京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21626号 / 医博第4432号 / 新制||医||1033(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 宮本 享, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Social isolation during adolescence can have long lasting negative effects in both humans and animal models. In mice, post-weaning social isolation leads to increased addiction-like behaviors in adulthood. However, little is known about how post-weaning social isolation alters the brain. Stress during development can lead to persistent restructuring of neurons. Changes in dendritic spines can be long-lasting and have been theorized to play an important role in the maintenance of cocaine craving. We found that post-weaning isolation led to a persistent increase in spine density in adulthood within both the core and shell regions of the nucleus accumbens in male mice, but not female mice. In contrast, in the infralimbic cortex, post-weaning social isolation led to an increase in spine density only in female mice. This study highlights the long-lasting, sex-specific effects of post-weaning isolation. Microglia have been shown to assist in both the formation and elimination of dendritic spines, and are activated following exposure to stress and cocaine. Therefore, we hypothesized that microglia may be involved in the restructuring of dendritic spines during post-weaning isolation, and contribute to addiction-like behavior in adulthood. We examined whether inhibiting microglia with minocycline during the first three weeks of post-weaning isolation altered the impact of isolation in cocaine seeking. Isolated animals that received minocycline showed increased cocaine seeking in adulthood compared to group housed mice and isolated mice that received saline. Minocycline and isolation also caused sex-specific alterations in spine density. The findings of these studies provide insight into the mechanisms by which social isolation during adolescence increases vulnerability to addiction later in life. / Psychology

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Traumatic brain injury (TBI) induces a state of microglialactivation, which includes upregulation of macrophage functions and release inflammatory mediators such as certain inflammatory cytokines. Current literature suggests that interleukin-1Beta is an important cytokine mediator, which is dramatically increased after brain injury. Previous studies indicate that ATP is released by traumatically injured astrocytes and serves as a cell-to-cell mediator through purinergic receptors after in vitro injury. However, the mechanism of interleukin-1Beta release after traumatic brain injury remains poorly defined and is difficult to study using in vivo models. Using an in vitro model for traumatic brain injury (cell strain or stretch), we investigated the role of the extracellular nucleotides (ATP) in regulation of interleukin-1Beta release in rat cortical brain cells. We now report that activated microglia constitute the major source of interleukin-1Beta release after in vitro trauma. ATP is a powerful stimulus for interleukin-1Beta release from microglial cultures. Glutamate inhibits interleukin-1Beta release. ATP-induced interleukin-1Beta release was blocked completely by the P2X7 receptor antagonist, oxidized ATP, and partially by the P2X7 receptor antagonist suramin, suggesting that ATP stimulates interleukin-1Beta release from microglia via purinergic receptor and the P2X7 receptor is responsible for the interleukin-1Beta release. Blockage of interleukin-1Beta release by the purinergic receptor antagonists oATP and suramin decreased cell damage in uninjured mixed organotypic brain cell culture exposed to activated microglia. Taken together, these results suggest that interleukin-1Beta mediated inflammatory events are regulated in activated microglia by extracellular nucleotides (ATP) via purinergic receptors in central nervous system after in vitro trauma.

BACKGROUND A subarachnoid hemorrhage (SAH) is a bleed into the subarachnoid space surrounding the brain. This disease affects roughly 30,000 Americans each year and approximately one in six affected individuals die at the time of the ictal event. Individuals that do survive suffer from many complications including delayed cerebral vasospasm (DCV), cerebral edema, fever, and increased intracranial pressure (ICP) amongst others. These patients often suffer from brain damage due to neuronal apoptosis as a consequence of excess neuroinflammation. Microglia, the resident macrophage of the central nervous system, and Toll-like Receptor-4 (TLR4), a pro-inflammatory transmembrane receptor, have both been shown to play a role in the neuroinflammation seen in SAH. RBC components have been shown to activate microglial TLR4, and this event is suggested to trigger downstream mechanisms leading to neuronal apoptosis. The presented research takes a closer look at the role of microglial TLR4 in early neuronal apoptosis seen in an SAH model. METHODS All mice used were 10- to 12-week-old males on a C57BL/6 background: TLR4−/−, MyD88−/−, TRIF−/− and wild type (WT). To induce an SAH, a total of 60 ul of arterial blood from a donor WT mouse was injected for over 30 seconds into another mouse. For in vitro experiments, either primary microglia (PMG) or murine microglial BV2 cells were used. Microglia were separated from murine neuronal HT22 cells by 3um cell culture inserts or transwells, before being stimulated with lipopolysaccharide (LPS), red blood cells (RBCs), or RBC components including hemin (structurally similar to heme) and hemoglobin. In vivo samples were studied using either immunohistochemistry (IHC) or Fluorescence Activated Cell Sorting (FACS), and in vitro cells were studied using IHC and Light Microscopy. Neuronal cell death was measured using TUNEL and/or FloroJade C (FJC) assays. Cognitive function after SAH was measured using the Barnes Maze protocol. RESULTS In a 24-hour time course, more death occurred in the HT22 cells associated with BV2s treated with RBCs for 12-hour and 24-hour incubation time points as compared to 1-hour and 3-hour time points. Similar results were seen in the WT PMGs, as HT22 apoptosis increased in the RBC treated WT groups as the incubation time points increased. The WT PMG and MyD88−/− RBC treated PMGs showed significant death as compared to a WT untreated control (p<0.05) using a FJC assay, and both showed more death in a TUNEL assay as compared to an untreated control. WT mice treated with whole blood and hemoglobin had significantly more apoptosis as compared with a normal saline (NS)-treated control mouse (p<0.05). WT PMGs treated with whole blood and hemoglobin had more apoptosis as compared with an untreated control. MyD88-/- treated with RBC, hemoglobin, and hemin had more HT22 cell death compared with other genotypes treated with the same component. For the Barnes Maze, TLR4−/− mice performed significantly less total errors than WT mice on POD5 and 6 (p<0.01), and took significantly less time to reach the goal chamber on POD4, POD5 (p<0.05), and POD6 (p<0.01). CONCLUSION Our experimental results suggest that a microglial TLR4-dependent, MyD88-independent pathway is involved in neuronal apoptosis very early in an SAH model via RBC and hemoglobin activation, and that neuronal cell apoptosis due to TLR4 expression may be related to SAH-related cognitive and behavioral deficits. Our results suggest that TRIF may be the intracellular adaptor that is involved in this mechanism, but further experiments are needed to confirm this.

Multiple Alzheimer’s disease (AD) clinical trials target pathogenic amyloid-β (Aβ) species using therapeutic anti-Aβ antibodies. However, failures from recent clinical trials investigating passive anti-Aβ antibody immunization demonstrate a continued gap in our understanding of AD pathogenesis. Hence, there is an immediate need to develop new safe therapeutic approaches that can be applicable at an early stage of the disease. We developed superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with anti-Aβ antibodies, which bind to Aβ peptides and aggregated Aβ species in vitro and in vivo. We hypothesized that acute and rapid removal of pathogenic Aβ species using our antibody-conjugated magnetic nanoparticles can block Aβ-driven pathogenic cascades, including Aβ-driven tau pathology in human neurons. To test this hypothesis, we applied our conjugated SPIONs in our 3D human neural cell culture model of AD, followed by rapid removal of SPION-Aβ complex by an external magnet force in real-time. We detected a 25% reduction in soluble and insoluble Aβ species including Thioflavin-S (ThioS) positive Aβ. We also showed that our targeted SPIONs could efficiently remove ThioS positive Aβ aggregates from 5XFAD AD mouse brain slices and frozen AD patient brain sections. More importantly, we found a 16% reduction in pathogenic phosphorylated-tau species after acute removal of Aβ species in our 3D human neural cell model. Our results demonstrate the therapeutic potential of SPION-assisted immunotherapy to acutely reduce both Aβ accumulation and tau pathology without chronic exposure to anti-Aβ antibodies that leads to amyloid-related imaging abnormality (ARIA) side effects. We next explored the in vivo application of conjugated SPIONs in a transgenic AD mouse model. We found that remote alternating magnetic field treatment at lower frequencies enhanced antibody delivery across the blood-brain barrier. We also observed increased microglial activation without inducing neuroinflammation using this methodology. Taken together, this work demonstrates proof of concept for applying nanomedicine and neurostimulation as a tool to remotely modulate AD pathology and improve cerebral AD drug bioavailability. / 2025-01-23T00:00:00Z