Parasympathetic neurons derived from human pluripotent stem cells model human diseases and development.

Autonomic parasympathetic neurons (parasymNs) control unconscious body responses, including "rest-and-digest." ParasymN innervation is important for organ development, and parasymN dysfunction is a hallmark of autonomic neuropathy. However, parasymN function and dysfunction in humans are vastly understudied due to the lack of a model system. Human pluripotent stem cell (hPSC)-derived neurons can fill this void as a versatile platform. Here, we developed a differentiation paradigm detailing the derivation of functional human parasymNs from Schwann cell progenitors. We employ these neurons (1) to assess human autonomic nervous system (ANS) development, (2) to model neuropathy in the genetic disorder familial dysautonomia (FD), (3) to show parasymN dysfunction during SARS-CoV-2 infection, (4) to model the autoimmune disease Sjögren's syndrome (SS), and (5) to show that parasymNs innervate white adipocytes (WATs) during development and promote WAT maturation. Our model system could become instrumental for future disease modeling and drug discovery studies, as well as for human developmental studies.

Regulation of Primary Cilium Length by O-GlcNAc during Neuronal Development in a Human Neuron Model.

The primary cilium plays critical roles in the homeostasis and development of neurons. Recent studies demonstrate that cilium length is regulated by the metabolic state of cells, as dictated by processes such as glucose flux and O-GlcNAcylation (OGN). The study of cilium length regulation during neuron development, however, has been an area left largely unexplored. This project aims to elucidate the roles of O-GlcNAc in neuronal development through its regulation of the primary cilium. Here, we present findings suggesting that OGN levels negatively regulate cilium length on differentiated cortical neurons derived from human-induced pluripotent stem cells. In neurons, cilium length increased significantly during maturation (after day 35), while OGN levels began to drop. Long-term perturbation of OGN via drugs, which inhibit or promote its cycling, during neuron development also have varying effects. Diminishing OGN levels increases cilium length until day 25, when neural stem cells expand and undergo early neurogenesis, before causing cell cycle exit defects and multinucleation. Elevating OGN levels induces greater primary cilia assembly but ultimately results in the development of premature neurons, which have higher insulin sensitivity. These results indicate that OGN levels and primary cilium length are jointly critical in proper neuron development and function. Understanding the interplays between these two nutrient sensors, O-GlcNAc and the primary cilium, during neuron development is important in paving connections between dysfunctional nutrient-sensing and early neurological disorders.

O-GlcNAcylation is crucial for sympathetic neuron development, maintenance, functionality and contributes to peripheral neuropathy.

O-GlcNAcylation is a post-translational modification (PTM) that regulates a wide range of cellular functions and has been associated with multiple metabolic diseases in various organs. The sympathetic nervous system (SNS) is the efferent portion of the autonomic nervous system that regulates metabolism of almost all organs in the body. How much the development and functionality of the SNS are influenced by O-GlcNAcylation, as well as how such regulation could contribute to sympathetic neuron (symN)-related neuropathy in diseased states, remains unknown. Here, we assessed the level of protein O-GlcNAcylation at various stages of symN development, using a human pluripotent stem cell (hPSC)-based symN differentiation paradigm. We found that pharmacological disruption of O-GlcNAcylation impaired both the growth and survival of hPSC-derived symNs. In the high glucose condition that mimics hyperglycemia, hPSC-derived symNs were hyperactive, and their regenerative capacity was impaired, which resembled typical neuronal defects in patients and animal models of diabetes mellitus. Using this model of sympathetic neuropathy, we discovered that O-GlcNAcylation increased in symNs under high glucose, which lead to hyperactivity. Pharmacological inhibition of O-GlcNAcylation rescued high glucose-induced symN hyperactivity and cell stress. This framework provides the first insight into the roles of O-GlcNAcylation in both healthy and diseased human symNs and may be used as a platform for therapeutic studies.

Low glucose induced Alzheimer's disease-like biochemical changes in human induced pluripotent stem cell-derived neurons is due to dysregulated O-GlcNAcylation.

INTRODUCTION: Sporadic Alzheimer's disease (sAD) is the leading type of dementia. Brain glucose hypometabolism, along with decreased O-GlcNAcylation levels, occurs before the onset of symptoms and correlates with pathogenesis. Heretofore, the mechanisms involved and the roles of O-GlcNAcylation in sAD pathology largely remain unknown due to a lack of human models of sAD. METHODS: Human cortical neurons were generated from pluripotent stem cells (PSCs) and treated with glucose reduction media. RESULTS: We found a narrow window of glucose concentration that induces sAD-like phenotypes in PSC-derived neurons. With our model, we reveal that dysregulated O-GlcNAc, in part through mitochondrial dysfunction, causes the onset of sAD-like changes. We demonstrate the therapeutic potential of inhibiting O-GlcNAcase in alleviating AD-like biochemical changes. DISCUSSION: Our results suggest that dysregulated O-GlcNAc might be a direct molecular link between hypometabolism and sAD-like alternations. Moreover, this model can be exploited to explore molecular processes and for drug development. HIGHLIGHTS: Lowering glucose to a critical level causes AD-like changes in cortical neurons. Defective neuronal structure and function were also recapitulated in current model. Dysregulated O-GlcNAcylation links impaired glucose metabolism to AD-like changes. Mitochondrial abnormalities correlate with O-GlcNAcylation and precede AD-like phenotype. Our model provides a platform to study sAD as a metabolic disease in human neurons.

Dual-specificity RNA aptamers enable manipulation of target-specific O-GlcNAcylation and unveil functions of O-GlcNAc on ß-catenin.

O-GlcNAc is a dynamic post-translational modification (PTM) that regulates protein functions. In studying the regulatory roles of O-GlcNAc, a major roadblock is the inability to change O-GlcNAcylation on a single protein at a time. Herein, we developed a dual RNA-aptamer-based approach that simultaneously targeted O-GlcNAc transferase (OGT) and ß-catenin, the key transcription factor of the Wnt signaling pathway, to selectively increase O-GlcNAcylation of the latter without affecting other OGT substrates. Using the OGT/ß-catenin dual-specificity aptamers, we found that O-GlcNAcylation of ß-catenin stabilizes the protein by inhibiting its interaction with ß-TrCP. O-GlcNAc also increases ß-catenin's interaction with EZH2, recruits EZH2 to promoters, and dramatically alters the transcriptome. Further, by coupling riboswitches or an inducible expression system to aptamers, we enabled inducible regulation of protein-specific O-GlcNAcylation. Together, our findings demonstrate the efficacy and versatility of dual-specificity aptamers for regulating O-GlcNAcylation on individual proteins.

Altered O-GlcNAcylation and mitochondrial dysfunction, a molecular link between brain glucose dysregulation and sporadic Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disease that affected over 6.5 million people in the United States in 2021, with this number expected to double in the next 40 years without any sort of treatment. Due to its heterogeneity and complexity, the etiology of Alzheimer's disease, especially sporadic Alzheimer's disease, remains largely unclear. Compelling evidence suggests that brain glucose hypometabolism, preceding Alzheimer's disease hallmarks, is involved in the pathogenesis of Alzheimer's disease. Herein, we discuss the potential causes of reduced glucose uptake and the mechanisms underlying glucose hypometabolism and Alzheimer's disease pathology. Specifically, decreased O-GlcNAcylation levels by glucose deficiency alter mitochondrial functions and together contribute to Alzheimer's disease pathogenesis. One major problem with Alzheimer's disease research is that the disease progresses for several years before the onset of any symptoms, suggesting the critical need for appropriate models to study the molecular changes in the early phase of Alzheimer's disease progression. Therefore, this review also discusses current available sporadic Alzheimer's disease models induced by metabolic abnormalities and provides novel directions for establishing a human neuronal sporadic Alzheimer's disease model that better represents human sporadic Alzheimer's disease as a metabolic disease.

Norepinephrine transporter defects lead to sympathetic hyperactivity in Familial Dysautonomia models.

Familial dysautonomia (FD), a rare neurodevelopmental and neurodegenerative disorder affects the sympathetic and sensory nervous system. Although almost all patients harbor a mutation in ELP1, it remains unresolved exactly how function of sympathetic neurons (symNs) is affected; knowledge critical for understanding debilitating disease hallmarks, including cardiovascular instability or dysautonomic crises, that result from dysregulated sympathetic activity. Here, we employ the human pluripotent stem cell (hPSC) system to understand symN disease mechanisms and test candidate drugs. FD symNs are intrinsically hyperactive in vitro, in cardiomyocyte co-cultures, and in animal models. We report reduced norepinephrine transporter expression, decreased intracellular norepinephrine (NE), decreased NE re-uptake, and excessive extracellular NE in FD symNs. SymN hyperactivity is not a direct ELP1 mutation result, but may connect to NET via RAB proteins. We found that candidate drugs lowered hyperactivity independent of ELP1 modulation. Our findings may have implications for other symN disorders and may allow future drug testing and discovery.

The Beginner's Guide to O-GlcNAc: From Nutrient Sensitive Pathway Regulation to Its Impact on the Immune System.

The addition of N-acetyl glucosamine (GlcNAc) on the hydroxy group of serine/threonine residues is known as O-GlcNAcylation (OGN). The dynamic cycling of this monosaccharide on and off substrates occurs via O-linked ß-N-acetylglucosamine transferase (OGT) and O-linked ß-N-acetylglucosaminase (OGA) respectively. These enzymes are found ubiquitously in eukaryotes and genetic knock outs of the ogt gene has been found to be lethal in embryonic mice. The substrate scope of these enzymes is vast, over 15,000 proteins across 43 species have been identified with O-GlcNAc. OGN has been known to play a key role in several cellular processes such as: transcription, translation, cell signaling, nutrient sensing, immune cell development and various steps of the cell cycle. However, its dysregulation is present in various diseases: cancer, neurodegenerative diseases, diabetes. O-GlcNAc is heavily involved in cross talk with other post-translational modifications (PTM), such as phosphorylation, acetylation, and ubiquitination, by regulating each other's cycling enzymes or directly competing addition on the same substrate. This crosstalk between PTMs can affect gene expression, protein localization, and protein stability; therefore, regulating a multitude of cell signaling pathways. In this review the roles of OGN will be discussed. The effect O-GlcNAc exerts over protein-protein interactions, the various forms of crosstalk with other PTMs, and its role as a nutrient sensor will be highlighted. A summary of how these O-GlcNAc driven processes effect the immune system will also be included.

Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine.

O-GlcNAc is a common post-translational modification of nuclear, mitochondrial, and cytoplasmic proteins that regulates normal physiology and the cell stress response. Dysregulation of O-GlcNAc cycling is implicated in the etiology of type II diabetes, heart failure, hypertension, and Alzheimer's disease, as well as cardioprotection. These protocols cover simple and comprehensive techniques for detecting proteins modified by O-GlcNAc and studying the enzymes that add or remove O-GlcNAc. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Increasing the stoichiometry of O-GlcNAc on proteins before analysis Basic Protocol 2: Detection of proteins modified by O-GlcNAc using antibodies Basic Protocol 3: Detection of proteins modified by O-GlcNAc using the lectin sWGA Support Protocol 1: Control for O-linked glycosylation Basic Protocol 4: Detection and enrichment of proteins using WGA-agarose Support Protocol 2: Digestion of proteins with hexosaminidase Alternate Protocol: Detection of proteins modified by O-GlcNAc using galactosyltransferase Support Protocol 3: Autogalactosylation of galactosyltransferase Support Protocol 4: Assay of galactosyltransferase activity Basic Protocol 5: Characterization of labeled glycans by ß-elimination and chromatography Basic Protocol 6: Detection of O-GlcNAc in 96-well plates Basic Protocol 7: Assay for OGT activity Support Protocol 5: Desalting of O-GlcNAc transferase Basic Protocol 8: Assay for O-GlcNAcase activity.

Nutrient regulation of the flow of genetic information by O-GlcNAcylation.

O-linked-ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification (PTM) that is actively added to and removed from thousands of intracellular proteins. As a PTM, O-GlcNAcylation tunes the functions of a protein in various ways, such as enzymatic activity, transcriptional activity, subcellular localization, intermolecular interactions, and degradation. Its regulatory roles often interplay with the phosphorylation of the same protein. Governed by 'the Central Dogma', the flow of genetic information is central to all cellular activities. Many proteins regulating this flow are O-GlcNAc modified, and their functions are tuned by the cycling sugar. Herein, we review the regulatory roles of O-GlcNAcylation on the epigenome, in DNA replication and repair, in transcription and in RNA processing, in protein translation and in protein turnover.

Excessive O-GlcNAcylation Causes Heart Failure and Sudden Death.

BACKGROUND: Heart failure is a leading cause of death worldwide and is associated with the rising prevalence of obesity, hypertension, and diabetes. O-GlcNAcylation (the attachment of O-linked ß-N-acetylglucosamine [O-GlcNAc] moieties to cytoplasmic, nuclear, and mitochondrial proteins) is a posttranslational modification of intracellular proteins and serves as a metabolic rheostat for cellular stress. Total levels of O-GlcNAcylation are determined by nutrient and metabolic flux, in addition to the net activity of 2 enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Failing myocardium is marked by increased O-GlcNAcylation, but whether excessive O-GlcNAcylation contributes to cardiomyopathy and heart failure is unknown. METHODS: We developed 2 new transgenic mouse models with myocardial overexpression of OGT and OGA to control O-GlcNAcylation independent of pathologic stress. RESULTS: We found that OGT transgenic hearts showed increased O-GlcNAcylation and developed severe dilated cardiomyopathy, ventricular arrhythmias, and premature death. In contrast, OGA transgenic hearts had lower O-GlcNAcylation but identical cardiac function to wild-type littermate controls. OGA transgenic hearts were resistant to pathologic stress induced by pressure overload with attenuated myocardial O-GlcNAcylation levels after stress and decreased pathologic hypertrophy compared with wild-type controls. Interbreeding OGT with OGA transgenic mice rescued cardiomyopathy and premature death, despite persistent elevation of myocardial OGT. Transcriptomic and functional studies revealed disrupted mitochondrial energetics with impairment of complex I activity in hearts from OGT transgenic mice. Complex I activity was rescued by OGA transgenic interbreeding, suggesting an important role for mitochondrial complex I in O-GlcNAc-mediated cardiac pathology. CONCLUSIONS: Our data provide evidence that excessive O-GlcNAcylation causes cardiomyopathy, at least in part, attributable to defective energetics. Enhanced OGA activity is well tolerated and attenuation of O-GlcNAcylation is beneficial against pressure overload-induced pathologic remodeling and heart failure. These findings suggest that attenuation of excessive O-GlcNAcylation may represent a novel therapeutic approach for cardiomyopathy.

Analytical and Biochemical Perspectives of Protein O-GlcNAcylation.

Protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) is a unique monosaccharide modification discovered in the early 1980s. With the technological advances in the past several decades, great progress has been made to reveal the biochemistry of O-GlcNAcylation, the substrates of O-GlcNAcylation, and the functional importance of protein O-GlcNAcylation. As a nutrient sensor, protein O-GlcNAcylation plays important roles in almost all biochemical processes examined. Although the functional importance of O-GlcNAcylation of proteins has been extensively reviewed previously, the chemical and biochemical aspects have not been fully addressed. In this review, by critically evaluating key publications in the past 35 years, we aim to provide a comprehensive understanding of this important post-translational modification (PTM) from analytical and biochemical perspectives. Specifically, we will cover (1) multiple analytical advances in the characterization of O-GlcNAc cycling components (i.e., the substrate donor UDP-GlcNAc, the two key enzymes O-GlcNAc transferase and O-GlcNAcase, and O-GlcNAc substrate proteins), (2) the biochemical characterization of the enzymes with a variety of chemical tools, and (3) exploration of O-GlcNAc cycling and its modulating chemicals as potential biomarkers and therapeutic drugs for diseases. Last but not least, we will discuss the challenges and possible solutions for basic and translational research of protein O-GlcNAcylation in the future.

Targeting O-GlcNAcylation to develop novel therapeutics.

O-linked ß-D-N-acetylglucosamine (O-GlcNAc) is an abundant post-translational modification (PTM) that modifies the serine or threonine residues of thousands of proteins in the nucleus, cytoplasm and mitochondria. Being a major "nutrient sensor" in cells, the O-GlcNAc pathway is sensitive to cellular metabolic states. Extensive crosstalk is observed between O-GlcNAcylation and protein phosphorylation. O-GlcNAc regulates protein functions at multiple levels, including enzymatic activity, transcriptional activity, subcellular localization, intermolecular interactions and degradation. Abnormal O-GlcNAcylation is associated with many human diseases including cancer, diabetes and neurodegenerative diseases. Though research on O-GlcNAc is still in its infantry, accumulating evidence suggest O-GlcNAcylation to be a promising therapeutic target. In this review, we briefly discuss the basic features of this PTM, the O-GlcNAc signaling pathway, its regulatory functions on different proteins, and its involvement in human diseases. We hope this review will provide insights to researchers who study human disease, as well as researchers who are interested in the fundamental roles of O-GlcNAcylation in all cells.

Oxidized CaMKII and O-GlcNAcylation cause increased atrial fibrillation in diabetic mice by distinct mechanisms.

Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

TATA-Box Binding Protein O-GlcNAcylation at T114 Regulates Formation of the B-TFIID Complex and Is Critical for Metabolic Gene Regulation.

In eukaryotes, gene expression is performed by three RNA polymerases that are targeted to promoters by molecular complexes. A unique common factor, the TATA-box binding protein (TBP), is thought to serve as a platform to assemble pre-initiation complexes competent for transcription. Here, we describe a novel molecular mechanism of nutrient regulation of gene transcription by dynamic O-GlcNAcylation of TBP. We show that O-GlcNAcylation at T114 of TBP blocks its interaction with BTAF1, hence the formation of the B-TFIID complex, and its dynamic cycling on and off of DNA. Transcriptomic and metabolomic analyses of TBPT114A CRISPR/Cas9-edited cells showed that loss of O-GlcNAcylation at T114 increases TBP binding to BTAF1 and directly impacts expression of 408 genes. Lack of O-GlcNAcylation at T114 is associated with a striking reprogramming of cellular metabolism induced by a profound modification of the transcriptome, leading to gross alterations in lipid storage.

CBS homogenization mutation strategy narrows the glycan binding profile of a GlcNAc-specific lectin AANL.

Glycosylation plays important roles in many cellular processes, such as signal transduction, cell cycle progression and transcriptional regulation. However, the identification and analysis of glycosylation are severely hampered by the low specificity or avidity of antiglycan antibodies and lectins. We have reported that a lectin AANL, which has high specificity for terminal GlcNAc glycans and contains six carbohydrate binding sites (CBSs), was used to enrich O-GlcNAcylated peptides. To further improve AANL binding specificity, we designed a CBS-homogenization strategy and restructured six mutant lectins, known as AANL1-AANL6. Affinity chromatography with GlcNAc and isothermal titration calorimetry analysis indicated that the two mutants (AANL3 and AANL6) all maintained GlcNAc binding activity. AANL6 and AANL3 showed higher specificity for terminal GlcNAc glycans than AANL, as shown by the hemagglutination assay, cell binding assays and glycan microarray analysis, and AANL6 exhibited the highest specificity. The binding activity of AANL6 for O-GlcNAcylated peptides was shown by surface plasmon resonance assays. By AANL6 affinity chromatography enrichment and mass spectrometry analysis, 79 high-confidence and 21 putative O-GlcNAcylated sites were identified on 85 peptides mapped onto 54 proteins. Most of these sites were new sites compared with reported data. These results indicate that the enrichment capacity of AANL6 is higher than that of wild-type AANL. In conclusion, the CBS-homogenization mutation strategy was successful, and AANL6 was identified as a powerful tool for O-GlcNAcylation enrichment. Our research suggests that the CBS-homogenization strategy is valuable for improving the specificity of lectins with multiple CBSs.

O-GlcNAcylation and phosphorylation of ß-actin Ser199 in diabetic nephropathy.

The function of actin is regulated by various posttranslational modifications. We have previously shown that in the kidneys of nonobese type 2 diabetes model Goto-Kakizaki rats, increased O-GlcNAcylation of ß-actin protein is observed. It has also been reported that both O-GlcNAcylation and phosphorylation occur on Ser199 of ß-actin. However, their roles are not known. To elucidate their roles in diabetic nephropathy, we examined the rat kidney for changes in O-GlcNAcylation of Ser199 (gS199)-actin and in the phosphorylation of Ser199 (pS199)-actin. Both gS199- and pS199-actin molecules had an apparent molecular weight of 40 kDa and were localized as nonfilamentous actin in both the cytoplasm and nucleus. Compared with the normal kidney, the immunostaining intensity of gS199-actin increased in podocytes of the glomeruli and in proximal tubules of the diabetic kidney, whereas that of pS199-actin did not change in podocytes but decreased in proximal tubules. We confirmed that the same results could be observed in the glomeruli of the human diabetic kidney. In podocytes of glomeruli cultured in the presence of the O-GlcNAcase inhibitor Thiamet G, increased O-GlcNAcylation was accompanied by a concomitant decrease in the amount of filamentous actin and in morphological changes. Our present results demonstrate that dysregulation of O-GlcNAcylation and phosphorylation of Ser199 occurred in diabetes, which may contribute partially to the causes of the morphological changes in the glomeruli and tubules. gS199- and pS199-actin will thus be useful for the pathological evaluation of diabetic nephropathy.

Nutrient regulation of signaling and transcription.

In the early 1980s, while using purified glycosyltransferases to probe glycan structures on surfaces of living cells in the murine immune system, we discovered a novel form of serine/threonine protein glycosylation (O-linked ß-GlcNAc; O-GlcNAc) that occurs on thousands of proteins within the nucleus, cytoplasm, and mitochondria. Prior to this discovery, it was dogma that protein glycosylation was restricted to the luminal compartments of the secretory pathway and on extracellular domains of membrane and secretory proteins. Work in the last 3 decades from several laboratories has shown that O-GlcNAc cycling serves as a nutrient sensor to regulate signaling, transcription, mitochondrial activity, and cytoskeletal functions. O-GlcNAc also has extensive cross-talk with phosphorylation, not only at the same or proximal sites on polypeptides, but also by regulating each other's enzymes that catalyze cycling of the modifications. O-GlcNAc is generally not elongated or modified. It cycles on and off polypeptides in a time scale similar to phosphorylation, and both the enzyme that adds O-GlcNAc, the O-GlcNAc transferase (OGT), and the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), are highly conserved from C. elegans to humans. Both O-GlcNAc cycling enzymes are essential in mammals and plants. Due to O-GlcNAc's fundamental roles as a nutrient and stress sensor, it plays an important role in the etiologies of chronic diseases of aging, including diabetes, cancer, and neurodegenerative disease. This review will present an overview of our current understanding of O-GlcNAc's regulation, functions, and roles in chronic diseases of aging.