Alpha-fodrin is cleaved by caspase-3 in a chronic ocular hypertensive (COH) rat model of glaucoma.

PURPOSE: alpha-Fodrin is a neuronal cytoskeletal protein and a known caspase-3 target. We sought to determine whether caspase-3 cleaves alpha-fodrin in COH rat retinas and whether this process is reduced by adeno-associated virus (AAV)-induced retinal ganglion cell expression of baculovirus inhibitory repeat-containing 4 (BIRC4), a potent caspase-3 inhibitor. METHODS: Ocular hypertension was induced unilaterally in five rat eyes by limbal injection of hypertonic saline. In a similar experiment, ocular hypertension was induced in four eyes pre-treated with an intravitreal injection of AAV-BIRC4 to assess alpha-fodrin cleavage. Western immunoblotting was performed on all retinas. RESULTS: Caspase-3 cleavage of alpha-fodrin yields a specific 120kDa protein fragment. COH retina immunoblots indicated significantly more caspase-3 cleavage of alpha-fodrin than controls (P < 0.01, paired t-test). Inhibition of retinal caspase-3 activity with BIRC4 reduced caspase-3-mediated alpha-fodrin cleavage compared to controls. CONCLUSIONS: This confirms our previous finding of caspase-3 cleavage of alpha-fodrin in COH retinas and parallels pathology seen in Alzheimer's disease, in which neurons undergo chronic caspase activation, slow build-up of cleavage products, and delayed apoptosis. If caspase activation in glaucoma leads to protracted rather than rapid retinal ganglion cell apoptosis, a much longer therapeutic window exists for apoptosis inhibition with caspase inhibitors such as BIRC4.

Apoptosis in the murine rd1 retinal degeneration is predominantly p53-independent.

PURPOSE: To determine if p53 mediates apoptosis in photoreceptors of retinal degeneration, rd1, mice. METHODS: The rd1/rd1 mice were interbred with p53 null mice to generate p53-/- rd1/rd1 and p53+/+ rd1/rd1 mice. Rates of loss and incidence of apoptosis in rod photoreceptors were analyzed at appropriate ages (postnatal days 12, 14 and 16). RESULTS: The extent and kinetics of photoreceptor cell loss in rd1 mice were nearly indistinguishable in the p53+/+ and p53 null mice. CONCLUSIONS: Photoreceptor cell apoptosis in the rd1 mouse model occurs by a predominantly p53-independent molecular pathway.

Ultrastructural features of retinal dystrophy in mutant vitiligo mice.

Ultrastructural features of the retinal pigment epithelium and photoreceptor cells were studied in vitiligo (C57BL/6 mivit/mivit) mice. Eyes from 12-day- to 56-week-old animals were analysed. Abnormal photoreceptors were seen in 12-day-old mice. By 3 weeks malformed outer segments were evident in the posterior and equatorial retina, but normal photoreceptors were present in the periphery. By 28 weeks, a marked gradient in cell loss was evident, with a progressive increase in cell viability along the posterior-peripheral axis. Viable intact photoreceptors were still present in the peripheral retina of 56-week-old mice. Melanosome content varied between adjacent pigment epithelium cells in both the posterior and peripheral retina. In the choroid, however, a steep posterior-peripheral gradient in melanosome content was evident with highest pigmentation in the periphery. In the optic nerve head region abnormal development of photoreceptors was correlated with proliferation of abnormal pigment epithelium cells. Accumulation of rod outer segment debris in the posterior and peripheral subretinal space preceded photoreceptor cell death. Short pigment epithelial microvilli without proper attachment to photoreceptors are suggestive of alterations in pigment-epithelium-photoreceptor interaction, which might affect photoreceptor differentiation and phagocytosis of rod outer segments by pigment epithelium cells.

rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis.

Small GTP-binding protein rab8 regulates transport from the TGN to the basolateral plasma membrane in epithelial cells and to the dendritic plasma membrane in cultured hippocampal neurons. In our approach to identify proteins involved in rhodopsin transport and sorting in retinal photoreceptors, we have found, using [32P]GTP overlays of 2D gel blots, that six small GTP-binding proteins are tightly bound to the post-Golgi membranes immunoisolated with a mAb to the cytoplasmic domain of frog rhodopsin. We report here that one of these proteins is rab8. About 50% of photoreceptor rab8 is membrane associated and approximately 13% is tightly bound to the post-Golgi vesicles. By confocal microscopy, antibody to rab8 specifically labels calycal processes and the actin bundles of the photoreceptor inner segment that extend inward to the junctional complexes that comprise the outer limiting membrane. Anti-rab8 shows a striking periodicity of high density labeling at 1 +/- 0.12 microns intervals along the actin bundles. Rhodopsin-bearing post-Golgi membranes cluster around the base of the cilium where rab8 and actin are also co-localized, as revealed by confocal microscopy of retinal sections double labeled with anti-rab8 and phalloidin. Microfilaments have been implicated in rod outer segment (ROS) disk morphogenesis. Our data suggest that rab6, which we have previously localized to the post-Golgi compartment, and rab8 associate with the post-Golgi membranes sequentially at different stages of transport. rab8 may mediate later steps that involve interaction of transport membranes with actin filaments and may participate in microfilament-dependent ROS disk morphogenesis.

The case for pedicle fixation of the lumbar spine.

STUDY DESIGN: This was a retrospective review of one surgeon's results using three different lumbosacral arthrodesis techniques: Group 1, no instrumentation; Group 2, Luque Rod and sublaminar wire technique; and Group 3, AO intrapedicular screw and plate technique. OBJECTIVE: To determine whether the use of metal implants results in a higher fusion rate. Once a solid arthrodesis is achieved, is this correlated with a good clinical result? SUMMARY OF BACKGROUND DATA: Controversy persists regarding the value of the use of intrapedicular fixation to augment arthrodesis of the lumbosacral junction. Controversy also exists regarding the correlation of solid arthrodesis with relief of preoperative symptoms. METHODS: Three serial sequential populations (50 subjects each) undergoing varied primary multiple-level lumbosacral arthrodesis procedures were studied retrospectively. The ultimate clinical results of these three different surgical populations were studied after prolonged follow-up. RESULTS: Group one had a 14% fusion rate and a 4% complication rate. Group two had a 36% fusion rate and an 8% complication rate. Group three had a 64% fusion rate and an 18% complication rate. Complications were intraoperative dural tears and nerve root injuries. Patient satisfaction with each operative procedure to relieve preoperative low back pain was statistically correlated with whether a solid arthrodesis was obtained. CONCLUSION: Intrapedicular fixation technique is the most reliable method for obtaining a solid multiple-level lumbosacral arthrodesis. Solid arthrodesis is correlated with a successful clinical result. Complications associated with the use of intrapedicular fixation were frequent but their occurrence demonstrated a "learning curve pattern."

Apoptosis or retinoblastoma: alternative fates of photoreceptors expressing the HPV-16 E7 gene in the presence or absence of p53.

A transgenic mouse model for retinoblastoma was produced previously by directing SV40 T antigen expression to retinal photoreceptor cells using the promoter of the interstitial retinol-binding protein (IRBP) gene. This gene becomes active prior to the terminal differentiation of photoreceptors. Because T antigen-transforming activity is attributable, at least in part, to the inactivation of the retinoblastoma (pRb) and p53 tumor suppressor proteins, we addressed the role of p53 in the development of retinoblastoma in mice. Transgenic mice expressing HPV-16 E7 under the control of the IRBP promoter were generated to inactivate pRb in photoreceptors while leaving p53 intact. Rather than developing retinoblastomas, the retinas of these mice degenerate due to photoreceptor cell death at a time in development when photoreceptors are normally undergoing terminal differentiation. The dying cells exhibit the histological and ultrastructural features of apoptosis and contain fragmented DNA. p53 is required for the induction of apoptosis in this model, because mice expressing E7 in a p53 nullizygous background develop retinal tumors instead of undergoing retinal degeneration.

Ultrastructural analysis of arrestin distribution in mouse photoreceptors during dark/light cycle.

Arrestin localization was studied in BALB/c mice retinas during a 12-hr dark/light diurnal cycle and under various light/dark interruptions. Intracellular distribution of arrestin in photoreceptor cells was determined by immunocytochemistry and electron microscopy. During the light phase of the diurnal cycle, arrestin was localized mostly in the rod outer segments. During the dark phase of the cycle, arrestin was localized mostly in the inner segment, nuclei and synaptic terminals. The disc domains of the rod outer segments were labeled at a low density, but conspicuous cytoplasmic regions in the outer segment were labeled at a very high density. These cytoplasmic regions were not labeled in our illuminated retinas. Hence, intrasegmental segregation within the outer segment may be influenced by environmental lighting. During dark adaptation, increase in inner segment labeling density was observed. In previous studies, decrease in outer segment and increase in inner segment labeling density in the dark, as determined by light microscopy, was interpreted as movement of arrestin from the outer to inner segments. Our present ultrastructural analysis of arrestin distribution indicates that yet undetermined amounts of arrestin accumulate in localized regions of the outer segment in the dark. The extent of movement of arrestin to the inner segment, if it occurs, remains to be established. Localization of arrestin in phagosomes indicates that at least part of the arrestin is being degraded in the pigment epithelium.

S-antigen in rods and cones of the primate retina: different labeling patterns are revealed with antibodies directed against specific domains in the molecule.

S-antigen (arrestin) is a soluble 48 KD protein of the retinal photoreceptor cells. It has been found to have a function in regulation of the phototransduction cascade. Previous labeling experiments with anti-S-antigen (SAg) antibodies have yielded conflicting reports as to the presence of SAg in cone photoreceptor cells. In the present study we employed five monoclonal anti-SAg antibodies (MAb) directed against different known domains in the SAg molecule. MAb A9C6, D9F2, and C10C10 are directed against sequences in the carboxy half of the SAg molecule. MAb 5C6.47 and F4C1 are directed against the amino terminal. Immunoelectron microscopy was used in the localization of SAg in LR Gold-embedded baboon retinas. Green/red and blue cones were identified with MAb COS-1 and OS-2, respectively. MAb A9C6, D9F2, and C10C10 densely labeled rods and blue cones but not green/red cones. MAb 5C6.47 and F4C1 labeled rods and both blue and green/red cones. It appears that, in the baboon retina, different SAg molecules are present in the blue and green/red cones. Whereas the blue cone SAg shares common antigenic determinants with rods, both in the amino and carboxy terminals, the green/red cone SAg contains different antigenic determinants at the carboxy half of the molecule.