Microglia-specific NF-kappaB signaling is a critical regulator of prion-induced glial inflammation and neuronal loss

Authors: HAY, ARIELLE - Colorado State University; POPICHAK, KATRINA - Colorado State University; GENOVA, MUMFORD - Colorado State University; PAYTON, SHIRLEY - Colorado State University; Bian, Jifeng; WOLFRATH, LAUREN - University Of Minnesota; EGGERS, MICHAEL - Oak Ridge Institute For Science And Education (ORISE); Nicholson, Eric; TJALKENS, RONALD - Colorado State University; ZABEL, MARK - Colorado State University; MORENO, JULIE - Colorado State University

Submitted to: PLoS Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Prions are infectious and neurotoxic proteins that can cause a group of always-fatal neurodegenerative diseases in animals and people. These infectious protein aggregates continuously grow by recruiting and reshaping the normal prion protein (PrPC) in the brain and eventually cause irreversible neuronal death. Even though we know that the transformation of the prion protein conformation is key to the disease, we're still not sure exactly how it leads to brain cell death, especially when it comes to the role of glial cells, which outnumber the neurons. To figure this out, we studied the nuclear factor kappa B (NF-'B) signaling pathway, the inhibitor of NF-'B kinase complex (IKK), and other markers of inflammation in cell culture and animal models infected with mouse prions. NF-'B is an important gene transcription factor that plays crucial roles in immune response and inflammation, and the IKK complex is a master regulator of the NF-kB signaling pathway. We utilized genetically engineered mice and microglial cells lacking the IKK gene, and exposed them to mouse prions. We checked the expression levels of various markers by using RT-PCR, Western blot and immunohistochemistry assay. Our discoveries include: 1) When we removed IKK gene from microglial cells in cell culture, the levels of the misfolded protein (PrPSc) increased; 2) In the brains of mice with these microglial cells lacking IKK gene, we noticed elevated levels of GFAP and active astrocytes during prion infection; 3) NF-'B-associated genes are also significantly downregulated in infected mixed glial cultures containing IKK KO microglial cells; and 4) Mice with these microglial cells lacking IKK developed prion disease more faster than control mice did. Our findings show that the IKK complex and the NF-'B signaling pathway in microglial cells in the brain play an important role in protecting against prion infections. The understanding of these pathways in microglia could potentially uncover both biomarkers and therapeutic targets for prion diseases.

Technical Abstract: Prion diseases are a group of rare and fatal neurodegenerative diseases caused by the cellular prion protein, PrPC, misfolding into the infectious form, PrPSc, which forms aggregates in the brain. This leads to activation of glial cells, neuroinflammation, and irreversible neuronal loss, however, the role of glial cells in prion disease pathogenesis and neurotoxicity is poorly understood. Microglia can phagocytose PrPSc, leading to the release of inflammatory signaling molecules, which subsequently induce astrocyte reactivity. Animal models show highly upregulated inflammatory molecules that are a product of the Nuclear Factor-kappa B (NF-'B) signaling pathway, suggesting that this is a key regulator of inflammation in the prion-infected brain. The activation of the I'B kinase complex (IKK) by cellular stress signals is critical for NF-'B-induced transcription of a variety of genes, including pro-inflammatory cytokines and chemokines, and regulators of protein homeostasis and cell survival. However, the contribution of microglial IKK and NF-'B signaling in the prion-infected brain has not been evaluated. Here, we characterize a primary mixed glial cell model containing wild-type (WT) astrocytes and IKK knock-out (KO) microglia. We show that, when exposed to prion-infected brain homogenates, NF-'B-associated genes are significantly downregulated in mixed glial cultures containing IKK KO microglia. Mice with IKK KO microglia show rapid disease progression when intracranially infected with prions, including an increase in microglia and reactive astrocytes, and accelerated loss of hippocampal neurons and associated behavioral deficits. These animals display clinical signs of prion disease early and have a 22% shorter life expectancy compared to infected wild-type mice. Intriguingly, PrPSc accumulation was significantly lower in the brains of infected animals with IKK KO microglia compared to age-matched controls, suggesting that accelerated disease is independent of PrPSc accumulation, highlighting a glial-specific pathology. Conversely, primary mixed glia with IKK KO microglia have significantly more PrPSc accumulation when exposed to infected brain homogenates. Together, these findings present a critical role in NF-'B signaling from microglia in host protection suggesting that microglial IKK may be involved in sufficient clearance of prions.