Sylvia Bolz

A homologous genetic basis of the murine cpfl1 mutant and human achromatopsia linked to mutations in the PDE6C gene

Retinal cone photoreceptors mediate fine visual acuity, daylight vision, and color vision. Congenital hereditary conditions in which there is a lack of cone function in humans cause achromatopsia, an autosomal recessive trait, characterized by low vision, photophobia, and lack of color discrimination. Herein we report the identification of mutations in the PDE6C gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase as a cause of autosomal recessive achromatopsia. Moreover, we show that the spontaneous mouse mutant cpfl1 that features a lack of cone function and rapid degeneration of the cone photoreceptors represents a homologous mouse model for PDE6C associated achromatopsia.

Subcellular distribution of glucose transporter (GLUT-1) during development of the blood-brain barrier in rats

Abstract.Electron microscopy was used to quantify the subcellular distribution of the GLUT-1 isoform of the glucose transporter in developing microvessels of the brain of embryonic rats from E (embryonic stage) 13 to E19 and in adult rats. Gold-conjugated secondary antibodies were used to localize, on ultrathin sections of brain, a rabbit polyclonal antiserum (anti-GLUT-1) raised against a synthetic peptide encoding 13 amino acids of the C-terminus of the human glucose transporter. Staining was weak at E13 but increased in density during development into adulthood. The increase represented an increase in the absolute amount of transporter per vessel profile, with a concomitant decrease in vessel size with the narrowing of the wall. At early stages, the percentages of total particles per profile of lumenal membrane, ablumenal membrane, and cytoplasm were approximately equivalent. The ratio of lumenal to ablumenal particle density then shifted from below 1 at E13 to above 2 at E19 and to 4 in the adult. In contrast, vessels of the choroid plexus were devoid of labeling, but the choroid plexus epithelium stained as early as E15. In the brain, no astrocytes, neurons, or pericytes were stained at any stage examined. Developmental upregulation of the GLUT-1 glucose transporter therefore seems to occur at the blood-brain barrier, and the modulation of the subcellular distribution of the transporter can be correlated with other observed changes in the microvessels as they develop the blood-brain barrier phenotype.

Structural and functional protein network analyses predict novel signaling functions for rhodopsin

Orchestration of signaling, photoreceptor structural integrity, and maintenance needed for mammalian vision remain enigmatic. By integrating three proteomic data sets, literature mining, computational analyses, and structural information, we have generated a multiscale signal transduction network linked to the visual G protein‐coupled receptor (GPCR) rhodopsin, the major protein component of rod outer segments. This network was complemented by domain decomposition of protein–protein interactions and then qualified for mutually exclusive or mutually compatible interactions and ternary complex formation using structural data. The resulting information not only offers a comprehensive view of signal transduction induced by this GPCR but also suggests novel signaling routes to cytoskeleton dynamics and vesicular trafficking, predicting an important level of regulation through small GTPases. Further, it demonstrates a specific disease susceptibility of the core visual pathway due to the uniqueness of its components present mainly in the eye. As a comprehensive multiscale network, it can serve as a basis to elucidate the physiological principles of photoreceptor function, identify potential disease‐associated genes and proteins, and guide the development of therapies that target specific branches of the signaling pathway.

Gene expression profiling of the retina after transcorneal electrical stimulation in wild-type Brown Norway rats.

PURPOSE Transcorneal electrical stimulation (TES) has been beneficial in several neurodegenerative ocular diseases, but the exact mechanisms remain to be elucidated. This study was conducted to investigate the effects of TES on the retinas of wild-type Brown Norway (BN) rats by gene expression profiling and to assess its effects on retinal function and morphology. METHODS TES was applied to BN wild-type rat retinas in vivo for 1 hour (1-ms biphasic pulses at 20 Hz; 200 μA). RNA was isolated and processed for microarray-based profiling 4 hours after TES; differentially expressed genes from TES compared with those from sham-treated animals were validated by quantitative real-time polymerase chain reaction. Furthermore, the effect of TES was assessed at the structural and functional levels using electroretinography, confocal scanning laser ophthalmoscopy, optical coherence tomography, and immunohistochemistry. RESULTS Transcriptome changes associated with TES versus sham-stimulated BN wild-type retina were identified. Four hundred ninety genes were differentially expressed in TES and included potentially neuroprotective genes such as Bax or members of the tumor necrosis factor family (Tnfrsf11b, Tnrsf12a, Tnfsf13b, Tnfsf13). ERG recordings showed physiological retinal function after TES, and structural in vivo and ex vivo studies revealed intact retinal anatomy. CONCLUSIONS These results demonstrate that TES applied to the retina of the wild-type BN rats induces distinct transcriptome level changes and may help in the understanding of the mechanisms underlying TES. In addition, TES treatment indicates no negative effect on structure and function of the wild-type BN retina up to 35 hours after application.

Toxicity testing of the VEGF inhibitors bevacizumab, ranibizumab and pegaptanib in rats both with and without prior retinal ganglion cell damage

Purpose:  To evaluate the effects of intravitreally introduced vascular endothelial growth factor (VEGF) inhibitors in rat eyes with healthy retinal ganglion cells (RGC) and into others with N‐methyl‐D‐aspartate (NMDA)‐induced RGC damage.

Transcorneal electrical stimulation shows neuroprotective effects in retinas of light-exposed rats

PURPOSE To examine the effects of transcorneal electrical stimulation (TES) on retinal degeneration of light-exposed rats. METHODS Thirty-three Sprague Dawley albino rats were divided into three groups: STIM (n = 15) received 60 minutes of TES, whereas SHAM (n = 15) received identical sham stimulation 2 hours before exposure to bright light with 16,000 lux; healthy animals (n = 3) served as controls for histology. At baseline and weekly for 3 consecutive weeks, dark- and light-adapted electroretinography was used to assess retinal function. Analysis of the response versus luminance function retrieved the parameters Vmax (saturation amplitude) and k (luminance to reach ½Vmax). Retinal morphology was assessed by histology (hematoxylin-eosin [HE] staining; TUNEL assay) and immunohistochemistry (rhodopsin staining). RESULTS Vmax was higher in the STIM group compared with SHAM 1 week after light damage (mean intra-individual difference between groups 116.06 μV; P = 0.046). The b-wave implicit time for the rod response (0.01 cd.s/m²) was lower in the STIM group compared with the SHAM group 2 weeks after light damage (mean intra-individual difference between groups 5.78 ms; P = 0.023); no other significant differences were found. Histological analyses showed photoreceptor cell death (TUNEL and HE) in SHAM, most pronounced in the superior hemiretina. STIM showed complete outer nuclear layer thickness preservation, reduced photoreceptor cell death, and preserved outer segment length compared with SHAM (HE and rhodopsin). CONCLUSIONS This sham-controlled study shows that TES can protect retinal cells against mild light-induced degeneration in Sprague Dawley rats. These findings could help to establish TES as a treatment in human forms of retinal degenerative disease.

Effects of Combined Ketamine/Xylazine Anesthesia on Light Induced Retinal Degeneration in Rats

Objectives To explore the effect of ketamine-xylazine anesthesia on light-induced retinal degeneration in rats. Methods Rats were anesthetized with ketamine and xylazine (100 and 5 mg, respectively) for 1 h, followed by a recovery phase of 2 h before exposure to 16,000 lux of environmental illumination for 2 h. Functional assessment by electroretinography (ERG) and morphological assessment by in vivo imaging (optical coherence tomography), histology (hematoxylin/eosin staining, TUNEL assay) and immunohistochemistry (GFAP and rhodopsin staining) were performed at baseline (ERG), 36 h, 7 d and 14 d post-treatment. Non-anesthetized animals treated with light damage served as controls. Results Ketamine-xylazine pre-treatment preserved retinal function and protected against light-induced retinal degeneration. In vivo retinal imaging demonstrated a significant increase of outer nuclear layer (ONL) thickness in the non-anesthetized group at 36 h (p<0.01) and significant reduction one week (p<0.01) after light damage. In contrast, ketamine-xylazine pre-treated animals showed no significant alteration of total retinal or ONL thickness at either time point (p>0.05), indicating a stabilizing and/or protective effect with regard to phototoxicity. Histology confirmed light-induced photoreceptor cell death and Müller cells gliosis in non-anesthetized rats, especially in the superior hemiretina, while ketamine-xylazine treated rats showed reduced photoreceptor cell death (TUNEL staining: p<0.001 after 7 d), thicker ONL and longer IS/OS. Fourteen days after light damage, a reduction of standard flash induced a-wave amplitudes and a-wave slopes (p = 0.01) and significant alterations in parameters of the scotopic sensitivity function (e.g. Vmax of the Naka Rushton fit p = 0.03) were observed in non-treated vs. ketamine-xylazine treated animals. Conclusions Our results suggest that pre-treatment with ketamine-xylazine anesthesia protects retinas against light damage, reducing photoreceptor cell death. These data support the notion that anesthesia with ketamine-xylazine provides neuroprotective effects in light-induced cell damage.

Caspase inhibitors protect against NMDA-mediated retinal ganglion cell death

Background:  Apoptosis is a major mechanism of cell death in glutamate‐induced excitotoxicity and caspases as the executors of apoptosis play an important role in the development of various central nervous system and eye diseases. We studied the involvement of certain caspases in excitotoxic retinal ganglion cell death, which was experimentally induced in Brown Norway Rats by application of the glutamate receptor agonist N‐methyl‐D‐aspartate (NMDA).

First appearance of orthogonal arrays of particles in Müller cell membranes depends on retina maturation.

A freeze-fracture analysis was undertaken to investigate both the appearance of orthogonal arrays of particles (OAP) in the Müller cell endfoot membrane at the retina-vitreous border and the endfoot size during development of two avian species. Chickens and pigeons have a common length of development until hatching (21 days), but differ in maturation velocity. In the chicken, the first OAP appear in the central retina at embryonic day 18 (E18), in the peripheral retina at E19. The OAP density increases rapidly. In the pigeon, the first OAP appear 6 days after hatching when the eyes are opened. In both species, the size of endfeet is maximal when the OAP first appear and is reduced thereafter. In the chicken, during the time up to adulthood both the mean endfoot size and the retina area increase 6-fold suggesting that the number of endfeet remains constant during growth of their individual area. In the pigeon, after an initial increase of mean endfoot area this decreases until adulthood, despite continuous growth of the eye. This strongly suggests that during retina growth the number of Müller cell endfeet increases. It is concluded that these alterations of Müller cell membranes reflect some functional aspects of retina maturation.

Targeted Ablation of the Pde6h Gene in Mice Reveals Cross-species Differences in Cone and Rod Phototransduction Protein Isoform Inventory

Background: Phosphodiesterase-6 (PDE6) is a multisubunit enzyme essential for visual signal processing. Rare mutations in the human PDE6H gene result in incomplete color blindness. Results: Pde6h-deficient mice exhibit no signs of photoreceptor dysfunction. Conclusion: PDE6 configurations differ between species and are more interchangeable than previously thought. Significance: Presence of related isoforms in the retina may allow adjustments of the phototransduction components thereby preventing the occurrence of pathological conditions. Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3′,5′-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h−/−) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h−/− retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h−/− mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system.