Quantification of amyloid beta in the eye: Novel biomarkers for Alzheimer’s disease

Abstract

Purpose : Alzheimer’s disease (AD) is the most common cause of dementia. It accounts for 60 to 80% of dementias in the elderly and affects approximately 500,000 Canadians. The current clinical tests to confirm AD diagnosis is expensive and can only be used to confirm AD in individuals already having cognitive memory impairment. Developing novel non-invasive and inexpensive techniques that can further identify those who may have incipient but asymptomatic AD is greatly needed. The eye has the same embryonic origin as the brain and they remain tightly interconnected physiologically and functionally. The relationship of amyloid beta (Aβ) load between in the eye and the brain was previously suggested but remains uncertain. In this study, we quantify the Aβ load in the eye and establish proof-of-concept of the relationship between the Aβ load in the eye and concomitant buildup in the brain of transgenic mouse models of AD.

Methods : To detect specific species of Aβ, the retina/choroid or brain tissues from transgenic mice (APPswe, PSEN1dE9) were homogenized in ice-cold RIPA buffer containing protease inhibitor cocktail or in 2% SDS alone. Blotting procedures followed our previously established western blot (WB) protocol. The reducing condition is prepared by mixing tissue lysates with 2× reducing loading buffer containing 2% β-mercaptoethanol and boiling for 5 minutes. The two primary antibodies, 6E10 and MOAB2 were used for the detection of Aβ in WB. GAPDH was used as a loading control.

Results : Both MOAB2 and 6E10 antibodies revealed several bands in WB results. As expected, MOAB2 did not reveal a band at around 100kDa, the predicted molecular weight (MW) of amyloid precursor protein (APP), while 6E10 detected a band at MW≈100kDa. Protein extraction by RIPA and SDS yielded similar results; while band definition was clearer with reducing compared to non-reducing conditions.

Conclusions : We optimized the protocol of WB to detect Aβ species in eye tissues. MOAB2 did not detect APP and yielded more specific results than 6E10. Future work will test the hypothesis that Aβ deposition in the eye is concomitant with the Aβ load in the brain of the mouse model of AD. These findings will drive future work on the role of Aβ on retinal homeostasis as well as the development of a new imaging device for non-invasive detection of retinal Aβ in vivo.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.