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

2.2. Discoidin Domain

The dominant feature of the RS1 polypeptide is the 157 amino acid discoidin (DS) domain or F5/8 type C domain which comprises over 75% of the mature polypeptide chain. DS domains are present in a wide range of membrane and extracellular proteins where they mediate a variety of cell adhesion, cell signaling and developmental processes (Baumgartner et al., 1998; Vogel, 1999). Examples of DS domain containing proteins are Factors V and VIII involved in blood coagulation, neuropilins 1 and 2, which mediate nervous system regeneration and degeneration, discoidin domain receptors DDR1 and DDR2 implicated in cancer metastasis, and discoidin I involved in cellular adhesion during slime mold development.

High-resolution structural studies indicate that DS domains consist of eight antiparallel b-strands arranged in a barrel-like structure with several loops or spikes, projecting from one end of the core barrel (Pratt et al., 1999). The DS domain of RS1 has been modeled after the C2 DS domain of Factors V and VIII and shown to consist of the core beta barrel conformation with 3 spike regions (Wu and Molday, 2003). Conserved cysteine residues at the beginning and end of the DS domains (C63 and C219 in RS1) form an intramolecular disulfide bond important in protein folding. A second intramolecular disulfide bond, absent in DS domains of other proteins, joins C110 in spike 2 to C142 in spike 3. The function of the DS domain of RS1 is not known although it has been implicated in cell adhesion.

Most disease-linked missense mutations occur within the DS domain of RS1 with over a quarter resulting in a loss or gain of a cysteine (Consortium, 1998). These disease-linked mutant proteins are expressed in culture cells at relatively normal levels, but unlike wildtype (WT) RS1, they are not secreted from cells. Instead, they are retained in the ER as misfolded, aggregated proteins (Wang et al., 2002; Wu and Molday, 2003).

2.3. Rs1 Domain and C-Terminal Segment

Two additional modules flank the DS domain. The 38 amino acid Rs1 domain resides just upstream of the DS domain, while a 5 amino acid C-terminal segment lies just downstream of the DS domain. Cysteine mutagenesis indicate that C59 of the Rs1 domain forms an intermolecular disulfide bond with C223 of the C-terminal segment of another RS1 subunit resulting in a disulfide-linked homo-octameric complex (Wu and Molday, 2003). Disease-causing mutations in these cysteines (C59S and C223R) are expressed and secreted from culture cells indicating that these mutant proteins fold into a native-like conformation. However, unlike WT RS1, the mutants fail to assemble into a disulfide-linked octameric complex indicating that multimeric assembly is critical for the function of RS1 as a cell adhesion protein (Wu and Molday, 2003).

3. GENE THERAPY IN THE RS1H-DEFICIENT MOUSE

Since female carriers of RS are asymptomatic, the lack of a functional RS1 protein and not the presence of a mutated protein is responsible for RS. Therefore, delivery of the normal RS1 gene to retinal cells, and in particular photoreceptor cells, using established gene therapy approaches (Acland et al., 2001; Flannery et al., 1997) could lead to an improved outcome for RS patients.

Figure 39.2. ERG recordings of AAV5-mOPs-RS1 treated and untreated eyes. (a). Bilateral full-field scotpic ERG recordings of a treated (R) and untreated (L) Rs1h-deficient mouse at 1 and 3 months posttreatment. Traces are the average of 5 responses to a stimulus intensity of 0.173 log cd m-2. (b) Maximum ERG amplitudes of WT and treated and untreated mice. Bar is the mean amplitude ± SEM for 7 eyes.

To explore the feasibility of using gene therapy as a treatment for RS, we have delivered an AAV serotype 5 vector containing the human RS1 cDNA under the control of the mouse opsin promoter (AAV5-mOPs-RS1) into the subretinal space of the right eyes of Rs1h-deficient 15-day old mice. The left eyes were not injected and served as controls. ERG recordings and immunocytochemical studies were carried out to determine the effect of gene delivery on the recovery of retinal structure and function.

3.1. ERG Recordings

Full-field scotopic ERG recordings of untreated and treated eyes of Rs1h-deficient mice were made at various times after a single subretinal injection with AAV5-mOPs-RS1. Figure 39.2a shows typical ERG recordings at 1 and 3 months posttreatment and Figure 39.2b displays the mean amplitudes of the a and b waves for the treated and untreated eyes at 1, 2 and 3 months. At 1 month, the amplitudes of the b-wave for the treated and untreated mice were similar and significantly reduced relative to WT mice. After 2 and 3 months there was a significant increase in b-wave amplitude for the treated eye, while the untreated eye showed a modest decline. The a-wave of the treated eye also showed an increase in amplitude at 3 months. Photopic responses were also significantly improved in the treated eye. There was a recovery of the oscillatory potentials of light-adapted single flash ERGs and a marked improvement in the responses to stimulus frequencies.

3.2. Immunocytochemical and Morphological Studies

The effect of AAV5-mOPs-RS1 treatment on the expression and tissue distribution of RS1 was studied by immuncytochemical labeling of retinal cryosections of mice 5 month

Figure 39.3. Immunoflurorescence microscopy of retinas from AAV-mOPs-RS1 treated and untreated Rs1h- deficient mice 5 month posttreatment. (a-c) Cryosections labeled with the 3R10 anti-RS1 monoclonal antibody. (d-f ) Sections imaged with DIC and stained with DAPI to highlight onl, inl and gcl layers. Arrows show gaps in the untreated mice. rpe, retinal pigment epithelium; os, outer segment; is, inner segment; onl, outer nuclear layer; opl, outer plexiform layer; inl, inner nuclear layer, ipl, inner plexiform layer; gcl, ganglion cell layer.

posttreatment. As shown in confocal micrographs of Figure 39.3a-c, retina tissue from a treated eye showed a RS1 immunostaining pattern similar to that of a wild-type retina. Intense RS1 labeling was present in the inner segment layer and more moderate labeling was seen in the outer nuclear and outer plexiform layers. The inner nuclear layer and to a lesser degree the inner plexiform layer was also labeled indicating that RS1 secreted from photoreceptors was able to move into the inner retina and bind to the surface of bipolar cells. Analysis of the whole retina of the treated eye showed that over 85% of the retina was labeled indicating that the RS1 protein spread laterally from the site of injection and expression. As previously reported (Weber et al., 2002), no RS1 labeling was observed for the untreated Rs1h-deficient retina.

The expression of RS1 coincided with a marked improvement in the structural organization of the retinal layers as visualized in DIC images merged with DAPI nuclear stain (Figure 39.3d-f ). The retina was organized into characteristic layers with a distinct separation of the inner and outer nuclear layers and an absence of gaps between bipolar cells. An increased thickness of the outer nuclear layer indicative of enhanced photoreceptor survival was also seen in the treated retina. In contrast, the untreated eye showed a pronounced disorganization of the retinal cell layers, reduced outer nuclear layer thickness, shortened outer segments and gaps between bipolar cells. Immunofluorescence labeling studies using antibodies specific for the synaptic layers, bipolar cells and cone photoreceptors further showed

a significant improvement in the structural integrity of the outer plexiform layer and inner nuclear layers and a 2-fold increase in the number of middle wavelength cones (data not shown).

Finally, glial fibrillary acidic protein (GFAP) expression and localization was studied to evaluate the pathological state of the retina. GFAP expression and distribution in the AAV5-mOPs-RS1 treated retina was comparable to that of a wild-type retina with labeling restricted to the basal region of the Müller cells. In contrast, the untreated retina showed increased GFAP expression and localization throughout the Müller cells.

In one series of studies, we also evaluated the long-term effect of AAV5-mOPs-RS1 treatment. RS1 expression and recovery of retinal structure and function persisted for at least l year following treatment.

Recently, Zeng et al. (2004) reported on AAV-mediated delivery of the mouse Rs1h gene under the control of a CMV promoter into the eye of a 13 week old Rs1h-deficient mouse. In this preliminary study, an improvement in the scotopic ERG b wave for the treated eye was observed, but no improvement in retina tissue organization or photoreceptor cell survival was demonstrated using this gene delivery protocol.

4. CONCLUSIONS

RS1 is a multisubunit, discoidin domain containing protein implicated in retinal cell adhesion. Disease-causing mutations cause defective protein biosynthetic processing, folding or subunit assembly resulting in an inactive protein. Delivery of the human RS1 gene via the AAV5 vector to photoreceptor cells of Rs1h-deficient mice results in near normal RS1 expression and tissue localization and a substantial recovery of retinal structure and visual function over the long term. These studies suggest that AAV-mediated RS1 gene delivery to photoreceptors may be an effective treatment for RS.

5. ACKNOWLEDGEMENTS

This work was supported by grants from the Foundation Fighting Blindness, Macular Vision Research Foundation, and the National Eye Institute.

6. REFERENCES

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of the UCP gene family in photoreceptors. It is likely that the high energy requirements of photoreceptors require very tight coupling of energy sources to ATP production and that any uncoupling of oxidative phosphorylation would be deleterious to photoreceptor function. Nevertheless, molecules that activate uncoupling proteins, such as the Coenzyme Q co-factor, may be useful in preventing damage to other retinal cells such as RPE and Müller cells.

3. CNTF HAS BOTH POSITIVE AND NEGATIVE EFFECTS ON ROD PHOTORECEPTORS

Ciliary Neurotrophic Factor (CNTF), an endogeneous retinal protein (Walsh et al., 2001), has long been known to protect a variety of neurons, including rod photoreceptors, from pathological stimuli (LaVail et al., 1992; Tao et al., 2002; Bok et al., 2002). CNTF binds to a receptor complex found on a number of retinal cells types, particularly Müller glial cells. Activation of the CNTF receptor, in turn, activates two signal transduction pathways, the JAK/STAT and the Erk1/2 MAPK pathways. Within the retina, there is some evidence that activation of one of these pathways is restricted to specific cell types, although both can be activated in adult Müller glial cells. Although Müller glial cells are one of the strongest candidates to respond to and transmit CNTF signals to other cell types of the retina, there is still ongoing debate about which cells actually mediate CNTF neuroprotective actions in the retina.

We have shown that CNTF can block the formation of rod photoreceptors in retinal explants, using opsin as a specific marker to monitor rod cell number (Zhang et al., 2004). This effect depends on the activation of the STAT3 and not the MAPK pathway. Recent experiments have shown that in retinas from animals with a retina specific STAT3 knockout, there is an apparent increase in thickness of the Outer Nuclear Layer, suggesting that CNTF/STAT3 inhibition of rod formation is part of normal retinal development.

To test whether CNTF had an effect on specific rod genes we carried out a microarray analysis of retinal explants treated with CNTF for various periods of time. CNTF cause a reduction in expression of a range of genes involved in visual transduction, including cGMPphosphodiesterase, recoverin, transducin and Abca4. On the other hand, we found that other genes such as Crx, Chx10 and Nr2E3 were not changed. The results suggest that CNTF has a very specific negative effect on rod development and may not always be an ideal therapeutic agent to combat retinal degenerations.

4. PEDF IS A POTENT NEUROPROTECTIVE FACTOR

PEDF is a novel neuroprotective factor that has proven therapeutic potential for a number of retinal diseases. PEDF is a 50 kD protein of the serpin family that was first isolated from medium conditioned by human RPE cells (Tombran-Tink and Johnson, 1989; Tombran-Tink et al., 1991). PEDF is an effective neuroprotective factor in many parts of the nervous system. In the eye, PEDF reduces apoptosis induced by H2O2 or light damage in rat photoreceptors (Cao et al., 1989, 2001), preserves the spatial organization, morphology, and function of photoreceptors after RPE detachment in a Xenopus model of retinal degeneration (Jablonski et al.) and protects retinal neurons from injuries caused by increased

Figure 40.1. Increased survival of human RPE cells treated with H2O2. Cells were pretreated with neuroprotective factor for 24 hrs before being treated with 60 mM H2O2 for I hour. 3 hr later, cell survival was measured by labeling cells with Calcein AM and measuring fluorescence. Concentrations of PEDF (P) and CNTF (CNTF) in ng/ml are marked on the axes.

intraocular pressure from transient ischemic reperfusion.11 In cells of other parts of the nervous system, such as cerebellar granule cells, hippocampal neurons and spinal cord motor neurons, nanogram amounts of PEDF provide protection from the damaging effects of glutamate toxicity.12-14

We have compared the efficacy of CNTF and PEDF to protect cells subjected to toxic levels of H2O2. As shown in the 3-D histogram of Figure 40.1, increasing concentrations of PEDF (left to right) or CNTF (front to back) increased the proportion of surviving cells. Saturation of the effect of each neuroprotective factor occurred at concentrations of approximately 100 ng/ml. Interestingly though even at saturating concentrations of one factor, addition of the other factor increases cell survival.

To test whether CNTF and PEDF might be acting in the same way at a molecular level we used a microarray analysis to compare changes in gene expression induced by treatment with each factor. Retinal explants were cultured in the presence or absence of each factor for 12, 24 or 48 hr. RNA from each group of retinas was used to prepare Cy3 and Cy5 labeled cDNA probes. These probes were hybridized to a microarray containing 12,000 spots of non-redundant retinal cDNA. Of the almost 10,000 retinal genes on these arrays CNTF induced changes in 62 genes and PEDF in only 30 genes. Very few of these genes were the

same, suggesting that the two neuroprotective factors act, at least in part, via different mechanisms. The results also emphasize that neuroprotective factors have very specific actions on target cells.

5. CONCLUSIONS

There is growing evidence that a variety of intrinsic and extrinsic factors can increase a neuron’s ability to withstand the episodic spikes in levels of toxic insults that occur in many neurodegenerative diseases. This suggests that neuroprotective agents can be used therapeutically for a range of complex retinal disorders such as Macular Degeneration and glaucoma, though they may be less efficacious over the long term against monogenic disorders with high penetrance such as many forms of retinitis pigmentosa. PEDF is an attractive candidate for neuroprotective therapies because it has no known harmful effects and is the only neuroprotective factor that also has antiangiogenic activity. Our findings suggest that different neuroprotective factors act via different pathways and thus that combinations of factors may be the most effective way of combating these retinal diseases.

6. ACKNOWLEDGEMENTS

We thank Drs. Samuel Shao-Min Zhang and Bing Chen for ongoing collaborations and discussions. Work in our laboratories has been supported by grants from the NIH and the David Woods Kemper Memorial Foundation.

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