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 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, we 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.
Edler, Melissa K.; Sherwood, Chet C.; Meindl, Richard S.; Munger, Emily L.; Hopkins, William D.; Ely, John J.; Erwin, Joseph M.; Perl, Daniel P.; Mufson, Elliott J.; Hof, Patrick R.; et al(
, Journal of Comparative Neurology)
Abstract
In Alzheimer's disease (AD), the brain's primary immune cells, microglia, become activated and are found in close apposition to amyloid beta (Aβ) protein plaques and neurofibrillary tangles (NFT). The present study evaluated microglia density and morphology in a large group of aged chimpanzees (n = 20, ages 37–62 years) with varying degrees of AD‐like pathology. Using immunohistochemical and stereological techniques, we quantified the density of activated microglia and morphological variants (ramified, intermediate, and amoeboid) in postmortem chimpanzee brain samples from prefrontal cortex, middle temporal gyrus, and hippocampus, areas that show a high degree of AD pathology in humans. Microglia measurements were compared to pathological markers of AD in these cases. Activated microglia were consistently present across brain areas. In the hippocampus, CA3 displayed a higher density than CA1. Aβ42 plaque volume was positively correlated with higher microglial activation and with an intermediate morphology in the hippocampus. Aβ42‐positive vessel volume was associated with increased hippocampal microglial activation. Activated microglia density and morphology were not associated with age, sex, pretangle density, NFT density, or tau neuritic cluster density. Aged chimpanzees displayed comparable patterns of activated microglia phenotypes as well as an association of increased microglial activation and morphological changes with Aβ deposition similar to AD patients. In contrast to human AD brains, activated microglia density was not significantly correlated with tau lesions. This evidence suggests that the chimpanzee brain may be relatively preserved during normal aging processes but not entirely protected from neurodegeneration as previously assumed.
Mantle, Jennifer L.; Lee, Kelvin H.(
, Biotechnology and Bioengineering)
Abstract
Immunotherapies are a promising strategy for the treatment of neurological diseases such as Alzheimer's disease (AD), however, transport of antibodies to the brain is severely restricted by the blood–brain barrier (BBB). Furthermore, molecular transport at the BBB is altered in disease, which may affect the mechanism and quantity of therapeutic antibody transport. To better understand the transport of immunotherapies at the BBB in disease, an in vitro BBB model derived from human induced pluripotent stem cells (iPSCs) was used to investigate the endocytic uptake route of immunoglobulin G (IgG). In this model, uptake of fluorescently labeled IgGs is a saturable process. Inhibition of clathrin‐mediated endocytosis, caveolar endocytosis, and macropinocytosis demonstrated that macropinocytosis is a major transport route for IgGs at the BBB. IgG uptake and transport were increased after the addition of stimuli to mimic AD (Aβ1–40and Aβ1–42) and neuroinflammation (tumor necrosis factor‐α and interleukin‐6). Lastly, caveolar endocytosis increased in the AD model, which may be responsible for the increase in IgG uptake in disease. This study presents an iPSC‐derived BBB model that responds to disease stimuli with physiologically relevant changes to molecular transport and can be used to understand fundamental questions about transport mechanisms of immunotherapies in health and neurodegenerative disease.
Ma, Fulin; Akolkar, Himanshu; Xu, Jianquan; Liu, Yang; Popova, Dina; Xie, Jiaan; Youssef, Mark M.; Benosman, Ryad; Hart, Ronald P.; Herrup, Karl(
, The Journal of Neuroscience)
The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the β-amyloid (Aβ) peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aβ are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase inAppgene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. The GCaMP8f Ca2+reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown ofAppblock the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aβ, either fibrillar or oligomeric, has no effect. In culture, APPSwe(a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and βIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model, both females and males, neurons have elevated levels of APP protein that invade the AIS.In vivoasin vitro, this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD.
SIGNIFICANCE STATEMENTWhile the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity.In vivoandin vitro, modest amounts of excess APP alter the properties of the axon initial segment. The β-amyloid peptide derived from APP is without effect. Consistent with the observed changes in the axon initial segment which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the axon initial segment, suggesting a relationship with AD dementia.
Dysfunction of the aquaporin‐4 (AQP4)‐dependent glymphatic waste clearance pathway has recently been implicated in the pathogenesis of several neurodegenerative diseases. However, it is difficult to unravel the causative relationship between glymphatic dysfunction, AQP4 depolarization, protein aggregation, and inflammation in neurodegeneration using animal models alone. There is currently a clear, unmet need for in vitro models of the brain's waterscape, and the first steps towards a bona fide “glymphatics‐on‐a‐chip” are taken in the present study. It is demonstrated that chronic exposure to lipopolysaccharide (LPS), amyloid‐β(1‐42) oligomers, and an AQP4 inhibitor impairs the drainage of fluid and amyloid‐β(1‐40) tracer in a gliovascular unit (GVU)‐on‐a‐chip model containing human astrocytes and brain microvascular endothelial cells. The LPS‐induced drainage impairment is partially retained following cell lysis, indicating that neuroinflammation induces parallel changes in cell‐dependent and matrisome‐dependent fluid transport pathways in GVU‐on‐a‐chip. Additionally, AQP4 depolarization is observed following LPS treatment, suggesting that LPS‐induced drainage impairments on‐chip may be driven in part by changes in AQP4‐dependent fluid dynamics.
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.},
}
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