Efficacy and Safety of a Brain-Penetrant Biologic TNF-α Inhibitor in Aged APP/PS1 Mice.

Tumor necrosis factor alpha (TNF-α) plays a vital role in Alzheimer's disease (AD) pathology, and TNF-α inhibitors (TNFIs) modulate AD pathology. We fused the TNF-α receptor (TNFR), a biologic TNFI that sequesters TNF-α, to a transferrin receptor antibody (TfRMAb) to deliver the TNFI into the brain across the blood-brain barrier (BBB). TfRMAb-TNFR was protective in 6-month-old transgenic APP/PS1 mice in our previous work. However, the effects and safety following delayed chronic TfRMAb-TNFR treatment are unknown. Herein, we initiated the treatment when the male APP/PS1 mice were 10.7 months old (delayed treatment). Mice were injected intraperitoneally with saline, TfRMAb-TNFR, etanercept (non-BBB-penetrating TNFI), or TfRMAb for ten weeks. Biologic TNFIs did not alter hematology indices or tissue iron homeostasis; however, TfRMAb altered hematology indices, increased splenic iron transporter expression, and increased spleen and liver iron. TfRMAb-TNFR and etanercept reduced brain insoluble-amyloid beta (Aß) 1-42, soluble-oligomeric Aß, and microgliosis; however, only TfRMAb-TNFR reduced Aß peptides, Thioflavin-S-positive Aß plaques, and insoluble-oligomeric Aß and increased plaque-associated phagocytic microglia. Accordingly, TfRMAb-TNFR improved spatial reference memory and increased BBB-tight junction protein expression, whereas etanercept did not. Overall, despite delayed treatment, TfRMAb-TNFR resulted in a better therapeutic response than etanercept without any TfRMAb-related hematology- or iron-dysregulation in aged APP/PS1 mice.

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections.

Extracellular accumulation of amyloid-beta (Aß) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral Aß plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the Aß load in brain tissues derived from AD transgenic mice. Though the methods for Aß detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on Aß load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the Aß load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of Aß load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on Aß-positive area quantification, and show a strong correlation between the Aß-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread Aß deposition.