Author’s Name(s): Printha Wijesinghe, Amir Hosseini, Justin Haynes, Ian Mackenzie, Veronica HirschReinshagen, Ging-Yuek Hsiung, Benjamin Spiller, Brian Wadzinski, Wellington Pham, Joanne Matsubara 

Author’s Disclosure Block: Printha Wijesinghe, none; Amir Hosseini, none; Justin Haynes, none; Ian Mackenzie, none; Veronica Hirsch-Reinshagen, none; Ging-Yuek Hsiung, none; Benjamin Spiller, none; Brian Wadzinski, none; Wellington Pham, none; Joanne Matsubara, none 

Abstract Body 

Purpose: An imbalance in amyloid beta (Aβ) production and clearance has emerged as a key factor in sporadic Alzheimer’s disease (AD). Approximately 40-60% of brain-derived Aβ diffuses into the bloodstream and is cleared through the peripheral system. Recent studies suggest that soluble Aβ oligomers (SAβOs) accumulate early in AD, likely impairing memory before plaque formation, making SAβOs early pathogenic factors in AD. Nanobodies, one-tenth the size of conventional antibodies, can cross the blood-brain barrier (BBB) and have been used for diagnostic and treatment purposes. The retina, an extension of the central nervous system, shares embryological origins and vasculature with the brain, utilizing similar molecules, growth factors, neurotransmitters, and cellular structures. Retinal wholemount studies can reveal cell-specific involvement in Aβ clearance mechanisms that cannot be observed in brain tissue ex vivo. Study Design:  This retrospective autopsy study used wholemount neuroretinas from AD (n=10, mean age=76.8, 4 females) and control (n=10, mean age=72.5, 5 females) donor eyes, focusing on the temporal peripheral region to investigate SAβO clearance mechanisms ex vivo. Methods: A novel anti-SAβO (E3) nanobody, generated from an alpaca immunized with solubilized human Aβ1-42 peptide, was screened alongside the 12F4 antibody, specific to human Aβ1-42 residues. Fluorescein (FAM)- and Near Infrared (NIR)-conjugated E3 nanobodies were used with a modified nanobody staining protocol, followed by high-resolution confocal fluorescence microscopy. Results: E3-positive SAβOs were predominantly identified within retinal blood vessels, confirming the efficacy of the E3 nanobody in crossing the inner-retinal blood barrier (iBRB), while the 12F4 antibody failed to do so. We observed a significant difference between control and AD samples, with NIR-E3positive macrophage-like Aβ-binding monocytes significantly more abundant in controls (P < 0.0001, Mann-Whitney U test). Additionally, both FAM-E3 and NIR-E3 nanobodies confirmed their localization and distribution at the retina-vitreous interface, along with 12F4. At this interface, the retina and vitreous appeared to host a network, likely composed of capillary beds, involved in SAβO clearance in controls. In contrast, AD retinas exhibited a disrupted SAβO clearance network, resulting in significantly reduced signal detection by FAM-E3 and Alexa 546-labeled 12F4 (P = 0.024 and P = 0.045, respectively, unpaired t-test). Conclusion: This study confirms the existence of an impaired SAβO clearance mechanisms in human AD eyes and highlights new possibilities for retinal imaging in early diagnostic and prognostic applications.