Glycoproteomics Landscape of Asymptomatic and Symptomatic Human Alzheimer's Disease Brain.

Molecular changes in the brain of individuals afflicted with Alzheimer's disease (AD) are an intense area of study. Little is known about the role of protein abundance and posttranslational modifications in AD progression and treatment, in particular large-scale intact N-linked glycoproteomics analysis. To elucidate the N-glycoproteome landscape, we developed an approach based on multi-lectin affinity enrichment, hydrophilic interaction chromatography, and LC-MS-based glycoproteomics. We analyzed brain tissue from 10 persons with no cognitive impairment or AD, 10 with asymptomatic AD, and 10 with symptomatic AD, detecting over 300 glycoproteins and 1900 glycoforms across the samples. The majority of glycoproteins have N-glycans that are high-mannosidic or complex chains that are fucosylated and bisected. The Man5 N-glycan was found to occur most frequently at >20% of the total glycoforms. Unlike the glycoproteomes of other tissues, sialylation is a minor feature of the brain N-glycoproteome, occurring at <9% among the glycoforms. We observed AD-associated differences in the number of antennae, frequency of fucosylation, bisection, and other monosaccharides at individual glycosylation sites among samples from our three groups. Further analysis revealed glycosylation differences in subcellular compartments across disease stage, including glycoproteins in the lysosome frequently modified with paucimannosidic glycans. These results illustrate the N-glycoproteomics landscape across the spectrum of AD clinical and pathologic severity and will facilitate a deeper understanding of progression and treatment development.

N-glycans show distinct spatial distribution in mouse brain.

The development and function of the brain requires N-linked glycosylation of proteins, which is a ubiquitous modification in the secretory pathway. N-glycans have a distinct composition and undergo tight regulation in the brain, but the spatial distribution of these structures remains relatively unexplored. Here, we systematically employed carbohydrate binding lectins with differing specificities to various classes of N-glycans and appropriate controls to identify glycan expression in multiple regions of the mouse brain. Lectins binding high-mannose-type N-glycans, the most abundant class of brain N-glycans, showed diffuse staining with some punctate structures observed on high magnification. Lectins binding specific motifs of complex N-glycans, including fucose and bisecting GlcNAc, showed more partitioned labeling, including to the synapse-rich molecular layer of the cerebellum. Understanding the spatial distribution of N-glycans across the brain will aid future studies of these critical protein modifications in development and disease of the brain.

The Inflammation Biomarker GlycA Reflects Plasma N-Glycan Branching.

BACKGROUND: GlycA is a nuclear magnetic resonance (NMR) signal in plasma that correlates with inflammation and cardiovascular outcomes in large data sets. The signal is thought to originate from N-acetylglucosamine (GlcNAc) residues of branched plasma N-glycans, though direct experimental evidence is limited. Trace element concentrations affect plasma glycosylation patterns and may thereby also influence GlycA. METHODS: NMR GlycA signal was measured in plasma samples from 87 individuals and correlated with MALDI-MS N-glycomics and trace element analysis. We further evaluated the genetic association with GlycA at rs13107325, a single nucleotide polymorphism resulting in a missense variant within SLC39A8, a manganese transporter that influences N-glycan branching, both in our samples and existing genome-wide association studies data from 22 835 participants in the Women's Health Study (WHS). RESULTS: GlycA signal was correlated with both N-glycan branching (r2 ranging from 0.125-0.265; all P < 0.001) and copper concentration (r2 = 0.348, P < 0.0001). In addition, GlycA levels were associated with rs13107325 genotype in the WHS (ß [standard error of the mean] = -4.66 [1.2674], P = 0.0002). CONCLUSIONS: These results provide the first direct experimental evidence linking the GlycA NMR signal to N-glycan branching commonly associated with acute phase reactive proteins involved in inflammation.

The schizophrenia-associated variant in SLC39A8 alters protein glycosylation in the mouse brain.

A missense mutation (A391T) in SLC39A8 is strongly associated with schizophrenia in genomic studies, though the molecular connection to the brain is unknown. Human carriers of A391T have reduced serum manganese, altered plasma glycosylation, and brain MRI changes consistent with altered metal transport. Here, using a knock-in mouse model homozygous for A391T, we show that the schizophrenia-associated variant changes protein glycosylation in the brain. Glycosylation of Asn residues in glycoproteins (N-glycosylation) was most significantly impaired, with effects differing between regions. RNAseq analysis showed negligible regional variation, consistent with changes in the activity of glycosylation enzymes rather than gene expression. Finally, nearly one-third of detected glycoproteins were differentially N-glycosylated in the cortex, including members of several pathways previously implicated in schizophrenia, such as cell adhesion molecules and neurotransmitter receptors that are expressed across all cell types. These findings provide a mechanistic link between a risk allele and potentially reversible biochemical changes in the brain, furthering our molecular understanding of the pathophysiology of schizophrenia and a novel opportunity for therapeutic development.

Aberrant glycosylation in schizophrenia: a review of 25 years of post-mortem brain studies.

Glycosylation, the enzymatic attachment of carbohydrates to proteins and lipids, regulates nearly all cellular processes and is critical in the development and function of the nervous system. Axon pathfinding, neurite outgrowth, synaptogenesis, neurotransmission, and many other neuronal processes are regulated by glycans. Over the past 25 years, studies analyzing post-mortem brain samples have found evidence of aberrant glycosylation in individuals with schizophrenia. Proteins involved in both excitatory and inhibitory neurotransmission display altered glycans in the disease state, including AMPA and kainate receptor subunits, glutamate transporters EAAT1 and EAAT2, and the GABAA receptor. Polysialylated NCAM (PSA-NCAM) and perineuronal nets, highly glycosylated molecules critical for axonal migration and synaptic stabilization, are both downregulated in multiple brain regions of individuals with schizophrenia. In addition, enzymes spanning several pathways of glycan synthesis show differential expression in brains of individuals with schizophrenia. These changes may be due to genetic predisposition, environmental perturbations, medication use, or a combination of these factors. However, the recent association of several enzymes of glycosylation with schizophrenia by genome-wide association studies underscores the importance of glycosylation in this disease. Understanding how glycosylation is dysregulated in the brain will further our understanding of how this pathway contributes to the development and pathophysiology of schizophrenia.

Glycobiology and schizophrenia: a biological hypothesis emerging from genomic research.

Advances in genomics are opening new windows into the biology of schizophrenia. Though common variants individually have small effects on disease risk, GWAS provide a powerful opportunity to explore pathways and mechanisms contributing to pathophysiology. Here, we highlight an underappreciated biological theme emerging from GWAS: the role of glycosylation in schizophrenia. The strongest coding variant in schizophrenia GWAS is a missense mutation in the manganese transporter SLC39A8, which is associated with altered glycosylation patterns in humans. Furthermore, variants near several genes encoding glycosylation enzymes are unambiguously associated with schizophrenia: FUT9, MAN2A1, TMTC1, GALNT10, and B3GAT1. Here, we summarize the known biological functions, target substrates, and expression patterns of these enzymes as a primer for future studies. We also highlight a subset of schizophrenia-associated proteins critically modified by glycosylation including glutamate receptors, voltage-gated calcium channels, the dopamine D2 receptor, and complement glycoproteins. We hypothesize that common genetic variants alter brain glycosylation and play a fundamental role in the development of schizophrenia. Leveraging these findings will advance our mechanistic understanding of disease and may provide novel avenues for treatment development.

N-glycome analysis detects dysglycosylation missed by conventional methods in SLC39A8 deficiency.

Congenital disorders of glycosylation (CDG) are a growing group of inborn metabolic disorders with multiorgan presentation. SLC39A8-CDG is a severe subtype caused by biallelic mutations in the manganese transporter SLC39A8, reducing levels of this essential cofactor for many enzymes including glycosyltransferases. The current diagnostic standard for disorders of N-glycosylation is the analysis of serum transferrin. Exome and Sanger sequencing were performed in two patients with severe neurodevelopmental phenotypes suggestive of CDG. Transferrin glycosylation was analyzed by high-performance liquid chromatography (HPLC) and isoelectric focusing in addition to comprehensive N-glycome analysis using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS). Atomic absorption spectroscopy was used to quantify whole blood manganese levels. Both patients presented with a severe, multisystem disorder, and a complex neurological phenotype. Magnetic resonance imaging (MRI) revealed a Leigh-like syndrome with bilateral T2 hyperintensities of the basal ganglia. In patient 1, exome sequencing identified the previously undescribed homozygous variant c.608T>C [p.F203S] in SLC39A8. Patient 2 was found to be homozygous for c.112G>C [p.G38R]. Both individuals showed a reduction of whole blood manganese, though transferrin glycosylation was normal. N-glycome using MALDI-TOF MS identified an increase of the asialo-agalactosylated precursor N-glycan A2G1S1 and a decrease in bisected structures. In addition, analysis of heterozygous CDG-allele carriers identified similar but less severe glycosylation changes. Despite its reliance as a clinical gold standard, analysis of transferrin glycosylation cannot be categorically used to rule out SLC39A8-CDG. These results emphasize that SLC39A8-CDG presents as a spectrum of dysregulated glycosylation, and MS is an important tool for identifying deficiencies not detected by conventional methods.

Rhes, a striatal-selective protein implicated in Huntington disease, binds beclin-1 and activates autophagy.

The protein mutated in Huntington disease (HD), mutant huntingtin (mHtt), is expressed throughout the brain and body. However, the pathology of HD is characterized by early and dramatic destruction selectively of the striatum. We previously reported that the striatal-specific protein Rhes binds mHtt and enhances its cytotoxicity. Moreover, Rhes-deleted mice are dramatically protected from neurodegeneration and motor dysfunction in mouse models of HD. We now report a function of Rhes in autophagy, a lysosomal degradation pathway implicated in aging and HD neurodegeneration. In PC12 cells, deletion of endogenous Rhes decreases autophagy, whereas Rhes overexpression activates autophagy. These effects are independent of mTOR and opposite in the direction predicted by the known activation of mTOR by Rhes. Rhes robustly binds the autophagy regulator Beclin-1, decreasing its inhibitory interaction with Bcl-2 independent of JNK-1 signaling. Finally, co-expression of mHtt blocks Rhes-induced autophagy activation. Thus, the isolated pathology and delayed onset of HD may reflect the striatal-selective expression and changes in autophagic activity of Rhes.

Rhes deletion is neuroprotective in the 3-nitropropionic acid model of Huntington's disease.

Although the mutated protein causing Huntington's disease (HD) is expressed throughout the body, the major pathology of HD is localized to the striatum of the brain. We previously reported that the striatal-enriched protein Rhes binds the mutated huntingtin protein and enhances its cytotoxicity. We now demonstrate that Rhes-deleted mice are dramatically protected from neurotoxicity and motor dysfunction in a striatal-specific model of HD elicited by 3-nitropropionic acid. This finding suggests that Rhes may, in part, determine the striatal selectivity of HD.

N-glycans show distinct spatial distribution in mouse brain.

Protein N-linked glycosylation is a ubiquitous modification in the secretory pathway that plays a critical role in the development and function of the brain. N-glycans have a distinct composition and undergo tight regulation in the brain, but the spatial distribution of these structures remains relatively unexplored. Here, we systematically employed carbohydrate binding lectins with differing specificities to various classes of N-glycans and appropriate controls to identify multiple regions of the mouse brain. Lectins binding high-mannose-type N-glycans, the most abundant class of brain N-glycans, showed diffuse staining with some punctate structures observed on high magnification. Lectins binding specific motifs of complex N-glycans, including fucose and bisecting GlcNAc, showed more partitioned labeling, including to the synapse-rich molecular layer of the cerebellum. Understanding the distribution of N-glycans across the brain will aid future studies of these critical protein modifications in development and disease of the brain.

Mammalian brain glycoproteins exhibit diminished glycan complexity compared to other tissues.

Glycosylation is essential to brain development and function, but prior studies have often been limited to a single analytical technique and excluded region- and sex-specific analyses. Here, using several methodologies, we analyze Asn-linked and Ser/Thr/Tyr-linked protein glycosylation between brain regions and sexes in mice. Brain N-glycans are less complex in sequence and variety compared to other tissues, consisting predominantly of high-mannose and fucosylated/bisected structures. Most brain O-glycans are unbranched, sialylated O-GalNAc and O-mannose structures. A consistent pattern is observed between regions, and sex differences are minimal compared to those in plasma. Brain glycans correlate with RNA expression of their synthetic enzymes, and analysis of glycosylation genes in humans show a global downregulation in the brain compared to other tissues. We hypothesize that this restricted repertoire of protein glycans arises from their tight regulation in the brain. These results provide a roadmap for future studies of glycosylation in neurodevelopment and disease.

Rhes, a physiologic regulator of sumoylation, enhances cross-sumoylation between the basic sumoylation enzymes E1 and Ubc9.

We recently reported that the small G-protein Rhes has the properties of a SUMO-E3 ligase and mediates mutant huntingtin (mHtt) cytotoxicity. We now demonstrate that Rhes is a physiologic regulator of sumoylation, which is markedly reduced in the corpus striatum of Rhes-deleted mice. Sumoylation involves activation and transfer of small ubiquitin-like modifier (SUMO) from the thioester of E1 to the thioester of Ubc9 (E2) and final transfer to lysines on target proteins, which is enhanced by E3s. We show that E1 transfers SUMO from its thioester directly to lysine residues on Ubc9, forming isopeptide linkages. Conversely, sumoylation on E1 requires transfer of SUMO from the thioester of Ubc9. Thus, the process regarded as "autosumoylation" reflects intermolecular transfer between E1 and Ubc9, which we designate "cross-sumoylation." Rhes binds directly to both E1 and Ubc9, enhancing cross-sumoylation as well as thioester transfer from E1 to Ubc9.

L-Fucose treatment of FUT8-CDG.

FUT8-CDG is a severe multisystem disorder caused by mutations in FUT8, encoding the α-1,6-fucosyltransferase. We report on dizygotic twins with FUT8-CDG presenting with dysmorphisms, failure to thrive, and respiratory abnormalities. Due to the severe phenotype, oral L-fucose supplementation was started. Glycosylation analysis using mass spectrometry indicated a limited response to fucose therapy while the clinical presentation stabilized. Further research is needed to assess the concept of substrate supplementation in FUT8-CDG.

The schizophrenia risk locus in SLC39A8 alters brain metal transport and plasma glycosylation.

A common missense variant in SLC39A8 is convincingly associated with schizophrenia and several additional phenotypes. Homozygous loss-of-function mutations in SLC39A8 result in undetectable serum manganese (Mn) and a Congenital Disorder of Glycosylation (CDG) due to the exquisite sensitivity of glycosyltransferases to Mn concentration. Here, we identified several Mn-related changes in human carriers of the common SLC39A8 missense allele. Analysis of structural brain MRI scans showed a dose-dependent change in the ratio of T2w to T1w signal in several regions. Comprehensive trace element analysis confirmed a specific reduction of only serum Mn, and plasma protein N-glycome profiling revealed reduced complexity and branching. N-glycome profiling from two individuals with SLC39A8-CDG showed similar but more severe alterations in branching that improved with Mn supplementation, suggesting that the common variant exists on a spectrum of hypofunction with potential for reversibility. Characterizing the functional impact of this variant will enhance our understanding of schizophrenia pathogenesis and identify novel therapeutic targets and biomarkers.

Rhes, a striatal-enriched small G protein, mediates mTOR signaling and L-DOPA-induced dyskinesia.

L-DOPA-induced dyskinesia, the rate-limiting side effect in the therapy of Parkinson's disease, is mediated by activation of mammalian target of rapamycin (mTOR) signaling in the striatum. We found that Ras homolog enriched in striatum (Rhes), a striatal-specific protein, binds to and activates mTOR. Moreover, Rhes(-/-) mice showed reduced striatal mTOR signaling and diminished dyskinesia, but maintained motor improvement on L-DOPA treatment, suggesting a therapeutic benefit for Rhes-binding drugs.