Differential effects of interleukin-1beta and S100B on amyloid precursor protein in rat retinal neurons

Abstract

Purpose: Interleukin-1beta (IL-1beta) and S100B calcium binding protein B (S100B) have been implicated in the pathogenesis of Alzheimer's disease. Both are present in and around senile plaques and have been shown to increase levels of amyloid precursor protein (APP) mRNA in vitro. However, it is not known how either of these substances affects APP in vivo.

Methods: We have studied the effects of IL-1beta and S100B on the expression and processing of APP using a retinal-vitreal model. We have also investigated the effect of amyloid beta peptide (Abeta) on APP in the same system and the regulation of S100B production by Abeta and IL-1beta from retinal glial cells.

Results: Retinal ganglion cells constitutively express APP. However, after intravitreal injection of IL-1beta or Abeta there was a marked reduction in APP levels as detected by Western blotting and IL-1beta produced a decrease in APP immunoreactivity (IR). Nissl staining showed that the integrity of the injected retinas was unchanged after injection. Two days after S100B injection, there was a small reduction in APP-IR but this was accompanied by the appearance of some intensely stained large ganglion cells and there was some up-regulation in APP holoprotein levels on Western blot. Seven days post-S100B injection, these large, highly stained cells had increased in number throughout the retina. Injection of Abeta and IL-1beta also caused an increase in S100B production within the retinal Müller glial cells.

Conclusion: These results support the hypothesis that S100B (a glial-derived neurotrophic factor) and IL-1beta (a pro-inflammatory cytokine) can modulate the expression and processing of APP in vivo and so may contribute to the progression of Alzheimer's disease.

Keywords: Alzheimer’s disease; S100B; amyloid precursor protein; amyloid-β; interleukin 1β; retina.

Figure 1

Photomicrographs showing APP immunoreactivity in rat retinas 2 days post-PBS injection at low (a) and high (b) magnification. Note staining is seen in the GCL with some axonal staining. Low magnification photomicrograph showing that APP immunoreactivity is decreased in retinas injected with IL-1β after 2 days (c) compared with low magnification micrograph the retina from the corresponding noninjected eye (d). Low (e) and high (f) magnifications of retinas 7 days post-IL-1β injection show recovery of APP staining of RGCs. Low and high magnifications of retinas 2 days post-S100B injection show sporadic RGCs strongly stained for APP. Low (i) and high (j) magnifications of retina 7 days post-S100B injection indicating an increase in RGC immunoreactivity. Note: Scale bar = 50 μm. Abbreviations: APP, amyloid precursor protein; GCL, granule cell layer; IL, interleukin; RGC, retinal ganglion cells; PBS, phosphate-buffer solution.

Figure 2

Nissl staining of (a) normal retina, (b) retina 2 days after injection with S100B and (c) retina 2 days after injection with IL-1β. Note that the tissue architecture is not affected by the injection and that the GCL layer appears intact without loss of the large ganglion cells. Abbreviations: GCL, granule cell layer; IL, interleukin.

Figure 3

Western blotting of APP in retinas injected with (a) IL-1β, (b) S100B, (c) Aβ_1–42 peptide, and (d) PBS 2 days after injection. Each panel shows 2 animals per treatment. Both IL-1β and Aβ_1–42 show a marked reduction in APP levels in the injected left eye (L) compared with the noninjected right eye (R), particularly in the 54 kDa band. There was no down-regulation after injection with S100B or PBS. Note also the appearance of bands around 13 kDa and 82 Kda after injection with Aβ_1–42. Abbreviations: APP, amyloid precursor protein; IL, interleukin; PBS, phosphate-buffer solution.

Figure 4

S100B immunostaining in (a) noninjected rat retina shows that there is normal expression of the protein in the Müller cell endfeet. (b) S100B is only slightly increased after injection of PBS. (c) 2 days after injection of Aβ_1–42 there is a marked up-regulation of S100B in the Müller cell endfeet, cell bodies (arrowhead) and processes (arrows) throughout the retina. (d) There was also a marked increase in S100B immunoreactivity 7 days after injection of IL-1β. (e) The increase in S100B after Aβ_1–42 injection was confirmed by Western blotting. Recombinant bovine S100B is shown in lanes 1–3 and forms two bands at 7 and 19 kDa. Injection of Aβ_1–42 caused strong expression of a single 15 kDa band in the injected left eye (L) compared with a comparatively weak expression in the noninjected, right eye (R). Abbreviations: PBS, phosphate-buffer solution.