alström syndrome, as in other ciliopathies penetrance of the ocular phenotype is variable (Malm et al., 2008). Most infants and young children present with nystagmus and extreme photophobia or light sensitivity (Marshall et al., 2011). Common for a cone-rod dystrophy, cone function is diminished first, often within the first six months and rod function is initially retained. By 40

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five years most patients still have some rod dysfunction but that is almost completely lost in the second decade of life (Russell-Eggitt et al., 1998). Other ocular abnormalities include cataracts, attenuation of retinal vessels, optic disc pallor, and increasingly significant RPE atrophy (Russell-Eggitt et al., 1998). Asteroid hyalosis, optic disc drusen, and bone spicules have also been observed upon histology (Sebag et al., 1984). Such diverse ocular abnormalities affecting numerous ocular cell types support the hypothesis that ocular cilia are required for various aspects of visual function. Intriguingly, optical coherence tomography (OCT) imaging in a young child showed signs of retinal immaturity and an early arrest of macular degeneration (Vingolo et al., 2010). This might suggest that ALMS1 is also required for the development and maturation of the retina. Bardet-Biedl syndrome (BBS): At least 20 different disease causing BBS genes have been identified so far (Suspitsin and Imyanitov, 2016). Many of the BBS molecules identified are components of the BBSome (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9), which is thought to act as an adaptor to IFT trafficking (Nachury et al., 2007). Three other BBS proteins (MKKS, BBS10, BBS12) are predicted to function in a chaperone complex required for the formation of the BBSome (Zhang et al., 2012). The remaining BBS molecules are all related to cilia function. In a seminal report defining diagnostic criteria for Bardet-Biedl Syndrome, it was reported that 93% of patients were diagnosed with rod-cone dystrophy with the remaining 7% being under the age of eight (Beales et al., 1999). Average age of onset was around eight years old, with a seven-year progression to being registered blind. Thus, the phenotype is observed earlier and has a faster progression than in cases of isolated typical retinitis pigmentosa. A more recent study, looking only at BBS affected adults, showed median age of onset of symptoms or signs of visual loss as 12 years (Denniston et al., 2014). With increasing age came more advanced stages of retinopathy, worsening visual acuity and nystagmus. Other abnormalities described in the initial report included astigmatism, strabismus, cataracts, colour blindness, macular oedema and degeration, and optic atrophy (Beales et al., 1999). Now with the advent of genetic testing, we are able to observe the progression in more detail (B. Brooks, personal communication). It has been documented that patients present with either rod-cone or cone-rod degeneration, usually in the first two decades of life. Those with a cone-rod degeneration notice problems with central vision and visual acuity earlier. The BBS fundus does not look like the textbook pictures of retinitis pigmentosa (bone spicule pigmentation). Retinal degeneration is early onset and atrophy can be subtle on physical exam, even when the electroretinograms shows widespread loss of photoreceptors. Closer examination of colourblindness shows that it does not fall neatly into one specific dyschromatopsia. An element of tritan deficiency (blue-yellow color blindness) has been shown to be associated with the phenotype although this is not uncommon in patients with retinitis pigmentosa because the rods and blue cones share downstream cellular pathways and post-synaptic connections. 41

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A few subtle genotype to phenotype correlations have been identified in severity of visual symptoms (Denniston et al., 2014). Patients with BBS1 mutations have been shown to display a milder phenotype than patients with mutations in other BBS genes. Clinically, this manifests as significantly better visual acuity and larger ERG amplitudes. Visual acuity or ERG amplitude did not depend on the type of mutation present (missense or null). BBS2 mutations were associated with significantly worse visual acuity than BBS1 mutations, although the age of affected patients was older. In contrast mutations in BBS10 were associated with a significantly earlier age of visual decline than BBS1 (Denniston et al., 2014). Joubert Syndrome: Mutations in up to 28 different genes have been identified in Joubert syndrome patients (https://www.omim.org/entry/213300)(Szymanska et al., 2015). The precise molecular function of Joubert molecules is varied, with some proteins localizing to the transition zone (Garcia-Gonzalo et al., 2011), while others are found at the basal body (Veleri et al., 2014). Others localize to the ciliary axoneme and may be involved in membrane biogenesis (Lu et al., 2015; Xu et al., 2016). Ocular abnormalities are common in Joubert Syndrome patients (70-100%) (Khan et al., 2008; Maria et al., 1999; Schild et al., 2010). Abnormal eye movements are varied and include nystagmus, strabismus, oculomotor apraxia, and vertical gaze palsy. Retinal degeneration and chorioteinal coloboma is also present, yet the extent to which these occur is not clear. In a small study of eight unrelated patients under the age of 10, the most common findings were saccadic dysfunction (typically with head thrusts) and primary position nystagmus (typically seesaw) (Khan et al., 2008). Three individuals showed signs of retinal dystrophy, yet surprisingly all eight patients had normal ERGs. Asymmetric flash visual-evoked potentials (fVEPs) were present and together with the presence of see-saw nystagmus suggest an abnormality of chiasmal decussation. In a separate study, 14 out of 18 patients had abnormal ERGs (Hodgkins et al., 2004), suggesting that the extent of retinal involvement is variable. Lowes Syndrome has been classified as ‘ciliopathy-like’ syndrome and results from a loss of oculocerebrorenal syndrome of Lowe (OCRL1). Patients are characterized by the presence of glaucoma and cataracts, learning disabilities and renal dysfunction, which often causes mortality in childhood (Coon et al., 2012; Luo et al., 2012). Although the cause of glaucoma and cataracts in these patients is not clear, OCRL localizes to the ciliated trabecular meshwork (Luo et al., 2013), were it is thought to be required for primary cilia to respond to pressure stimulation via the transient receptor potential vanilloid 4 (TRPV4) channel (Luo et al., 2014). Mechanotransduction by primary cilia in trabecular meshwork cells could therefore be one of the ways in which the eye is able to monitor and regulate intraocular pressure. Meckel Syndrome is one of the most severe ciliopathies, in which classical ciliopathy symptoms are accompanied with central nervous system malformation, most commonly occipital encephalocele. Most of the proteins associated with Meckle syndrome localize to the transition zone where they are thought to function as ciliary gate keepers (Garcia42

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Gonzalo et al., 2011; Liyun Sang et al., 2011). To date 13 different genes have been identifies as causative for Meckel syndrome (Szymanska et al., 2015). Most affected individuals die in utero or shortly after birth. For this reason, not much is known about the ocular phenotype associated with Meckel syndrome. However, in a report from 1972, ocular manifestations in three individuals with Meckel syndrome were described. The patients died shortly after birth and all studies were done histopathologically. All six eyes exhibited ocular abnormalities, which included cryptophthalmos, clinical anophthalmos, microphthalmos, sclerocornea, microcornea, abnormal iridocorneal angle, partial aniridia, cataract, persistent tunica vasculosa lentis, retinal dysplasia, posterior staphyloma, and hypoplasia of the optic nerve (MacRae et al., 1972). Senior-Loken syndrome is characterized by the presence of retinal degeneration in combination with nephronophthisis. Mutations in many of the same genes that cause nephronophthisis also result in Senior-Loken syndrome. Similar to Meckel syndrome and Joubert syndrome, most Senior-Loken syndrome proteins are localized to the ciliary transition zone (Ronquillo et al., 2012). The retinal degeneration associated with the disease has been described in many different ways, and as with other ciliopathies can be variable even among family members. Retinitis pigmentosa (RP) is the commonest form of ocular disorders and it can include bone spicule degeneration that begins from the periphery of the retina and extends to involve the entire retino-choroid (Fillastre et al., 1976). LCA is also common (Ronquillo et al., 2012). Other ocular findings include cataracts, Coat’s disease and keratoconus (Aggarwal et al., 2013). Usher syndrome (USH) is the most common form of combined hereditary deafblindness affecting the ciliated sensory cells of the inner ear and the retina (Reiners et al., 2006). USH is a complex, clinically and genetically heterogeneous ciliopathy disease. It is divided into 3 clinical types, USH1, USH2 and USH3 which differ in onset time, progression and severity of symptoms (Davenport and Omenn, 1977). USH1 is the most severe form, characterized by profound congenital deafness, vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa (RP), whereas USH2 is the most prevalent form with moderate hearing impairment without vestibular dysfunction and variable onset of RP. USH3 is characterized by progressive hearing loss combined with variable vestibular and retinal dysfunction. So far, 10 USH genes from at least 13 loci have been identified. They encode for rather diverse proteins from different families: a molecular motor myosin VIIa (MYO7A, USH1B), the Ca2+-binding protein CIB2 (USH1J), cadherins, cadherin 23 (CDH23, USH1D) and prodocadherin 15 (PCDH15, USH1F), and other transmembrane adhesion proteins, namely chlarin 1 (CLRN1, USH3A), USH2A (Usherin) and the very large adhesion GPCR VLGR1 (ADRRV, GPR98, USH2C) and the scaffold proteins harmonin (USH1C), SANS (USH1G) and whirlin (WHRN, USH2D) (Mathur and Yang, 2015). In addition, defects in the PDZD7, HARS, CEP250 and C2orf7 associate with USH (Aparisi et al., 2014).

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The decipherment of the protein networks related to USH has strongly contributed to the understanding the pathophysiology of the USH disease (Wolfrum, 2011). In both the inner ear and the eye, USH proteins are integrated into membrane adhesion complexes (El-Amraoui and Petit, 2005; Wolfrum, 2011). Intense work on mouse models revealed that USH proteins function as core components of the tip-link complexes at the tip of the stereocilia (or better: actin filament-based stereovilli) which form the hair bundles of hair cells and are essential for the mechanosensation (El-Amraoui and Petit, 2005). During development of hair bundles, USH2 proteins are essential for the formation of filamentous links, e.g. the lateral links between neighboring stereovilli and connectors between the longest stereocilia and the single kinocilium (Mathur and Yang, 2015). Similar linkers are found between the membranes of the inner segment and the connecting cilium of photoreceptor cells (Maerker et al., 2008; Mathur and Yang, 2015). This pericilary USH membrane adhesion complex is thought to define the target membrane for intracellular transport of cargo vesicles to the photoreceptor cilium (Maerker et al., 2008; Overlack et al., 2011). Myosin VIIa (USH1B) is additionally localized underneath the connecting ciliary membrane (Liu et al., 1997) and there is evidence for a role in opsin transport across the connecting cilium (Liu et al., 1999; Wolfrum and Schmitt, 2000). Furthermore, in the cells of RPE myosin VIIa is essential for melanosome movements and participates in phagocytosis of outer segment tips (Liu et al., 1998) At the specialized ribbon synapses of both hair cells and photoreceptor cells USH proteins are found and may play roles in channel regulation and the synchronization of exocytosis (Gregory et al., 2013). However, unlike USH patients, Ush mouse models develops relatively mild phenotype, in the case of Ush2 mice, or even display no retinal degeneration, which certainly hindered causal functional analysis USH defects in the retina (Williams, 2008). The reason for this discrepancy is reasoned by a USH protein network present in the calyceal processes, which extend from the inner segment and enclose the outer segment base in primates and other vertebrates, but are absent from photoreceptor cells of rodents (Sahly et al., 2012; Wolfrum et al., 2010).

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5. Treatment approaches for ocular phenotype in ciliopathies The eye is an ideal organ for the application of therapeutics and subsequent evaluation of therapeutic success. Due to the size and structure of the retina, only small volumes of therapeutics and/or number of cells need to be applied into an already immune-privileged site. After treatment the retina can be visualized using a variety of imaging modalities assessing both structure (e.g. optical coherence tomography) and function (e.g. ERG), without the need for tissue biopsy. Thus, restored functionality after therapeutic application can be quantified using a variety of noninvasive functional measures including microperimetry, visual field testing, visual acuity, color vision, and autofluorescence (Schwartz et al., 2016). Although no standardized treatment of vision

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loss in ciliopathies or any other ocular disorder exists to date, in the last years enormous efforts have been made in the evaluation of various therapeutic strategies. Since the nineties, growth factors have been tested for their ability to protect retinal cells from cell death. To date several factors, such as the ciliary neurotrophic factor (CNTF), Rod-derived cone viability factor (RdCVF), and Brain-derived neurotrophic factor (BDNF), have been shown to slow down photoreceptor death when delivered locally into the eye in various rodent models of retinitis pigmentosa (Guadagni et al., 2015; LaVail et al., 1998; Wen et al., 2012). Published data on previous phase II/III trials showed no therapeutic benefits to visual field sensitivity and best-corrected visual acuity. However a pilot study using adaptive optics imaging to investigate in vivo cone structure found that cone density remained stable in eyes with a CNTF implant (Tee et al., 2016). To date several clinical trials involving patients with achromatopsia (NCT01648452), glaucoma (NCT01408472) and Ischemic Optic Neuropathy/Optic Nerve Stroke (NCT01411657) (Guadagni et al., 2015) are ongoing. Since continuous secretion of these neurotrophic factors is required, these current clinical trials utilize CNTF-releasing implants embedded in the patient´s eye. Alternatively, the implantation of genetically engineered microencapsulated human stem cells (MicroBeads) recombinantly expressing neurotrophic factors (Fischer et al., 2013) or gene addition based intraocular adenoassociated viruses carrying the neurotrophic factor gene (Dalkara et al., 2015) are currently being evaluated for continuous expression of the neurotrophic factor. Since the mode of action of neurotrophic factors is not gene specific, this strategy may be a therapeutic option for several ciliopathy patients. The combination of molecular diagnosis with basic research has provided detailed knowledge about the molecular mechanism underlying ciliopathies. Generally speaking, in recessive forms of ciliopathies the lack of the gene product is disease causing, therefore reinstating gene expression should result in recovery of cilia function. These therapeutic interventions include gene addition, exon skipping and pharmacological intervention such as read-through of nonsense mutations (Figure 9). In dominant forms of ciliopathies the expressed mutant protein could interfere with the function of the wild type protein or - even worse - have a toxic gain of function effect. Whereas in the case of interference, increased expression of the wild type protein may be therapeutic, in the case of toxicity, suppression of the mutant protein may also be needed in addition to reexpression of the wild type protein (Lewin et al., 2014). Downregulation could be achieved by allele specific catalytic RNAs, including short hairpin RNA, ribozymes or molecular scissors, such as CRISPR/Cas9, zinc finger nucleases (ZFN) or Tal effector nucleases (TALENs) (Millington-Ward et al., 2011; Mussolino et al., 2011; Trapani et al., 2015) (Figure 9D) 5.1 Advancements in gene addition Gene addition is currently the most advanced therapeutic strategy for ocular disorders. In this approach wild-type cDNA of the mutated gene is delivered to the retina. Viral 45

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vectors, mostly adeno-associated viruses (AAV) or lentiviruses are used for gene delivery. Alternatively, nanoparticles can be employed (Figure 9A). To date gene addition using AAVs represent the most promising approach to prevent photoreceptor cell degeneration in retinal disorders (Colella et al., 2014). The first clinical trials using AAVs were initiated in LCA type 2 patients carrying mutations in RPE65 (Buch et al., 2008; Cideciyan et al., 2008; Hauswirth et al., 2008; Maguire et al., 2008). Several other clinical trials with AAVs on retinal disease genes have since been completed, are currently ongoing, or are actively recruiting patients (https://clinicaltrials.gov/ct2/results?term=AAV+retina&Search=Search). All completed studies demonstrated AAVs to be safe and effective (Buch et al., 2008; Cepko and Vandenberghe, 2013; Cideciyan et al., 2008; Hauswirth et al., 2008; Kiyota et al., 2012; MacLaren et al., 2014; Maguire et al., 2008). One drawback of using AAVs is their limited transgene-carrying capacity (packaging size) 4.7 kb. Particularly considering that many ciliopathy related genes, such as CEP290 (~8 kb) (Burnight et al., 2014) or myosin VIIa (USH1B; ~6.7 kb), are far too large for US Food and Drug Administration (FDA)approved AAV packaging. Various strategies have been implemented to overcome this limitation. One promising example is the generation of dual AAVs, in which two AAVs each containing one half of a large transgene are co-delivered and the transgene is reconstituted either by splicing (trans-splicing), homologous recombination (overlapping) or a combination of the two (hybrid) AAVs (Colella et al., 2014). In a proof of concept study, a significant improvement of vison in two mouse models using dual AAVs was observed, although the levels of transgene expression achieved with dual AAVs are lower than those achieved with regular AAVs (Colella et al., 2014). Lentiviral vectors have a 2fold larger transgene-carrying capacity than AAVs (8–10 kb) (Balaggan and Ali, 2012). Therefore, lentiviruses offer an alternative to AAVs for the delivery of larger cDNA, such as for MYO7A (USH1B) (Zallocchi et al., 2014) and CEP290 (Burnight et al., 2014). In collaboration with Sanofi, Phase I and II studies with Usher syndrome Type 1B patients using lentivirus for the delivery of myoIIa are underway (NCT01505062, NCT02065011). The lentiviral delivery of CEP290 successfully transduced patientderived induced pluripotent stem cells (iPSCs) and rescued the ciliogenesis defect in these cells (Burnight et al., 2014). However, there are some concerns regarding the immune response to AAVs and lentiviruses. The transgene-carrying capacity of currently used viruses is still too small for some exceptionally large ciliopathy genes, such as VLGR1/GPR98 (19.3 kb) or USH2A (McMillan et al., 2002). These limitations have encouraged evaluation of nonviral alternatives such as nanoparticles (Zulliger et al., 2015a). Nanoparticles have a transgene-carrying capacity of up to 20 kb (Fink et al., 2006). To date several types of nanoparticles exist, including liposomes, polymers or peptide compacted DNA. Several types of nanoparticles have already been tested in mouse models of ocular diseases (Adijanto and Naash, 2015; Zulliger et al., 2015a). Unfortunately, no clinical trial has been initiated yet. 46

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In summary, initial clinical trials yielded promising results for transgene delivery via AAVs. However, follow-up studies of the initial AAV-RPE65 treatment of RPE65LCA2 patients, showed slow emergence of pseudo-foveas and continued retinal degeneration, despite substantial visual improvement at first (Cideciyan et al., 2015, 2013). Therefore, improvement in visual function cannot imply protection from degeneration (Megaw et al., 2015). In addition, the genetic heterogeneity of ciliopathies combined with the high costs of virus generation have to be considered. Furthermore, several ciliopathy genes are alternatively spliced (e.g. USH1C) (Reiners et al., 2006) and to date it is often elusive which isoform must be replaced (Nagel-Wolfrum et al., 2014). Thus, for the near future at least, it seems unlikely that gene addition will turn out to become a widespread treatment for recessive RP (Guadagni et al., 2015). Furthermore, this approach is inapplicable for dominant forms of RP. Therefore, the development of alternative gene-based strategies and/or a combinatorial approach of gene addition and alternative gene-based strategies for treating retinal dystrophies are necessary (Schwartz et al., 2016).

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5.2 Read-through of nonsense mutations In-depth genotyping revealed that ~12% of all described mutations causing human inherited diseases are nonsense mutations (Mort et al., 2008). Nonsense mutations introduce a premature translational termination codon and thereby terminate protein translation. Recently, the so-called read-through therapy has emerged as a promising gene-based therapeutic approach for genetic diseases caused by nonsense mutations (Nagel-Wolfrum et al., 2016). This therapy is based on the discovery that molecules, known as translational read-through-inducing drugs (TRIDs), allow the translation machinery to suppress a nonsense mutation, thereby inducing the elongation of the nascent peptide chain and consequently resulting in the synthesis of the full-length protein (Figure 9B). Since only small amounts of functional protein are expected to be sufficient to be therapeutically relevant, reversal of the clinical phenotype can be expected (Pérez et al., 2012), making this approach both practical and economical. The ability of aminoglycoside antibiotics to induce read-through of nonsense mutations has been known for more than 50 years (Lederberg et al., 1964). However, due to severe side effects e.g. nephro-, retinal and ototoxicity their long-term clinical use is not practical (Goldmann et al., 2012, 2010; Lopez-Novoa et al., 2011; Nagel-Wolfrum et al., 2016). Several attempts have been initiated to identify improved TRIDs. One approach is to alleviate the toxicity associated with aminoglycosides and improve read-through activity by the generation of aminoglycoside analogues, so-called designer aminoglycosides (summarized in Nagel-Wolfrum et al., 2016). The approach is based on the hypothesis that the structural elements of aminoglycosides causing toxicity can be separated from those inducing read-through. To date, several designer aminoglycosides based on the modification of neomycin (“TC” compounds), kanamycin B analogues (“JL” compounds) and paromomycin (“NB” compounds) have been developed (summarized in 47

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Nagel-Wolfrum et al., 2016). The efficacy of NB compounds has been demonstrated on several disorders, including subtypes of the human Usher syndrome, such as disease causing nonsense mutations of the PCDH15 gene (USH1F) and USH1C gene (Goldmann et al., 2012, 2010; Lee et al., 2011; Nudelman et al., 2009; Rebibo-Sabbah et al., 2007). Furthermore, NB compounds recovered full-length USH1C/harmonin protein in cell culture, organotypic retina cultures and in vivo in transgenic USH1C/hamonin a1 mice (Goldmann et al., 2012, 2010). Remarkably, NB30 and NB54 recovered functional activity of the different harmonin isoform a1 and b3 in treated cells (Goldmann et al., 2012, 2010). In order to identify non-aminoglycoside compounds with read-trough activity, several high throughput screens have been performed (Welch et al., 2007). Through such screening, PTC-Therapeutics identified Ataluren (PTC124, Translarna, 3-[5-(2fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoic acid), and most recently its analog PTC-414, which has better pharmacokinetic properties (Moosajee et al., 2016). To date Ataluren has been authorized for the treatment of Duchene muscular dystrophy (DMD) and Cystic fibrosis (CF) in the US. Furthermore, Ataluren induced read-though of an USH1C causing nonsense mutation in cell culture, ex vivo in retinal explants and in vivo in murine retina (Goldmann et al., 2012, 2011). Encouragingly, Ataluren has also shown excellent biocompatibility in the retina (Goldmann et al., 2011) . More recently, the read-through activity of Ataluren was demonstrated on a retinitis pigmentosa (RP) causing nonsense mutation in RP2 patient-derived fibroblasts. The levels of restored RP2 protein following Ataluren (and the aminoglycoside G418) was sufficient to cause reversal of the pathogenic cellular phenotype in patient-derived cells, such as the correct localization of biomarkers such as IFT20 (cilia marker), GM130 (Golgi apparatus maker) and the G protein subunit β1 (Schwarz et al., 2015). Patients with anirida, a genetic disorder often caused by nonsense mutations and characterized by iris hypoplasia associated with additional ocular abnormalities, are currently being recruited for a Phase II clinical study involving oral application of Ataluren (NCT02647359). The next steps will be to test a topical application of Ataluren termed “START” (Gregory-Evans et al., 2014). “START” (0.9% Sodium chloride, 1% Tween80, 1% Ataluren, 1% carboxy methylcellulose) is a specifically derived formula designed to enhance particle dispersion and increase viscosity for delivery of Ataluren to the eye (Gregory-Evans et al., 2014). An additional screen identified a further drug candidate Amlexanox (Gonzalez-Hilarion et al., 2012). In addition to read-through activity, Amlexanox is also an attenuator of nonsense-mediated mRNA decay (NMD). NMD is a translation-coupled quality control system that recognizes and degrades nonsense mutation carrying mRNAs (NagelWolfrum et al., 2016; Wang and Gregory-Evans, 2015). The degradation of nonsense mutation containing mRNAs by NMD, and consequent lack of transcripts available for protein synthesis, significantly reduces read-through efficacy of TRIDs. This might depreciate the outcome of therapeutic intervention. Thus, attenuating NMD to increase the abundance of nonsense mutation containing mRNAs in combination with TRIDs 48

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might have a synergistic effect and thereby increase the expression of full-length functional protein (Nagel-Wolfrum et al., 2016). Thus far, nothing has been published regarding the efficacy of Amlexanox in ciliopathies caused by nonsense mutations. Nevertheless, recent findings involving TRIDs have raised hope for the usage of translational read-through therapy as a gene-based pharmacogenetic therapy for nonsense mutations in various genes responsible for a variety of genetic ocular diseases.

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5.3 Exon skipping A large proportion of ciliopathy-causing mutations have been shown to affect pre-mRNA splicing mechanisms. Most recently, NGS data from a cohort of Swiss patients with retinal dystrophies predicted that 10.8% of mutations affect splicing (Tiwari et al., 2016). There are different types of splice site mutations and therefore different approaches in order to ameliorate faulty splicing events are currently being investigated (ArechavalaGomeza et al., 2014; Havens et al., 2013). Inactivation of a canonical splice site in the exonic region, and cryptic splice site mutations that create de novo splice sites, result in the inclusion of intronic sequences (pseudoexons) in the mature mRNA. Consequently, the resulting aberrant mRNA may often have an altered reading frame resulting in incorporation of faulty amino acids or a shortened protein due to premature termination codons. Deletion of a splice site in the intronic region results in skipping of the subsequent exon (exon skipping), thus the resulting proteins may lack amino acids that might be important for functionality, may include faulty amino acids or could be shortened due to frameshifts. Antisense oligonucleotides (AON, ASO) are short RNA-oligonucleotides of 15-25 bases in length, which are complementary to the reverse sequence of the mutated target pre-mRNA. Upon binding of an ASO, the targeted region of the pre-mRNA is no longer available for splicing which results in the restoration of normal splicing by suppression of cryptic splicing (Garanto et al., 2016). To date several approaches using ASO have been tested to target ciliopathy causing mutations in vitro, both in patient-derived cell lines and animal models (Garanto et al., 2016; Lentz et al., 2013). In all Louisiana Acadian cases of USH1C the USH1C216G>A mutation within exon 3 creates a cryptic splice site leading to the deletion of the last part of the exon 3 in the mRNA and a frameshift that results in premature termination of translation and thereby a truncated USH1C transcript harmonin. In 2013, Lentz and co-workers developed an ASO targeted to the cryptic splice site in USH1C. In vivo application in a mouse model of USH1C c.216G>A corrected the splicing of the USH1C transcript, restored harmonin expression, and rescued cochlear and vestibular hair cell function for at least 6 months (Lentz et al., 2013), In CEP290, the intronic mutation c.2991+1655A > G creates a cryptic splice donor site resulting in the insertion of a pseudoexon into the mRNA (den Hollander et al., 2006; Den Hollander et al., 1999). ASOs targeted against this cryptic splice site results in pseudoexon skipping in patient derived lymphocytes (Collin et al., 2012) and fully restored CEP290 pre-mRNA splicing, thereby significantly increasing CEP290 protein 49

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levels and rescuing the ciliary phenotype in patient-derived fibroblast cells (Garanto et al., 2016). Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized Cep290lca/lca mouse model carrying the human intronic mutation, showed a statistically significant reduction of pseudoexon-containing Cep290 transcripts and had no severe side effects (Gérard et al., 2015). Unfortunately no statement on the efficacy or improvement to visual function could be made, since these mice do not develop retinal degeneration (Garanto et al., 2016). A further strategy is the modification of the U1 small nuclear RNA splicing factor. U1 is a splicing factor required for recognition of the splice donor site in pre-mRNAs and initializes the splicing process (Wahl et al., 2009) (Figure. 9C). Splice site mutations are often the result of disrupted U1 binding to mutated splice donor sites, thereby resulting in aberrant splicing. Increasing the affinity of U1 to the mutated premRNA sequence restores splice site recognition. Efficient correction of splicing defects using mutation-adapted U1 has already been demonstrated in RPGR in patient-derived fibroblasts (Glaus et al., 2011) and in RHO in photoreceptor cells of mouse retinal explants (Tanner et al., 2009).

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5.4 Stem cell therapy In recent years tremendous efforts in the development of cell-based (cell-replacement) therapies towards the treatment of ocular disorders have been made (Tucker et al., 2015; Wiley et al., 2015). Cell-based therapy is simply defined as replacing damaged or dead cells (e.g. photoreceptor or RPE cells) with healthy cells to restore the function within a tissue (Wiley et al., 2015). Currently, adult stem cells, embryonic stem cells (ESC), and induced pluripotent stem cells (iPSCs) are being used. Adult stem cells are multipotent, giving rise to just a limited number of cell types dependent on the tissue from which they were derived. ESC are pluripotent cells that give rise to all cell types, however, because they are harvested from the inner cell mass of the blastocyst during embryonic development this restricts their clinical practicality due to limited availability and ethical concerns. iPSCs are pluripotent cells and can be generated by dedifferentiation of any cell type in the body such as adult dermal fibroblasts via viral transduction of four transcription factors Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2016). Remarkable progress has been made in differentiating ESCs and iPSCs into photoreceptor precursor and RPE cells, which in turn can be transplanted into the retina (Leach et al., 2016; Osakada et al., 2008). Transplantation of ESC-derived RPE in mouse models for RPE dystrophies resulted in an improvement of vision in mice (Barber et al., 2013; Pearson et al., 2012; Singh et al., 2013). ESC-derived RPE for cell replacement in RPE dystrophies is already in clinical trials for patients with age-related macular degeneration and Stargardt´s macula dystrophy and appears to be safe (Schwartz et al., 2016). Most recently, Sanges et al demonstrated the suitability of hematopoietic stem and progenitor cells (HSPCs) to induce reprogramming of retinal Müller glia in vivo into retinal precursors which can 50

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then be differentiated into photoreceptors (Sanges et al., 2016). For this, HSPCs were transplanted into retinas of rd10 mice, a model for inherited RP. Following transplantation spontaneous cell fusion events between Müller glia and the transplanted cells was observed. Re-activation of Wnt signalling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids, their reprogramming into intermediate photoreceptor precursors and differentiation into photoreceptors. Finally, transplantation of Wnt-activated HSPCs functionally rescued the retinal degeneration phenotype in rd10 mice. This study suggest that reprogramming Müller glia into photoreceptor cells may have potential as a strategy for reversing retinal degeneration (Sanges et al., 2016). iPSCs are currently in the spotlight of regenerative medicine, since they can be dedifferentiated directly from adult cells. Ideally, these cells can be directly obtained from the patient, dedifferentiated into iPSCs and redifferentiated into the target cells before transplanted into the same individual. This should reduce the risk of immune rejection. However, a major obstacle in the use of patient-derived iPSCs, is that any mutation present in the patient will also be present in the patient derived iPSC progeny, thereby decreasing the functionality of transplanted cells. However, for cases of slow retinal degeneration this would be partially beneficial. Alternatively, the development of techniques to repair disease-causing genes, such as CRISPR/Cas9, ZFN or TALENs, may permit the use of gene-corrected, genetically matched donor cells for autologous transplantation (Li et al., 2016) (Figure. 9D). This gene editing approach has also been tested to repair disease-causing mutations in vivo in the eye. A first proof of principle that the CRISPR/Cas9 system can be successfully used for genome editing in the eye was demonstrated in the rd1 mouse model (Wu et al., 2016). In addition, gene-correction is a promising therapeutic strategy for autosomal dominant RP. The allele-specific depletion (knock-out) of the mutated gene should eliminate expression of the toxic mutant protein and in turn allow the native wild-type protein to restore vision (Bakondi et al., 2016). Most recently, Bakondi et al., demonstrated the ability of CRIPR/Cas9 to selectively ablate the mutant rhodosin gene (RhoS334ter) in vivo in rats. Although no visual improvement was mesuarable by electroretinagraphy an 53% increase of visual acuity was detected by optokinetic response in the treated eye compared to the untreated eye. In summary, gene-repair might be an upcoming tool to treat inherited autosomal-dominant RP. 5.5. Retinal implants and optogenetics If retinal cells are fully degenerated, any gene-based therapies cannot be considered, in these cases, alternative strategies become necessary in an attempt to restore some form of visual function. One such option is optogenetic therapy (Packer et al., 2013), or, at a very advanced stage of the disease, electronic prostheses (Yue et al., 2016; Zrenner et al., 2011).

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Three types of electronic prostheses have been generated: epiretinal prostheses anchored to the innermost layer of the retina, subretinal prostheses implanted between the retina and RPE/choroid and suprachoroidal prostheses embedded between the choroid and sclera (Yue et al., 2016). In human electronic prostheses have already been successfully tested (Stingl and Zrenner, 2013) and several clinical trials are recruiting patients or are ongoing. An alternative strategy is based on so-called optogenetics. This relatively new method combines optics and genetics to photosensitize specific retinal neurons. Light sensitivity is conferred to retinal cells via transgenic expression of light-sensitive proteins, such as channelrhodopsin and halorhodopsin (Yue et al., 2016). Both proteins are ionic channels and thus simultaneously perform phototransduction and electric excitation (Sahel and Roska, 2013). Several studies demonstrated the efficacy of optogenetic approaches in restoring photosensory responses and improved locomotor behaviors in mouse models of RP (Yue et al., 2016). Improvements in optogenetics are necessary in order to improve the level of transfection efficacy, increase targeting of the transgene to specific retinal cells and decrease the intensity of light and wavelengths needed to produce sufficient activation of the transfected ionic channels. If these adjustments can be achieved, optogenetics may open a completely new avenue for the treatment of visual impairments. Both strategies, electronic prostheses and optogenetics, relies on the viability of the inner retina and has the noticeable advantage of being gene- and mutation-independent (Guadagni et al., 2015) they thereby offer hope for ciliopathy patients with late stage RP.

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6. Future directions As can be seen from the weighting of this review there has been a tremendous amount of research into the retinal photoreceptor, arguably the most important ciliated cell type in the ocular system. However, with the rise of discoveries involving primary cilia and their roles in other ocular tissues, it is safe to assume that the importance of primary cilia function in other aspects in the visual system will be unraveled in the coming years. One important place to start is to further dissect the ocular phenotype of cilia disease genes. Currently little is known about the differences among the various ciliopathies in terms of ocular structures affected and severity of disease in different ocular tissues. Albeit subtle, these differences may help uncover cilia functions in the eye that extends from the role of cilia genes in the photoreceptor cell. An example for this may be the role of the primary cilium in the trabecular meshwork. Whether here the cilium may also be taking on a mechanosensory role in regulating ocular pressure remains to be seen. Most ciliary proteins were initially identified based upon their association with cilia structure or function. In recent years, it has become evident that many of these proteins also have additional functions. For example IFT proteins have been reported at nonciliary locations including the membranous Golgi and dendrites of retinal neurons (Finetti et al., 2009; Sedmak and Wolfrum, 2010; Yuan and Sun, 2013). Furthermore, BBS proteins have been implicated in regulation of the actin cytoskeleton (Hernandez52

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Hernandez et al., 2013; Tobin et al., 2008) and are localized at actin-rich structures in cultured cells and mouse cochleae (Hernandez-Hernandez et al., 2013; May-Simera et al., 2009), similar to the known roles of some of the Usher molecules. Considering that a primary role for ciliary proteins is the movement of cargo along microtubules it is highly likely that these proteins may also regulate aspects of intracellular trafficking along the cytoskeleton such as vesicular transport (Delaval et al., 2011; Kim and Tsiokas, 2011; Robert et al., 2007) (Follit et al., 2008; Follit et al., 2006; Pedersen et al., 2008; Sedmak and Wolfrum, 2010). Another commonality is the tight association of microtubules with the actin cytoskeleton. Actin-microtubule interactions in the context of cell adhesion and cell migration has been known for some time, yet the association between the two cytoskeletal systems in the context of ciliogenesis and cilia function is only now coming to light (May-Simera et al., 2016; Yan and Zhu, 2013). With the advent of more advanced ‘omic’ screens, we can expect to identify more novel putative ciliary proteins related to unexpected novel functions. This kind of work has already identified a link between ciliogenesis genes and mRNA processing, protein translation, DNA damage repair, folding and degradation. These molecular processes may be directly linked to cilia function, or they may be further examples of additional functions of ‘traditional’ cilia proteins.

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Acknowledgements We thank Viola Kretschmer, Nasrin Sorusch, Kirsten Wunderlich and Elisabeth Sehn for help with generation of the figures. We thank Viola Kretschmer, Tiziana Coglati and Sarita Patnaik for critical reading of the manuscript. We thank Y. Sun, Y. Sugiyama and Stephan Neuhaus for kindly providing images used in Figure 3 and Figure 5, respectively.

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Funding sources The authors are supported by the Alexander von Humboldt foundation (HMS), German Research Council (DFG) FOR 2149, Wo548/8 (UW), FAUN (KNW, UW), Foundation Fighting Blindness (KNW, UW), European Community FP7/2009/241955 (SYSCILIA), BMBF, under the frame of E-RARE2 (EUR-USH, KNW), BMBF (HOPE2, UW), ProRetina Deutschland e.V. (KNW, UW), USHER2020 (KNW, UW), Inneruniversitäre Forschungsförderung Stufe 1 (KNW, UW). References Abd-El-Barr, M.M., Sykoudis, K., Andrabi, S., Eichers, E.R., Pennesi, M.E., Tan, P.L., Wilson, J.H., Katsanis, N., Lupski, J.R., Wu, S.M., 2007. Impaired photoreceptor protein transport and synaptic transmission in a mouse model of Bardet-Biedl syndrome. Vision Res. 47, 3394–407. doi:10.1016/j.visres.2007.09.016 Adijanto, J., Naash, M.I., 2015. Nanoparticle-based technologies for retinal gene therapy. Eur. J. Pharm. Biopharm. 95, 353–367. doi:10.1016/j.ejpb.2014.12.028 Aggarwal, H.K., Jain, D., Yadav, S., Kaverappa, V., Gupta, A., 2013. Senior-loken 53

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doi:10.1016/j.preteyeres.2016.05.002 Zallocchi, M., Binley, K., Lad, Y., Ellis, S., Widdowson, P., Iqball, S., Scripps, V., Kelleher, M., Loader, J., Miskin, J., Peng, Y.-W., Wang, W.-M., Cheung, L., Delimont, D., Mitrophanous, K.A., Cosgrove, D., 2014. EIAV-based retinal gene therapy in the shaker1 mouse model for usher syndrome type 1B: development of UshStat. PLoS One 9, e94272. doi:10.1371/journal.pone.0094272 Zhang, H., Constantine, R., Vorobiev, S., Chen, Y., Seetharaman, J., Huang, Y.J., Xiao, R., Montelione, G.T., Gerstner, C.D., Davis, M.W., Inana, G., Whitby, F.G., Jorgensen, E.M., Hill, C.P., Tong, L., Baehr, W., 2011. UNC119 is required for G protein trafficking in sensory neurons. Nat. Neurosci. 14, 874–80. doi:10.1038/nn.2835 Zhang, H., Fan, J., Li, S., Karan, S., Rohrer, B., Palczewski, K., Frederick, J.M., Crouch, R.K., Baehr, W., 2008. Trafficking of membrane-associated proteins to cone photoreceptor outer segments requires the chromophore 11-cis-retinal. J. Neurosci. 28, 4008–14. doi:10.1523/JNEUROSCI.0317-08.2008 Zhang, H., Hanke-Gogokhia, C., Jiang, L., Li, X., Wang, P., Gerstner, C.D., Frederick, J.M., Yang, Z., Baehr, W., 2015. Mistrafficking of prenylated proteins causes retinitis pigmentosa 2. FASEB J. 29, 932–942. doi:10.1096/fj.14-257915 Zhang, H., Li, S., Doan, T., Rieke, F., Detwiler, P.B., Frederick, J.M., Baehr, W., 2007. Deletion of PrBP/delta impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments. Proc. Natl. Acad. Sci. U. S. A. 104, 8857–62. doi:10.1073/pnas.0701681104 Zhang, Q., Yu, D., Seo, S., Stone, E.M., Sheffield, V.C., 2012. Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable bardet-biedl syndrome protein complex, the BBSome. J. Biol. Chem. 287, 20625–35. doi:10.1074/jbc.M112.341487 Zheng, J., Trudeau, M.C., Zagotta, W.N., 2002. Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit. Neuron 36, 891–6. Zimmermann, K.W., 1898. Beiträge zur Kenntniss einiger Drüsen und Epithelien. Arch. für Mikroskopische Anat. 52, 552–706. doi:10.1007/BF02975837 Zrenner, E., Bartz-Schmidt, K.U., Benav, H., Besch, D., Bruckmann, A., Gabel, V.-P., Gekeler, F., Greppmaier, U., Harscher, A., Kibbel, S., Koch, J., Kusnyerik, A., Peters, T., Stingl, K., Sachs, H., Stett, A., Szurman, P., Wilhelm, B., Wilke, R., 2011. Subretinal electronic chips allow blind patients to read letters and combine them to words. Proc. Biol. Sci. 278, 1489–97. doi:10.1098/rspb.2010.1747 Zulliger, R., Conley, S.M., Naash, M.I., 2015a. Non-viral therapeutic approaches to ocular diseases: An overview and future directions. J. Control. Release 219, 471–487. doi:10.1016/j.jconrel.2015.10.007 Zulliger, R., Naash, M.I., Rajala, R.V.S., Molday, R.S., Azadi, S., 2015b. Impaired association of retinal degeneration-3 with guanylate cyclase-1 and guanylate cyclase-activating protein-1 leads to leber congenital amaurosis-1. J. Biol. Chem. 290, 3488–99. doi:10.1074/jbc.M114.616656 Figure legends

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Figure 1) Schematic representation of a prototypic cilium. Structure of a prototypic cilium. The axoneme is composed of nine microtubule (MT) doublets, which extend from the microtubule triplet of the mother centriole. In motile cilia are characterized by the central pair of MT doublets, radial spokes, and the inner and outer dynein arms which are essential to drive cilia motility. The mother centriole is characterized by distal and subdistal appendages, which connect to the plasma membrane and facilitate ciliary trafficking. Transition fibers termed Y-linkers (YL) connect the microtubule axoneme to the ciliary membrane in the region of the transition zone. Invagination of the plasma membrane (PM) around the base of the cilium forms the ciliary pocket (CP). The basal body complex (BBC) encompasses both the mother centriole (basal body) and the daughter centriole. The striated ciliary rootlet (CR) extends away from the basal body complex towards the nucleus. Right: Cross sections through ciliary compartments: axoneme (9x2+0 or 9x2+2 microtubule arrangement), transition zone (9x2+0 microtubule arrangement with Y-linkers (YL)) and centrioles (9x3+0 microtubule arrangement) of the basal body. DA: Distal appendages, SA: Sub-distal appendages, > <: start central microtubule pair (basal plate).

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Figure 2) Schematic representation of different regions in the vertebrate eye that contain cilia. Cross section through the vertebrate eye. Light enters the anterior section of the eye were it travels through the lens and vitreous before it hits the retina (R) at the back of the eye. The choroid (C) and sclera (S) surround the retina and retinal pigment epithelial (RPE) layers. The optic nerve (ON) bundles the axons of the retinal ganglion cells (GCL) and connects to the brain. Number of different cell types is not proportionally representative. (A) Cross section of the retina highlighting ciliated (red projection) Müller glia cells and rod and cone photoreceptors. RPE cells have been included to emphasize the relationship between these two tissues. The retina is composed of numerous cell layers consisting of various different cell types. (B) Close up view of the anterior segment of the vertebrate eye. The cornea (C) covers the iris (I) and the lens (L). L is attached to the ciliary body (CB) via zonular fibres (Zf). The ciliated (red projections) trabecular meshwork (TM) is shown in more detail below. (C) Close up view of the RPE/photoreceptor outer segment (OS) interface. The apical processes of the RPE surround and engulf the distal end of the outer segments (OS). Melanosomes (black dots) are only found in the pigmented RPE. V: Vitreous, IS: inner segment, CC: Connecting cilium, ST: Synaptic terminus, OLM: Outer limiting membrane, ONL/INL: Outer/inner nuclear layer, OPL/IPL: Outer/inner plexiform layer. Figure 3) Examples of cilia in ocular tissues. (A) Left: Schematic representation of the trabecular meshwork. Each cell has a cilium projecting into the lumen. Right: Immunohistochemistry using antibodies against γ-tubulin (basal body, green) and Arl13b (ciliary axoneme, red) and dapi (blue) in a section of human trabecular meshwork. Two examples of cilia can been seen (white arrows). Image courtesy of Y. Sun, Indiana 92

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University-Purdue University Indianapolis. (B) Left: Schematic of rat lens fibre cells. Cilia are uniformly polarized to the edge of each cell. Right: Immunohistochemistry using antibodies against β-catenin (cell membrane, magenta) and pericentrin (basal body, green) in postnatal day (PN) 34 rat lens fibre cells. Inset: Transmission electron micrograph of PN34 rat lens fibre cell showing extension of a ciliary axoneme. Images courtesy of Y. Sugiyama, Sydney University. (C) Left: Schematic representation of a retinal pigment epithelial (RPE) cell with a cilium projecting from the apical surface. Right: Transmission electron micrograph of a P3 mouse RPE cell with a protruding primary cilium. (D) Left: Schematic representation the apical projection of a Müller glia cell in between photoreceptor inner segments. A primary cilium extends from the surface. Right: Transmission electron micrograph of a Müller cell cilium (arrow). Photoreceptor nuclei (N) can be seen as well as the electron density at the adherens junctions of the outer limiting membrane (asterix). Scale bars: (A) 5 µm; (B) 5 µm; (C) 500 nm; (D) 500 nm.

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Figure 4) Compartmentalization of ciliated photoreceptors. (A, B) Schematic representation of a rod photoreceptor cilium. The inner segment (IS) and outer segments (OS) are connected by the connecting cilium (CC), analogous to the transition zone of prototypic cilia. Axonemal microtubules project from the IS into the OS. The CC, containing Y-linkers (YL), corresponds to the transition zone of a prototypic cilium. The basal body complex (BBC) is localized to the apical region of the inner segment (IS) and give arise to the ciliary rootlet (CR). Calyceal processes (CaP) extend from the inner segment. (C) Transmission electronmicrograph from a longitudinal section through a human rod photoreceptor cell demonstrating the complex basal body composed of the mother and daughter centriole (m/dCE) which are linked by branches of the striated ciliary rootlet (CR). (D) Immunofluorescence localization of the transition zone/CC and centriole marker centrin 3 (Cen3) in a mouse photoreceptor cell. Post-embedding immunoelectron microscopy labeling of mouse and human photoreceptor cilium: Cen3 is localized at the microtubule doublets (inner side) of the connecting cilium (orange), in the distal part of the basal body and the adhesion centriole (red) in mouse and human rod cells. CR: ciliary rootlet, Ax: axoneme, CJ: Connecting junctions, CP: ciliary pocket, DA: Distal appendages, ER: endoplasmatic reticulum, FL: fiber links, MI: mitochondria, RPE: Retinal pigment epithelium, SA: Sub-distal appendages, ST: Synaptic terminal. Scale bars: (C) 250 nm; (D) 200 nm.

Figure 5) Transmission electron microscopy of photoreceptor cells. (A) Longitudinal section of a murine rod photoreceptor cell. The outer segment (OS) is linked by the connecting cilium (CC) with the inner segment (IS). From the CC the axoneme extends into the OS. A striated ciliary rootlet (CR) extends from the basal body (BB) and projects into the IS. (B) Part of a mouse rod OS. Disk stacks are interrupted by incisures indicted by arrows. (C) Part of a human rod OS. Calyceal processe (CaP) are present in cross 93

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section. *ciliary pocket (D) Longitudinal section of a human rod photoreceptor cell. A prominent calyceal process (CaP) projects parallel to the OS in longitudinal orientation (left). On the right, CaPs are seen in cross section. (E) Slightly oblique cross section through a connecting cilium of a mouse rod. Y-links and the characteristic ring-likestructure of the axoneme are colorized in E`. (F) Scanning electron micrograph of adult zebrafish outer retina. The accessory outer segment (AOS) extends from the CC parallel to the cone outer segment (COS). CaPs (arrows) can also be detected. (G) Transmission electron micrograph of adult zebrafish cone photoreceptor stained with a zebrafishspecific antibody against Myo7a, which predominantly localizes to the AOS. Cone inner segment (CIS). Images F and G courtesy of S. Neuhaus, Univ. Zurich (Hodel et al., 2014). (H) Schematic representation of the basal portion of a zebrafish cone photoreceptor. Both COS and AOS extend from the distal end of the connecting cilium. Scale bars: (A) 300 nm; (B) 900 nm; (C) 1.5 µm; (D) 200 nm; (E) 100 nm; (F) 1 µm; (G) 250 nm.

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Figure 6) Ciliogenesis in the photoreceptor cells. (A) Schematic representation of postnatal differential stages S1 to S6 of photoreceptor cell ciliogenesis in the retina of postnatal day 0 (PN 0), PN3, PN7 mice. Stage S1: A primary vesicle (PV), which begins to enlarge by repeated fusion of post-Golgi vesicles encloses the distal end of the mother centriole. Stage 2: As the PV expands it forms the ciliary vesicle (CV), into which the ciliary bud elongates. This becomes the ciliary shaft. From then on, the mother centriole is termed the basal body. Stage 3: The CV fuses with the plasma membrane of the inner segment. The newly assembled cilium appears from the cell surface. Stage 4: The elongating cilium is divided into the proximal (pC) and distal cilium (dC). The proximal cilium is characterized by periodic pearl-like densities along the plasma membrane, which are absent from the distal cilium. Stage 5: The pC becomes the connecting cilium (CC). The dC forms the outer segment (OS). Stage 6: The ciliary axoneme (arrow) extends into the OS. The first stacks of membrane disks begin to appear. Symbols ‘>’ and ‘<’ indicate adherens junctions at the outer limiting membrane. IS: Inner segment. (B) Transmission electron microscopy of a PN3 mouse retina demonstrates that photoreceptor cell ciliogenesis is not synchronized. Yellow dashed lines encircle the different stages (S1/S2, S2 and S4) of ciliogenesis. (C) Immunofluorescence double labelling of IFT20 (red) and the ciliary/centriole marker centrin 3 (green) in a PN3 mouse retina. (D) Immunoelectronmicroscopy pre-embedding labelling of IFT88 in a S1 cilium of a PN0 mouse retina. IFT88 is localized at the distal end of the mother centriole surrounding vesicles pseudo-colored in blue. (E) TEM of a late photoreceptor differentiation stage S6 in a PN7 mouse retina. First membrane stacks appear in the OS. RPE: retinal pigment epithelium. Scale bars: (B) 600 nm; (C) 1 µm; (D) 200 nm; (E) 500 nm. Schema is modified from Sedmak and Wolfrum (2011).

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Figure 7) Schematic representation of transport modules in photoreceptor cells. All components required for outer segment (OS) function are transported from the site of synthesis in the inner segment (IS) to the OS. Transmembrane proteins are synthesized at the rough ER and subsequently processed via the trans-Golgi network (TGN) (ER-Golgi transection). All cellular cargos are shipped via intracellular transport modules along microtubules towards the base of the photoreceptor cilium. After the cargo is reloaded, IFT-trafficking transports cargo destined for the OS across the connecting cilium (CC) and along the axoneme. A subset of IFT molecules are also involved in retrograde transport back towards the IS. Others release their cargo, which is required for disk neogenesis, at the base of the OS. At the tip of the OS the disks are shed and phagocytosed by the retinal pigment epithelium (RPE). For details see text.

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Figure 8) Subciliary localization of IFT molecules and BBS components in mouse photoreceptor cells. (Left panel) Immunofluorescence co-staining of IFT57 and IFT88 (red) with the ciliary marker centrin (green). Both IFTs localize to the ciliary base and the compartment of disk neogenesis at the outer segment (OS) base indicated by the #. IFT88 staining additionally extends into the axoneme of the OS (arrow). (Middle image) Immunoelectronmicroscopy pre-embedding labelling of IFT140 in a longitudinal section through a mouse rod photoreceptor cell. IFT140 localizes to the periciliary region, the ciliary base (yellow) in the apical inner segment (IS), parts of the connecting cilium (CC), the compartment of disk neogenesis (#) and the axoneme (arrow). (Right panel) Immunofluorescence co-staining of the BBSome components BBS4 and BBS5 (green) with the ciliary marker centrin (red) of mouse photoreceptor cells. Both BBS molecules localize to the basal body and the adjacent centriole (arrowhead). BBS5 is additionally found along the axoneme of the OS (arrows). Scale bars, left panel: 1 µm; middle TEM image 200 nm; right panel: 1.2 µm.

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Figure 9) Gene-based therapeutic approaches for the ocular phenotype of ciliopathies. (A) Gene addition by adeno-associated virus (AAV), lentivirus (LV) and nanoparticles. All vectors carry a wildtype cDNA sequence of the mutated gene. Upon transduction of the target cells, the exogenously introduced wildtype cDNA is expressed. (B) Translational read-through of nonsense mutations. Nonsense mutations introduce a premature stop in the mRNA (red X) resulting in shortened non-functional proteins. Translational read-through inducing drugs (green star) induce the integration of an amino acid at the triplet of the stop codon. Although the novel amino acid (yellow) might differ from the one in the wildtype protein, a full-length protein is still produced. (C) Targeting splice site mutations. For protein synthesis, intronic sequences (grey lines) are spliced out of the pre-mRNA. The resulting mRNA contains all exons (blue, red orange boxes) and is translated into the protein. Splice site mutations (red arrow) disturb splicing; resulting in the integration of intronic sequences (grey) and often introduces frameshifts in the mRNA sequence (faint red and orange boxes). Following translation an altered, often 95

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shortened protein is produced. As an example to target splice site mutations, the use of modified U1 (mU1) is shown. The modified U1 is able to recognize the mutated splice site and subsequently recruits the spliceosome to the pre-mRNA. Correct splicing takes place and full-length functional protein is produced. (D) Genome editing as a method to correct disease-causing mutations. Precise molecular scissors are necessary to generated double strand breaks at a specific DNA locus close to the mutation (red line). Currently used molecular scissors are zinc finger nucleases (ZFN), Tal effector nucleases (TALENs) and CRISPR/Cas9. Upon induction of a double strand break the endogenous homologous recombination machinery is activated. An exogenously delivered rescue DNA carrying the wildtype sequence of the mutated gene serves as template to repair the break, thereby replacing the mutated sequence with the wildtype sequence.

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