54. Development of therapeutics for neurodegenerative diseases via CTL1-targeted induction of M1-to-M2 microglial polarization

Abstract

Abstract Background Microglia are central regulators of neuroinflammation in neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). In these conditions, pathological stimuli including lipopolysaccharide (LPS) and amyloid-β (Aβ) peptides promote a phenotypic switch from neuroprotective M2 microglia to proinflammatory M1 microglia, thereby accelerating neuronal injury and disease progression. Therapeutic strategies that restore microglial homeostasis by inducing M1-to-M2 repolarization are considered promising. Choline uptake and metabolism have been implicated in cytokine regulation in macrophages, suggesting that choline transport may modulate microglial inflammatory responses. The choline transporter-like protein 1 (CTL1) is expressed in various cell types and functions as an intermediate-affinity choline transporter. Meanwhile, Licochalcones (Licos), a group of flavonoids, are known to exert antioxidant and anti-amyloid effects. Aims & Objectives Here, we investigated whether pharmacological inhibition of CTL1 by Licochalcones could suppress M1 activation, promote M2 polarization, and inhibit Aβ aggregation in microglia. Method Mouse microglial SIM-A9 cells were used as an in vitro model. Cells were stimulated with LPS or Aβ1-42 to induce proinflammatory activation, or with IL-4 to promote M2 polarization. Expression of M1 and M2 markers was quantified by RT-qPCR. Choline uptake was measured under basal and stimulated conditions, and the effects of choline deprivation and CTL1 inhibition were evaluated. In addition, the inhibitory activity of Licochalcones A–E on Aβ1-42 aggregation was assessed using Thioflavin T fluorescence, and their cytotoxicity was compared. Results SIM-A9 cells expressed functional CTL1, and choline uptake was upregulated by both LPS and IL-4 stimulation. LPS markedly increased M1 markers IL-1β and IL-6, while choline deprivation or pharmacological inhibition of CTL1 significantly suppressed these responses, indicating that CTL1-mediated choline uptake is essential for proinflammatory activation. Similarly, Aβ1-42 stimulation elevated TNF-α expression, which was also attenuated by choline deprivation. In contrast, IL-4 stimulation upregulated the M2 marker Arg-1, and this effect was further enhanced by CTL1 inhibition, suggesting that suppression of CTL1 activity facilitates M2 polarization. Screening of Licochalcones revealed that their inhibitory potency against Aβ aggregation ranked D > E > C > B > A. Among them, Lico E exhibited lower cytotoxicity than Lico D, while effectively inhibiting choline uptake. Functionally, Lico E suppressed LPS- and Aβ-induced TNF-α expression and enhanced IL-4-induced Arg-1 expression, thereby promoting M1-to-M2 repolarization under both inflammatory and amyloidogenic conditions. Discussion & Conclusions Our findings demonstrate that SIM-A9 microglia utilize a CTL1-dependent choline uptake system, which is critical for M1 polarization triggered by LPS and Aβ stimulation. Pharmacological inhibition of CTL1 by Lico E attenuates proinflammatory cytokine production, enhances M2 polarization, and directly inhibits Aβ aggregation. This dual mechanism highlights Lico E as a promising lead compound for developing novel therapeutics that restore microglial homeostasis and confer neuroprotection in AD and PD.