Periaxin is required for hexagonal geometry and membrane organization of mature lens fibers

AbstractTransparency of the ocular lens depends on symmetric packing and membrane organization of highly elongated hexagonal fiber cells. These cells possess an extensive, well-ordered cortical cytoskeleton to maintain cell shape and to anchor membrane components. Periaxin (Prx), a PDZ domain protein involved in myelin sheath stabilization, is also a component of adhaerens plaques in lens fiber cells. Here we show that Prx is expressed in lens fibers and exhibits maturation dependent redistribution, clustering discretely at the tricellular junctions in mature fiber cells. Prx exists in a macromolecular complex with proteins involved in membrane organization including ankyrin-B, spectrin, NrCAM, filensin, ezrin and desmoyokin. Importantly, Prx knockout mouse lenses were found to be softer and more easily deformed than normal lenses, revealing disruptions in fiber cell hexagonal packing, membrane skeleton and membrane stability. These observations suggest a key role for Prx in maturation, packing, and membrane organization of lens fiber cells. Hence, there may be functional parallels between the roles of Prx in membrane stabilization of the myelin sheath and the lens fiber cell

Deformation of Optic Nerve Head and Peripapillary Tissues by Horizontal Duction.

PURPOSE: To ascertain deformation of the optic nerve head (ONH) and peripapillary tissues caused by horizontal duction. DESIGN: Prospective, experimental study. METHODS: Optical coherence tomography of the ONH region was performed in 23 eyes of 12 normal volunteers in central gaze and increasing (10, 20, and 30 degrees) adduction and abduction. Main outcome measures were changes from central gaze in the configuration of the ONH and peripapillary tissues in eccentric gazes. RESULTS: Adduction but not abduction was associated with significant, progressive relative posterior displacement of the temporal peripapillary retinal pigment epithelium (tRPE) from its position in central gaze reaching 49 ± 10 μm in 30-degree adduction (standard error of mean, P < .0001). Absolute (anterior or posterior) optic cup displacement (OCD) averaged 41 ± 7 μm in 30-degree adduction. Linear regression showed significant effect of adduction on absolute OCD (slope 1.09 ± 0.36 μm/degree, P = .0037). In 20-degree and 30-degree adduction, all eyes exhibited significant progressive temporal ONH tilting reaching 3.1 ± 0.4 degrees in 30-degree adduction (P < .0001). Abduction was not associated with significant peripapillary RPE displacement, OCD, or ONH tilt. Both nasal and temporal peripapillary choroid averaged 9-19 μm thinner in adduction and abduction than in central gaze (P < .02). CONCLUSIONS: Adduction temporally tilts and displaces the prelaminar ONH and peripapillary tissues. Both adduction and abduction compress the peripapillary choroid. These effects support magnetic resonance imaging and biomechanical evidence that adduction imposes strain on the ONH and peripapillary tissues. Repetitive strain from eye movements over decades might in susceptible individuals lead to optic neuropathies such as normal tension glaucoma

Autotaxin-Lysophosphatidic Acid Axis Is a Novel Molecular Target for Lowering Intraocular Pressure

<div><p>Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP) resulting from diminished aqueous humor (AH) drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX), a secretory protein and a major source for extracellular lysophosphatidic acid (LPA), in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non-glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD) activity of ATX was found to be significantly elevated (by ∼1.8 fold; n = 20) in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM) cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826), which inhibited AH LysoPLD activity <em>in vitro</em> (by >90%), led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (∼2 µM) led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA) caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients.</p> </div

Elevated levels of LysoPLD activity in AH derived from primary open angle glaucoma human patients.

<p>A. Details of the demographics for the Cataract control and POAG patients. B. LysoPLD activity (expressed per unit of AH total protein) in the AH of POAG patients (n = 20) revealed a significant (* p< = 0.005) increase relative to age-matched cataract control specimens based on the Wilcoxon Rank Sum test. C. Box-and-whisker plot of the AH LysoPLD activity of glaucoma and cataract controls with box showing median and interquartile range.</p

Detection of ATX in the AH from human and other species.

<p><b>A</b>. Immunoblot analysis of AH (10 µg protein) derived from two different human donors (aged 40 and 52 years) revealed multiple immunopositive bands with molecular mass ranging between 65 to 250 kDa. B. Detection of ATX by immunoblot analysis of mouse and porcine AH (10 µg protein). Similar to human AH, both mouse and porcine exhibit ATX immunopositive bands with molecular mass ranging from 95 to 250 kDa.</p

ATX inhibitor-S32826 induced changes in cultured human TM cells.

<p>A. Primary cultures of human TM cells cultured either on gelatin-coated glass coverslips or in plastic petridishes were treated with S32826 (10 µM) in the absence of serum or in the presence of 0.5% fetal bovine serum for 24 hrs. Based on phase contrast microscopic examination, treatment of TM cells cultures with drug caused the appearance of membranous floating clumps (arrows) in culture medium, without any noticeable changes in cell morphology and cell adhesion relative to control cells treated with DMSO vehicle. This response appeared to increase progressively with time. B. The membranous floating material generated from drug (S32826) treated cells (A) was extracted from the cell culture media by centrifugation, rinsed with PBS, dissolved in Laemmli sample buffer, separated by SDS-PAGE and stained with gelcode blue as shown in the figure (two independent samples are shown from each treatment). Subsequently, the predominant protein bands were gel extracted and subjected to mass spectrometry-based identification. This analysis revealed presence of proteins involved in lysosomal exocytosis, trafficking and membrane repair as discussed in the results section.</p

Suppression of ATX expression in human TM cells induces changes in actin cytoskeletal organization and myosin II phosphorylation.

<p>A. Primary cultures of human TM cells treated with ATX-specific siRNA revealed significant suppression of ATX expression (different isoforms) relative to cells treated with FITC-conjugated scrambled siRNA control. β-tubulin was used as a loading control. B. Protein levels of one of the variants (∼125 kDa) of ATX were significantly reduced (by >75%) in ATX siRNA treated HTM cells based on densitometric semi-quantification. C. The ATX siRNA treated human TM cells (after 72 hrs) maintained under serum starved conditions for 24 hrs revealed a marked decrease in actin stress fibers (labelled using rhodamine-phalloidin) relative to cells treated with scrambled siRNA (FITC-siRNA). D. ATX siRNA treated human TM cells (for 72 hrs) maintained under serum starved conditions for 24 hrs showed a significant decrease in the levels of phosphorylated myosin light chain (pMLC), a regulatory subunit of myosin II, as compared to cells treated with scrambled siRNA (FITC-siRNA). β-tubulin was blotted as a loading control. E. Changes in the levels of phospho-MLC in the ATX siRNA treated cells compared to the scrambled siRNA treated control, based on densitometric semi-quantitative analysis.</p

Topical application of ATX inhibitor (S32826) decreases IOP in rabbits.

<p>Topical application of S32826 (dissolved in water, pH∼8–9) decreased IOP in a dose- and time- dependent manner in Dutch-Belted adult male rabbits, as determined using pneumotonometer under waking conditions. A. 10 mM S32826 exhibited the maximum and statistically significant effect in lowering IOP (by 5–6 mm Hg; n = 9) with drug application at 0 and 5 hrs (indicated with arrows, 35 µl×2) relative to sham controls receiving pH adjusted water. The IOP-lowering effect of drug lasted for a minimum of 24 hrs, after which, the changes in IOP began to normalize and were similar to control values by 48 hrs after initial drug application. B. The effects of S32826 (10 mM) on net change in IOP from basal values in the same animals. Net IOP values were significantly reduced from basal values, and between 2 to 24 hours following drug administration, based on a paired t-test. Further, IOP in the drug treated eyes was significantly reduced as compared to IOP in the control eyes based on the Wilcoxon Rank Sum test of differences in median's (n = 9). C and D. Effects of 5 and 2 mM S32826 on IOP with a daily (single morning dose, 35 µl×2; n = 4) dose of drug application, respectively. Relative to the 2 mM concentration of drug, administration of 5 mM drug resulted in a significant drop in IOP starting from 2 hrs post drug injection as compared the control values, with the IOP lowering effect lasting till the end of the study (5 days later). While eyes treated with 2 mM S32826 exhibited IOP values that were significantly reduced relative to control values throughout the 5-day study period, based on the Wilcoxon Rank Sum test of differences in medians, significance was not consistent across all time points as shown in the figure. In all these experiments, sham treatment alone increased IOP; the difference was not significant relative to the basal IOP values in any of the experiments. *P<0.05.</p