5.2.5. Category 05: Synapse

PRs absorb light that passes through the anterior portion of the eye and convert the light to electrochemical signals that are transmitted through the neuroretina via synaptic connections to the optic nerve and visual cortex [305]. Thus, synapses, necessary for proper cell-to-cell communication, are critical for vision. Discussion of the complexity of PR synaptic development and function is reviewed in [306–308], and is beyond the scope of this review. Suffice to say that mutations in many of the components of synapses, such as presynaptic exocytotic proteins, endocytic proteins, calcium channels, postsynaptic receptors, and associated elements, must be properly organized to mediate transmission of signals, or can lead to visual problems [307]. It is interesting to note that disruption of some synaptic components of the secondary neurons (e.g., GRM6, GPR179, TRPM1, NYX, GNAO1, GNB5, and GNB3), while affecting function as assessed by ERG response, does not normally lead to PR degeneration [309]. This is also true of some presynaptic proteins, such as dystrophin [310] or dystroglycan [311]. However, disruption of some synaptic genes such as *Ache, Cabp4, Cacna1f, Cacna2d4,* and *Unc119* does lead to PR degeneration. For example, a null allele of *Ache* [312], causes a 50% loss of PR nuclei between 1.5 and 2 months and >80% by 6–8 months. Although it was initially determined that the ACHE protein played an important role in hydrolyzing acetylcholine at synapses, its isoforms are now recognized to have far reaching structural functions [313]. Additionally, it has been shown that the loss of secondary neurons in the null allele model is likely to cause secondary PR cell loss [312]. Null or spontaneous alleles of synaptic genes that encode subunits of calcium channels that regulate the release of neurotransmitters, and the development and maturation of exocytic function of PR ribbon synapses, *Cacna1f* [314] and *Cacna2d4* [315,316], respectively, show a slower rate of degeneration. By two months, there are approximately 10–25% of PR nuclei that have degenerated. CABP4, a protein that regulates calcium levels and neurotransmitter release at PR synapses, and modulates CACNA1F and other calcium channel activity shows a similar rate of PR degeneration of 10–25% loss at 2 months [317]. UNC119, which localizes to PR synapses (and IS) and is hypothesized to play a role in neurotransmitter release, also leads to a relatively late onset, slower rate of PR degeneration [318]. Interestingly, Haeseleer has described an interaction between synaptic genes *Cabp4* and *Unc119* [319]. It is likely that other synaptic proteins will also lead to PR degeneration through either a primary or secondary effect and that the interactions among the synaptic proteins will play a significant role in determining the relative rate of the degenerative process.

### 5.2.6. Category 06: Channels and Transporters

Ions such as sodium, potassium, and chloride, play important roles in the visual circuitry [320]. Their intracellular concentrations and movements within the cell, and between cells and the environment are exquisitely regulated by channels and transporters. Due to the importance of maintaining appropriate levels of these ions for proper function and maintenance of PRs, it is not surprising that disruption of these genes can lead to PR degeneration. Members of the ClC family of chloride channels, such as *Clcn2, Clcn3,* and *Clcn7,* show particularly early and significant PR degeneration. Compared to other channels in this section, they appear to have an enriched expression in the RPE. A 50% PR cell loss can be seen as early as 14–16 days in certain models with disruptions in these genes [231,321–323]. Indeed, rapid progression of PR cell loss is observed in mice carrying any of the following alleles: *Clcn2nmf240*, *Clcn2tm1Tjj*, *Clcn3tm1Lamb*, *Clcn3tm1Tjj*, *Clcn7tm1Tjj*, *Clcn7tm2Tjj*, and *Clcn7tm4.1Tjj* [231,321,322,324–327]. A similar rapid and complete loss of PRs is seen when *Atp1b2*, a Na+/K<sup>+</sup>-ATPase thought to play a role in cell adhesion, is inactivated [328]. A targeted mutation in *Slc6a6,* which encodes a taurine/beta-alanine transporter, also leads to rapid, complete degeneration [329]. In contrast, inactivation of the bicarbonate, amino acid transporters, and Na+/H<sup>+</sup> exchangers, *Slc4a7, Slc7a14,* and *Slc9a8* results in a later onset, but still severe PR degeneration [330–332]. Mouse models bearing mutations affecting BSG, a protein that has a role in targeting monocarboxylate transporters such as SLC16A1 to the plasma membrane, or REEP6, which mediates trafficking of clathrin-coated vesicles from the ER to the plasma membrane at outer plexiform layer sites enriched for synaptic ribbon protein STX3, also fall in this latter late onset/severe category [333,334]. *Asic3*, an acid sensing Na<sup>+</sup> channel, *Clcc1*, an intracellular chloride channel, and *Slc7a14*, an intracellular arginine transporter, all cause moderately slow degeneration when mutated [331,335,336]. Slowly progressive PR cell loss is also caused by mutation of *Mfsd2a*, which encodes a sodium-dependent lipid transporter responsible for maintaining a high DHA concentration in the retina is important for OS homeostasis, as discussed in Category 03 [337]. More data are needed to see if sensing and intracellular channels/transporters generally have milder phenotypes.

### 5.2.7. Category 07: Adhesion and Cytoskeletal

Proper structure of the retina is developed through protein interactions between cells and within cells. The spatial and laminar organization of the retina is maintained through junctional interactions between cells that impart mechanical support to maintain retinal architecture, a means for bidirectional communication (e.g., extracellular changes to the cell and from the cell to its environment), and together can form diffusion barriers. Within cells, cytoskeletal architecture is maintained through interactions of proteins with actin, intermediate filaments, and microtubules that serve to maintain cell morphology and polarity, and as discussed elsewhere, intracellular trafficking, contractility, motility, and cell division. Examples of disrupted proteins that lead to gaps between cell layers, presumably through aberrant adhesion, are mutations in *Adam9* and *Rs1*. The null allele of *Adam9*, a single pass transmembrane protein with disintegrin and metalloprotease domains that has been shown to interact with a number of integrins [338], leads to aberrant adhesion between the apical processes of the RPE and OS and to late onset PR degeneration [339]. Likewise, mutations in RS1, a protein with a discoid domain, which has been implicated in cell adhesion and cell–cell interactions [340] lead to a splitting of the inner retinal layer and progressive PR loss [341–343]. RS1 binding to phospholipids on the membrane surface, together with other proteins [340] may provide a stabilizing scaffold that is important in cell–matrix, cell–cell, and cytoskeletal organization.

CRB1 and its interacting partners, such MPP3, MPP5, and PARD6A, have been shown to be important in establishing proper retinal lamination presumably through their essential roles in establishing cellular apical basal polarity [344]. A primary defect of a disruption in CRB1 is the fragmentation of the outer limiting membrane [345,346]. As reviewed previously [344,347], the outer limiting membrane consists of adherens/tight junctions formed in part by the CRB1 complexes between Müller glia and the rod or cone IS that form a diffusion barrier. Loss of components of the CRB1

complexes (CRB1-MPP5-PATJ, CRB1-MPP5-MPDZ, and CRB1-PARD6A-MPP5-MPP3/MPP4) leads to lamination defects with formation of rosettes and a progressive loss of PRs. Although ye<sup>t</sup> to be reported, it is likely that mutations in PATJ, PARD3, MPDZ, and MPP4 will lead to similar disease phenotypes, as a reduction in ERG response in MPDZ mutants [348] and a reduced ERG with abnormal retinal morphology have been indicated for PARD3 mutants [41,43].

Equally important to the function and maintenance of the retina are the intracellular components that make up the cytoskeletal cell structure. Disruption of proteins that interact with actin intracellularly have been shown to lead to PR degeneration. For example, CDC42, a small GTPase that is a key regulator of actin dynamics [349] leads to an early onset, progressive PR degeneration when disrupted. Models caused by mutations in FSCN2, an actin crosslinking protein [350,351], and by a hypomorphic variant of CTNNA1, a protein that coordinates cell surface cadherins with the intracellular actin filament network [352], show slow-paced PR cell loss. The proper localization of organelles within the cell is also mediated by the cytoskeletal architecture and can have an untoward e ffect when disrupted. For example, SYNE2, a nuclear outer membrane protein that binds to F-actin, tethers the nucleus to the cytoskeleton and is necessary for the structural integrity of the nucleus [353,354]. Without it, early onset, moderately paced PR cell loss occurs.
