Biology subjects

Bennett, F. C.

Publications and source records attributed to Bennett, F. C..

6 recordsLinked to original sources

bioRxiv2026

Brain-Resident CD8+ T Cells Regulate Neuronal Activity and Behavior via Interferon-Gamma

Maintaining brain homeostasis is crucial for proper function of the central nervous system and has traditionally been attributed to neuronal and glial interactions. However, recent research highlights the essential role of brain-resident immune cells in this process. Our study characterizes brain-specific CD8+ T cells and elucidates their significant contribution to brain homeostasis and behavior. We identified a distinct population of CD8+ T cells that infiltrates the brain during early development, undergoes clonal expansion, and acquires effector memory-like characteristics through interactions with microglia. Notably, the absence of these cells results in hyperactivation of neuronal activity and abnormal behaviors, due to loss of regulation of interferon-gamma (IFN-{gamma}) secreted by CD8+ T cells. Our findings demonstrate that IFN-{gamma} secreting brain-specific CD8+ T cells are crucial for maintaining the physiological level of neuronal excitability and normal behavioral patterns. This study provides novel insights into neuroimmune interactions, emphasizing the critical role of CD8+ T cells in sustaining brain function and behavior.

immunology↗

bioRxiv2026

Naked antisense oligonucleotides remain endolysosomally sequestered despite induced membrane damage

Antisense oligonucleotides (ASOs) enter cells efficiently, but the compartment from which productive escape occurs remains uncertain. We used live-cell microscopy, ratiometric pH measurements and 3D focused ion beam scanning electron microscopy (FIB-SEM) in U2OS cells to track a Malat1-targeting ASO from uptake to delivery. The ASO entered by endocytosis and accumulated in late endosomes, endolysosomes and lysosomes, where it induced luminal neutralization without galectin-3 recruitment or limiting-membrane rupture. Under conditions that reduced Malat1-RNA by >90%, quantitative imaging showed that less than 4% of internalized ASOs reached the nucleus. L-leucyl-L-leucine methyl ester (LLOMe)-induced membrane damage released co-internalized dextran but not ASOs, showing that ASOs remain sequestered even in damaged late endocytic compartments. In apilimod-expanded organelles, ASOs concentrated at limiting membranes and intraluminal foci with constrained motion, consistent with association with membrane and luminal structures. Although G3BP1/2 has been proposed to plug damaged endocytic membranes, we detected no recruitment of G3BP1 to endosomes or lysosomes; loss of G3BP1 and G3BP2 increased functional delivery modestly. We therefore propose that productive escape occurs earlier in endocytosis, most likely in early or recycling endosomes, where ASOs would still be unbound within the lumen and where membrane fusion and fission could generate perforations permitting release.

cell biology↗

bioRxiv2024

Microglia replacement by ER-Hoxb8 conditionally immortalized macrophages provides insight into Aicardi-Goutieres Syndrome neuropathology

Microglia, the brains resident macrophages, can be reconstituted by surrogate cells - a process termed "microglia replacement." To expand the microglia replacement toolkit, we here introduce estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages, a cell model for generation of immune cells from murine bone marrow, as a versatile model for microglia replacement. We find that ER-Hoxb8 macrophages are highly comparable to primary bone marrow-derived (BMD) macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells. Furthermore, ER-Hoxb8 progenitors are readily transducible by virus and easily stored as stable, genetically manipulated cell lines. As a demonstration of this systems power for studying the effects of disease mutations on microglia in vivo, we created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi-Goutieres Syndrome (AGS), an inherited interferonopathy that primarily affects the brain and immune system. We find that Adar1 knockout elicited interferon secretion and impaired macrophage production in vitro, while preventing brain macrophage engraftment in vivo - phenotypes that can be rescued with concurrent mutation of Ifih1 (MDA5) in vitro, but not in vivo. Lastly, we extended these findings by generating ER-Hoxb8 progenitors from mice harboring a patient-specific Adar1 mutation (D1113H). We demonstrated the ability of microglia-specific D1113H mutation to drive interferon production in vivo, suggesting microglia drive AGS neuropathology. In sum, we introduce the ER-Hoxb8 approach to model microglia replacement and use it to clarify macrophage contributions to AGS.

neuroscience↗

bioRxiv2024

Microglia are not required for maintenance of blood-brain barrier properties in health, but PLX5622 alters brain endothelial cholesterol metabolism

Microglia are resident immune cells of the central nervous system, yet their functions far exceed those related to immunology. From pruning neural synapses during development to preventing excessive neural activity throughout life, microglia are intimately involved in the brains most basic processes. Studies have reported a close interaction between microglia and endothelial cells, as well as both helpful and harmful roles for microglia at the blood-brain barrier (BBB) in the context of disease. However, much less work has been done to understand microglia-endothelial cell interactions in the healthy brain. Here, we aim to determine the role of microglia at the healthy BBB. We used the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia and analyzed BBB ultrastructure, permeability, and transcriptome. Interestingly, we found that, despite their direct contact with endothelial cells, microglia are not necessary for maintenance of BBB structure, function, or gene expression in the healthy brain. However, we found that PLX5622 treatment alters brain endothelial cholesterol metabolism, and this effect was independent from microglial depletion, suggesting PLX5622 has off-target effects on brain vasculature.

neuroscience↗

bioRxiv2020

ASO-based PKM Splice-switching Therapy Inhibits Hepatocellular Carcinoma Cell Growth

The M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in the Warburg effect, which is characterized by the preference for aerobic glycolysis for energy metabolism. PKM2 is an alternative-splice isoform of the PKM gene and is a potential therapeutic target. Previously, we developed antisense oligonucleotides (ASOs) that switch PKM splicing from the cancer-associated PKM2 to the PKM1 isoform and induce apoptosis in cultured glioblastoma cells. Here, we explore the potential of ASO-based PKM splice-switching as a targeted therapy for liver cancer. We utilize a more potent lead cEt/DNA ASO and demonstrate that it induces PKM splice-switching and inhibits the growth of cultured hepatocellular-carcinoma (HCC) cells. This PKM isoform switch increases pyruvate-kinase activity and alters glucose metabolism. The lead ASO and a second ASO targeting a non-overlapping site inhibit tumorigenesis in an orthotopic-xenograft HCC mouse model. Finally, a surrogate mouse-specific ASO induces Pkm splice-switching and inhibits HCC growth, without observable toxicity, in a genetic HCC mouse model. These results lay the groundwork for a potential ASO therapy for HCC. Statement of significanceAntisense oligonucleotides are used to force a change in PKM isoform usage in HCC, reversing the Warburg effect and inhibiting tumorigenesis.

cancer biology↗

bioRxiv2019

Neuroinflammation and EIF2 signaling persist in an HiPSC tri-culture model of HIV infection despite antiretroviral treatment

HIV-Associated Neurocognitive Disorders (HAND) affect over half of HIV-infected individuals worldwide, despite antiretroviral therapy (ART). Therapeutically targetable mechanisms underlying HAND remain elusive. We developed a human-induced pluripotent stem cell (HiPSC) based model; whereby, we independently differentiate HiPSCs into neurons, astrocytes, and microglia and systematically combine to generate a tri-culture with or without HIV-infection and ART. scRNAseq analysis on tri-cultures including HIV-infected microglia revealed inflammatory signatures in the microglia and EIF2 signaling in all three cell types. Remarkably, EFZ alone induced a similar response to infection. Treatment with the antiretroviral compound Efavirenz (EFZ) mostly resolved these signatures; However, EFZ increased RhoGDI and CD40 signaling in the HIV-infected microglia. This activation was associated with a persistent increase in TNFa expression. This work establishes a tri-culture that recapitulates key features of HIV infection in the CNS and provides a new model to examine the effects of HIV infection and its treatment with antiretrovirals.

pathology↗