280 - Targeting Apoptosis-Associated Speck-Like Protein Containing a Caspase Recruitment Domain (ASC) Alleviates Hyperoxia-Induced Brain Injury in Neonatal Mice

Publication Number: 4276.280

Noor Mehandi, University of Miami Miller School of Medicine, Miami, FL, United States; Huijun Yuan, University of Miami Leonard M. Miller School of Medicine, miami, FL, United States; Shaoyi Chen, University of Miami, Miller school of Medicine, Miami, FL, United States; Matthew R. Duncan, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Juan Pablo de Rivero Vaccari, University of Miami, Miami, FL, United States; Robert Keane, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Merline Benny, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Karen C. Young, Division of Neonatology, Department of Pediatrics, Leonard M. Miller School of Medicine, University of Miami, Miami, FL, United States; Augusto F. Schmidt, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Shu Wu, University of Miami, miami, FL, United States

Background: Neurodevelopmental impairment is a common long-term consequence of extreme prematurity. One proposed underlying mechanism involves hyperoxia-induced neuroinflammation, which disrupts neuronal and oligodendrocyte maturation and alters cerebral vascularization. Emerging evidence implicates inflammasome activation in mediating neonatal brain injury. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) serves as a key adaptor in inflammasome assembly, orchestrating inflammatory signaling and cell death. IC100 is a humanized IgG4 monoclonal antibody that targets ASC, disrupting ASC oligomerization and speck formation, and inhibiting IL-1β release.

Objective: This study aimed to:
1. Determine if hyperoxia induces ASC speck formation in the neonatal brain, and
2. Evaluate the therapeutic efficacy of IC100, in mitigating hyperoxia-induced brain injury.

Design/Methods: 1. Assessment of ASC speck formation in the brain: ASC-citrine reporter mice expressing an ASC fusion protein with a C-terminal Citrine (fluorescent GFP isoform) were exposed to 85% O2 from postnatal day (P)1 to P14 to induce hyperoxia-mediated brain injury. The brain was dissected on P14 to examine ASC speck formation.

2. Therapeutic evaluation of IC100: Newborn mice (C57BL/6J) were randomized into 3 groups from P1 to P14:room air (RA), 85% O2 (O2), 85% O2 + IC100 intraperitoneal injection (O2+IC100). Brains were dissected for evaluation on P14. The hippocampus was assessed for microglial activation with AIF-1 staining, cell proliferation with Ki67 staining and apoptosis with TUNEL.
Data are presented as mean ± SD and analyzed using one-way ANOVA.

Results: As per Figure 1: Immunostaining showed a significantly higher ASC speck formation in microglial cells within the hippocampus specifically in hyperoxia-exposed neonatal mice.

As per Figure 2: The O2+IC100 group had a reduced number of activated microglia, reflecting attenuation of hyperoxia-induced neuroinflammation by IC100. Increased cellular proliferation was observed in various brain regions in the O2+IC100 group compared to O2-exposed group. TUNEL staining revealed a marked reduction in apoptotic cell death in O2+IC100 group, demonstrating IC100's neuroprotective efficacy against hyperoxia-induced brain injury.

Conclusion(s): These findings identify ASC as a pivotal mediator of hyperoxia-induced neuroinflammation and neurodegeneration in the neonatal brain. Moreover, they highlight the therapeutic potential of the humanized anti-ASC monoclonal antibody IC100 in ameliorating hyperoxia-related brain injury in neonates.

Hyperoxia induces ASC specks in the brain
ASC reporter mice were exposed to 85% O2 from P1 to P14. ASC specks were detected primarily as perinuclear aggregate within microglia.

Demonstrating effect of IC100 in hyperoxia exposed brain.
Treatment with IC100 A. reduced microglial inflammation, B. enhanced cell proliferation and C. decreased apoptosis. n=5/group. Scale bar: 50µm. *p <0.05, **p <0.01