The Role of Hippocampal Endothelin B Receptors in Amyloid-β-Induced Neuroinflammation using Mouse and Patient-Derived iPSC Models of Alzheimer’s disease

BACKGROUND: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline, synaptic loss, and the accumulation of amyloid-beta (Aβ) plaques. Growing evidence suggests that neuroinflammation, particularly reactive astrocytes, plays a crucial role in AD progression and may precede Aβ deposition. Endothelin-1 (ET-1), a proinflammatory peptide, is elevated in AD and linked to neuroinflammation through its interaction with endothelin B receptors (ETBR), which are predominantly expressed in astrocytes. ETBR promotes astrocyte reactivity and inflammatory cytokine production. Moreover, excitatory and inhibitory neurons play an essential role in modulating astrocyte function and neuroinflammation. Thus, understanding the interaction between these neuronal subtypes and ETBR signaling in AD could provide valuable insights into disease mechanisms and potential therapeutic targets. I hypothesize that targeting ETBR could offer a promising strategy to modulate neuroinflammation and mitigate AD pathology. METHOD: Using cell biology and neuroanatomical procedures combined with confocal microscopy, we investigated changes in the ETBR system during the progression of AD. To assess whether AD-driven neuroinflammation could be modulated, we conducted pharmacological manipulations with the ETBR agonist IRL-1620 and antagonist IRL-2500. These experiments were performed using brain slices of APPNL-F/NL-F mouse model of AD and age-matched to wild-type littermates (12–16 months), as well as post-mortem brain tissue confirmed cases of AD patients and their control counterparts. Furthermore, human induced pluripotent stem cells (hiPSCs) carrying the APP V717I mutation along with isogenic controls were differentiated into excitatory neurons, astrocytes, parvalbumin (PV)- and somatostatin (SST)-expressing interneurons. RESULTS: Our study revealed a significant upregulation of ETBR expression in astrocytes of APPNL-F/NL-F mouse models, particularly within the hippocampal CA1 region. Colocalization analysis demonstrated increased overlap between ETBR and SOX9+/GFAP+ reactive astrocytes that correlated with AD neuroinflammation. This pattern was consistently observed in post-mortem AD brain tissue, where ETBR colocalization with astrocytes was markedly higher than in mouse models. The hypertrophic morphology of GFAP-expressing astrocytes across these systems further corroborated the involvement of ETBR in astrocyte activation.In APPNL-F/NL-F mouse models, 7-day in vivo pharmacological modulation showed distinct outcomes that ETBR antagonism significantly reduced both reactive astrocytes and amyloid-associated neuroinflammation in brain slices, whereas ETBR agonism exacerbated astrocyte activation, the expression of GFAP highly increased compared to vehicle controls. These anti-inflammatory effects were replicated in iPSC models following 24-hour in vitro pharmacological modulation, with ETBR antagonism suppressing reactive phenotypes and agonism conversely inducing astrocytic hyperactivation. CONCLUSION: Our findings suggest that ETBR expression is significantly upregulated in astrocytes in AD models, contributing to neuroinflammation and disease progression. Pharmacological antagonism of ETBR effectively reduces reactive astrocytes and amyloid-associated neuroinflammation, highlighting ETBR antagonism as a potential therapeutic strategy for AD. Furthermore, the role of excitatory and inhibitory neurons in modulating astrocyte function and neuroinflammation underscores the importance of studying these neuronal subtypes in the context of AD pathology.