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Burda JE, O'Shea TM, Ao Y, Suresh KB, Wang S, Bernstein AM, Chandra A, Deverasetty S, Kawaguchi R, Kim JH, McCallum S, Rogers A, Wahane S, Sofroniew MV. Divergent transcriptional regulation of astrocyte reactivity across disorders. Nature. 2022 Jun;606(7914):557-564. Epub 2022 May 25 PubMed.
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Johns Hopkins University
Within the complex system of the brain, the non-neuronal cells do make a great impact on the origin of neurodegenerative diseases. Recent experimental reports have evidently proved that the reactive form of astrocyte activates via releasing factors such as IL-1α, TNF, and C1q secreted by active microglia, and results in the demise of neuronal and oligodendrocyte cells leading to the cause of neurodegenerative diseases such as PD, AD, and ALS. The reactive astrocyte loses its homeostatic functions such as neuron survival, synaptogenesis, phagocytosis, etc., while impacting neighboring supporting neuronal and non-neuronal cells (Liddelow et al., 2017). Further, blocking reactive astrocytes by prohibiting microglial activation via the GLP-1R agonist NLY01 showed neuroprotective effects in in vitro and in vivo models of PD and AD respectively (Yun et al., 2018; Park et al., 2021). Yet, it is still uncovered by which cellular mechanisms reactive astrocytes lose homeostatic functions and gain harmful outputs.
The new findings here by the Kampmann lab have tried to identify cellular pathways controlling inflammatory astrocyte reactivity in a unique manner, by employing the latest and most robust technology of single-cell transcriptomics with CRISPR interference (CRISPRi) screening in human iPSC (hiPSC)-derived astrocytes. Currently, the methods being utilized in vitro and in vivo to understand astrocyte reactivity are not clearly established due to limitations such as isolation of primary astrocytes or long procedure of generation of hiPSC-derived astrocytes. Moreover, molecular profiling approaches such as RNA-Seq, which is presently being used to determine altered cellular mechanisms in inflammatory reactive astrocytes, are also limited as the methodology could not explain the causative pathways for controlling inflammatory reactivity.
Within this study, the authors have developed a scalable method and utilized the latest application of CRISPR-based functional genomics (Kampmann, 2020). CRISPRi screens were combined with hiPSC screens to uncover the cellular pathways responsible for IL-1α, TNF, and C1q- induced inflammatory astrocyte reactivity within this study. Furthermore, the scalability and homogeneity of hiPSC-derived iAstrocytes were vitally important to performing pooled screens. The phenotypes of multiple hiPSC-derived iAstrocytes were maintained throughout the whole study. The authors performed the reactivity evaluation based on CRISPRi screens and the computational master regulator analysis (MRA) platform, where they preferentially considered the CRISPRi screens due to MRA application in astrocyte-diverse phenotypes.
The results obtained from CRISPRi screens with single transcriptomics data on the influence of IL-1α+TNF+C1q are that they induce two distinct inflammatory reactive astrocyte signatures (IRAS) forms, i.e., IRAS1 and IRAS2, dependent upon canonical NF-κB driving through IL-6 and interferon signaling. The transcription factors (CEBP/D, NF-κB, and STAT3) at the upstream promote IRAS1 while inhibiting IRAS2 through IL-6. IFN-β acts through STAT1/2 and IRF1 to partially influence both IRAS1 and IRAS2. The obtained findings state that IL-1/IL-6 are IRAS1-responsive, while IRAS2 is referred to as TNF/IFN-responsive signaling.
Furthermore, several studies are well-corroborated with this study, supporting its finding of STAT3’s role in inflammatory astrocyte reactivity (Herrmann et al., 2008; Kim et al., 2022; Ben Haim et al., 2015), while potential inhibition or deletion prevents the neurodegenerative disease, spinal cord injury, and its progression in vitro and in vivo (Reichenbach et al., 2019; Anderson et al., 2016). This new study also found upregulated vascular cell adhesion molecule 1(VCAM1) transcript-level expression in response to the IL-1α+TNF+C1q, and results in support of previous findings with a pro-inflammatory environment (Rubio et al., 2010; Labib et al., 2022).
Along with the inflammatory astrocyte reactivity cellular signaling in vitro data, Leng and co-workers performed a few in vivo mouse model experiments and also studied human AD and hypoxic-ischaemic encephalopathy, and obtained the overlapped inflammatory profile.
This iAstrocytes platform is unique and will be greatly beneficial in future investigations using patient-derived hiPSCs in dissecting the impact of disease-associated mutations on inflammatory astrocyte reactivity. This appreciable work acts as an important reference to future approaches in characterizing and validating the practical outputs of IL-1/IL-6-responsive versus TNF/IFN-responsive astrocyte reactivity in animal models of neuroinflammation and neurodegeneration.
The significant part of this advanced technical approach is an application that accelerates drug discovery. Furthermore, it can help in searching for novel cellular proteins involved in diverse cellular signaling using CRISPR pooled-screen with single-cell transcriptomics in various cell types such as microglia, oligodendrocytes, endoderm development, neuronal development, etc.
Moreover, this will serve as a tool to characterize β-cell type function and mechanism and selectively monitor the development of novel therapeutics against several neurodegenerative diseases.
References:
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