Targeting amyloid-beta in glaucoma treatment

Abstract

The development of the devastating neurodegenerative condition, Alzheimer's disease, is strongly associated with amyloid-beta (Abeta) deposition, neuronal apoptosis, and cell loss. Here, we provide evidence that implicates these same mechanisms in the retinal disease glaucoma, a major cause of irreversible blindness worldwide, previously associated simply with the effects of intraocular pressure. We show that Abeta colocalizes with apoptotic retinal ganglion cells (RGC) in experimental glaucoma and induces significant RGC apoptosis in vivo in a dose- and time-dependent manner. We demonstrate that targeting different components of the Abeta formation and aggregation pathway can effectively reduce glaucomatous RGC apoptosis in vivo, and finally, that combining treatments (triple therapy) is more effective than monotherapy. Our work suggests that targeting the Abeta pathway provides a therapeutic avenue in glaucoma management. Furthermore, our work demonstrates that the combination of agents affecting multiple stages in the Abeta pathway may be the most effective strategy in Abeta-related diseases.

Fig. 1.

Aβ and RGC apoptosis. (A–D) Experimental glaucoma model. (E and F) Control. Aβ deposition was labeled by Aβ antibody (A and E, red) colocalized with RGC apoptosis labeled by fluorescent-labeled annexin 5 (B and E, green), from an OHT eye at 2 weeks (A–D). Composite (C and E) and transmission (D and F) images of the retinal cross-section show colocalization to retinal ganglion cell layer of Aβ deposition and RGC apoptosis only in the OHT eye. (G) APP deposition was found to decrease over time (P < 0.01) as assessed by histological grading. (Scale bar: 50 μm.)

Fig. 2.

Effects of Aβ on RGC apoptosis in vivo. Both Aβ_1–42 and Aβ_25–35 induced time- (A) and dose- (B) dependent levels of RGC apoptosis in vivo. (A) Aβ_1–42 appeared more toxic to RGCs than Aβ_25–35 at much reduced concentrations (0.55 versus 50 nmol, respectively). (B) RGC apoptosis count was found to increase with increasing doses of Aβ_25–35. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3.

Approaches for targeting Aβ. APP, a transmembrane protein, has two identified processing pathways: a nonamyloidogenic pathway, where APP is cleaved by α- and γ-secretases producing α-APP and p3, respectively, and an amyloidogenic pathway associated with β- and γ-secretase-mediated cleavage of APP. Aβ may aggregate, deposit, and form ion channels in cell plasma membrane, leading to neuronal death. The βSI is believed to block Aβ formation by inhibiting β-secretase activity. The Aβab is not only able to clear preexisting Aβ but also to block further Aβ aggregation. CR is thought to block Aβ aggregation and neurotoxicity by interfering with protein misfolding and preventing ion channel formation. ⊢, blocking effects.

Fig. 4.

Targeting Aβ in experimental glaucoma. In vivo DARC images show the effects of different approaches targeting Aβ on RGC apoptosis in OHT rats. Eyes were assessed at 3 (A, D, G, and J), 8 (B, E, H, and K), and 16 (C, F, I, and L) weeks after IOP elevation with treatments of Aβab (D–F), CR (G–I), and βSI (J–L), respectively, compared with control (IgG1, no antibody; A–C). The white spots represent apoptotic RGCs labeled by annexin 5.

Fig. 5.

Effects of targeting Aβ on RGC apoptosis. (A) All treatments reduced levels of RGC apoptosis at 3 weeks. Aβ antibody (Aβ/Ab) resulted in a significant reduction of RGC apoptosis at 3, 8, and 16 weeks compared with control. CR showed a significant reduction of RGC apoptosis at 3 but not 8 and 16 weeks. The βSI showed a modest (no significant) decrease of RGC apoptosis at 3 but not 8 and 16 weeks. (B) RGC apoptosis as a percentage of total RGC count with time after IOP elevation. All three treatments delayed peak RGC apoptosis from 3 to 8 weeks with reduced peak levels of RGC apoptosis from 15% to at least 3%. (C) Comparing time of administration in terms of efficacy, OHT animals treated with the Aβ/Ab at the time of IOP elevation (0 weeks) as opposed to 2 weeks later showed a significant reduction in RGC apoptosis at 16 weeks after IOP elevation. **, P < 0.01.

Fig. 6.

Effect of combining agents targeting Aβ in glaucoma. In vivo DARC images show the effects of triple (C and D) and dual (E and F) therapies on prevention of RGC apoptosis at 3 weeks after IOP elevation compared with control (A) and Aβab monotherapy (B). (G) Triple therapy (triple Aβab) significantly reduced RGC apoptosis compared with Aβab alone (*, P < 0.05). In fact, the triple therapy resulted in 84% mean reduction of RGC apoptosis compared with 74% by Aβab monotherapy. All other combining therapies showed significant reduction of RGC apoptosis compared with control, although there was no statistic significance compared with Aβab monotherapy. For comparison, the dashed line represents the results of our previous study, where we had combined two different glutamate modulators (MK801 and an mGlut agonist) (22) and shown a 60% reduction of RGC apoptosis at 3 weeks after IOP elevation.