Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes

Kantzer, Christina Geraldine; Boutin, Camille; Herzig, Ina D.; Wittwer, Carolina; Reiß, Sandy; Tiveron, Marie-Catherine; Drewes, J; Rockel, Thomas Dino; Ohlig, Stefanie; Ninkovic, Jovica; Cremer, Harold; Pennartz, Sandra; Jungblut, Melanie; Bosio, Andreas

doi:10.1002/glia.23140

Cited by 80 publications

(75 citation statements)

References 59 publications

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“…As an initial validation of the model, Aldh1l1-NuTRAP mice were systemically delivered Tam for 5 consecutive days and a week after induction, brains were dissected for flow cytometry (FC) and immunohistochemistry (IHC) analyses. Single-cell suspensions of brains immunostained with ACSA-2 antibody, a pan-astrocytic marker 15 , revealed a distinct EGFP + cell population present in the Aldh1l1-NuTRAP brains but not in the cre negative counterparts, consistent with the reported 10-20% astroglial cellularity in the rodent brain 16, 17 , Almost the entirety of the EGFP + cell population co-expressed ACSA-2, supporting that cre-mediated recombination upon Tam induction specifically targeted astrocytes (Figure 1A-B) .…”

Section: Resultsmentioning

confidence: 99%

“…Subsequent gating based on CD11b or ACSA-2 expression was done with 640 nm laser and 676/629 filter, or with 488 nm laser and 740 LP filter combinations, respectively. The antibodies used were anti-mouse CD11b: APC (#17-0112, clone M1/70) (eBioscience, San Diego, CA), and ACSA-2: PE-Vio770 (#130-116-246, Milteny Biotec) 15 . Isotype controls for each antibody and unstained cells were used for proper post-color compensation (Supplemental Figures 14-15) .…”

Section: Methodsmentioning

confidence: 99%

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Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

Chucair-Elliott

Ocañas

Stanford

et al. 2019

Preprint

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23Epigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type 24 specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and 25 introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and 26Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling 27 and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific CNS cell types. 28Paired analysis of the transcriptome and DNA modifications in astrocytes and microglia 29 demonstrates differential usage of DNA methylation and hydroxymethylation in CG and non-CG 30 contexts that corresponds to cell type-specific gene expression. Application of this approach in 31 LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in 32 inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model 33 and the validation approaches presented can be applied to any CNS cell type for which a cell 34 type-specific cre is available. 36Significant advances are being made in understanding the epigenome and its relationship with 37 gene expression in the brain 1-3 . However, the lack of approaches for paired analysis of DNA and 38RNA profiles at the cell type-specific level within the same animal is a significant limitation for the 39 field, given that epigenetic processes differ across CNS cell types at the level of chromatin 40 organization and DNA modifications 1,4 . Obtaining enriched cell populations by flow sorting 41 requires cell surface markers but these markers can change with experimental conditions and cell 42 sorting causes molecular, morphological, and functional changes, such as cell activation, that 43 could confound studies 3,5,6 . Single cell approaches 7 may overcome some of the challenges of 44 cell sorting but the scale of such studies, partial genomic coverage, restriction to only certain types 45 of endpoints, and continued potential for brain dissociation artifacts are limitations. 46This has led to development of transgenic labeling approaches to isolate RNA or DNA from 47 specific cell types. Ribosome labeling and RNA isolation methods, such as Translating Ribosome 48Affinity Purification (TRAP 8 ), and ribosome tagging (RiboTag 9 ), are gaining acceptance across 49 neuroscience studies examining the transcriptome. Similar approaches have been developed to 50 transgenically tag and allow isolation of nuclei and thus DNA (Isolation of Nuclei TAgged in 51 Specific Cell Types, INTACT) 10 . However, using separate transgenic mouse strains for DNA and 52RNA endpoints is a complicated and resource intensive approach. 53Here we describe an approach where Nuclear Tagging and Translating Ribosome Affinity 54Purification (NuTRAP) 11 is combined with well-established cell-specific inducible cre-recombinase 55 expressing systems 12,13 to perform paired transcriptomic and epigenomic analyses of specific 56 CNS cell types in a temporally controllable manner from a single mouse. ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

Chucair-Elliott

Ocañas

Stanford

et al. 2019

Preprint

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“…These include the well-established antibodies against CD45 (36) and O4 (37,38), the astrocyte-specific anti-ACSA-2 antibody that has been recently validated by ourselves and independent groups (27,39,40), (Supplemental Figure 2), and the anti-CD49a antibody ( Figure 1B, Figure 2, and Supplemental Figure 2). Employing the commonly used ALDH1l1-EGFP reporter (41) believed to mark the majority of astrocytes, we found that the anti-ACSA-2 antibody labeled all GFP + astrocytes in addition to a sizable GFP -astrocyte population (Supplemental Figure 3A), suggesting that this reporter line does not mark all astrocytes in adult brains.…”

Section: Concurrent Brain Cell Type Acquisition From a Single Adult Mmentioning

confidence: 99%

Concurrent cell type–specific isolation and profiling of mouse brains in inflammation and Alzheimer’s disease

Swartzlander¹,

Propson²,

Roy³

et al. 2018

JCI Insight

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“…S1B-C). Astrocytes and Müller cells were isolated using ASCA2+ selection, as reported previously (Kantzer et al, 2017). By 14 days post-injection, ACSA2+ cells exhibited increased levels of pan reactive and A1 astrocyte specific markers ( Fig.…”

Section: Elevated Intraocular Pressure Induces A1 Astrocyte Reactivitmentioning

confidence: 76%

GLP-1R agonist NLY01 reduces retinal inflammation, astrocyte reactivity, and retinal ganglion cell death secondary to ocular hypertension

Sterling

Adetunji

Guttha

et al. 2020

Preprint

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Glaucoma is the leading cause of irreversible blindness worldwide and is characterized by the death of retinal ganglion cells. Reduction of intraocular pressure (IOP) is the only therapeutic mechanism available to slow disease progression. However, glaucoma can continue to progress despite normalization of IOP. New treatments are needed to reduce vision loss and improve outcomes for patients who have exhausted existing therapeutic avenues. Recent studies have implicated neuroinflammation in the pathogenesis of neurodegenerative diseases of both the retina and the brain, including glaucoma and Parkinson's disease. Pro-inflammatory A1 astrocytes contribute to neuronal cell death in multiple disease processes and have been targeted therapeutically in mouse models of Parkinson's disease. Microglial release of pro-inflammatory cytokines C1q, IL-1α, and TNF-α is sufficient to drive the formation of A1 astrocytes. The role of A1 astrocytes in glaucoma pathogenesis has not been explored. Using a mouse model of glaucoma, we demonstrated that IOP elevation was sufficient to trigger production of C1q, IL-1α, and TNF-α by infiltrating macrophages followed by resident microglia. These three cytokines drove the formation of A1 astrocytes in the retina. Furthermore, cytokine production and A1 astrocyte transformation persisted following IOP normalization. Ablation of this pathway, by either genetic deletions of C1q, IL-1α, and TNF-α, or treatment with glucagon-like peptide-1 receptor agonist NLY01, reduced A1 astrocyte transformation and RGC death. Together, these results highlight a new neuroinflammatory mechanism behind glaucomatous neurodegeneration that can be therapeutically targeted by NLY01 administration.

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Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes

Cited by 80 publications

References 59 publications

Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

Concurrent cell type–specific isolation and profiling of mouse brains in inflammation and Alzheimer’s disease

GLP-1R agonist NLY01 reduces retinal inflammation, astrocyte reactivity, and retinal ganglion cell death secondary to ocular hypertension

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