Dataset Information

Astrocytes express aberrant immunoglobulins as putative gatekeeper of astrocytes to neuronal progenitor conversion

ABSTRACT: Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and resembles aberrant immunoglobulins as observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in astrocytes adult rats, whereas in primary astrocytes from human fetal astrocytes we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 IgG2B constant heavy chain (IgH6), overexpression, proximal labeling of rat astrocytes IgH6 and targets identification through 2D gels were performed. In CRISPR-Cas9 IgG2B KO, overexpression of factors involved in hematopoietic cells, neural stem cells and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis whereas CRISPR-Cas9 IgG2B KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex; and behaves partially like CD98hc through its association with LAT1. Proximity-dependent biotin labeling experiments suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypotheses that aberrant neural IgG chains may act as gatekeepers of astrocytes fate.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Cell Culture

SUBMITTER: Capuz Alice

LAB HEAD: Michel Salzet

PROVIDER: PXD040562 | Pride | 2023-05-10

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	CRISPR2-CTL-P9.pdResultView	Other
	CRISPR2-CTL-P9.raw	Raw
	CRISPR2-CTL-P9_ProteinGroups.txt	Txt
	CRISPR2-CTL-POLY-P9.pdResultView	Other
	CRISPR2-CTL-POLY-P9.raw	Raw

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Publications

Astrocytes express aberrant immunoglobulins as putative gatekeeper of astrocytes to neuronal progenitor conversion.

Capuz Alice A Osien Sylvain S Karnoub Mélodie Anne MA Aboulouard Soulaimane S Laurent Estelle E Coyaud Etienne E Raffo-Romero Antonella A Duhamel Marie M Bonnefond Amélie A Derhourhi Mehdi M Trerotola Marco M El Yazidi-Belkoura Ikram I Devos David D Zilkova Monika M Kobeissy Firas F Vanden Abeele Fabien F Fournier Isabelle I Cizkova Dasa D Rodet Franck F Salzet Michel M

Cell death & disease 20230404 4

Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In additi ...[more]

PMID: 37015912

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Project description:The dogma “One gene, one protein” is clearly obsolete since cells use alternative splicing and generate multiple transcripts which are translated into protein isoforms, but also use alternative translation initiation sites (TISs) and termination sites on a given transcripts. Alternative open reading frames for individual transcripts give proteins originate from the 5'- and 3'- UTR mRNA regions, frameshifts of mRNA ORFs or from non-coding RNAs. Longtime considered as non-coding, recent in-silico translation prediction methods enriched the protein databases allowing the identification of new target structures that have not been identified previously. To gain insight into the role of these newly identified alternative proteins in the regulation of cellular functions, it is crucial to assess their dynamic modulation within a framework of altered physiological modifications such as experimental spinal cord injury (SCI). Here, we carried out a longitudinal proteomic study on rat SCI from 12h to 10 days. Based on the alternative protein predictions, it was possible to identify a plethora of newly predicted protein hits. Among these proteins, some presented a special interest due to high homology with variable chain regions of immunoglobulins. We focus our interest on the one related to Kappa variable light chains which is similarly highly produced by B-cells in the Bence jones disease, but here expressed in astrocytes. This protein, name Heimdall is an Intrinsically disordered protein which is secreted under inflammatory conditions. Immunoprecipitation experiments showed that the Heimdall interactome contained proteins related to astrocyte fate keepers such as “NOTCH1, EPHA3, IPO13” as well as membrane receptor protein including “CHRNA9; TGFBR, EPHB6, and TRAM”. However, when Heimdall protein was neutralized utilizing a specific antibody or its gene knocked out by CRISPR-Cas9, sprouting elongations were observed in the corresponding astrocytes. Interestingly, depolarization assays and intracellular calcium measurements in Heimdall KO, established a depolarization effect on astrocyte membranes KO cells were more likely that the one found in neuroprogenitors. Proteomic analyses performed under injury conditions or under lipopolysaccharides (LPS) stimulation, revealed the expression of neuronal factors, stem cell proteins, proliferation, and neurogenesis of astrocyte convertor factors such as EPHA4, NOTCH2, SLIT3, SEMA3F, suggesting a role of Heimdall could regulate astrocytic fate. Taken together, Heimdall could be a novel member of the gatekeeping astrocyte-to-neuroprogenitor conversion factors.