Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

or neurotrophin-4/5 (NT-4/5), applied intravitreally (Cui and Harvey, 1994; 1995) or distally to RGC terminals/axons (Ma et al., 1998; Spalding et al., 1998). However, the protective effect of these molecules is transient, delaying but not preventing RGC loss (Cui and Harvey, 1995).

To understand why neurotrophins only temporarily reduce neonatal RGC death after target ablation we analysed changes in neurotrophin receptor expression and possible changes in growth factor dependency (Spalding et al., 2005a). Neurotrophins mediate many of their effects via receptor tyrosine kinases (trkA, trkB and trkC), BDNF and NT-4/5 signalling primarily through the trkB receptor (Huang and Reichardt, 2003). Consistent with RGC sensitivity to BDNF and NT-4/5, these neurons express the trkB receptor (Jelsma et al., 1993; Perez and Caminos, 1995; Vecino et al., 2002).

In unlesioned rats, trkB immunohistochemical analysis revealed no change in the number of trkB positive cells in the RGC layer 24 hrs after intraocular NT-4/5 injection. However, after SC lesions there were less immunoreactive cells and even fewer cells in NT- 4/5 injected eyes (Spalding et al., 2005a). Semi-quantitative confocal analysis of immunofluorescence intensity revealed an increase in trkB staining in the RGC layer in unlesioned rats 24 hrs after NT-4/5 injection, whereas in SC-lesioned animals exposed to NT-4/5 there was a significant decrease in staining. In summary, application of neurotrophins caused a down-regulation of the cognate trkB receptor, presumably altering the long-term responsiveness of neonatal RGCs to exogenous neurotrophins.

Injured neonatal RGCs in P4/P5 retinas do not appear to shift their trophic dependence to other survival factors (Meyer-Franke et al., 1995). Different doses of ciliary neurotrophic factor (CNTF) were given intraocularly, either alone or combined with NT-4/5. We also tested an SC-derived chondroitin sulfate proteoglycan that has been reported to promote adult RGC survival after injury (Huxlin et al., 1995). None of these interventions reduced lesion-induced RGC death 24 hrs or 36 hrs after SC ablation (Spalding et al., 2005a).

3. SOURCES OF TROPHIC SUPPORT FOR RETINAL GANGLION CELLS

Consistent with the neurotrophic hypothesis of RGC dependence on target-derived factors, BDNF is produced in the SC and is retrogradely transported by RGCs (Ma et al., 1998). However using 125I-labelled peptides we recently showed that there was also substantial and rapid anterograde transport of BDNF and, to a lesser extent, neurotrophin-4/5 (NT-4/5) to central visual target areas in the neonatal rat brain (Spalding et al., 2002). Six hours after unilateral intraocular injection, all retino-recipient regions in the thalamus and midbrain were heavily labelled. Neonatal eye removal results in increased cell death in the SC (Lund et al., 1973), suggesting an anterograde trophic influence on tectal cells. We found that, 24 hrs after intraocular application of physiologically relevant doses of neurotrophin, there was significantly decreased neuronal death in the contralateral SC. Our new data thus support the proposal that BDNF and NT-4/5 can be anterograde survival factors for postsynaptic cells in the developing rat nervous system (cf. Caleo et al., 2000).

BDNF and NT-4/5 protein and mRNA have now been identified not only in central sites such as the superficial layers of the SC but also in the retina itself. Indeed, BDNF levels in neonatal rodents are greater in the SC than in the retina (Ma et al., 1998; Frost et al., 2001; Seki et al., 2003) and cells of the Xenopus and chick RGC layer have been reported to receive BDNF trophic support predominantly from intra-retinal, rather than collicular

sources (Cohen-Cory et al., 1996; Herzog and von Bartheld, 1998). In mammals there is now evidence that RGCs may be supported not only by target-derived neurotrophins but also by retinally-derived neurotrophins (de Araujo and Linden, 1993; Cohen-Cory et al., 1996; Ary-Pires et al., 1997).

To determine whether neonatal rat RGC viability depends on intra-retinally derived neurotrophins, we measured RGC death 24 hrs following injections of a mixture of BDNF and NT-4/5 blocking antibodies into the eye. RGC death was also assessed 24 hrs and 48 hrs after injection of these same antibodies into the SC (Spalding et al., 2004). It was found that collicular injections of BDNF and NT-4/5 blocking antibodies significantly increased RGC death in the neonatal rat 24 hrs post injection, death rates returning to normal by 48 hrs. The increase in death was greatest following SC injections, but death was also significantly increased 24 hrs following intravitreal antibody injection, providing further evidence for a survival-promoting role for intraretinally derived neurotrophic factors.

The mechanisms whereby RGCs respond differentially to target-derived versus locally supplied neurotrophins are unclear, although there is evidence that intracellular signalling pathways activated by growth factors can differ depending upon where the neurotrophin binds, e.g. at the cell body, dendrite, or distally at the nerve terminal (Heerssen and Segal, 2002). There is also evidence that trophic factors derived from local and target sources have differing effects on RGC development (Lom et al., 2002). Taken together, many issues concerning the development of retinofugal connections remain to be resolved. BDNF and NT- 4/5 are produced in the retina and in central targets, they are transported in both directions along RGC axons, and these neurotrophins increase the viability of both retinal and target neurons. Understanding the bi-directional relationship between developing RGCs and central neurons, and how these cells distinguish between local paracrine/autocrine neurotrophin expression and factors derived from more distant sources, are clearly important areas for future research.

4. MECHANISMS OF NEONATAL RETINAL GANGLION CELL DEATH

Compared to injury in neonates, ON injury in adult rodents results in RGC death but at a much slower rate (Misantone et al., 1984; Villegaz-Perez et al., 1993). Nonetheless, injury-induced RGC death in both the adult (Berkelaar et al., 1994; Garcia-Venezuela et al., 1994; Isenmann et al., 1997) and neonatal rat (Harvey et al., 1994; Rabacchi et al., 1994; Cui and Harvey, 1995) has primarily been ascribed to apoptosis. There is evidence in adults that RGC death after ON injury is due to caspase activation. In vivo studies have shown that caspases-3, -8, and -9 become activated in adult rat RGCs following optic nerve axotomy and intraocular injection of relevant caspase inhibitors can reduce RGC death, at least for a certain period of time (Kermer et al., 1998; Chaudhary et al., 1999; Kermer et al., 1999; Weishaupt et al., 2003).

We recently examined whether blocking caspases in vivo reduces neonatal RGC death after SC lesions (Spalding et al., 2005b). Surprisingly, intraocular injection of general and specific caspase inhibitors did not increase neonatal RGC survival 6 hrs and 24 hrs after SC ablation. These inhibitors were, however, effective in blocking caspases in another welldefined in vitro apoptosis model, the ovarian corpus luteum. Retinal caspase-3 protein and mRNA levels were assessed 3, 6 and 24 hrs after SC removal using immunohistochemistry, western and northern blots, and quantitative real-time PCR. Terminal deoxynucleotidyl

transferase-mediated dUTP nick-end labelling (TUNEL) was used to independently monitor retinal cell death. The PCR data showed a small but not significant increase in caspase-3 mRNA in retinas 24 hrs PL, however Western blot analysis did not reveal a significant shift to cleaved (activated) caspase-3 protein. There was a small increase in the number of cleaved caspase-3 immunolabelled cells in the ganglion cell layer 24 hrs after SC removal, but this represented only a fraction of the death revealed by TUNEL.

In summary, the inability of caspase inhibitors to reduce lesion-induced RGC death 6 hrs or 24 hrs after neonatal SC ablation, and the lack of major activation of caspase-3 mRNA and production of cleaved protein 24 hrs PL, strongly suggests that most lesioninduced RGC death in immature rats is not caspase-dependent. Within a given neuronal population, it appears that different cell death cascades can be initiated at different maturational stages during the lifetime of a cell.

5. REFERENCES

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PART VIII

BASIC SCIENCE UNDERLYING RETINAL DEGENERATION

quence of arrestin movement, or whether de novo protein synthesis coupled with arrestin degradation plays a significant role in the apparent redistribution of arrestin. In addition, we utilize a variant of arrestin that is unable to bind to rhodopsin to investigate the role of arrestin binding to light-activated phosphorhodopsin in the translocation of arrestin. Finally, we address whether vertebrate arrestins rely on an affinity for phosphoinositol lipids for translocation.

2. METHODS AND MATERIALS

2.1. Cycloheximide Treatment of Transgenic Tadpoles

Transgenic Xenopus expressing a fusion of GFP at the C-terminus of Xenopus arrestin (xAr-GFP) were obtained from breeding pairs of adult transgenic Xenopus as previously described (Peterson et al. 2003) and dark-adapted overnight. Tadpoles were placed in 0.1x tadpole Ringers with or without 100 mM cycloheximide (CHI) for 1 h. Tadpoles then either remained in the dark, or were exposed to laboratory lighting for 45 min or for 240 min. At this point, tadpoles were euthanized in 0.025% benzocaine, one eye removed, the cornea punctured with a scalpel, and placed in 100 mL 1x tadpole Ringers with 20 mM glucose and 70 mCi 35S-labeled methionine and cysteine (Amersham) for 2 h at room temperature. After rinsing, the eye was disrupted by vigorous pipetting and vortexing in 50 mL 1x Laemmli sample buffer (Laemmli 1970), and viscosity reduced by the addition of 25 units of benzonase (Novagen). An aliquot of the extract was separated on 12% SDS-PAGE, the gel stained with Coomassie Brilliant blue, and the dried gel exposed to x-ray film to detect incorporated radiolabeled proteins.

The contralateral eye was fixed in methanolic formaldehyde as described (Peterson et al. 2003), and processed for confocal microscopy to detect the localization of the arrestin/GFP fusion using the endogenous fluorescence of GFP.

2.2. Myc-Tagged Arrestin

The ten amino acid myc tag (EQKLISEEDL) was incorporated into Xenopus arrestin using overlapping PCR products that introduced the cDNA for the myc-tag between the codons for Leu-76 and Thr-77. The overlapping products were combined and the complete cDNA amplified using primers against the 5¢ and 3¢ ends of the arrestin cDNA, incorporating XhoI and NotI sites, respectively. This product was cloned into the XhoI and NotI sites of the XOPS1.3 vector under the control of the 1.3 kb Xenopus rod opsin promoter (Tam et al. 2000). The cDNA for GFP was inserted immediately prior to the arrestin stop codon at an introduced NheI site. Transgenic animals expressing this myc-tagged xAr-GFP (xAr- myc-GFP) were prepared by nuclear transplantation (Kroll and Amaya 1996).

For in vitro studies, a His(6) tag was incorporated by PCR at the 5¢ end of the cDNA immediately after the initiating ATG for xAr-GFP and xAr-myc-GFP, and then cloned into the shuttle vector pPIC-ZA for heterologous expression in Pichia pastoris (Dinculescu et al. 2002). Expressed proteins were purified over nickel-agarose (Ni-NTA, Qiagen), followed by heparin agarose. In vitro binding assays with rhodopsin were performed using unphosphorylated and phosphorylated rod disc membranes prepared from Rana catesbeiana retinas as described for bovine disc membranes (McDowell 1993).

2.3. Four Lysine Mutants of Arrestin

Multiple alignment of Drosophila, bovine, and Xenopus arrestins was performed using ClustalX (Thompson et al. 1997). Two of the three lysines (Lys-228 and Lys-231) identified as important for promoting an association with inositol phospholipids in Drosophila (Lee et al. 2003) are conserved in bovine arrestin and were changed to alanines by sitedirected mutagenesis. Because sequence conservation is not perfect, two additional lysines were substituted with alanines to insure disruption of the potential inositol phospholipid binding site, creating the four lysine mutant K232A/K235A/K236A/K238A (4K Æ A) in bovine visual arrestin. The 4K Æ A bovine arrestin mutant was expressed in Pichia, purified to homogeneity, and tested for binding to inositol phospholipids immobilized on nitrocellulose strips (PIP Strips, Echelon, Inc.). PIP-Strips were blocked for 60 min with 1% gamma globulin-free horse serum in phosphate-buffered saline (PBS). Wild-type arrestin, 4K Æ A arrestin, and the PH domain of phospholipase C d1 fused to glutathione-S- transferase (GST-GRIP; Echelon, Inc) (0.5 mg/mL) were then incubated with the PIP strips for 4 h. After washing with 0.05% Tween-20 in PBS, the blots were immunoprobed to detect binding using anti-arrestin monoclonal SCT-128 for bovine arrestin and anti-GST monoclonal for the GRIP positive control. Binding of the primary antibody was detected using an anti-mouse antibody conjugated to alkaline phosphatase with nitroblue tetrazolium/5- bromo-4-chloro-3-indoyl phosphate as the substrate.

Relative affinity of bovine arrestin and 4K Æ A arrestin for phytic acid was assessed by competitive elution from heparin agarose. Arrestin and 4K Æ A (1 mg) was immobilized on a 1 mL heparin sepharose column (Amersham) in 10 mM HEPES/30 mM NaCl, pH 7.0. Aliquots of phytic acid (1 mM - 10 mM) in the same mobile phase were added to the column and the amount of arrestin eluted in each aliquot measured by absorbance at 278 nm.

Homologous mutations were also created in Xenopus arrestin by PCR site-directed mutagenesis, substituting lysines 232, 235, 236, and 267 with glutamine (4K Æ Q). This substituted arrestin was fused with GFP as previously described and used to create transgenic Xenopus tadpoles. Translocation of the Ar(4K Æ Q)-GFP was assessed by confocal microscopy using tadpoles that were dark-adapted for 3 days, then exposed to laboratory lighting for 50 min, or for 240 min.

3. RESULTS AND DISCUSSION

3.1. Does Arrestin Translocate from the Outer Segments?

Our previously published work clearly demonstrates that arrestin translocates from the inner segments (RIS) of dark-adapted rod photoreceptors, moving to the rod outer segments (ROS) upon exposure to light (Peterson et al. 2003). In these studies, it also appears that arrestin leaves the rod outer segments and returns to the inner segments in response to either dark adaptation or extended light adaptation. However, another equally tenable explanation is that the arrestin that translocates to the outer segments is proteolyzed and that the arrestin subsequently seen in the inner segment is a result of newly synthesized arrestin (Azarian et al. 1995).

To discriminate between these two mechanisms, transgenic Ar-GFP tadpoles were treated with cycloheximide (CHI) to inhibit protein synthesis (Obrig et al. 1971), and

Figure 63.1. Inhibition of protein synthesis in the eyes of tadpoles treated with 100 mM cycloheximide (CHI). Treated (+) or untreated (-) tadpoles were dark adapted overnight (DA) or exposed to light for 45 min (LA45) or for 240 min (LA240). One eye was removed and new protein synthesis assessed as described in Methods. Proteins extracted from eye homogenates were separated by 12% SDS-PAGE and stained with Coomassie blue (A). Newly synthesized proteins that incorporated radiolabeled cysteine and methionine are revealed in the autoradiograph of the same gel (B).

Figure 63.2. Arrestin-GFP translocation in rods from transgenic tadpoles treated or not treated with CHI. Contralateral eyes from the animals used in Figure 63.1 were cryosectioned and the endogenous fluorescence of the arrestin/GFP fusion protein visualized using confocal microscopy. Tadpoles were dark adapted overnight (DA), exposed to light for 45 min (LA 45 min), or to light for 240 min (LA 240 min). See also color insert.

translocation subsequently assessed. Figure 63.1 shows total homogenates prepared from one eye from each of these tadpoles, separated on 12% SDS-PAGE. Protein staining of total homogenates prepared from eyes treated with 100 mM CHI revealed a similar profile as the untreated animals, although the total protein content extracted was slightly less in the treated animals (Figure 63.1A). Autoradiography of this same gel indicates that there was substantial incorporation of the radiolabeled cysteine and methionine in the untreated animals, but that new protein synthesis was largely blocked in the tadpoles treated with cycloheximide (Figure 63.1B).

The contralateral eyes from the same tadpoles used in Figure 63.1 were fixed in formaldehyde and processed for confocal microscopy to show the distribution of the ArGFP fusion protein, using the endogenous fluorescence of GFP (Figure 63.2). In both the