Klotho controls the brain-immune system interface in the choroid plexus.

Located within the brain's ventricles, the choroid plexus produces cerebrospinal fluid and forms an important barrier between the central nervous system and the blood. For unknown reasons, the choroid plexus produces high levels of the protein klotho. Here, we show that these levels naturally decline with aging. Depleting klotho selectively from the choroid plexus via targeted viral vector-induced knockout in Klotho flox/flox mice increased the expression of multiple proinflammatory factors and triggered macrophage infiltration of this structure in young mice, simulating changes in unmanipulated old mice. Wild-type mice infected with the same Cre recombinase-expressing virus did not show such alterations. Experimental depletion of klotho from the choroid plexus enhanced microglial activation in the hippocampus after peripheral injection of mice with lipopolysaccharide. In primary cultures, klotho suppressed thioredoxin-interacting protein-dependent activation of the NLRP3 inflammasome in macrophages by enhancing fibroblast growth factor 23 signaling. We conclude that klotho functions as a gatekeeper at the interface between the brain and immune system in the choroid plexus. Klotho depletion in aging or disease may weaken this barrier and promote immune-mediated neuropathogenesis

FGF2 is expressed in human and murine embryonic choroid plexus and affects choroid plexus cell behaviour

<p>Abstract</p> <p>Background</p> <p>Although fibroblast growth factor (Fgf) signalling plays crucial roles in several developing and mature tissues, little information is currently available on expression of Fgf2 during early choroid plexus development and whether Fgf2 directly affects the behaviour of the choroid plexus epithelium (CPe). The purpose of this study was to investigate expression of Fgf2 in rodent and human developing CPe and possible function of Fgf2, using <it>in vitro </it>models. The application of Fgf2 to brain <it>in vivo </it>can affect the whole tissue, making it difficult to assess specific responses of the CPe.</p> <p>Methods</p> <p>Expression of Fgf2 was studied by immunohistochemistry in rodent and human embryonic choroid plexus. Effects of Fgf2 on growth, secretion, aggregation and gene expression was investigated using rodent CPe vesicles, a three-dimensional polarized culture model that closely mimics CPe properties <it>in vivo</it>, and rodent CPe monolayer cultures.</p> <p>Results</p> <p>Fgf2 was present early in development of the choroid plexus both in mouse and human, suggesting the importance of this ligand in Fgf signalling in the developing choroid plexus. Parallel analysis of Fgf2 expression and cell proliferation during CP development suggests that Fgf2 is not involved in CPe proliferation <it>in vivo</it>. Consistent with this observation is the failure of Fgf2 to increase proliferation in the tri-dimensional vesicle culture model. The CPe however, can respond to Fgf2 treatment, as the diameter of CPe vesicles is significantly increased by treatment with this growth factor. We show that this is due to an increase in cell aggregation during vesicle formation rather than increased secretion into the vesicle lumen. Finally, Fgf2 regulates expression of the CPe-associated transcription factors, <it>Foxj1 </it>and <it>E2f5</it>, whereas transthyretin, a marker of secretory activity, is not affected by Fgf2 treatment.</p> <p>Conclusion</p> <p>Fgf2 expression early in the development of both human and rodent choroid plexus, and its ability to modulate behaviour and gene expression in CPe, supports the view that Fgf signalling plays a role in the maintenance of integrity and function of this specialized epithelium, and that this role is conserved between rodents and humans.</p

Distinct morphologies of fusion and closure of the choroid fissure

Coloboma is a disorder characterized by the lack of fusion of the embryonic choroid fissure, a transient cleft along the ventral midline of the eye. Traditionally, work in this field has treated the appearance of continuous tissue along the ventral midline as a fused eye. We report that along the proximodistal axis of the chick and the mouse eye, there are different morphologies accounting for this ventrally continuous tissue. We show that the eye has to modes of achieving ventral continuity: closure, involving a ventrally continuous tissue because of the intercalation of the optic nerve between the choroid fissure folds, and fusion, involving the fusion of the basement membranes of the fissure margins and/or the fusion of the margins themselves. We demonstrate that the chick exhibits this closure morphology proximally, fused basement membranes medially and unfused margins because of the intercalated pecten, and fused margins distally. We show that for most of the mouse eye, the ventral midline is fused, except at the optic disc, where optic nerve intercalation yields a closed eye. Thus, choroid fissure closure results in different morphologies that are species-dependent and that vary along the axis of a single eye.Molecular Bioscience

Tissue- and Species-Specific Patterns of RNA metabolism in Post-Mortem Mammalian Retina and Retinal Pigment Epithelium.

Accurate analysis of gene expression in human tissues using RNA sequencing is dependent on the quality of source material. One major source of variation in mRNA quality is post-mortem time. While it is known that individual transcripts show differential post-mortem stability, few studies have directly and comprehensively analyzed mRNA stability following death, and in particular the extent to which tissue- and species-specific factors influence post-mortem mRNA stability are poorly understood. This knowledge is particularly important for ocular tissues studies, where tissues obtained post-mortem are frequently used for research or therapeutic applications. To directly investigate this question, we profiled mRNA levels in both neuroretina and retinal pigment epithelium (RPE) from mouse and baboon over a series of post-mortem intervals. We found substantial changes in gene expression as early as 15 minutes in the mouse and as early as three hours in the baboon eye tissues. Importantly, our findings demonstrate both tissue- and species- specific patterns of RNA metabolism, by identifying a set of genes that are either rapidly degraded or very stable in both species and/or tissues. Taken together, the data from this study lay the foundation for understanding RNA regulation post-mortem and provide novel insights into RNA metabolism in the tissues of the mammalian eye

Long-term visual and microperimetry outcomes following autologous retinal pigment epithelium choroid graft for neovascular age-related macular degeneration

To describe the 2- to 4-year visual and microperimetry outcomes of autologous retinal pigment epithelium (RPE)-choroid graft in patients with neovascular age-related macular degeneration (AMD).In this retrospective cohort study, 12 patients with subfoveal neovascular AMD who had undergone autologous RPE-choroid graft between August 2004 and June 2005 were reviewed. Change in visual acuity (VA), contrast sensitivity (CS), fixation stability and retinal sensitivity on microperimetry after 2-3 years and the rates of late postoperative complications were examined.Patients were followed for 26-48 months (mean, 39). Median preoperative VA (logMAR) was 0.87 but declined to 1.43 (1 year), 1.46 (2 years) and 1.38 (3 years), P = 0.001. Median CS (logCS) was 0.75 preoperatively but declined to 0.45 at 2 years. Six patients had serial microperimetry. Fixation stability declined in 1 but improved in 2 patients. All 6 had decline in retinal sensitivity over the graft during follow up. Retinal detachment did not occur after 12 months but 8 developed epiretinal membrane, 12 had cystic retinal change over the graft and 4 developed recurrent choroidal neovascularization. However, 10 grafts retained autofluorescence signal at 18-48 months of follow up.Autologous RPE-choroid graft can maintain VA, stable fixation and retinal sensitivity in some patients for over 3 years. The spatial correlation between graft autofluorescence, outer retinal structures on optical coherence tomography and retinal sensitivity are consistent with photoreceptor cell rescue. However, we caution the use of this technique as there is high complication rate and delayed loss of retinal function

Peripapillary and macular choroidal thickness in glaucoma.

PurposeTo compare choroidal thickness (CT) between individuals with and without glaucomatous damage and to explore the association of peripapillary and submacular CT with glaucoma severity using spectral domain optical coherence tomography (SD-OCT).MethodsNinety-one eyes of 20 normal subjects and 43 glaucoma patients from the UCLA SD-OCT Imaging Study were enrolled. Imaging was performed using Cirrus HD-OCT. Choroidal thickness was measured at four predetermined points in the macular and peripapillary regions, and compared between glaucoma and control groups before and after adjusting for potential confounding variables.ResultsThe average (± standard deviation) mean deviation (MD) on visual fields was -0.3 (±2.0) dB in controls and -3.5 (±3.5) dB in glaucoma patients. Age, axial length and their interaction were the most significant factors affecting CT on multivariate analysis. Adjusted average CT (corrected for age, axial length, their interaction, gender and lens status) however, was not different between glaucoma patients and the control group (P=0.083) except in the temporal parafoveal region (P=0.037); nor was choroidal thickness related to glaucoma severity (r=-0.187, P=0.176 for correlation with MD, r=-0.151, P=0.275 for correlation with average nerve fiber layer thickness).ConclusionsChoroidal thickness of the macular and peripapillary regions is not decreased in glaucoma. Anatomical measurements with SD-OCT do not support the possible influence of the choroid on the pathophysiology of glaucoma

Cyclic AMP modulation of ion transport across frog retinal pigment epithelium. Measurements in the short-circuit state.

In the frog retinal pigment epithelium (RPE), the cellular levels of cyclic AMP (cAMP) were measured in control conditions and after treatment with substances that are known to inhibit phosphodiesterase (PDE) activity (isobutyl-1-methylxanthine, SQ65442) or stimulate adenylate cyclase activity (forskolin). The cAMP levels were elevated by a factor of 5-7 compared with the controls in PDE-treated tissues and by a factor of 18 in forskolin-treated tissues. The exogenous application of cAMP (1 mM), PDE inhibitors (0.5 mM), or forskolin (0.1 mM) all produced similar changes in epithelial electrical parameters, such as transepithelial potential (TEP) and resistance (Rt), as well as changes in active ion transport. Adding 1 mM cAMP to the solution bathing the apical membrane transiently increased the short-circuit current (SCC) and the TEP (apical side positive) and decreased Rt. Microelectrode experiments showed that the elevation in TEP is due mainly to a depolarization of the basal membrane followed by, and perhaps also accompanied by, a smaller hyperpolarization of the apical membrane. The ratio of the apical to the basolateral membrane resistance increased in the presence of cAMP, and this increase, coupled with the decrease in Rt and the basolateral membrane depolarization, is consistent with a conductance increase at the basolateral membrane. Radioactive tracer experiments showed that cAMP increased the active secretion of Na (choroid to retina) and the active absorption of K (retina to choroid). Cyclic AMP also abolished the active absorption of Cl across the RPE. In sum, elevated cellular levels of cAMP affect active and passive transport mechanisms at the apical and basolateral membranes of the bullfrog RPE

Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques.

Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive 'fit-free' approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580-625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550-585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells