Kim, Jungsu

Increasing evidence strongly links neuroinflammation to Alzheimer’s disease (AD) pathogenesis. Peripheral monocytes are crucial components of the human immune system, but their contribution to AD pathogenesis is still largely understudied partially due to limited human models. Here, we introduce human cortical organoid microphysiological systems (hCO-MPSs) to study AD monocyte-mediated neuroinflammation. By culturing doughnut-shape organoids on 3D-printed devices within standard 96-well plates, we generate hCO-MPSs with reduced necrosis, minimized hypoxia, and improved viability. Using these models, we found that monocytes from AD patients exhibit increased infiltration ability, decreased amyloid-β clearance capacity, and stronger inflammatory response than monocytes from age-matched control donors. Moreover, we observed that AD monocytes induce pro-inflammatory effects such as elevated astrocyte activation and neuronal apoptosis. Furthermore, the marked increase in IL1B and CCL3 expression underscores their pivotal role in AD monocyte-mediated neuroinflammation. Our findings provide insight into understanding monocytes’ role in AD pathogenesis, and our lab-compatible MPS models may offer a promising way for studying various neuroinflammatory diseases.

Abstract Increasing evidence suggests that Alzheimer’s disease (AD) pathogenesis strongly correlates with neuroinflammation. Peripheral monocytes are crucial components of the human immune system that may play a role in neuroinflammation, but their contribution to AD pathogenesis is largely understudied partially due to the lack of appropriate human models. Here, we present human cortical organoid microphysiological systems (hCO-MPSs) for modeling dynamic AD neuroinflammation mediated by monocytes. By incorporating 3D printed devices into an existing cortical organoid protocol, 96 hCO-MPSs can be established with significantly reduced necrosis and hypoxia as well as enhanced viability within a commonly used 96 well plate, and each hCO-MPS consists of a doughnut-shaped hCO and a 3D printed device per well. Using this approach, monocytes from AD patients exhibit higher infiltration, decreased amyloid-beta (Aβ) clearance, and stronger inflammatory responses compared to monocytes from age-matched control donors. Moreover, pro-inflammatory effects such as elevated astrocyte activation and neuronal apoptosis were observed to be induced by AD monocytes. Furthermore, the significant increase in the expression of IL1B and CCL3, both at the transcriptional and protein levels, indicated the pivotal role of these cytokine and chemokine in monocyte-mediated AD neuroinflammation. Our findings provide insight for understanding monocytes’ role in AD pathogenesis, and the user-friendly MPS models we present are compatible with existing laboratory settings, highlighting their potential for modeling neuroinflammation and developing new therapeutics for various neuroinflammatory diseases.

Abstract Background: Levels of plasma proteins are under control of environmental and genetic factors. To use plasma proteins in biomarker studies, we need to understand how genetic modifiers influence their abundance. Although there has been expression quantitative trait loci (eQTL) studies on a few limited numbers of proteins, the effect of genetic variants on the levels of multiple plasma proteins still warrants more systematic investigation.Results: To identify genetic modifiers that influence the levels of clinically relevant plasma proteins, we performed protein quantitative trait locus (pQTL) mapping on the 92 proteins present in the Olink Mouse Exploratory Panel using the Diversity Outbred (DO) mouse population. We identified 12 significant pQTL that were located in cis and 6 that were in trans. Among them, we discovered that the presence of coding variants in the gene encoding for the Aryl Hydrocarbon Receptor (Ahr) had a significant effect on its abundance in plasma. Most interestingly, we identified variants in the Regulatory Factor X1 (Rfx1) gene that influence the abundance of the IL-17A protein in plasma.Conclusion: Our study reports an innovative pipeline for the identification of genetic modifiers that may be targeted for drug development.

Abstract Waldenströms Macroglobulinemia (WM) is a unique form of lymphoplasmacytic lymphoma, in which the tumor compartment is comprised of B-lymphocytes, plasmacytoid lymphocytes and plasma cells. The majority of WM cases are immunophenotypically characterized by expression of surface antigens that are present on both B-cells (CD19/20) and plasma cells (CD38/138). However, in advanced stage disease, which has been treated with various chemoimmunotherapeutics the molecular profile of WM cells can drastically shift resulting in a majority of the WM tumor compartment exhibiting loss of CD20 and concurrent upregulation of atypical surface antigens (Barakat et al, Am J Clin Pathol 2011; Morice et al Mod Pathol 2009). These external immunophenotypic changes are associated with underlying epigenetic and genomic alterations, whose integrated study may provide further insight into the biology of advanced stage WM. Moreover, such an analysis holds clinical potential by uncovering of therapeutic vulnerabilities, which can direct the selection of appropriate therapeutics for patients with CD20- WM. With this in mind, we conducted comprehensive molecular testing coupled with functional studies to understand if underlying genomic/epigenetic changes correlated with drug sensitivity using appropriate preclinical models of CD20+ WM (BCWM.1 cell line) and CD20- WM (RPCI-WM1 cell line). Importantly, the index patient from whom the CD20- cells were developed had received a total of 6 lines of treatment, two of which contained rituximab and was documented as being refractory to rituximab. Immunophenotyping of CD20- cells showed differential downregulation of 19 and upregulation of 7 CD antigens relative to CD20+ WM cells (cutoff at 1.5 fold; >20% gated expression). Upregulated antigens were associated with a phenotype reminiscent of a memory B-cell with plasmacytoid features. Next, we conducted RNA-Seq analysis (Illumina HiSeq), which revealed downregulation of 6,673 and upregulation of 4,594 genes (>1.5 fold cutoff) in CD20- WM cells compared with CD20+ cells. Gene pathway enrichment analysis (NextBio, Illumina Inc.) of mRNA differentially expressed in CD20- cells showed significant enrichment of upregulated genes corresponding to Protein Transport (p<1.8E-98) and Response to Unfolded Protein Gene Ontology groups (p<5.9E-20). Interestingly, pathway analysis of downregulated genes in CD20- WM cells showed enrichment within the B-cell receptor-signaling (BCR) pathway (Broad MSigDB) (p<6.1E-14) relative to CD20+ WM cells. BCR signaling has been linked to dysregulated expression of several micro-RNAs (miRs). Consistent with pathway analysis results, we observed decreased levels of numerous miRs (Exiqon qPCR) in CD20- vs. CD20+ WM cells, which target and regulate components involved in BCR activation. To examine further epigenetic changes that may account for decreased BCR signaling, we examined the global methylation profile (Infinium HumanMethylation 450K array) of CD20- and CD20+ WM cells and noted hypermethylation of the promoter sites (200 base pairs upstream) for a number of transcription factors involved in the BCR pathway. We therefore examined sensitivity of CD20- WM cells to the BTK inhibitor ibrutinib, which abrogates BCR signaling. As anticipated, CD20- WM cells retained >90% viability and demonstrated significantly less apoptosis in presence of ibrutinib (4uM) compared to CD20+ cells, suggesting decreased reliance on canonical BCR signaling for survival. In parallel and guided by pathway analysis results, we exposed both CD20- and CD20+ WM cells to a novel 19S proteasome inhibitor (VLX1570), which caused profound proteotoxicity and ER stress at concentrations ranging between 250 – 500nM. We noted CD20- cells were significantly more sensitive (p<0.01) to 19S proteasome inhibition than CD20+ WM cells indicating the formers critical dependence on tightly regulated protein homeostasis. In summary we describe for the first time, a comprehensive molecular and immunophenotypic investigation of CD20- WM cells compared to CD20+ WM cells, using validated preclinical models of the two disease phenotypes. Overall our data suggests significant architectural differences between CD20+ and CD20- WM, which can potentially impact the behavior of the cells and their sensitivity towards novel therapeutics being developed for treatment of advanced stage WM. Disclosures Martin: Janssen: Honoraria.

Purpose of review APOE4 genotype is the strongest genetic risk factor for Alzheimer's disease. Prevailing evidence suggests that amyloid β plays a critical role in Alzheimer's disease. The objective of this article is to review the recent findings about the metabolism of apolipoprotein E (ApoE) and amyloid β and other possible mechanisms by which ApoE contributes to the pathogenesis of Alzheimer's disease. Recent findings ApoE isoforms have differential effects on amyloid β metabolism. Recent studies demonstrated that ApoE-interacting proteins, such as ATP-binding cassette A1 (ABCA1) and LDL receptor, may be promising therapeutic targets for Alzheimer's disease treatment. Activation of liver X receptor and retinoid X receptor pathway induces ABCA1 and other genes, leading to amyloid β clearance. Inhibition of the negative regulators of ABCA1, such as microRNA-33, also induces ABCA1 and decreases the levels of ApoE and amyloid β. In addition, genetic inactivation of an E3 ubiquitin ligase, myosin regulatory light chain interacting protein, increases LDL receptor levels and inhibits amyloid accumulation. Although amyloid β-dependent pathways have been extensively investigated, there have been several recent studies linking ApoE with vascular function, neuroinflammation, metabolism, synaptic plasticity, and transcriptional regulation. For example, ApoE was identified as a ligand for a microglial receptor, TREM2, and studies suggested that ApoE may affect the TREM2-mediated microglial phagocytosis. Summary Emerging data suggest that ApoE affects several amyloid β-independent pathways. These underexplored pathways may provide new insights into Alzheimer's disease pathogenesis. However, it will be important to determine to what extent each mechanism contributes to the pathogenesis of Alzheimer's disease.

AbstractAccumulation of amyloid β (Aβ) in the brain is a key pathological hallmark of Alzheimer's disease (AD). Because aging is the most prominent risk factor for AD, understanding the molecular changes during aging is likely to provide critical insights into AD pathogenesis. However, studies on the role of miRNAs in aging and AD pathogenesis have only recently been initiated. Identifying miRNAs dysregulated by the aging process in the brain may lead to novel understanding of molecular mechanisms of AD pathogenesis. Here, we identified that miR‐186 levels are gradually decreased in cortices of mouse brains during aging. In addition, we demonstrated that miR‐186 suppresses β‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1) expression by directly targeting the 3′UTR of Bace1 mRNA in neuronal cells. In contrast, inhibition of endogenous miR‐186 significantly increased BACE1 levels in neuronal cells. Importantly, miR‐186 over‐expression significantly decreased Aβ level by suppressing BACE1 expression in cells expressing human pathogenic mutant amyloid precursor protein. Taken together, our data demonstrate that miR‐186 is a potent negative regulator of BACE1 in neuronal cells and it may be one of the molecular links between brain aging and the increased risk for AD during aging. image We identified that miR‐186 levels are gradually decreased in mouse cortices during aging. Furthermore, we demonstrated that miR‐186 is a novel negative regulator of beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1) expression in neuronal cells. Therefore, we proposed that reduction in miR‐186 levels during aging may lead to the up‐regulation of BACE1 in the brain, thereby increasing a risk for Alzheimer's disease in aged individuals. Read the Editorial Highlight for this article on page 308.