Age-related loss of neural stem cell O-GlcNAc promotes a glial fate switch through STAT3 activation

Abstract

Increased neural stem cell (NSC) quiescence is a major determinant of age-related regenerative decline in the adult hippocampus. However, a coextensive model has been proposed in which division-coupled conversion of NSCs into differentiated astrocytes restrict the stem cell pool with age. Here we report that age-related loss of the posttranslational modification, O-linked β-N-acetylglucosamine (O-GlcNAc), in NSCs promotes a glial fate switch. We detect an age-dependent decrease in NSC O-GlcNAc levels coincident with decreased neurogenesis and increased gliogenesis in the mature hippocampus. Mimicking an age-related loss of NSC O-GlcNAcylation in young mice reduces neurogenesis, increases astrocyte differentiation, and impairs associated cognitive function. Using RNA-sequencing of primary NSCs following decreased O-GlcNAcylation, we detected changes in the STAT3 signaling pathway indicative of glial differentiation. Moreover, using O-GlcNAc–specific mass spectrometry analysis of the aging hippocampus, together with an in vitro site-directed mutagenesis approach, we identify loss of STAT3 O-GlcNAc at Threonine 717 as a driver of astrocyte differentiation. Our data identify the posttranslational modification, O-GlcNAc, as a key molecular regulator of regenerative decline underlying an age-related NSC fate switch.

Towards structure-focused glycoproteomics

“…( m/z 138 + m/z 168) and ( m/z 126 + m/z 144), referred as the GlcNAc/GalNAc ratio, generated using ion trap CID- or HCD-MS/MS, is reportedly able to discriminate between GlcNAc- and GalNAc-containing N - and O -glycopeptides [ 226 , 227 ]. In support, multiple glycoproteomics studies have applied this approach to distinguish extended α-linked GalNAc and β-linked GlcNAc O -glycopeptides [ 36 , 131 , 236 , 237 ], glycopeptides carrying either a single O -GlcNAc or O -GalNAc residue [ 238 ] or, more crudely, to simply classify O - and N -glycan types on peptides [ 41 , 197 ]. Furthermore, α2,3- and α2,6-sialyl linkage information was obtained by determining the signal intensity ratio of the HCD-MS/MS-derived oxonium ions specific for LacNAc ( m/z 204 + m/z 366) and NeuAc ( m/z 274 + m/z 292) following an elaborate signal normalisation process that considers both the glycan composition and the LacNAc structure (Galβ1,3GalNAc or Galβ1,4GlcNAc) [ 228 ].…”

Novel antibodies for the simple and efficient enrichment of native O-GlcNAc modified peptides

“…In fact, we found that increasing HCD collision energies led to higher scoring O-HexNAc-modified PSMs in both HCD and EThcD. This suggests that HCD "scouting" scan to trigger EThcD will be compatible with isobaric labeling for quantitation (iTRAQ, TMT) of O-GlcNAcylation (9,15,27) and is a subject of our future studies.…”

“…Wheat germ agglutinin (WGA), a lectin that has a weak affinity for GlcNAc, has been used successfully for O-GlcNAc-modified peptide enrichment across a wide range of cells and tissues (23)(24)(25). Although WGA-based lectin weak affinity chromatography (LWAC) has provided some of the largest datasets of O-GlcNAc-modified peptides (24,26,27) its lack of specificity is well-documented, and the amount of input peptide needed can be prohibitive (26,28,29).…”

Targeted inhibition of STAT3 in neural stem cells promotes neuronal differentiation and functional recovery in rats with spinal cord injury

“…However, accumulating evidence has suggested that NSC-based therapy cannot achieve optimal results, mainly due to the limited differentiation potential of neurons and excessive differentiation of astrocytes, which contributes to glial scarring ( 13 ). Natarajan et al ( 14 ) demonstrated that treatment with an inhibitor of STAT3 promoted NSC differentiation into neurons and suppressed glial differentiation in vitro , a finding that was also subsequently confirmed by White et al ( 15 ). However, few studies have assessed the effects of STAT3 silencing on NSCs transplantation and its functional outcomes downstream in vivo .…”

“…NSCs were cultured in 5% CO 2 at 37˚C and passaged weekly by digesting with Accutase (EMD Millipore) in the aforementioned medium. NSCs at passages 2-4 were used for subsequent experiments ( 15 , 16 ).…”