Chemical Modulation of Protein O-GlcNAcylation via OGT Inhibition Promotes Human Neural Cell Differentiation

Abstract

The enzymes that determine protein O-GlcNAcylation, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), act on key transcriptional and epigenetic regulators, and both are abundantly expressed in the brain. However, little is known about how alterations in O-GlcNAc cycling affect human embryonic stem cell (hESC) neural differentiation. Here, we studied the effects of perturbing O-GlcNAcylation during neural induction of hESCs using the metabolic inhibitor of OGT, peracetylated 5-thio-N-acetylglucosamine (Ac4-5SGlcNAc). Treatment of hESCs with Ac4-5SGlcNAc during induction limited protein O-GlcNAcylation and also caused a dramatic decrease in global levels of UDP-GlcNAc. Concomitantly, a subpopulation of neural progenitor cells (NPCs) acquired an immature neuronal morphology and expressed early neuronal markers such as β-III tubulin (TUJ1) and microtubule associated protein 2 (MAP2), phenotypes that took longer to manifest in the absence of OGT inhibition. These data suggest that chemical inhibition of OGT and perturbation of protein O-GlcNAcylation accelerate the differentiation of hESCs along the neuronal lineage, thus providing further insight into the dynamic molecular mechanisms involved in neuronal development.

Metabolic Inhibitors of O‐GlcNAc Transferase That Act In Vivo Implicate Decreased O‐GlcNAc Levels in Leptin‐Mediated Nutrient Sensing

“…Notably, others have shown that Ac 4 5SGlcNAc ( 3 ‐OAc) appears to be able to inhibit other glycosyltransferases in cell lines . We found, however, by lectin blot assessment that 12 induced no apparent changes in other forms of protein glycosylation in all tissues tested (Figure S7) even at a dose of 300 mg kg −1 after 48 h, which is consistent with a recent report on the use of the parent compound Ac 4 5SGlcNAc ( 3 ‐OAc) in cells …”

Metabolism in pluripotency: Both driver and passenger?

“…The hexosamine biosynthesis pathway (HBP) is a nutrient-sensing pathway that depends on glucose, glutamine, and cytosolic Ac-CoA levels (Figure 2 Conversely, a decrease in O-GlcNAcylation disrupts self-renewal and reprogramming (57), but promotes differentiation in neuronal cells (63)(64)(65). Moreover, CPTFs such as OCT4 and SOX2 harbor an O-GlcNAcylation motif that is quickly erased upon differentiation (57).…”

O-GlcNAc cycling in the developing, adult and geriatric brain

“…Total O-GlcNAc in brain homogenate is the highest during pre- and perinatal development and decreases during later development (Yanagisawa and Yu 2009 ; Liu et al 2012 ). In vitro , early neuronal differentiation also has been associated with a decrease in global O-GlcNAcylation, albeit the global O-GlcNAc levels appear to cycle from day to day (Andres et al 2017 ; Speakman et al 2014 ). At around one month after birth, overall O-GlcNAc plateaus at a relatively low level and then remains stable for most of adulthood (Liu et al 2012 ).…”

“…Overall OGT protein levels and activity in vivo are mostly stable during development through adulthood (Yanagisawa and Yu 2009 ; Liu et al 2012 ). Neuronal differentiation in vitro , in contrast, has been associated with a small decrease in OGT mRNA and protein abundance (Andres et al 2017 ; Maury et al 2013 ). In the very old brain, the total protein level may go down slightly (Fulop et al 2008 ).…”

“…As for OGT, the expression and specific activity of OGA in the brain are among the highest of all organs (Dong and Hart 1994 ; Gao et al 2001 ). Total activity appears to decrease postnatally and, in vitro , neuronal differentiation is associated with lower total OGA protein levels (Liu et al 2012 ; Maury et al 2013 ; Andres et al 2017 ). There are two splice variants of OGA, full-length OGA (OGA) and short OGA (sOGA).…”