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Mohand Sa|«d S, Deudon-Combe A, Hicks D et al 1998 Normal retina releases a di¡usible factor stimulating cone survival in the retinal degeneration (rd) mouse. Proc Natl Acad Sci USA 95:8357^8362

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DISCUSSION

Hauswirth:What does the human version of this gene look like and where does it map relative to RD?

Sahel: We recently looked at the intron/exon sequence of the gene and there is a human counterpart as well as a rat counterpart. We are now de¢ning the chromosomal location. We have plans to work on trying to ¢nd a mutation in cone degeneration, for example.

Nathans: Can you tell us something about what the protein looks like?

Sahel: The sequence doesn’t look like a neurotrophin, for example. From looking at the sequence and doing some bioinformatics analysis, we haven’t found an obvious function for this protein.

Ali: Don’t you think that it is strange that it is localized in the outer segments, as opposed to the cell body? Can you speculate a little about this?

Sahel: I can’t explain this. There was a nice paper by Cideciyan et al (1998) in which they showed by psychophysical studies that cones start to degenerate once you lose 75% of the outer segments of a photoreceptor. It is possible it is expressed at this level.

128 DISCUSSION

Hauswirth: Just because you detect the protein by antibody localization in the outer segments it doesn’t mean that it is made in the rods. Have you looked at RNA in situ to see where it is made?

Sahel: No, but if we cut the retina in slices (and we get 99% photoreceptor purity in these preparations), the RNA expression is in the photoreceptor layer.

Travis: From the primary sequence does the protein look like a secreted protein?

Sahel: Yes.

LaVail: Did you say that conditioned medium from wild-type mice was e¡ective whereas that from rd1 mice was not?

Sahel: Yes.

LaVail: What was the age of the rd mouse?

Sahel: 5 weeks. At 8 days you get the e¡ect but at 5 weeks there is no e¡ect. Hauswirth: We have tried many times to express and secrete a vectored gene

product in a photoreceptor-speci¢c way using a rod promoter and vector. We can’t get anything secreted for rods. It may not apply to this situation, but I don’t think a rod photoreceptor is a great source of secreted anything.

Farber: I don’t agree. You can get things secreted from rods or cones. Hauswirth: I stated our experience. There are a lot of endogenously secreted

proteins in photoreceptors but we can’t seem to engineer them get to secrete a vectored gene product under these circumstances.

Bok: It is a little weird to have a protein secreted by the outer segments. There is no secretory mechanism that I know of for proteins in the outer segment.

Dryja: Are you sure it is in the outer segments?

Sahel: It might be at the surface, or inside the outer segment. Kaleko: Couldn’t it be secreted from elsewhere in the photoreceptor?

Bok: IRBP is a good example. If you stain a retina for IRBP using the method that you have employed here, it would look as though the protein is within the outer segments. However, IRBP is extracellular and the zonulae adherens, which collectively form the outer limiting membrane, hold the IRBP in the subretinal space. It can’t di¡use past that. If the protein that you describe is extracellular it must be bound to something, because it is a small protein of 25 kDa which could escape the con¢nes of the outer limiting membrane. That is to say, it is small enough to get through the zonula adherens that form the outer limiting membrane. It is likely that it is bound to something in the extracellular space, which would make sense. Many neurotrophic factors bind there and it acts like a slow release capsule. This would make sense to me.

Travis: Is this protein glycosylated?

Sahel: We haven’t looked yet. We are currently making transgenic animals overexpressing the factor in the RPE and we are crossing back to the rd1 mouse to see whether we can rescue them.

Kaleko: In your assays can you distinguish a survival factor from a growth factor?

Sahel: No. We decided to call this a viability factor and not a survival factor. The reason for this is that cones are not directly undergoing any degeneration. They just need something to make them viable.

Kaleko: Is that similar to GDNF and CNTF? Do they really cause improved viability?

LaVail: That’s a semantic distinction. The term ‘survival’ factor is used in neurotrophic factor parlance as distinct from a di¡erentiation factor.

Kaleko: Do any of them cause cell proliferation in the retina? LaVail: They do, under certain circumstances.

Kaleko: I was thinking more along the lines of RPE or photoreceptors. Can these factors reverse a disease phenotype or are they more likely to slow the disease course?

LaVail: People haven’t studied this as thoroughly as we would like. There have not been many cases where we see the sort of overabundance that might result from a reinstitution of proliferation at a later stage.

Travis: In addition to this very interesting protein did you ¢nd any growth factors such as CNTF in your clones?

Sahel: CNTF is not one of the 24. In the di¡erential expression in the chips, there is a 10-fold increase in CNTF mRNA expression after rod degeneration rather than the loss of expression. One of the criteria we used for identifying the clone was loss of expression after the rods have degenerated. CNTF did not ¢t this criterion. Our hypothesis was that there was loss of something.

LaVail: CNTF is up-regulated in almost all kinds of injury. Even normal cell death can cause its expression in surrounding cells. We should recognize that in some species neural cells begin to proliferate in response to injury and loss of cells, such as retinal detachment. It is not out of the question that some retinal cells might proliferate.

Swaroop: When are the rods completely degenerated? Is this factor still detectable after all rods have died?

Sahel: Yes, which is why I discussed the possibility that it is a photoreceptor factor. In the rd1 mouse there are still a few rods remaining at the periphery.

Dryja: I remember hearing of an ophthalmologist who had decided to treat RP patients by laser. He/she photocoagulated the boundary between the degenerated retina and the remaining viable retina. If your data are correct about these survival factors, then killing o¡ even more photoreceptors can’t be of any bene¢t.

Sahel: CNTF might be released by the cells.

LaVail: And also FGF. In almost every case where there is injury, FGF is upregulated (reviewed by Gao & Holly¢eld 1996). It is the prime candidate for the

molecule that is e¡ecting neurotrophic factor rescue. In other words, any way you can up-regulate FGF will protect photoreceptor cells. With regard to Dr Dryja’s comment about laser treatment, it has been shown by Chu et al (1998) that in RCS rats with inherited retinal dystrophy, laser photocoagulation leads to FGF2 upregulation in surrounding regions at precisely the areas where photoreceptors are transiently protected from degeneration.

Dryja: Are you advocating this as a possible therapy?

LaVail: No, I’m just saying that there is a reason why it might work.

Sieving: The laser work in human retinitis pigmentosa patients was being done at Ohio State University by Dr Davidorf nearly two decades ago, and there was no positive bene¢t reported.

Dryja: I think a pharmaceutical high-throughput screen for a small molecule that mimics the function of the survival factor that could be systemically delivered would probably be quite valuable.

References

Chu Y, Humphrey MF, Alder VV, Constable IJ 1998 Immunocytochemical localization of basic ¢broblast growth factor and glial ¢brillary acidic protein after laser photocoagulation in the Royal College of Surgeons rat. Aust NZ J Ophthalmol 26:87^96

Cideciyan AV, Hood DC, Huang Y et al 1998 Disease sequence from mutant rhodopsin allele to rod and cone photoreceptor degeneration in man. Proc Natl Acad Sci USA 95:7103^7108 Gao H, Holly¢eld JG 1996 Basic ¢broblast growth factor: increased gene expression in inherited

and light-induced photoreceptor degeneration. Exp Eye Res 62:181^189