ABS 101: Investigating the Effects of Aging on Microglia Morphology and Gene Expression in Gpx4 Knockout Mice: Implications for Autism Spectrum Disorder and Neurodegeneration

Kyung-A Jennifer Hong , Izabella Bankowski , Evan Bordt

¹ Northeastern University
² Lurie Center for Autism at Massachusetts General Hospital

Van Wickle (2025) Volume 1, ABS 101

Introduction: Autism spectrum disorder (ASD) is a neurodevelopmental condition marked by persistent social communication deficits and repetitive behaviors. While ASD has been studied in childhood, little is known about its effects during aging, as the first diagnosed individuals are only now reaching old age. Aging is associated with increased risk of neurodegenerative diseases like Alzheimer’s, many of which involve chronic inflammation and elevated pro-inflammatory cytokines. A major contributor to these inflammatory processes is microglia which are immune cells in the brain that respond to injury or stress by altering their shape and function.
In their resting state, microglia have small cell bodies and long, branched processes, but upon activation, they adopt an amoeboid shape and perform tasks like phagocytosis. Activated microglia also release cytokines and reactive oxygen species (ROS), making them highly sensitive to oxidative stress.
There is growing evidence linking oxidative stress to ASD. Glutathione peroxidase 4 (Gpx4) is an antioxidant enzyme that protects against oxidative stress and prevents ferroptosis, a form of cell death. Gpx4 has been implicated in autism-related behaviors, and its loss may contribute to neurodegeneration.
This project investigates how aging affects microglial morphology and gene expression in Gpx4 conditional knockout (cKO) mice. The goal is to understand the role of Gpx4 in aging, inflammation, and potential links to ASD and Alzheimer’s disease. We expect to see changes in genes related to inflammation and oxidative stress, such as Sod2, Tnf, Il6, Il10, Cxcl10, Ndufs4, Gpx1, Drp1, Nos2, Nox1, App, and Psen2.
The central hypothesis is that Gpx4 cKO mice will show increased microglial activation, altered morphology, and upregulation of inflammatory and mitochondrial genes, driven by elevated oxidative damage due to the loss of Gpx4 protection.

Methods: To study changes in microglial morphology and gene expression, Gpx4 conditional knockout (cKO) mice were generated by breeding Gpx4 fully floxed Cre– mice with Gpx4 fully floxed Cre+ mice. At Postnatal Day 0 (PND 0), control (Cre–) and cKO (Cre+) mice were born. At PND 18, mice were ear-tagged and genotyped using tail tissue through DNA extraction, PCR, and gel electrophoresis. Mice were then divided into two cohorts for qPCR and immunohistochemistry (IHC).
At PND 280, mice were perfused to preserve brain tissue. For gene expression analysis, microglia were isolated from one cohort, followed by RNA extraction, cDNA synthesis, and qPCR. For morphology analysis, brains from the second cohort were frozen, sliced at 30 μm, mounted, and stained using IHC. Confocal microscopy and 3D reconstruction via Imaris were used to assess microglial size, shape, and branching patterns.

Results: Female Gpx4 cKO mice showed statistically significant downregulation of antioxidant genes (Gpx1, Sod2), mitochondrial genes (Ndufs4, Drp1), and the neurodegeneration-associated gene App. In males, only Psen1 was significantly downregulated. No significant changes were found in inflammatory or immune-related genes (Il10, Il6, Cxcl10, Tnf, Nos2) in either sex. These findings highlight sex-specific transcriptional effects of Gpx4 loss. We are currently still gathering data on microglial morphology to determine whether these gene expression changes correspond with structural alterations in microglia.

Discussion: This study found that conditional knockout of Gpx4 led to sex-specific downregulation of antioxidant (Gpx1, Sod2), mitochondrial (Ndufs4, Drp1), and neurodegeneration-related genes (App in females, Psen1 in males), with no changes in inflammatory markers. These findings suggest oxidative stress and mitochondrial dysfunction primarily affect females. Ongoing microglia morphology analysis will clarify structural consequences. Estrogen’s role in redox regulation may explain the sex differences. These results highlight early molecular signs of vulnerability, relevant to both Alzheimer’s and ASD, and emphasize the need for sex-specific approaches in understanding neurodegeneration and developing targeted interventions.