Significance O-GlcNAcylation is an abundant posttranslational modification suggested to have both neuroprotective and neurodegenerative functions. Although previous studies on O-GlcNAc have illustrated its potential to modulate preexisting phenotypes in neurodegenerative animal models, the importance of O-GlcNAcylation in both the induction of neuropathology and the functioning of healthy adult neurons remained unclear. We generated a forebrain-specific O-GlcNAc transferase conditional knockout mouse and found that diminution of O-GlcNAc signaling induced progressive neurodegeneration in vivo, including pathogenic processing of tau and amyloid precursor protein, widespread neuronal death, gliosis, and memory loss. These results indicate that O-GlcNAcylation regulates pathways critical for neuronal health and survival and that modulating O-GlcNAc signaling may represent a neuroprotective strategy for neurodegenerative diseases.

Abstract O-GlcNAc glycosylation (or O-GlcNAcylation) is a dynamic, inducible posttranslational modification found on proteins associated with neurodegenerative diseases such as α-synuclein, amyloid precursor protein, and tau. Deletion of the O-GlcNAc transferase (ogt) gene responsible for the modification causes early postnatal lethality in mice, complicating efforts to study O-GlcNAcylation in mature neurons and to understand its roles in disease. Here, we report that forebrain-specific loss of OGT in adult mice leads to progressive neurodegeneration, including widespread neuronal cell death, neuroinflammation, increased production of hyperphosphorylated tau and amyloidogenic Aβ-peptides, and memory deficits. Furthermore, we show that human cortical brain tissue from Alzheimer’s disease patients has significantly reduced levels of OGT protein expression compared with cortical tissue from control individuals. Together, these studies indicate that O-GlcNAcylation regulates pathways critical for the maintenance of neuronal health and suggest that dysfunctional O-GlcNAc signaling may be an important contributor to neurodegenerative diseases.

Despite the increasing prevalence of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease, there still are no effective treatments to prevent, cure, or stop neurons from degenerating. Neurodegeneration at the cellular level involves the dysfunction of several processes important for proper neuronal function and health, including synaptic plasticity (1), autophagy (2), mitochondrial dynamics (3), and cellular metabolism (4). An improved understanding of the protective factors responsible for maintaining neuronal health is essential for uncovering the biology of neuropathies and the development of more effective treatments for neurodegenerative conditions.

O-GlcNAc glycosylation (or O-GlcNAcylation) is a dynamic posttranslational modification that regulates important processes such as transcription (5, 6), proteostasis (7), the stress response (8), autophagy (9), and metabolism (6, 10). The O-GlcNAc transferase enzyme OGT catalyzes the covalent attachment of N-acetyl-d-glucosamine to serine or threonine residues of proteins, whereas β-N-acetylhexosaminidase (also known as “O-GlcNAcase,” OGA) removes it (6). Although OGT is ubiquitously expressed, it is particularly abundant in neurons, where it is enriched in the nucleus (6) and at synapses (11). OGT participates in specialized neuronal processes, including activity-dependent transcription and long-term depression via regulation of cAMP-responsive element binding protein (CREB) (5) and the AMPA receptor GluA2 subunit (12), respectively. O-GlcNAcylation also has been implicated in neurodegenerative diseases and is reported on proteins such as amyloid precursor protein (APP) (13), α-synuclein (14), neurofilament M (NFM) (15), and tau (16). Several studies have suggested neuroprotective roles for the modification. For example, increasing global levels of O-GlcNAcylation in neurons decreased the production of pathological forms of NFM and tau (15, 16) and directly inhibited the aggregation of both tau (17) and α-synuclein (18). Glycosylation of nicastrin, a component of the γ-secretase complex, enhanced the nonamyloidogenic processing of APP (13, 19). Moreover, raising O-GlcNAcylation levels by pharmacological inhibition of OGA attenuated amyloid-β deposition, tau phosphorylation, motor deficits, and memory impairments in certain AD mouse models (17, 19). On the other hand, studies also have suggested neurodegenerative roles for O-GlcNAcylation. For instance, increasing O-GlcNAcylation levels exacerbated neurodegenerative phenotypes of tauopathy, amyloid β-peptide, and polyglutamine expansion in Caenorhabditis elegans models, whereas decreasing O-GlcNAcylation levels rescued those phenotypes (20). Thus, it is unclear whether O-GlcNAcylation has neuroprotective or neurodegenerative functions and whether aberrant O-GlcNAc signaling plays a direct role in the induction of neuronal pathology in vivo.

Understanding the functions of O-GlcNAcylation in the mature brain and in neurodegenerative diseases has been complicated by the difficulty of knocking out the ogt gene. Because OGT is ubiquitously expressed and is essential for cell-cycle progression and proliferation (6), constitutive deletion of OGT in mice resulted in embryonic lethality, and neuron-specific OGT deletion using a Syn1-Cre transgene led to severe developmental defects, increased phosphorylation of tau, and early postnatal lethality (21). In this study, we produced a forebrain-specific OGT conditional knockout (OGT cKO) mouse to assess the effects of O-GlcNAc depletion on learning, memory, and neuronal function in adult mice. The mice were phenotypically normal at birth but at 2 mo of age developed signs of severe, progressive neurodegeneration, including loss of neurons, gliosis, increased hyperphosphorylated tau and Aβ−peptides, altered expression of genes involved in inflammation and cell-cycle arrest, and memory impairments. Together, our studies link OGT and diminished O-GlcNAcylation to the induction of neuropathological phenotypes and may have important implications for the study and treatment of neurodegenerative diseases.

Discussion In this study, we generated a forebrain-specific ogt cKO mouse to study the roles of O-GlcNAcylation in adult neurons. Because the mice survived to adulthood, the effects of ogt deletion on learning, memory, and adult neuronal function could be evaluated directly. Loss of OGT led to progressive neuronal death and other phenotypes associated with neurodegenerative diseases, including gliosis, activation of immune-response and cell-cycle genes, aberrant phosphorylation of tau, and amyloidogenic Aβ-42 peptides. OGT cKO mice also displayed behavioral deficits such as abnormal nesting behavior, memory impairments, and increased anxiety, which are characteristics observed in neurodegenerative mouse models of AD (31), obsessive-compulsive disorder (32), and schizophrenia (33). Notably, we found that human AD patients showed a significant decrease in OGT protein expression levels in the frontal cerebral cortex compared with control individuals, and this decrease correlated with progressive cognitive decline. Together, our studies reveal important neuroprotective roles for O-GlcNAcylation and suggest that alterations in O-GlcNAc signaling may contribute to neuronal pathology. Both tau phosphorylation and APP processing were significantly altered in OGT cKO mice. Upon ogt deletion, we observed enhanced tau aggregation and hyperphosphorylation at sites associated with neurofibrillary tangles. These findings further support previous studies suggesting a reciprocal relationship between O-GlcNAc and phosphorylation on tau. For example, increasing global O-GlcNAc levels reduced tau phosphorylation at Ser-199, Thr-212, Thr-217, Ser-262, Ser-396, and Ser-422 in mouse brain slices (16). Moreover, inhibition of OGA elevated O-GlcNAc levels and decreased tau hyperphosphorylation in an AD mouse model (17). Although we observed no significant change in the levels of tau O-GlcNAcylated at Ser-400, it is possible that O-GlcNAcylation of tau is differentially regulated at specific sites or that a small amount of residual OGT may be sufficient to maintain O-GlcNAc levels at this site. Previous studies have suggested that higher O-GlcNAcylation levels can enhance nonamyloidogenic processing of APP by raising α-secretase activity (13) and lowering γ-secretase activity (19). In accordance with these observations, we found that loss of OGT enhanced the pathological processing of APP in vivo, increasing the ratio of the amyloidogenic 42-mer Aβ-peptide to the 40-mer. Notably, accumulation of hyperphosphorylated tau and Aβ-peptides is not observed in many neurodegenerative mouse models, and the two phenotypes are rarely observed together. For example, 5XFAD mice that express five different familial AD mutations in APP and presenilin1 showed no accumulation of hyperphosphorylated tau (34), and mutant P301L tau transgenic mice displayed no increases in amyloid beta load (35) despite the presence of extensive neuronal death. Thus, the defects in tau phosphorylation and APP processing observed in OGT cKO mice are not likely to be indirect effects caused by a global requirement for OGT in proper neuronal function and survival. Together, the findings suggest that the O-GlcNAc modification plays a central role in regulating both APP and tau and that dysfunctional O-GlcNAc signaling may contribute to improper APP processing and tau pathology. We performed gene microarray analyses to obtain insights into the global, systems-level changes induced by loss of OGT. Our studies revealed that several cell-cycle genes, including Mki67, Prc1, and Spc25, were up-regulated in OGT cKO mice. Consistent with an important role for OGT in cell-cycle regulation, O-GlcNAcylation previously has been shown to modulate cell-cycle progression by prolonging cyclin A and B expression and catalyzing proteolytic maturation of the critical cell-cycle regulator, host cell factor-1 (HCF-1) (36, 37). Interestingly, several genes involved in controlling oxidative stress were also significantly up-regulated in OGT cKO mice, including heme oxygenase 1 (Hmox1), cyclooxygenase 1 (Ptgs1), and activity-dependent neuroprotective protein homeobox 2 (ADNP2). O-GlcNAcylation previously has been shown to be required for stress granule assembly in response to oxidative stress and to decrease oxidative stress in cardiomyocytes by reducing reactive oxygen species (8, 38). Thus, one important mechanism by which OGT may exert its neuroprotective effects is through the regulation of critical cell-cycle regulators and modulators of oxidative stress. The two-hit hypothesis of AD postulates that both oxidative stress and mitotic dysregulation are necessary and sufficient to cause the neurodegeneration associated with AD (39). Further supporting the gene-expression analyses, we found that cyclin A2, a marker of cell-cycle progression, was increased in OGT cKO mice, providing strong evidence for enhanced cell-cycle progression in the hippocampi of these mice. Moreover, CDK5 protein expression levels were significantly decreased upon loss of OGT. CDK5 represses neuronal cell-cycle advancement (40) and is an important upstream regulator of oxidative stress through phosphorylation of peroxiredoxin I/II and p53 (39). Interestingly, a recent paper suggests that blocking O-GlcNAcylation of CDK5 can lead to neuronal apoptosis by enhancing its association with the p53 pathway (41). Several other studies have implicated aberrant cell-cycle advancement in AD-associated neurodegeneration through deregulation of CDK5 levels and activity (40, 42). Together, these results suggest that a major mode by which OGT ablation leads to neurodegeneration is through cell-cycle dysfunction. Future studies will focus on understanding the detailed mechanisms by which OGT affects CDK5 function, the cell cycle, and oxidative stress in neurons. Lagerlöf et al. reported that the tamoxifen-induced deletion of ogt from αCaMKII+ neurons in adult mice leads to obesity caused by overeating (43). This phenotype was the result of reduced satiety caused by OGT ablation in the paraventricular nucleus (PVN) of the hypothalamus. Interestingly, no change in neuronal number in the hippocampus or PVN was noted upon quantification of DAPI+ cells, although the age of the mice analyzed was not reported. There could be several explanations for the phenotypic differences between Lagerlöf’s model and ours. First, there likely are differences in the timing of the OGT knockout in the αCaMKII-Cre versus the αCaMKII-CreERT2 tamoxifen-inducible systems. In our study, loss of OGT and O-GlcNAcylation began at 4 wk of age, and the neuronal loss and degenerative phenotypes were not significant until 8 wk of age. In the Lagerlöf study, tamoxifen injections were initiated at 6 wk of age, and phenotypic responses were monitored up to 4 wk later. Second, it is possible that the neurodegenerative phenotypes were not yet apparent during the window of their study, because tamoxifen-induced Cre recombination can take several days. Furthermore, variability in the location and timing of αCaMKII promoter-driven Cre recombination has been described previously and attributed to differences in the location of αCaMKII-Cre transgene insertion within the genome (44). Alternatively, excitatory forebrain neurons may be more vulnerable to OGT deletion at specific stages of maturation. Last, any effects on satiety in our model were likely obscured by the strong neurodegenerative phenotype. The phenotypic differences between the two OGT cKO models suggest that OGT plays specific roles in neuronal health and homeostasis during various stages, and they underscore the importance of the precise spatiotemporal coordination of O-GlcNAcylation. The O-GlcNAc modification appears to play multiple, complex roles in neurodegeneration, and previous studies paradoxically have suggested that decreasing O-GlcNAcylation has both neuroprotective and neurodegenerative effects (17, 20). We show that loss of O-GlcNAcylation in healthy neurons leads to progressive neurodegeneration in vivo, providing strong evidence that O-GlcNAc has neuroprotective functions in adult mammalian neurons. The neurodegenerative, transcriptional, and behavioral phenotypes observed in OGT cKO mice are shared by many AD mouse models, suggesting common mechanisms of neurodegeneration. It is noteworthy that loss of OGT alone is sufficient to induce neurodegenerative pathologies and that this neurodegeneration occurs relatively rapidly. Induction of such phenotypes in other mouse models generally requires mutation or overexpression of multiple familial AD-related proteins such as tau, APP, and presenilin (31), and the pathological phenotypes take 9–12 mo to progress (23, 31). The rapid course of neurodegeneration in OGT cKO mice underscores the critical importance of OGT in the maintenance of adult neuronal health and suggests that dysfunctional O-GlcNAc signaling may be an important contributor to neuronal pathology. Collectively, our studies provide a direct link between the ablation of O-GlcNAcylation and the induction of neurodegenerative phenotypes, suggesting that strategies to control O-GlcNAcylation and its neuroprotective effects may represent an approach for the treatment of neurodegenerative conditions.

Methods Human Tissue. Brain samples of frontal cortex from control (n = 6), Braak stage VI (n = 8), and Braak stage IV-V (n = 4) patients were obtained from the Alzheimer’s Disease Research Center at the University of California, Los Angeles. Samples were from patients with a similar age, sex, and postmortem interval. Mice. αCaMKII-Cre mice were provided by Mary Kennedy, California Institute of Technology, Pasadena, CA (22), and OGTfl mice (B6.129-Ogttm1Gwh/J) were purchased from Jackson Laboratories. Both lines were backcrossed six times into the C57/Bl6 background. Details of breeding are provided in SI Methods. All animal procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the California Institute of Technology. IHC. Mice were transcardially perfused with 4% (wt/vol) paraformaldehyde (PFA) and were fixed overnight in 4% (wt/vol) PFA at 4 °C, followed by cryoprotection with 15–30% (wt/vol) sucrose. Sagittal sections (20 μm) were obtained using a Leica CM1850 cryostat and were stored in an ethylene glycol solution at −20 °C. Slices were stained with primary antibody in 2% (vol/vol) goat serum, 0.1% Triton-X 100 in PBS overnight at 4 °C. Slices were mounted with Vectashield (Vector Labs, H1200), coded, and imaged with a Zeiss 700 confocal microscope, with the investigator blind to the genotype. Images were collected from slices spaced 100 μm apart through the region of interest. A minimum of 12 slices were quantified for each animal, and a minimum of four animals were evaluated for each genotype. Quantification of fluorescence intensity was performed as previously described (45) and was analyzed using a two-tailed, unpaired Student’s t test. Additional materials and methods, including detailed IHC procedures, behavioral experiments, ELISA, microarray, and qRT-PCR analyses, are provided in SI Methods. A list of primers used in this study can be found in Table S5.

SI Methods Antibodies. The following primary antibodies were used for immunohistochemistry (1:250 dilution unless otherwise stated) or Western blotting (1:1,000 unless otherwise stated): OGT antibodies: TI-14 (Sigma, O6014), AL-25 and AL-28, gift of G. W. Hart, Johns Hopkins University School of Medicine, Baltimore; Tubulin (Sigma, T9026); O-GlcNAc (RL2) (Pierce, MA1-072); Tau antibodies: pThr231-AT180 (Pierce, MN1040), pSer202/Thr205-AT8 (Pierce, MN1020), pThr205 (Life Technologies, 44738G), pThr231 (Millipore, AB9668), tau-5 (Millipore, MAB361), pSer396 (Abcam, 32057); O-GlcNAc Ser400 (AnaSpec, AS-55945, Western blotting: 1:200); NeuN (Millipore, MAB377); NeuN (Abcam, ab134014, IHC: 1:100); GFP (Life Technologies, G10362); Iba-1 (Wako, 019-19741); GFAP (Cell Signaling Technologies, 3670P); CDK5 (Santa Cruz Biotechnology, SC-173); Cyclin A2 (Abcam, ab32386, IHC: 1:50); PCNA (Abcam, ab18197, IHC: 1:100); BrdU (Abcam, ab6326, IHC: 1:100). The following secondary antibodies were used for IHC (1:400 dilution): goat anti-mouse Alexa Fluor-405 (Life Technologies, A-31553), goat anti-mouse Alexa Fluor-488 (Life Technologies, A-11001), goat anti-mouse Alexa Fluor-546 (Life Technologies, A-11003), goat anti-rabbit Alexa Fluor-488 (Life Technologies, A-11008); goat anti-rabbit Alexa Fluor-405 (Life Technologies, A-31556); goat anti-rabbit Alexa Fluor-546 (Life Technologies, A-11035); goat anti-chicken Alexa Fluor-546 (Life Technologies, A-11040); goat anti-rat Alexa Fluor-488 (Life Technologies, A-11006). The following secondary antibodies were used for Western blotting (1:10,000 dilution): goat anti-mouse Alexa Fluor-680, highly cross-adsorbed (Life Technologies, A21058); goat anti-mouse Alexa-Fluor-790 (Life Technologies, A11357); goat anti-rabbit Alexa Fluor-680 (Life Technologies, A21109); goat anti-rabbit Alexa Fluor-790 (Life Technologies, A11369). Mice. Mice were housed in groups whenever possible, and all animal procedures were performed in accordance with Institutional Animal Care and Use Committee guidelines of the California Institute of Technology. Only male mice were considered in this study because random X inactivation of the ogt gene in females would have complicated phenotype analysis. OGT cKO mice were produced at the expected frequencies and were indistinguishable from WT littermates at birth. Because OGT cKO mice did not breed well, OGTfl and αCaMKII-Cre mouse lines were maintained separately. By breeding homozygous OGTfl females (OGTfl−/−) with heterozygous αCaMKII-Cre males (αCaMKII-Cre+/−), OGT cKO (OGTfl -/Y;αCaMKII-Cre−/+) mice were produced, along with WT littermates (OGTfl -/Y; αCaMKII-Cre+/+). Because OGT cKO mice had an excessive grooming phenotype that resulted in skin lesions, the mice were culled at 6–7 mo for humane reasons. IHC. For Nissl staining, slices were incubated in 100% EtOH followed by Histo-Clear (Electron Microscopy Sciences, 641101-01) for 2 min each. Slices then were sequentially rehydrated in 100, 70, and 50% (vol/vol) aqueous EtOH for 2 min each. Slices were stained with a 0.1% cresyl violet solution for 10 min, rinsed in distilled water, and differentiated in 90% (vol/vol) EtOH with 1% acetic acid for 10 s. After a final washing in 100% EtOH, slices were cleared for 2 min in Histo-Clear and mounted with Vectashield. For IHC staining with Fluoro-Jade C (Millipore, AG325) and Thioflavin S (Sigma, T1892), slices were incubated in 0.06% potassium permanganate solution for 10 min, washed in double-distilled H 2 O, and transferred to a 0.0001% solution of Fluoro-Jade C in 0.1% acetic acid or a 0.05% Thioflavin S solution in 50% EtOH for 10 min. Following a 5-min washing in H 2 O (or 50% EtOH for Thioflavin S), slices were mounted with Vectashield. For Cyclin A2 and PCNA staining, slices underwent antigen retrieval for 1 h at 37 °C in citrate buffer (0.01 M sodium citrate in 0.1% Triton X-100 in H 2 O, pH 6.0) before blocking. For BrdU staining, slices were incubated in 2 N HCl for 20 min at 37 °C followed by 10 min of neutralization (two 5-min incubations) in 0.1 M sodium borate buffer, pH 8.5. Next, the slices were rinsed three times with PBS and then were blocked as normal. Brain samples of frontal cortex from control (n = 6), Braak stage VI (n = 8), and Braak stage IV-V (n = 4) patients were obtained from the Alzheimer’s Disease Research Center at the University of California, Los Angeles. Samples were from patients with a similar age, sex, and postmortem interval. Slices were pretreated with 0.01 M citrate buffer (37 °C, pH 3.5) followed by treatment with Sudan black [0.1% (wt/vol) in 70% EtOH] (Sigma, 199664) and were stained as described above. Four representative regions were imaged in each slice, and at least four slices were imaged per individual. Images were coded, and discrete quantitative scoring of the OGT protein signal (on a scale from 1 to 4) was carried out by researchers blind to sample ID. Behavioral Studies. Rotarod tests were performed as described previously (46). Briefly, the latency to fall from a rotarod (Ugo Basile) with rotation beginning at 5 rpm and accelerating to 40 rpm over 240 s was scored. Mice were allowed to stay on the rotarod for a maximum of 300 s. Mice were trained for two consecutive days, with two trials per day with a 10-min intertrial interval. On testing days, two trials were performed and averaged. Open field tests were performed as described previously (46). Briefly, mice were placed in the lower left corner of a 50 × 50 cm white box, and activity was recorded for 10 min by a ceiling-mounted video camera. The total distance traveled and center entries were scored. Fear conditioning was performed as described previously (47). Briefly, mice were placed in a chamber (Med Associates) for the first time and allowed to explore for 120 s, after which an audible tone was played for 30 s. During the last 2 s of tone, a 0.7-mA foot shock was applied. This 150-s sequence was repeated; then mice were returned to their home cages. After 24 h, mice were placed in the same chamber and monitored for 5 min to evaluate context-dependent memory. Mice were returned to their home cages for 1 h and then were placed into a chamber with altered scent, shape, and lighting for 5 min, with a tone playing for the last 180 s. All freezing quantification was done by Med Associates software, and freezing was normalized to pretone freezing percent for cued tests. No significant difference in pretone freezing was observed between WT and OGT cKO mice using an unpaired, two-tailed Student’s t test. TUNEL Labeling. TUNEL labeling was performed using the ApopTag Fluorescein Kit (Millipore, S7110) according to the manufacturer’s protocol with one modification. Briefly, slices were treated with proteinase-K for 15 min, washed in PBS, and microwaved for 30 s in 10 mM citrate buffer, pH 3. Slices then were incubated with terminal deoxynucleotidyl transferase (TdT) enzyme for 1 h at 37 °C. After a PBS washing step, an anti-digoxigenin antibody conjugated to fluorescein was applied for 1 h at room temperature. Slides then were washed in PBS and mounted with Vectashield. Costaining with an anti-O-GlcNAc antibody (RL2) was done after the anti-digoxigenin antibody incubation, as described in Methods, IHC in the main text. BrdU Assay for Neurogenesis. The BrdU assay for neurogenesis was performed as previously described. Briefly, 2-mo-old male mice were injected i.p. with 100–200 μL of sterile 10 mg/mL BrdU (Sigma Aldrich, B5002-1G) in PBS buffer to achieve a final BrdU concentration of 200 mg/kg. Twenty-four hours after the injection, the mice were transcardially perfused followed by dissection, fixation, and IHC staining as described previously (48). Aβ-Peptide ELISA. The Amyloid-beta ELISA Kits 1-40 and 1-42 (Covance, SIG-38954 and 38956) were used to quantify Aβ-peptide levels following the manufacturer’s protocol. Whole cortices were lysed in TBS [50 mM Tris⋅HCl (pH 7.5), 150 mM NaCl] containing 1% Triton-X100 and protease inhibitor mixture (Roche, 11697498001) and were centrifuged at 100,000 × g. The supernatant was assayed following the ELISA kit protocol. Microarray, WGCNA, and Gene Ontology Analysis. Hippocampal tissue was isolated from 3-wk-old or 2-mo-old mice, flash frozen, and stored at −80 °C in nuclease-free tubes until extraction. Total RNA was extracted using an RNeasy kit with RNase-free DNase treatment per the manufacturer’s instructions (Qiagen, 79254). Following RNA extraction, the samples were sent to Phalanx Biotech Group for microarray analysis. Briefly, a library was generated from the mRNA using the Ambion Amino Allyl MessageAmp II aRNA Amplification Kit (Life Technologies, AM1753) and applied to Mouse OneArray v2 containing probes for more than 23,000 genes (Phalanx Biotech). The microarray intensities were normalized using more than 800 control probes throughout the array. In addition to microarray analysis, the extracted mRNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, 1708891) for qRT-PCR. Following reverse transcription, the cDNA was used for qPCR analysis using PerfeCTa SYBR Green FastMix with ROX (VWR, 101414-280) on an AB7300 Real Time System (Applied Biosystems). The qRT-PCR expression values were calculated using the comparative cycle threshold (C T ) method, and expression values were normalized to the geometric mean of five different reference genes as previously described (49). A list of primers used in this study can be found in Table S5. Statistical significance of qRT-PCR results was determined using an unpaired t test. Reported P values for the microarray results from this study were calculated using a Bonferroni correction (50). Using the microarray data, we performed WGCNA in R package version 3.0.0 on all microarray-detected genes using the protocols previously described (51). Following hierarchical clustering and module assignment, gene ontology enrichment analysis was performed using Bioconductor packages GO and the Database for Annotation, Visualization and Integrated Discovery (DAVID) as previously described (52). The package VisANT version 5.0 was used to visualize WGCNA gene networks using a correlation cutoff of 0.1 (cor > 0.1) (53). Statistical Analyses. Unless otherwise stated, all results are expressed as the mean ± SEM and are representative of at least four experimental replicates. Statistical tests for significant deviation between samples were performed using an unpaired, two-tailed Student’s t test.

Acknowledgments We thank Dr. G. W. Hart for generously providing the AL-25 and AL-28 anti-OGT antibody; Dr. P. B. Dervan for quantitative PCR instrumentation; Dr. P. H. Patterson and Dr. K. Winden for helpful discussions; and John Thompson for careful reading of the manuscript. This research was supported by NIH Grant R01 GM084724-11 (to L.C.H-W.) and a National Defense Science and Engineering Graduate Fellowship (E.H.J.). H.V.V. was supported in part by National Institute on Aging Grant P50 AG16570.