Title: Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems
AbstractBackground
Alzheimer’s disease (AD) is a progressive neurodegenerative disease that impacts nearly 400 million people worldwide. The accumulation of amyloid beta (Aβ) in the brain has historically been associated with AD, and recent evidence suggests that neuroinflammation plays a central role in its origin and progression. These observations have given rise to the theory that Aβ is the primary trigger of AD, and induces proinflammatory activation of immune brain cells (i.e., microglia), which culminates in neuronal damage and cognitive decline. To test this hypothesis, many in vitro systems have been established to study Aβ-mediated activation of innate immune cells. Nevertheless, the transcriptional resemblance of these models to the microglia in the AD brain has never been comprehensively studied on a genome-wide scale.
Methods
We used bulk RNA-seq to assess the transcriptional differences between in vitro cell types used to model neuroinflammation in AD, including several established, primary and iPSC-derived immune cell lines (macrophages, microglia and astrocytes) and their similarities to primary cells in the AD brain. We then analyzed the transcriptional response of these innate immune cells to synthetic Aβ or LPS and INFγ.
Results
We found that human induced pluripotent stem cell (hIPSC)-derived microglia (IMGL) are the in vitro cell model more »
that best resembles primary microglia. Surprisingly, synthetic Aβ does not trigger a robust transcriptional response in any of the cellular models analyzed, despite testing a wide variety of Aβ formulations, concentrations, and treatment conditions. Finally, we found that bacterial LPS and INFγ activate microglia and induce transcriptional changes that resemble many, but not all, aspects of the transcriptomic profiles of disease associated microglia (DAM) present in the AD brain.
Conclusions
These results suggest that synthetic Aβ treatment of innate immune cell cultures does not recapitulate transcriptional profiles observed in microglia from AD brains. In contrast, treating IMGL with LPS and INFγ induces transcriptional changes similar to those observed in microglia detected in AD brains.
TheBIN1locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease.BIN1undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer’s disease, have not been examined in depth.
Methods
Microglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells.Bin1expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches.
Results
Here, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencingBin1expression in primary microglial cultures, wemore »demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specificBin1conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss ofBin1impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene,Ifitm3.
Conclusions
Our convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship betweenBin1andIfitm3, two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulateIfitm3upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases.
Emerging RNA viruses that target the central nervous system (CNS) lead to cognitive sequelae in survivors. Studies in humans and mice infected with West Nile virus (WNV), a re-emerging RNA virus associated with learning and memory deficits, revealed microglial-mediated synapse elimination within the hippocampus. Moreover, CNS-resident memory T (TRM) cells activate microglia, limiting synapse recovery and inducing spatial learning defects in WNV-recovered mice. The signals involved in T cell-microglia interactions are unknown.
Methods
Here, we examined immune cells within the murine WNV-recovered forebrain using single-cell RNA sequencing to identify putative ligand-receptor pairs involved in intercellular communication between T cells and microglia. Clustering and differential gene analyses were followed by protein validation and genetic and antibody-based approaches utilizing an established murine model of WNV recovery in which microglia and complement promote ongoing hippocampal synaptic loss.
Results
Profiling of host transcriptome immune cells at 25 days post-infection in mice revealed a shift in forebrain homeostatic microglia to activated subpopulations with transcriptional signatures that have previously been observed in studies of neurodegenerative diseases. Importantly, CXCL16/CXCR6, a chemokine signaling pathway involved in TRM cell biology, was identified as critically regulating CXCR6 expressing CD8+TRM cell numbers within the WNV-recovered forebrain. We demonstrate that CXCL16 is highlymore »expressed by all myeloid cells, and its unique receptor, CXCR6, is highly expressed on all CD8+T cells. Using genetic and pharmacological approaches, we demonstrate that CXCL16/CXCR6 not only is required for the maintenance of WNV-specific CD8 TRM cells in the post-infectious CNS, but also contributes to their expression of TRM cell markers. Moreover, CXCR6+CD8+T cells are required for glial activation and ongoing synapse elimination.
Conclusions
We provide a comprehensive assessment of the role of CXCL16/CXCR6 as an interaction link between microglia and CD8+T cells that maintains forebrain TRM cells, microglial and astrocyte activation, and ongoing synapse elimination in virally recovered animals. We also show that therapeutic targeting of CXCL16 in mice during recovery may reduce CNS CD8+TRM cells.
Crohn’s disease is a lifelong disease characterized by chronic inflammation of the gastrointestinal tract. Defining the cellular and transcriptional composition of the mucosa at different stages of disease progression is needed for personalized therapy in Crohn’s.
Methods
Ileal biopsies were obtained from (1) control subjects (n = 6), (2) treatment-naïve patients (n = 7), and (3) established (n = 14) Crohn’s patients along with remission (n = 3) and refractory (n = 11) treatment groups. The biopsies processed using 10x Genomics single cell 5' yielded 139 906 cells. Gene expression count matrices of all samples were analyzed by reciprocal principal component integration, followed by clustering analysis. Manual annotations of the clusters were performed using canonical gene markers. Cell type proportions, differential expression analysis, and gene ontology enrichment were carried out for each cell type.
Results
We identified 3 cellular compartments with 9 epithelial, 1 stromal, and 5 immune cell subtypes. We observed differences in the cellular composition between control, treatment-naïve, and established groups, with the significant changes in the epithelial subtypes of the treatment-naïve patients, including microfold, tuft, goblet, enterocyte,s and BEST4+ cells. Surprisingly, fewer changes in the composition of the immune compartment were observed; however, gene expression in the epithelial and immune compartment was different between Crohn’s phenotypes, indicating changes inmore »cellular activity.
Conclusions
Our study identified cellular and transcriptional signatures associated with treatment-naïve Crohn’s disease that collectively point to dysfunction of the intestinal barrier with an increase in inflammatory cellular activity. Our analysis also highlights the heterogeneity among patients within the same disease phenotype, shining a new light on personalized treatment responses and strategies.
Pollock, Tanner B.; Mack, Jacob M.; Day, Ryan J.; Isho, Noail F.; Brown, Raquel J.; Oxford, Alexandra E.; Morrison, Brad E.; Hayden, Eric J.; Rohn, Troy T.(
, Oxidative Medicine and Cellular Longevity)
Despite the fact that harboring the apolipoprotein E4 ( APOE4 ) allele represents the single greatest risk factor for late-onset Alzheimer’s disease (AD), the exact mechanism by which apoE4 contributes to disease progression remains unknown. Recently, we demonstrated that a 151 amino-terminal fragment of apoE4 (nApoE4 1-151 ) localizes within the nucleus of microglia in the human AD brain, suggesting a potential role in gene expression. In the present study, we investigated this possibility utilizing BV2 microglia cells treated exogenously with nApoE4 1-151 . The results indicated that nApoE4 1-151 leads to morphological activation of microglia cells through, at least in part, the downregulation of a novel ER-associated protein, CXorf56. Moreover, treatment of BV2 cells with nApoE4 1-151 resulted in a 68-fold increase in the expression of the inflammatory cytokine, TNF α , a key trigger of microglia activation. In this regard, we also observed a specific binding interaction of nApoE4 1-151 with the TNF α promoter region. Collectively, these data identify a novel gene-regulatory pathway involving CXorf56 that may link apoE4 to microglia activation and inflammation associated with AD.
Dhavale, Dhruva; Lai, Hy K.; Warwick, Paityn; Henry, James E.(
, Bulletin of the National Research Centre)
AbstractBackground
Amyloid beta peptide (Aβ) is the main component of plaques and is known to play a role in the development of Alzheimer's disease (AD). As a result, structures that can trap Aβ or disrupt the interaction between Aβ and cells have been researched as a way to lessen the pathological effects of Aβ. Particularly, sialylated compounds that exhibit clustering effects could be advantageous.
Results
Through the use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chemistry, sialic acid (N-acetylneuraminic acid) was used to decorate a chitosan backbone. The compounds were characterized using Fourier-transform infrared spectroscopy (FTIR) and colorimetric assays. Using the model neuroblastoma cell line SH-SY5Y, the ability of these compounds to lessen the toxicity of Aβ was examined in vitro. Successful in vitro mitigation of Aβ toxicity was found to be critically dependent on the degree of sialylation. In particular, a balance between the degree of sialylation and molecular flexibility was determined to be the criteria as it allows for natural clustering. Additionally, chitosan alone demonstrated low levels of cellular toxicity with moderate levels of toxicity mitigation (comparable to low degrees of labelling).
Conclusions
Compounds were successfully produced, and they varied in their effectiveness in reducing Aβ's toxicity to cells in culture. The effect of molecular flexibility and clustering on toxicity mitigation is explained inmore »this work. This shows the potential of polymeric sugars for the creation of AD treatments.
Quiroga, I. Y., Cruikshank, A. E., Bond, M. L., Reed, K. S. M., Evangelista, B. A., Tseng, J. H., Ragusa, J. V., Meeker, R. B., Won, H., Cohen, S., Cohen, T. J., and Phanstiel, D. H.. Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems. Journal of Neuroinflammation 19.1 Web. doi:10.1186/s12974-022-02459-1.
Quiroga, I. Y., Cruikshank, A. E., Bond, M. L., Reed, K. S. M., Evangelista, B. A., Tseng, J. H., Ragusa, J. V., Meeker, R. B., Won, H., Cohen, S., Cohen, T. J., & Phanstiel, D. H.. Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems. Journal of Neuroinflammation, 19 (1). https://doi.org/10.1186/s12974-022-02459-1
Quiroga, I. Y., Cruikshank, A. E., Bond, M. L., Reed, K. S. M., Evangelista, B. A., Tseng, J. H., Ragusa, J. V., Meeker, R. B., Won, H., Cohen, S., Cohen, T. J., and Phanstiel, D. H..
"Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems". Journal of Neuroinflammation 19 (1). Country unknown/Code not available: Springer Science + Business Media. https://doi.org/10.1186/s12974-022-02459-1.https://par.nsf.gov/biblio/10380081.
@article{osti_10380081,
place = {Country unknown/Code not available},
title = {Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems},
url = {https://par.nsf.gov/biblio/10380081},
DOI = {10.1186/s12974-022-02459-1},
abstractNote = {Abstract BackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disease that impacts nearly 400 million people worldwide. The accumulation of amyloid beta (Aβ) in the brain has historically been associated with AD, and recent evidence suggests that neuroinflammation plays a central role in its origin and progression. These observations have given rise to the theory that Aβ is the primary trigger of AD, and induces proinflammatory activation of immune brain cells (i.e., microglia), which culminates in neuronal damage and cognitive decline. To test this hypothesis, many in vitro systems have been established to study Aβ-mediated activation of innate immune cells. Nevertheless, the transcriptional resemblance of these models to the microglia in the AD brain has never been comprehensively studied on a genome-wide scale. MethodsWe used bulk RNA-seq to assess the transcriptional differences between in vitro cell types used to model neuroinflammation in AD, including several established, primary and iPSC-derived immune cell lines (macrophages, microglia and astrocytes) and their similarities to primary cells in the AD brain. We then analyzed the transcriptional response of these innate immune cells to synthetic Aβ or LPS and INFγ. ResultsWe found that human induced pluripotent stem cell (hIPSC)-derived microglia (IMGL) are the in vitro cell model that best resembles primary microglia. Surprisingly, synthetic Aβ does not trigger a robust transcriptional response in any of the cellular models analyzed, despite testing a wide variety of Aβ formulations, concentrations, and treatment conditions. Finally, we found that bacterial LPS and INFγ activate microglia and induce transcriptional changes that resemble many, but not all, aspects of the transcriptomic profiles of disease associated microglia (DAM) present in the AD brain. ConclusionsThese results suggest that synthetic Aβ treatment of innate immune cell cultures does not recapitulate transcriptional profiles observed in microglia from AD brains. In contrast, treating IMGL with LPS and INFγ induces transcriptional changes similar to those observed in microglia detected in AD brains.},
journal = {Journal of Neuroinflammation},
volume = {19},
number = {1},
publisher = {Springer Science + Business Media},
author = {Quiroga, I. Y. and Cruikshank, A. E. and Bond, M. L. and Reed, K. S. M. and Evangelista, B. A. and Tseng, J. H. and Ragusa, J. V. and Meeker, R. B. and Won, H. and Cohen, S. and Cohen, T. J. and Phanstiel, D. H.},
}
