Poster CC16 - Attenuating Alzheimer’s Associated Neuroinflammation with Novel Peptoids as RAGE Antagonists

Introduction: With over six million Americans currently living with Alzheimer’s disease (AD), and an expectation of this number to almost triple by 2060, the need for efficacious therapeutics is evident. While amyloid plaques, comprised of aggregated amyloid-β protein (Aβ), have proven a defining characteristic of prognosis, increasing evidence points to chronic neuroinflammation in AD pathogenesis. The receptor for advanced glycation end products (RAGE) is a key receptor in immediate inflammatory responses and is upregulated on neurons and microglia in areas of the brain impacted by AD. Activation of RAGE by these ligands induces inflammation and nonspecific toxicity via cytokine storms that could indicate early onset progression. With increased concentrations of RAGE ligands, including Aβ and HMGB1, in areas of the brain impacted by AD, antagonizing RAGE has garnered inquiry in the field of AD therapeutics. The inclusion of peptoid-based therapeutics in the treatment of AD appears favorable given their ability to evade protease degradation, cross the blood-brain barrier (BBB), and elicit low immunogenicity and toxicity. The peptoids examined throughout this study, JPT1 and JPT1a, were designed as RAGE antagonists via mimic of RAGE ligand. With modifications employed to enhance the peptoids’ abilities to act as effective RAGE antagonists, research suggests the chronic cytokine expression that is characteristic of AD is diminished in response to RAGE targeting by peptoids. This study aims to develop a robust model that verifies the peptoids’ ability to act as RAGE antagonists in the presence of HMGB1 and attenuate subsequent inflammatory cytokine release.

Materials and

Methods: Tohoku Hospital Pediatrics-1 (THP-1) monocyte cells were cultured in RPMI 1640 medium containing 10% FBS and 0.75 mL/100 mL penicillin (10,000 I.U./mL)/streptomycin (10,000 μg/mL). Cells were seeded on 6-well plates at 2.25E5 cells/well and differentiated into a macrophage phenotype using phorbol 12-myristate 13-acetate (PMA) (200 nM, 3 days). To determine optimal HMGB1-induced cytokine production, cells were treated (3 days) with a range of HMGB1 concentrations: 0, 2,000, 5,000, and 10,000 ng/mL. A sandwich ELISA was used to measure cytokine concentration and determine the HMGB1 treatment for significant cytokine production. Subsequently, cells were treated simultaneously with 50 µM of either JPT1 or JPT1a and 10,000 ng/mL HMGB1 to determine the cytokine-attenuation capabilities of the peptoids. Cells treated with 10,000 ng/mL HMGB1 served as a positive control, and cells treated with vehicle (DI H2O) and served as a negative control. Cell supernatants were harvested 72 h after treatment and measured for IL-1β and TNF-α, cytokines upregulated in AD brain, via sandwich ELISA.

Results, Conclusions, and Discussions: To determine the optimal HMGB1 treatment concentration, THP-1 supernatants were measured using a sandwich IL-1β and TNF-α ELISA 3 days after treatment with varying concentrations of HMGB1. A dose-dependent increase in cytokine release was observed from 2,000 to 10,000 ng/mL HMGB1. A robust response was observed following treatment with 10,000 ng/mL HMGB1, and this concentration was selected for future treatments. When THP-1 macrophages were treated simultaneously with 10,000 ng/mL HMGB1 and 50 µM peptoid JPT1 or JPT1a, supernatant levels of cytokines IL-1β and TNF-α were diminished 3 days post-treatment compared to cells treated with HMGB1 alone. Moreover, JPT1 exhibited a slightly more pronounced ability to attenuate cytokines than JPT1a. These results demonstrate the ability for peptoid antagonists of RAGE to reduce both IL-1β and TNF-α in the presence of HMGB1.
Brain inflammation is a neuropathological event characteristic of AD. The peptoids employed in this study are a treatment-driven step towards managing the persistent release of inflammatory mediators that progresses throughout AD. In determining effective HMGB1 treatment concentrations for cytokine production, the THP-1 treatment procedure emulates the AD microenvironment for efficacious peptoid experiments wherein 10,000 ng/mL HMGB1 treatment effectively elevated IL-1β and TNF-α cytokine production to model chronic neuroinflammation. When 50 µM of either peptoid was introduced simultaneously with the HMGB1 treatment, a noticeable reduction in the supernatant concentrations of both IL-1β and TNF-α was observed, with superior performance noted for JPT1.
Previous research has indicated that antagonizing RAGE can slow neurodegeneration and chronic inflammation in mice. This work has demonstrated the potential of peptoids JPT1 and JPT1a in this therapeutic context. The superior ability of JPT1 to reduce cytokine concentrations of both IL-1β and TNF-α may be attributed to the chirality of this peptoid, which allows helical formation that places aromatic residues to better mimic RAGE ligands. In contrast, while JPT1a is comprised of the same residue sequence, its achiral groups diminish helicity. Future experimentation will modify the side chains to ascertain the role of aromatic placement within JPT1.

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