Proteomic Profiling of Astrocytic O-GlcNAc Transferase-Related Proteins in the Medial Prefrontal Cortex

Abstract

The medial prefrontal cortex (mPFC), a key part of the brain networks that are closely related to the regulation of behavior, acts as a key regulator in emotion, social cognition, and decision making. Astrocytes are the majority cell type of glial cells, which play a significant role in a number of processes and establish a suitable environment for the functioning of neurons, including the brain energy metabolism. Astrocyte’s dysfunction in the mPFC has been implicated in various neuropsychiatric disorders. Glucose is a major energy source in the brain. In glucose metabolism, part of glucose is used to convert UDP-GlcNAc as a donor molecule for O-GlcNAcylation, which is controlled by a group of enzymes, O-GlcNAc transferase enzyme (OGT), and O-GlcNAcase (OGA). However, the role of O-GlcNAcylation in astrocytes is almost completely unknown. Our research showed that astrocytic OGT could influence the expression of proteins in the mPFC. Most of these altered proteins participate in metabolic processes, transferase activity, and biosynthetic processes. GFAP, an astrocyte maker, was increased after OGT deletion. These results provide a framework for further study on the role of astrocytic OGT/O-GlcNAcylation in the mPFC.

O-GlcNAc transferase in astrocytes modulates depression-related stress susceptibility through glutamatergic synaptic transmission

“…Following CSDS, the dynamic change in glutamate signaling was significantly increased in the control mice in the FIT, which is similar to what occurred in previous reports ( 61 , 64 ). In contrast, the deletion of OGT decreased GLT-1 O-GlcNAcylation and enhanced glutamate uptake under stress ( Figure 7 and Figure 8 ) without affecting the expression of GLT-1 in the mPFC, which is consistent with our previous study ( 41 ).…”

“…Furthermore, we employed astrocytic OGT-KO mice to investigate whether the O-GlcNAcylation of GLT-1 was regulated by astrocytic OGT in the mPFC. Tissue from the mPFC was collected, and the co-IP analysis showed that GLT-1 O-GlcNAcylation was significantly reduced in the mPFC in the astrocyte-specific cKO mice compared with that in the WT controls ( Figure 7, G and H ), but this reduction did not affect the total expression of GLT-1 ( Supplemental Figure 10 , D and E) as in our previous proteomic analysis ( 41 ). These results indicate that astrocytic OGT regulates the O-GlcNAcylation of GLT-1 in the mPFC.…”

“…The Ogt gene localizes at Xq13 , and global OGT deletion results in embryonic lethality ( 40 ). To investigate potential astrocyte- or neuron-specific effects associated with depressive-related phenotypes derived from Ogt gene deletion, we generated astrocyte-specific ( Figure 2A ) ( 41 ) and neuron-specific ( Supplemental Figure 5A ) conditional Ogt deletion mouse lines by crossing the floxed OGT allele with Fgfr3-iCreER T2 or CaMKIIα-creER T2 transgenic lines. Consistent with a previous report ( 15 ), the OGT neuron-specific conditional knockout (cKO) mice showed an increased growth rate and body size after tamoxifen (TAM) injection to induce the expression of Cre recombinase ( Supplemental Figure 5 , B–E).…”

“…The astrocyte-specific cKO animals exhibited normal growth rates and body sizes ( Supplemental Figure 3 , A–D). Additionally, the deletion of OGT in astrocytes did not lead to gross anatomical changes in the brain or the densities of astrocytes and neurons ( Supplemental Figure 3 , E–J) ( 41 ). To confirm an astrocyte-specific OGT reduction in the Fgfr3-iCreER T2 ; OGT fl/Y mice, we employed a specific OGT antibody and costained with glial fibrillary acidic protein (GFAP), a marker of astrocytes.…”

“…More than 4,000 proteins in all major cell compartments, including the membrane, cytoplasm, mitochondria, and nucleus, have been identified as being O-GlcNAcylated ( 8 , 53 ). In our previous study, we deleted Ogt in astrocytes and found alterations in metabolic processes, transferase activity, and biosynthetic processes ( 41 ), but the function of OGT in astrocytes remained unknown. Moreover, O-GlcNAcylation is highly dynamic and reversible and is involved in energy metabolism and stress responses.…”