Photoreceptor survival-promoting activity in interphotoreceptor matrix preparations: Characterization and partial purification

Abstract

It is well established that many types of cells are dependent on trophic factors for their survival. We are investigating whether photoreceptor survival may also be dependent upon such a factor, as well as the possibility that the interphotoreceptor matrix (IPM), which lies between the outer retina and the RPE, may be a source of photoreceptor survival-promoting activities. Well-characterized cultures of embryonic chick retinal neurons and photoreceptors, in which the photoreceptors spontaneously degenerate between 7 and 10 days in culture, were used to test this hypothesis. Crude extracts of IPM were found to support three- to fourfold increases in photoreceptor survival in 10-day cultures. This response was dose-dependent, saturable, and specific for photoreceptors, since there was no difference in the number of non-photoreceptor neurons between treated and control cultures. This photoreceptor survival-promoting activity (PSPA) may function when present either as a substratum-bound molecule or as a medium supplement. PSPA is heat labile, sensitive to freeze-thawing, and stable only within a very narrow pH range. The activity binds to heparin affinity columns but, in contrast to the behavior of growth factors such as FGF, it can be eluted from the columns at low salt concentrations (0·5 m NaCl). PSPA is eluted from a Sephacryl S-200 gel filtration column in two regions with M r = 400–450 kDa and 33 kDa, respectively. SDS-gel electrophoresis suggests that the high molecular weight activity is composed of aggregates of lower molecular weight molecules. While this factor has not been purified to homogeneity, the combination of heparin-agarose affinity chromatography and hydrophobic interaction chromatography on phenyl-Sepharose columns results in 720-fold purification of the activity, with one unit of PSPA activity corresponding to 40 ng protein. This partial purification is accompanied by substantial reduction in the complexity of protein patterns in silver-stained SDS-gels. These findings are potentially important since a macromolecular factor promoting photoreceptor survival could play a role in the normal development of the retina, and could also further our understanding of retinal degenerations such as retinitis pigmentosa.