Proteomic Alterations and Novel Markers of Neurotoxic Reactive Astrocytes in Human Induced Pluripotent Stem Cell Models

Abstract

Astrocytes respond to injury, infection, and inflammation in the central nervous system by acquiring reactive states in which they may become dysfunctional and contribute to disease pathology. A sub-state of reactive astrocytes induced by proinflammatory factors TNF, IL-1α, and C1q (“TIC”) has been implicated in many neurodegenerative diseases as a source of neurotoxicity. Here, we used an established human induced pluripotent stem cell (hiPSC) model to investigate the surface marker profile and proteome of TIC-induced reactive astrocytes. We propose VCAM1, BST2, ICOSL, HLA-E, PD-L1, and PDPN as putative, novel markers of this reactive sub-state. We found that several of these markers colocalize with GFAP+ cells in post-mortem samples from people with Alzheimer’s disease. Moreover, our whole-cells proteomic analysis of TIC-induced reactive astrocytes identified proteins and related pathways primarily linked to potential engagement with peripheral immune cells. Taken together, our findings will serve as new tools to purify reactive astrocyte subtypes and to further explore their involvement in immune responses associated with injury and disease.

CRISPRi screens in human astrocytes elucidate regulators of distinct inflammatory reactive states

“…1m-n, Extended Data Fig. 2d), corroborating previous reports in the literature demonstrating induction of VCAM1 in astrocytes under pro-inflammatory conditions 33–35 .…”

Distinct and Dynamic Transcriptome Adaptations of iPSC-Generated Astrocytes after Cytokine Stimulation

“…Our transcriptome analysis contained probes for three of these markers (VCAM-1, HLA-E and PDPN) which were all up-regulated by TNFα at 24 h (IFNγ failed to regulate PDPN). The response in our system was characterized by the expression of genes involved in immune effector recruitment and activation, similar to the findings by Labib et al [ 31 ]. The study on TIC-activated ACs identified proteomics-based activation markers.…”

“…A particular combination of inflammogens, TNFα, IL1α and C1q (termed TIC), has often been used to generate and characterize neurotoxic ACs [ 30 , 66 ]. A recent study on TIC-induced reactive ACs revealed a distinct signature of six cell-surface markers: VCAM-1, BST2, ICOSL, HLA-E, PD-L1 and PDPN after 24 h of stimulation [ 31 ]. Our transcriptome analysis contained probes for three of these markers (VCAM-1, HLA-E and PDPN) which were all up-regulated by TNFα at 24 h (IFNγ failed to regulate PDPN).…”

“…The study on TIC-activated ACs identified proteomics-based activation markers. Altogether, 24 of the most regulated markers (Figure 4D in [ 31 ]) were detectable by our transcriptome approach. In ACs stimulated with TNFα or TNFα plus IFNγ, we found 23 of them to be up-regulated ( Table S2 ).…”

“…The first whole transcriptome study that investigated cytokine effects (IL-1/TNFα) for at least two time points became available recently [ 29 ]. A little later, it became clear that different cocktails of inflammogens produce other phenotypes of reactive ACs [ 30 , 31 ]. Very recently, the availability of large amounts of homogeneous iPSC-derived ACs has been used to compare their resting state to the patterns and response dynamics of two distinct stimuli (synuclein; TNFα) [ 32 ].…”