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

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Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is typified by a robust microglial-mediated immune response. Genetic studies have demonstrated that many genes that alter AD risk are involved in the innate immune response and are primarily expressed in microglia. Among these genes is phospholipase C gamma 2 (PLCG2), a critical element for various immune receptors and a key regulatory hub for immune signaling. PLCG2 genetic variants are associated with altered AD risk. The primary objective of this thesis was to determine the role of PLCG2 in AD pathogenesis. We observed significant upregulation of PLCG2 expression in three brain regions of late-onset AD (LOAD) patients and a significant positive correlation of PLCG2 expression with amyloid plaque density. Furthermore, the differential gene expression analysis highlighted inflammatory response-related pathways. These results suggest that PLCG2 plays an important role in AD. We systematically investigated the impact of PLCG2 haploinsufficiency on the microglial response and amyloid pathology in the amyloidogenic 5xFAD mouse model. The results demonstrated that Plcg2 haploinsufficiency altered the phenotype of plaqueassociated microglia, suppressed cytokine levels, increased compact X34-positive plaque deposition, and downregulated the expression of microglial genes associated with immune cell activation and phagocytosis. Our study highlights the role of PLCG2 in immune responses; loss of function of PLCG2 exacerbates the amyloid pathology of AD. Genetic studies demonstrated that the hypermorphic P522R variant is protective and that the loss of function M28L variant confers an elevated risk for AD. Our results demonstrated that PLCG2 variants modulate disease pathologies through specific transcriptional programs. In the presence of amyloid pathology, the M28L risk variant impaired microglial response to plaques, suppressed cytokine release, downregulated disease-associated microglial genes, and increased plaque deposition. However, microglia harboring the P522R variant exhibit a transcriptional response endowing them with a protective immune response signature linked to their association with plaques and Aβ clearance, attenuating disease pathogenesis in an amyloidogenic mouse model of AD. Collectively, our study provides evidence that the M28L variant is associated with accelerated and exacerbated disease-related pathology, and conversely, the P522R variant appeared to attenuate disease severity and progression. / 2024-10-03

Immune-brain-endocrine communication influences behaviour and contributes to the development of the central nervous system (CNS) in a sexually dimorphic manner. The bed nucleus of the stria terminalis (BST) is a highly sexually dimorphic brain region; in most mammalian species the male BST is larger than the female BST. Previously, our lab has shown that male and female mice lacking T cells due to knock out of the beta (b) and delta (d) chains of the T cell receptor (TCRb-/-d-/-) have reduced anxiety-like behaviour. This was shown with increased time spent in the open arms of the elevated plus maze by TCRb-/-d-/- mice compared to wild type (WT) mice of both sexes. T cell deficient mice also show differences in brain volume compared to WT, including a lack of sexual dimorphism in volume of the BST. The present study explored the impact of T cell deficiency on immune and endocrine factors implicated in sex differences of the CNS. The first analysis was of serum Anti-Müllerian hormone (AMH). AMH is a key determinant of the male phenotype during fetal development. It has also been shown by others to contribute to sexual dimorphic development of the BST. Our postnatal analysis of serum AMH using ELISA demonstrated an age and genotype effect, where a peak in serum AMH levels in WT mice of both sexes was absent in both male and female TCRb-/-d-/- mice at postnatal day (P) 7. These results suggest that T cells have an impact on the endocrine system in early life but the process does not appear to be sexually dimorphic. The present study also explored the impact of TCR knockout on microglia, the resident immune cells of the brain. Other have shown microglia contribute to sexual dimorphic brain development. This contribution occurs through interaction with endocrine factors, making them a key player in the immune-brain-endocrine crosstalk. Using immunohistochemistry and the microglial marker, anti-Iba1, microglia were examined in adult and P7 WT and TCRb-/-d-/ mice. To quantify microglia, soma were traced using AxioVision microscope software, and microglia cell number, perimeter, radius, feret ratio, and area in dorsal and ventral BST were assessed. Our results show sex differences in microglia number in dorsal BST in adult WT mice, where female WT mice had a lower number of microglia compared to WT males, however this difference was absent in TCRb-/-d-/- adult mice. There were no effects on microglia number in the ventral BST and morphology analysis did not reveal any effects in the dorsal or ventral BST. Furthermore, the difference in microglia number was absent in all groups of P7 mice and analysis of soma morphology did not reveal any significant effects. This study explored the impact of TCR knockout on the BST by exploring the immune and endocrine factors shown to contribute to its sexual dimorphic development. The results suggest a non-dimorphic impact on the endocrine system in the postnatal period and a dimorphic impact on microglia that is age and region-specific. The findings reveal a complex network emphasizing the importance of a systems-wide approach to the study of sex differences in the CNS. / Thesis / Master of Science (MSc)

Classically, the following three morphological states of microglia have been defined: ramified, amoeboid and phagocytic. While ramified cells were long regarded as “resting”, amoeboid and phagocytic microglia were viewed as “activated”. In aged human brains, a fourth, morphologically novel state has been described, i.e., dystrophic microglia, which are thought to be senescent cells. Since microglia are not replenished by blood-borne mononuclear cells under physiological circumstances, they seem to have an “expiration date” limiting their capacity to phagocytose and support neurons. Identifying factors that drive microglial aging may thus be helpful to delay the onset of neurodegenerative diseases, such as Alzheimer’s disease (AD). Recent progress in single-cell deep sequencing methods allowed for more refined differentiation and revealed regional-, age- and sex-dependent differences of the microglial population, and a growing number of studies demonstrate various expression profiles defining microglial subpopulations. Given the heterogeneity of pathologic states in the central nervous system, the need for accurately describing microglial morphology and expression patterns becomes increasingly important. Here, we review commonly used microglial markers and their fluctuations in expression in health and disease, with a focus on IBA1 low/negative microglia, which can be found in individuals with liver disease.

Alzheimer’s Disease (AD) is a chronic neurodegenerative disease commonly characterized by the aggregation and deposition of insoluble amyloid beta plaques throughout the brain, and by an associated neuroinflammatory response to these plaques involving astrocytes and microglia. Choline is an essential nutrient with diverse functional roles that has emerged as a promising candidate for the treatment of AD. Localized plaque regions in the polymorphic layer in the medial dentate gyrus of the hippocampus and in the cortex were examined in 9-month-old APP-NL-G-F knock-in AD model mice to determine the effects of perinatal choline supplementation on astrocytosis and gliosis associated with amyloid beta. The size of ionized calcium-binding adaptor molecule 1 (Iba1)-positive cells and clusters were larger in control diet APPNL-G-F mice, although the number and total area covered by Iba1+ cells/clusters were decreased compared to those of control diet C57BL6/J mice. In comparison, choline supplementation significantly reduced the size of Iba1+ cells/clusters in APPNL-G-F mice. These results suggest that perinatal choline supplementation ameliorates neuroinflammatory processes associated with amyloid plaques in these 9-month-old APPNL-G-F mice, and that dietary supplementation of choline might serve as an effective treatment for AD. / 2026-02-14T00:00:00Z

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