Retinal degeneration 10 (rd10) mice are a model of autosomal recessive Retinitis Pigmentosa (RP), identified by Chang et al. in 2002. These mice carry a spontaneous mutation of the rodphosphodiesterase (PDE) gene, leading to a rod degeneration that starts around P18. Later, cones are also lost. Because of photoreceptor degeneration does not overlap with retinal development, and light responses can be recorded for about a month after birth, rd10 mice mimic typical human RP more closely than the well-known rd1 mutants. Aim of this study is to provide a comprehensive analysis of the morphology and function of the rd10 mouse retina during the period of maximum photoreceptor degeneration, thus contributing useful data for exploiting this novel model to study RP.We analyze the morphology and survival of retinal cells in rd10 mice of various ages with quantitative immunocytochemistry and confocal microscopy; we also study retinal function with the electroretinogram (ERG), recorded between P18 and P30.We find that photoreceptor death (peaking around P25) is accompanied and followed by dendritic retraction in bipolar and horizontal cells, which eventually undergo secondary degeneration. ERG reveals alterations in the physiology of the inner retina as early as P18 (before any obvious morphological change of inner neurons) and yet consistently with a reduced band amplification by bipolar cells.Thus, changes in the rd10 retina are very similar to what previously found in rd1 mutants. However, an overall slower decay of retinal structure and function predict that rd10 mice might become excellent models for rescue approaches.Keywords retinitis pigmentosa; bipolar cell; horizontal cell; confocal microscopy; immunocytochemistry; ERG A very high number of genetic mutations affect the eye. Those occurring in photoreceptor or pigment epithelium -specific genes often cause retinal degenerations (RDs), a family of inherited dystrophies characterized by photoreceptor dysfunction and death.It is estimated that more than 15 million people worldwide have vision loss due to inherited forms of RD. These include patients suffering from retinitis pigmentosa (RP), a disease for which there is no cure yet (Chader, 2002 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptFor over 30 years, the retina of rodents has provided an invaluable tool to study the dynamics and mechanisms of inherited RD, as mouse photoreceptors undergo dystrophies caused by spontaneous DNA mutations, closely related to those of humans.Among the mouse models, the best characterized is the retinal degeneration 1 (rd1) mouse, first described decades ago as a rodless phenotype (reviewed in Farber et al., 1994). This animal carries a spontaneous mutation of the β-subunit of the rod-phosphodiesterase (PDE) gene, causing the massive death of rods in the first weeks of postnatal life. As in typical RP, cones eventually die as well (LaVail et al., 1997;Pierce, 2001). The fast degeneration of photoreceptors is an obvious limit to the employment of r...

Retinitis pigmentosa (RP), a family of inherited disorders characterized by progressive photoreceptor death, is a leading cause of blindness with no available cure. Despite the genetic heterogeneity underlying the disease, recent data on animal models show that the degeneration of photoreceptors triggers stereotyped remodeling among their postsynaptic partners. In particular, bipolar and horizontal cells might undergo dendritic atrophy and secondary death. The aim of this study was to investigate whether or not concomitant changes also occur in retinal ganglion cells (RGCs), the only retinal projection neurons to the brain and the proposed substrate for various therapeutic approaches for RP. We assessed the retention of morphology, overall architecture, and survival of RGCs in a mouse model of RP at various stages of the disease. To study the morphology of single RGCs, we generated a new mouse line by crossing Thy1-GFP-M mice (Feng et al., 2000), which express GFP (green fluorescent protein) in a small number of heterogeneous RGCs types, and rd10 mutants, a model of autosomal recessive RP, which exhibit a typical rod-cone degeneration (Chang et al., 2002). We show remarkable preservation of RGC structure, survival, and projections to higher visual centers in the time span from 3 to 9 months of life, well beyond the death of photoreceptors. Thus, unlike second-order neurons, RGCs appear as a considerably stable population of cells, potentially constituting a favorable substrate for restoring vision in RP individuals by means of electronic prostheses or direct expression of photosensitive proteins.

A clear demonstration of the role of melatonin and its receptors in specific retinal functions is lacking. The present study investigated the distribution of MT1 receptors within the retina, and the scotopic and photopic electroretinograms (ERG) and retinal morphology in wildtype (WT) and MT1 receptor-deficient mice. MT1 receptor transcripts were localized in photoreceptor cells and in some inner retinal neurons. A diurnal rhythm in the dark-adapted ERG responses was observed in WT mice, with higher a-and b-wave amplitudes at night, but this rhythm was absent in mice lacking MT1 receptors. Injection of melatonin during the day decreased the scotopic response threshold and the amplitude of the a-and b-waves in the WT mice, but not in the MT1 ؊/؊ mice. The effects of MT1 receptor deficiency on retinal morphology was investigated at three different ages (3, 12, and 18 months). No differences between MT1 ؊/؊ and WT mice were observed at 3 months of age, whereas at 12 months MT1 ؊/؊ mice have a significant reduction in the number of photoreceptor nuclei in the outer nuclear layer compared with WT controls. No differences were observed in the number of cells in inner nuclear layer or in ganglion cells at 12 months of age. At 18 months, the loss of photoreceptor nuclei in the outer nuclear layer was further accentuated and the number of ganglion cells was also significantly lower than that of controls. These data demonstrate the functional significance of melatonin and MT1 receptors in the mammalian retina and create the basis for future studies on the therapeutic use of melatonin in retinal degeneration.electroretinogram ͉ neuroprotection ͉ visual sensitivity ͉ glaucoma

RPE cells are the most actively phagocytic cells in the human body. In the eye, RPE cells face rod and cone photoreceptor outer segments at all times but contribute to shedding and clearance phagocytosis of distal outer segment tips only once a day. Analysis of RPE phagocytosis in situ has succeeded in identifying key players of the RPE phagocytic mechanism. Phagocytic processes comprise three distinct phases, recognition/binding, internalization, and digestion, each of which is regulated separately by phagocytes. Studies of phagocytosis by RPE cells in culture allow specifically analyzing and manipulating these distinct phases to identify their molecular mechanisms. Here, we compare similarities and differences of primary, immortalized, and stem cell-derived RPE cells in culture to RPE cells in situ with respect to phagocytic function. We discuss in particular potential pitfalls of RPE cell culture phagocytosis assays. Finally, we point out considerations for phagocytosis assay development for future studies.

Melatonin modulates many important functions within the eye by interacting with a family of G-protein-coupled receptors that are negatively coupled with adenylate cyclase. In the mouse, Melatonin Receptors type 1 (MT1) mRNAs have been localized to photoreceptors, inner retinal neurons, and ganglion cells, thus suggesting that MT1 receptors may play an important role in retinal physiology. Indeed, we have recently reported that absence of the MT1 receptors has a dramatic effect on the regulation of the daily rhythm in visual processing, and on retinal cell viability during aging. We have also shown that removal of MT1 receptors leads to a small (3–4 mmHg) increase in the level of the intraocular pressure during the night and to a significant loss (25–30%) in the number of cells within the retinal ganglion cell layer during aging. In the present study we investigated the cellular distribution in the C3H/f+/+ mouse retina of MT1 receptors using a newly developed MT1 receptor antibody, and then we determined the role that MT1 signaling plays in the circadian regulation of the mouse electroretinogram, and in the retinal dopaminergic system. Our data indicate that MT1 receptor immunoreactivity is present in many retinal cell types, and in particular, on rod and cone photoreceptors and on intrinsically photosensitive ganglion cells (ipRGCs). MT1 signaling is necessary for the circadian rhythm in the photopic ERG, but not for the circadian rhythm in the retinal dopaminergic system. Finally our data suggest that the circadian regulation of dopamine turnover does not drive the photopic ERG rhythm.

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial‐specific clathrin adaptor AP‐1B. Some native epithelia lack AP‐1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP‐1B‐deficient epithelia to relocate AP‐1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP‐1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP‐1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus‐end kinesin KIF16B and non‐centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a‐dependent TfR recycling pathway of non‐polarized cells. They define a transcytotic pathway important for the physiology of native AP‐1B‐deficient epithelia and report the first microtubule motor involved in transcytosis.

Retinitis pigmentosa (RP) is a family of inherited diseases causing progressive photoreceptor death. Retinal ganglion cells (RGCs) form the biological substrate for various therapeutic approaches designed to restore vision in RP individuals. Assessment of survival and preservation of RGCs in animal paradigms mimicking the human disease is of key importance for appropriate implementation of vision repair strategies. Here we studied the survival of RGCs in the rd1 mutant mouse, a known model of early onset, autosomic recessive RP, at various stages of photoreceptor degeneration. Furthermore, we analyzed the morphology of various types of RGCs using the newly generated transgenic mouse rd1/Thy1-GFP, in which the rd1 mutation is associated with green fluorescent protein (GFP) expression in a small population of different RGCs. We found excellent survival of cells at up to 1 year of age, a time at which the inner retina is known to have severely reorganized and partially degenerated. However, 50% of the cells analyzed within all RGC types exhibit an undersized dendritic tree, spanning about half of the normal area. Undersized cells are found both in adult and in very young (1-month-old) mice. This suggests that their aberrant phenotype is due to incomplete dendritic development, possibly as a consequence of altered visual input at the time of dendritic arbor refinement. These data show the importance of the timing of photoreceptor death in RGC dendritic development.

There was an error published in J. Cell Sci. 125, 5937-5943.In Fig. 4B the same image was inadvertently presented as the xy section (top panels) of syntaxin 4 staining in MDCK wild-type and MDCK m1A-KD cells. In the correct version (shown below), the appropriate xy image for m1A-KD (top panel, middle) matches the corresponding xz panel (bottom panel) that was part of the original figure.The mistake in the figure did not affect the conclusions of the paper. Fig. 4B is shown below. The correctThe authors apologise for this mistake. Summary Fusion of lysosomes with the plasma membrane is a calcium-dependent process that is crucial for membrane repair, limiting pathogen entry and clearing cellular debris. In non-polarized cells, lysosome exocytosis facilitates rapid resealing of torn membranes. Here, we investigate the mechanism of lysosome exocytosis in polarized epithelia, the main barrier between the organism and the external environment and the first line of defense against pathogens. We find that in polarized Madin-Darby canine kidney (MDCK) cells, calcium ionophores or pore-forming toxins cause lysosomes to fuse predominantly with the basolateral membrane. This polarized exocytosis is regulated by the actin cytoskeleton, membrane cholesterol and the clathrin adaptor AP-1. Depolymerization of actin, but not microtubules, causes apical lysosome fusion, supporting the hypothesis that cortical actin is a barrier to exocytosis. Overloading lysosomes with cholesterol inhibits exocytosis, suggesting that excess cholesterol paralyzes lysosomal traffic. The clathrin adaptor AP-1 is responsible for accurately targeting syntaxin 4 to the basolateral domain. In cells lacking either the ubiquitous AP-1A or the epithelialspecific AP-1B, syntaxin 4 is non-polar. This causes lysosomes to fuse with both the apical and basolateral membranes. Consistent with these findings, RNAi-mediated depletion of syntaxin 4 inhibits basolateral exocytosis in wild-type MDCK, and both apical and basolateral exocytosis in cells lacking AP-1A or AP-1B. Our results provide fundamental insight into the molecular machinery involved in membrane repair in polarized epithelia and suggest that AP-1 is a crucial regulator of this process.