Relationships between gene expression and behavior in mice in response to systemic modulation of the O-GlcNAcylation pathway.

Enhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA), which removes the O-GlcNAc modification from proteins, has been explored in mouse models of amyloid-beta and tau pathology. However, the O-GlcNAcylation-dependent link between gene expression and neurological behavior remains to be explored. Using chronic administration of Thiamet G (TG, an OGA inhibitor) in vivo, we used a protocol designed to relate behavior with the transcriptome and selected biochemical parameters from the cortex of individual animals. TG-treated mice showed improved working memory as measured using a Y-maze test. RNA sequencing analysis revealed 151 top differentially expressed genes with a Log2fold change >0.33 and adjusted p-value <0.05. Top TG-dependent upregulated genes were related to learning, cognition and behavior, while top downregulated genes were related to IL-17 signaling, inflammatory response and chemotaxis. Additional pathway analysis uncovered 3 pathways, involving gene expression including 14 cytochrome c oxidase subunits/regulatory components, chaperones or assembly factors, and 5 mTOR (mechanistic target of rapamycin) signaling factors. Multivariate Kendall correlation analyses of behavioral tests and the top TG-dependent differentially expressed genes revealed 91 statistically significant correlations in saline-treated mice and 70 statistically significant correlations in TG-treated mice. These analyses provide a network regulation landscape that is important in relating the transcriptome to behavior and the potential impact of the O-GlcNAC pathway.

Alpha-synuclein preformed fibril-induced aggregation and dopaminergic cell death in cathepsin D overexpression and ZKSCAN3 knockout mice.

α-synuclein accumulation is recognized as a prominent feature in the majority of Parkinson's disease cases and also occurs in a broad range of neurodegenerative disorders including Alzheimer's disease. It has been shown that α-synuclein can spread from a donor cell to neighboring cells and thus propagate cellular damage, antagonizing the effectiveness of therapies such as transplantation of fetal or iPSC derived dopaminergic cells. As we and others previously have shown, insufficient lysosomal function due to genetic mutations or targeted disruption of cathepsin D can cause α-synuclein accumulation. We here investigated whether overexpression of cathepsin D or knockout (KO) of the transcriptional suppressor of lysosomal biogenesis ZKSCAN3 can attenuate propagation of α-synuclein aggregation and cell death. We examined dopaminergic neurodegeneration in the substantia nigra using stereology of tyrosine hydroxylase-immunoreactive cells 4 months and 6 months after intrastriatal injection of α-synuclein preformed fibrils or monomeric α-synuclein control in control, central nervous system (CNS)-cathepsin D overexpressing and CNS-specific ZKSCAN3 KO mice. We also examined pS129-α-synuclein aggregates in the substantia nigra, cortex, amygdala and striatum. The extent of dopaminergic neurodegeneration and pS129-α-synuclein aggregation in the brains of CNS-specific ZKSCAN3 knockout mice and CNS-cathepsin D overexpressing mice was similar to that observed in wild-type mice. Our results indicate that neither enhancing cathepsin D expression nor disrupting ZKSCAN3 in the CNS is sufficient to attenuate pS129-α-synuclein aggregate accumulation or dopaminergic neurodegeneration.

Optimization of measurement of mitochondrial electron transport activity in postmortem human brain samples and measurement of susceptibility to rotenone and 4-hydroxynonenal inhibition.

Mitochondrial function is required to meet the energetic and metabolic requirements of the brain. Abnormalities in mitochondrial function, due to genetic or developmental factors, mitochondrial toxins, aging or insufficient mitochondrial quality control contribute to neurological and psychiatric diseases. Studying bioenergetics from postmortem human tissues has been challenging due to the diverse range of human genetics, health conditions, sex, age, and postmortem interval. Furthermore, fresh tissues that were in the past required for assessment of mitochondrial respiratory function were rarely available. Recent studies established protocols to use in bioenergetic analyses from frozen tissues using animal models and cell cultures. In this study we optimized these methods to determine the activities of mitochondrial electron transport in postmortem human brain. Further we demonstrate how these samples can be used to assess the susceptibility to the mitochondrial toxin rotenone and exposure to the reactive lipid species 4-hydroxynonenal. The establishment of such an approach will significantly impact translational studies of human diseases by allowing measurement of mitochondrial function in human tissue repositories.

AMPA receptor subunit localization in schizophrenia anterior cingulate cortex.

The glutamate hypothesis of schizophrenia suggests that altered glutamatergic transmission occurs in this illness, although precise mechanisms of dysregulation remain elusive. AMPA receptors (AMPARs), a subtype of ionotropic glutamate receptor, are the main facilitators of fast, excitatory neurotransmission in the brain, and changes in AMPAR number or composition at synapses can regulate synaptic strength and plasticity. Prior evidence of abnormal expression of transmembrane AMPAR regulatory proteins (TARPs) in schizophrenia suggests defective trafficking of AMPARs, which we propose could lead to altered AMPAR expression at excitatory synapses. To test this hypothesis, we isolated subcellular fractions enriched for endoplasmic reticulum (ER) and synapses from anterior cingulate cortex (ACC) from schizophrenia (N = 18) and comparison (N = 18) subjects, and measured glutamate receptor subunits (GluA1, GluA2, GluA3, GluA4, NR1, NR2A, NR2B, and NR3A) and TARP member γ2 (stargazin) in homogenates and subcellular fractions by western blot analysis. We found decreased expression of stargazin and an increased ratio of GluA2:stargazin in ACC homogenates, while in the synapse fraction we identified a decrease in GluA1 and reduced ratios of GluA1:stargazin and GluA1:GluA2 in schizophrenia. The amount of stargazin in the ER fraction was not different, but the relative amount of ER/Total stargazin was increased in schizophrenia. Together, these findings suggest that associations between stargazin and AMPA subunits are abnormal, potentially affecting forward trafficking or synaptic stability of GluA1-containing AMPARs. These data provide evidence that altered interactions with trafficking proteins may contribute to glutamate dysregulation in schizophrenia.

Acute inhibition of OGA sex-dependently alters the networks associated with bioenergetics, autophagy, and neurodegeneration.

The accumulation of neurotoxic proteins characteristic of age-related neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases is associated with the perturbation of metabolism, bioenergetics, and mitochondrial quality control. One approach to exploit these interactions therapeutically is to target the pathways that regulate metabolism. In this respect, the nutrient-sensing hexosamine biosynthesis pathway is of particular interest since it introduces a protein post-translational modification known as O-GlcNAcylation, which modifies different proteins in control versus neurodegenerative disease postmortem brains. A potent inhibitor of the O-GlcNAcase enzyme that removes the modification from proteins, Thiamet G (TG), has been proposed to have potential benefits in Alzheimer's disease. We tested whether key factors in the O-GlcNAcylation are correlated with mitochondrial electron transport and proteins related to the autophagy/lysosomal pathways in the cortex of male and female mice with and without exposure to TG (10 mg/kg i.p.). Mitochondrial complex activities were measured in the protein homogenates, and a panel of metabolic, autophagy/lysosomal proteins and O-GlcNAcylation enzymes were assessed by either enzyme activity assay or by western blot analysis. We found that the networks associated with O-GlcNAcylation enzymes and activities with mitochondrial parameters, autophagy-related proteins as well as neurodegenerative disease-related proteins exhibited sex and TG dependent differences. Taken together, these studies provide a framework of interconnectivity for multiple O-GlcNAc-dependent pathways in mouse brain of relevance to aging and sex/age-dependent neurodegenerative pathogenesis and response to potential therapies.

Post-translational protein modifications in schizophrenia.

Research investigating the pathophysiology of schizophrenia has not yet precisely defined the molecular phenotype of this disorder. Many studies have investigated cellular dysfunction by examining expression levels of molecular targets in postmortem patient brain; however, inconsistencies between transcript and protein measures in schizophrenia are common in the field and represent a challenge to the identification of a unified model of schizophrenia pathogenesis. In humans, >4800 unique proteins are expressed, and the majority of these are modified by glycans and/or lipids. Estimates indicate ~70% of all eukaryotic proteins are modified by at least one type of glycosylation, while nearly 20% of all proteins are known to be lipid-modified. Protein post-translational modification (PTM) by glycosylation and lipidation rely on the spatiotemporal colocalization of enzyme, substrate, and glycan or lipid donor molecule and do not require an upstream "blueprint" or specialized processing machinery for synthesis. Glycan and lipid PTMs can thus facilitate cellular adaptation to environmental signals more rapidly than changes of gene or protein expression, and can significantly impact the localization, function, and interactions of modified substrates, though relatively few studies in schizophrenia have evaluated the PTM status of target proteins. A growing body of literature reports glycosylation and lipidation abnormalities in schizophrenia brain as well as in patient peripheral fluids. In this review, we explain the functional significance of key glycan and lipid PTMs and summarize current findings associated with abnormal glycosylation and lipidation in this illness.

Protein expression of prenyltransferase subunits in postmortem schizophrenia dorsolateral prefrontal cortex.

The pathophysiology of schizophrenia includes altered neurotransmission, dysregulated intracellular signaling pathway activity, and abnormal dendritic morphology that contribute to deficits of synaptic plasticity in the disorder. These processes all require dynamic protein-protein interactions at cell membranes. Lipid modifications target proteins to membranes by increasing substrate hydrophobicity by the addition of a fatty acid or isoprenyl moiety, and recent evidence suggests that dysregulated posttranslational lipid modifications may play a role in multiple neuropsychiatric disorders, including schizophrenia. Consistent with these emerging findings, we have recently reported decreased protein S-palmitoylation in schizophrenia. Protein prenylation is a lipid modification that occurs upstream of S-palmitoylation on many protein substrates, facilitating membrane localization and activity of key intracellular signaling proteins. Accordingly, we hypothesized that, in addition to palmitoylation, protein prenylation may be abnormal in schizophrenia. To test this, we assayed protein expression of the five prenyltransferase subunits (FNTA, FNTB, PGGT1B, RABGGTA, and RABGGTB) in postmortem dorsolateral prefrontal cortex from patients with schizophrenia and paired comparison subjects (n = 13 pairs). We found decreased levels of FNTA (14%), PGGT1B (13%), and RABGGTB (8%) in schizophrenia. To determine whether upstream or downstream factors may be driving these changes, we also assayed protein expression of the isoprenoid synthases FDPS and GGPS1 and prenylation-dependent processing enzymes RCE and ICMT. We found these upstream and downstream enzymes to have normal protein expression. To rule out effects from chronic antipsychotic treatment, we assayed FNTA, PGGT1B, and RABGGTB in the cortex from rats treated long-term with haloperidol decanoate and found no change in the expression of these proteins. Given the role prenylation plays in localization of key signaling proteins found at the synapse, these data offer a potential mechanism underlying abnormal protein-protein interactions and protein localization in schizophrenia.

New Insights Into the Biology of Protein O-GlcNAcylation: Approaches and Observations.

O-GlcNAcylation is a protein posttranslational modification that results in the addition of O-GlcNAc to Ser/Thr residues. Since its discovery in the 1980s, it has been shown to play an important role in a broad range of cellular functions by modifying nuclear, cytosolic, and mitochondrial proteins. The addition of O-GlcNAc is catalyzed by O-GlcNAc transferase (OGT), and its removal is catalyzed by O-GlcNAcase (OGA). Levels of protein O-GlcNAcylation change in response to nutrient availability and metabolic, oxidative, and proteotoxic stress. OGT and OGA levels, activity, and target engagement are also regulated. Together, this results in adaptive and, on occasions, detrimental responses that affect cellular function and survival, which impact a broad range of pathologies and aging. Over the past several decades, approaches and tools to aid the investigation of the regulation and consequences of protein O-GlcNAcylation have been developed and enhanced. This review is divided into two sections: 1) We will first focus on current standard and advanced technical approaches for assessing enzymatic activities of OGT and OGT, assessing the global and specific protein O-GlcNAcylation and 2) we will summarize in vivo findings of functional consequences of changing protein O-GlcNAcylation, using genetic and pharmacological approaches.

Fractionation of Subcellular Compartments from Human Brain Tissue.

Subcellular fractionation methods permit the isolation, purification, and/or enrichment of specific cellular compartments from complex tissue samples. Enrichment of multiple subcellular compartments from the same tissue sample permits comparisons of the spatial distribution of target proteins between specific intracellular compartments and, in some cases, can provide information about spatiotemporal processing of key cellular components. Here we describe a method to generate subcellular fractions enriched for heavy membranes and nuclei, rough and smooth endoplasmic reticulum membranes, light membranes and cytosol, synapses, and other intermediate cellular membranes from postmortem human brain tissue. These subcellular fractions can be used in a variety of downstream applications to assess the localization, relative abundance, and stoichiometry of glutamate receptor subunits along the forward trafficking pathway.

Altered fucosyltransferase expression in the superior temporal gyrus of elderly patients with schizophrenia.

Glycosylation is a post-translational modification that is an essential element in cell signaling and neurodevelopmental pathway regulation. Glycan attachment can influence the tertiary structure and molecular interactions of glycosylated substrates, adding an additional layer of regulatory complexity to functional mechanisms underlying central cell biological processes. One type of enzyme-mediated glycan attachment, fucosylation, can mediate glycoprotein and glycolipid cell surface expression, trafficking, secretion, and quality control to modulate a variety of inter- and intracellular signaling cascades. Building on prior reports of glycosylation abnormalities and evidence of dysregulated glycosylation enzyme expression in schizophrenia, we examined the protein expression of 5 key fucose-modifying enzymes: GDP-fucose:protein O-fucosyltransferase 1 (POFUT1), GDP-fucose:protein O-fucosyltransferase 2 (POFUT2), fucosyltransferase 8 (FUT8), fucosyltransferase 11 (FUT11), and plasma α-l-fucosidase (FUCA2) in postmortem superior temporal gyrus of schizophrenia (N=16) and comparison (N=14) subjects. We also used the fucose binding protein, Aleuria aurantia lectin (AAL), to assess α-1,6-fucosylated N-glycoprotein abundance in the same subjects. In schizophrenia, we found increased expression of POFUT2, a fucosyltransferase uniquely responsible for O-fucosylation of thrombospondin-like repeat domains that is involved in a non-canonical endoplasmic reticulum quality control pathway. We also found decreased expression of FUT8 in schizophrenia. Given that FUT8 is the only α-1,6-fucosyltransferase expressed in mammals, the concurrent decrease in AAL binding in schizophrenia, particularly evident for N-glycoproteins in the ~52-58kDa and ~60-70kDa molecular mass ranges, likely reflects a consequence of abnormal FUT8 expression in the disorder. Dysregulated FUT8 and POFUT2 expression could potentially explain a variety of molecular abnormalities in schizophrenia.

Actin polymerization is reduced in the anterior cingulate cortex of elderly patients with schizophrenia.

Recent reports suggest abnormalities in the regulation of actin cytoskeletal dynamics in schizophrenia, despite consistent evidence for normal actin expression. We hypothesized that this may be explained by changes in the polymerization state of actin, rather than in total actin expression. To test this, we prepared filamentous actin (F-actin, polymeric) and globular actin (G-actin, monomeric) fractions from postmortem anterior cingulate cortex from 16 patients with schizophrenia and 14 comparison subjects. Additionally, binding of fluorescently-labeled phalloidin, a selectively F-actin-binding peptide, was measured in unfractionated samples from the same subjects. Western blot analysis of fractions revealed decreased F-actin, increased G-actin, and decreased ratios of F-actin/total actin and F-actin/G-actin in schizophrenia. Decreased phalloidin binding to F-actin in parallel experiments in the same subjects independently supports these findings. These results suggest a novel aspect of schizophrenia pathophysiology and are consistent with previous evidence of reduced dendritic spine density and altered synaptic plasticity in schizophrenia, both of which have been linked to cytoskeletal abnormalities.

Listening to bowel sounds: an evidence-based practice project: nurses find that a traditional practice isn't the best indicator of returning gastrointestinal motility in patients who've undergone abdominal surgery.

Nurses' practice of listening to bowel sounds was first proposed in 1905 and continues today, largely unquestioned. The authors developed a project to determine whether any compelling evidence exists for using this method to assess for the return of gastrointestinal (GI) motility following abdominal surgery. Literature on the subject was evaluated and an assessment of nursing practice was conducted. Based on the literature review and the assessment, a nursing practice guideline was developed, implemented, and evaluated. (Note that the nursing practice guideline outlined in this article was evaluated for use with abdominal surgery patients only and has not been evaluated in and may not be appropriate for other patient populations). The results were positive and indicate that clinical parameters other than bowel sounds, such as the return of flatus and the first postoperative bowel movement, are appropriate in assessing for the return of GI motility after abdominal surgery. Bowel sound assessment was discontinued and patient outcomes were evaluated to make sure that the practice change had no adverse effect on patients' recovery.

Abnormal N-acetylglucosaminyltransferase expression in prefrontal cortex in schizophrenia.

Changes in the extent of the posttranslational modification glycosylation have been previously reported in several brain regions in schizophrenia. Quality control within the endoplasmic reticulum and Golgi, branching of glycans, intracellular trafficking and targeting, protein-protein interactions, and endocytosis are processes regulated by both N-linked and O-linked glycosylation. Previous studies in schizophrenia have found altered glycan biosynthesis and abnormal glycan levels in cerebrospinal fluid (CSF) and plasma, as well as altered expression in frontal cortex of glycosyltransferase transcripts encoding proteins associated with both N- and O-linked glycosylation. The N-acetylglucosaminyltransferases (GlcNAcTs) are glycosylating enzymes that play a key role in adding N-acetylglucosamine (GlcNAc) to substrates to facilitate their proper trafficking, intracellular targeting, and cellular function. Given previous results indicating abnormal glycosylation in schizophrenia, we hypothesized that these GlcNAcTs may be abnormally expressed in this illness. We measured protein expression of nine distinct GlcNAcTs by Western blot analysis in postmortem samples of dorsolateral prefrontal cortex (DLPFC) from twelve pairs of elderly patients with schizophrenia and comparison subjects. We found decreased protein expression of UDP-GlcNAc:BetaGal Beta-1,3 GlcNAcT 8 (B3GNT8) and mannosyl (alpha-1,3-)-glycoprotein beta-1,4 GlcNAcT (MGAT4A) expression in schizophrenia. These data provide further evidence that glycosylation is dysregulated in schizophrenia, and suggest a potential mechanism associated with alterations in protein function, trafficking, and intracellular targeting in this illness.

N-Glycosylation of GABAA receptor subunits is altered in Schizophrenia.

The molecular mechanisms of schizophrenia have been under investigation for decades; however, the exact causes of this debilitating neuropsychiatric disorder are still unknown. Previous studies have identified multiple affected neurotransmitter systems, brain regions, and cell types, each making a unique contribution to symptom presentation and pathophysiology. Numerous studies have identified gene and protein expression changes in schizophrenia, but the role of post-translational modifications, specifically N-glycosylation, has only recently become a target of investigation. N-glycosylation of molecules associated with glutamatergic neurotransmission is disrupted in schizophrenia, but it was unknown if these alterations are exclusive to the glutamatergic system or due to a more generalized deficit.In normal human cortex, we found evidence for N-glycosylation of the α1, ß1, and ß2 γ-aminobutyric type A receptor (GABAAR) subunits using deglycosylation protein shift assays. This was confirmed with lectin affinity assays that revealed glycan attachment on the α1, α4, and ß1-3 GABAAR subunits. Examining GABAAR subunit N-glycosylation in matched pairs of schizophrenia (N=14) and comparison (N=14) of superior temporal gyrus revealed a smaller molecular mass of immature N-glycans on the α1 subunit, more immature N-glycosylation of the 49-kDa ß1 subunit isoform, and altered total N-glycosylation of the ß2 GABAAR subunit in schizophrenia. Measures of altered N-glycosylation of the ß1 and ß2 subunits were confounded by an increased apparent molecular mass of all ß1 and ß2 subunit isoforms in schizophrenia. Although N-glycosylation of α1, ß1, and ß2 were all changed in schizophrenia, the concentrations of GABAAR subunits themselves were unchanged. These findings suggest that disruptions of N-glycosylation in schizophrenia are not exclusive to glutamate and may indicate a potential disruption of a central cell signaling process in this disorder.