Supplementary Figure 1. Expression levels of EGR-1, GRASP55 and eIF4G following kainic acid treatment of rats. Cortical neuronal lysates were obtained 6 h post-injection of kainic acid or PBS. EGR-1 expression changed by 1.8 ± 0.2, GRASP55 expression by 0.61 ± 0.09 and eIF4G expression by 1.5 ± 0.1. Data represent the mean ± s.d. for 3 experiments.

Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here, we describe a powerful method to visualize the O-GlcNAc-modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation, or expensive radiolabels. In addition, the glycosylation state (e.g., mono-, di-, tri-) of proteins is established, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation, and discover an unexpected reverse yin-yang relationship on the transcriptional repressor MeCP2, which was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs.

The transcription factor CREB is a key regulator of many neuronal processes, including brain development, circadian rhythm, and long-term memory. Studies of CREB have focused on its phosphorylation, although the diversity of CREB functions in the brain suggests additional forms of regulation. Here we expand on a chemoenzymatic strategy for quantifying glycosylation stoichiometries to characterize the functional roles of CREB glycosylation in neurons. We show that CREB is dynamically O-GlcNAc-modified in response to neuronal activity and glycosylation represses CREB-dependent transcription by impairing its association with the co-activator CRTC/TORC. Blocking glycosylation of CREB altered cellular function and behavioral plasticity, enhancing both axonal and dendritic growth and long-term memory consolidation. Our findings demonstrate a new role for O-glycosylation in memory formation and provide a mechanistic understanding of how glycosylation contributes to critical neuronal functions. Moreover, we identify a previously unknown mechanism for the regulation of activity-dependent gene expression, neural development, and memory.

Identifying the mechanisms through which genetic risk causes dementia is an imperative for new therapeutic development. Here, we apply a multi-stage, systems biology approach to elucidate disease mechanisms in frontotemporal dementia (FTD). We identify two gene co-expression modules that are preserved in mice harboring mutations in MAPT, GRN, and other dementia mutations on diverse genetic backgrounds. We bridge the species divide via integration with proteomic and transcriptomic data from human brain to identify evolutionarily conserved, disease-relevant networks. We find that overexpression of miR-203, a hub of a putative regulatory miRNA module, re-capitulates mRNA co-expression patterns associated with disease state and induces neuronal cell death, establishing this miRNA as a regulator of neurodegeneration. Using a database of drug-mediated gene expression changes, we identify small molecules that can normalize the disease-associated modules and validate this experimentally. Our results highlight the utility of an integrative, cross-species, network approach to drug discovery.