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Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAPTg;Gfap+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAPTg;Gfap+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAPTg;Gfap+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAPTg;Gfap+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAPTg;Gfap+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap+ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAPTg;Gfap+/R236H mice. Last, to determine whether the findings in GFAPTg;Gfap+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAPTg;Gfap+/R236H mice., Graphical abstract, Highlights • Models of AxD provide insight into functions of astrocytes related to neuropathology. • PPAR pathway found upregulated in AxD mice may be neuroprotective. Fatty acid binding protein (Fabp7) was upregulated in AxD mice and human CNS samples. • Depleted levels in the classical constituents of myelin were observed in AxD mice. • UGT8 may be related to myelin deficits in AxD., In Brief The article contains the first whole brain proteomic survey from a mouse model of Alexander disease (AxD). Several novel findings include activation of the PPAR signaling pathway, which has been reported to be protective in models of amyotrophic lateral sclerosis (ALS). Another finding related to the gliosis phenotype in AxD mice was the upregulation of fatty acid binding protein 7 (FABP7), which induces an NF-κB inflammatory response and counteracts the anti-inflammatory effects of PPAR signaling.

Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Enhancing activity of the erythrocyte glycolytic pathway has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, factors that decrease HbS polymerization and improve erythrocyte membrane integrity. These factors can be modulated by mitapivat, which activates erythrocyte pyruvate kinase (PKR) and improves sickling kinetics in SCD patients. We investigated mechanisms by which mitapivat may impact SCD by examining its effects in the Townes SCD mouse model. Control (HbAA) and sickle (HbSS) mice were treated with mitapivat or vehicle. Surprisingly, HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. These data suggest that, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels.

Intracellular oxidative stress and oxidative modification of sickle hemoglobin (HbS) play a role in sickle cell disease (SCD) pathogenesis. Recently, we reported that Hb-dependent oxidative stress induced post-translational modifications (PTMs) of Hb and red blood cell (RBC) membrane proteins of transgenic SCD mice. To identify the mechanistic basis of these protein modifications, we followed in vitro oxidative changes occurring in intracellular Hb obtained from RBCs and RBC-derived microparticles (MPs) from the blood of 23 SCD patients (HbSS) of which 11 were on, and 12, off hydroxyurea (HU) treatment, and 5 ethnic matched controls. We used mass spectrometry-based proteomics to characterize these oxidative PTMs on a cross-sectional group of these patients (n = 4) and a separate subgroup of patients (n = 2) studied prior to initiation and during HU treatment. Collectively, these data indicated that band-3 and its interaction network involved in MPs formation exhibited more protein phosphorylation and ubiquitination in SCD patients than in controls. HU treatment reversed these oxidative PTMs back to level observed in controls. These PTMs were also confirmed using orthogonal immunoprecipitation experiments. Moreover, we observed specific markers reflective of oxidative stress, including irreversible oxidation of βCys93 and ubiquitination of Hb βLys145 (and βLys96). Overall, these studies strongly suggest that extensive erythrocyte membrane protein phosphorylation and ubiquitination are involved in SCD pathogenesis and provide further insight into the multifaceted effects of HU treatment.

The overarching goal of this exploratory study is to link subcellular microdomain specific protein-protein interactomes with big data analytics. We isolated postsynaptic density-95 (PSD-95) complexes from four human brain regions and compared their protein interactomes using multiple bioinformatics techniques. We demonstrate that human brain regions have unique postsynaptic protein signatures that may be used to interrogate perturbagen databases. Assessment of our hippocampal signature using the iLINCS database yielded several compounds with recently characterized “off target” effects on protein-protein interactions in the posynaptic density compartment.

Alexander disease (AxD) is an often fatal astrogliopathy caused by dominant gain-of-function missense mutations in the glial fibrillary acidic protein (GFAP) gene. The mechanism by which the mutations produce the AxD phenotype is not known. However, the observation that features of AxD are displayed by mice that express elevated levels of GFAP from a human WT GFAP transgene has contributed to the notion that the mutations produce AxD by increasing accumulation of total GFAP above some toxic threshold rather than the mutant GFAP being inherently toxic. A possible mechanism for accumulation of GFAP in AxD patients is that the mutated GFAP variants are more stable than the WT, an attribution abetted by observations that GFAP complexes containing GFAP variants are more resistant to solvent extraction. Here we tested this hypothesis by determining the relative levels of WT and mutant GFAP in three individuals with AxD, each of whom carried a common but different GFAP mutation (R79C, R239H, or R416W). Mass spectrometry analysis identified a peptide specific to the mutant or WT GFAP in each patient, and we quantified this peptide by comparing its signal to that of an added [(15)N]GFAP standard. In all three individuals, the level of mutant GFAP was less than that of the WT. This finding suggests that AxD onset is due to an intrinsic toxicity of the mutant GFAP instead of it acting indirectly by being more stable than WT GFAP and thereby increasing the total GFAP level.

The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes-sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent β-globin posttranslational modifications, including the irreversible oxidation of βCys93 and the ubiquitination of βLys96 and βLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, βCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients.

Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.

Data-independent acquisition (DIA)-based proteomics has become increasingly complicated in recent years because of the vast number of workflows described, coupled with a lack of studies indicating a rational framework for selecting effective settings to use. To address this issue and provide a resource for the proteomics community, we compared 12 DIA methods that assay tryptic peptides using various mass-isolation windows. Our findings indicate that the most sensitive single injection LC-DIA method uses 6 m/z isolation windows to analyze the densely populated tryptic peptide range from 450 to 730 m/z, which allowed quantification of 4465 Escherichia coli peptides. In contrast, using the sequential windowed acquisition of all theoretical fragment-ions (SWATH) approach with 26 m/z isolation windows across the entire 400-1200 m/z range, allowed quantification of only 3309 peptides. This reduced sensitivity with 26 m/z windows is caused by an increase in co-eluting compounds with similar precursor values detected in the same tandem MS spectra, which lowers the signal-to-noise of peptide fragment-ion chromatograms and reduces the amount of low abundance peptides that can be quantified from 410 to 920 m/z. Above 920 m/z, more peptides were quantified with 26 m/z windows because of substantial peptide (13) C isotope distributions that parse peptide ions into separate isolation windows. Because reproducible quantification has been a long-standing aim of quantitative proteomics, and is a so-called trait of DIA, we sought to determine whether precursor-level chromatograms used in some methods rather than their fragment-level counterparts have similar precision. Our data show that extracted fragment-ion chromatograms are the reason DIA provides superior reproducibility. Copyright © 2015 John Wiley & Sons, Ltd.

HYDROXYUREA REVERSES DYSFUNCTIONAL UBIQUITIN-PROTEASOMAL SYSTEM IN SICKLE CELL DISEASE AND SUPPRESSES POSTTRANSLATIONAL ALTERATIONS IN HEMOGLOBIN AND CELL MEMBRANES Sirsendu Jana, PhD1, Michael Brad Strader, PhD1, Fantao Meng, PhD1,Michael Heaven, PhD2, Arun Shet, MD3, Swee Lay Thein, MD3, and Abdu I. Alayash, PhD1. 1Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), 2Vulcan Analytical, Birmingham Al, 3Sickle Cell Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH). Introduction: Intracellular oxidative stress and oxidative modifications of sickle cell hemoglobin (HbS) play an important role in the pathogenesis of sickle cell disease (SCD). We recently reported transgenic mice studies revealing microparticles (MP) proteome differences between SCD and control mice expressing human HbS and HbA, respectively. Hb-dependent oxidative reactions and consequent posttranslational modifications of Hb βCys93 were central to red cell membrane changes that included modification of band3, and ubiquitination of proteins (Jana S et al., JCI Insights, 2018 3:120451). Ubiquitination is an important post-translational modification required for several biological functions including degradation by the ubiquitin-proteasomal system (UPS) proteolytic pathway. Proteins susceptible to oxidative damage are therefore likely degraded by UPS machinery. When these animals were treated with hydroxyurea (HU) they were able to reduce oxidative stress by controlling Hb oxidation side reactions. As a follow-up study, we have characterized human RBC derived MP proteomes of control, untreated and HU treated SCD patient samples to identify the mechanistic basis of how HU treatment reduces oxidative stress. Methods: We used a variety of biochemical and hematological methods to investigate a group of sickle cell disease (SCD) patients (n= 22) who are either on HU treatment (n=10) or without HU treatment (n=12) and a group of ethnically matched controls (n=4). We also performed an additional proteomic analysis on a subset of these patients, including a separate longitudinal study in which SCD patients (n=2) were followed before and after treatment with HU. Results: Immunoprecipitation experiments on RBCs obtained from untreated SCD patients revealed the presence of extensive ubiquitination contrary to those samples obtained from HU treated patients and controls. High proteasomal activity was found in SCD RBCs suggesting accumulated polyubiquitinated proteins found in these samples were not a byproduct of proteasomal inhibition but rather due to imbalance in the redox state of SS RBCs. In addition to Hb oxidation and oxidative modifications (including βCys93), our results revealed differences in the SCD proteome (from both control and untreated groups) including upregulation of phosphorylation and ubiquitination of proteins that are known to interact directly with band3 and are functionally involved in MP formation. Ubiquitination of Hb βLys145 and βLys96 were more abundant in SS patient's samples as well as phosphorylation of band3 (a prerequisite process for band3 clustering and MPs release). As revealed by the separate longitudinal study, HU treatment uniformly reversed ubiquination and phosphorylation of proteins involved in SCD induced MP formation. Conclusion: These mechanistic analyses of SCD RBC derived MPs suggest a potential involvement of ubiquitination and phosphorylation in SCD pathogenesis and provide additional insight into the therapeutic mechanisms of HU treatment. Disclosures No relevant conflicts of interest to declare.

State-of-the-art strategies for proteomics are not able to rapidly interrogate complex peptide mixtures in an untargeted manner with sensitive peptide and protein identification rates. We describe a data-independent acquisition (DIA) approach, microDIA (μDIA), that applies a novel tandem mass spectrometry (MS/MS) mass spectral deconvolution method to increase the specificity of tandem mass spectra acquired during proteomics experiments. Using the μDIA approach with a 10 min liquid chromatography gradient allowed detection of 3.1-fold more HeLa proteins than the results obtained from data-dependent acquisition (DDA) of the same samples. Additionally, we found the μDIA MS/MS deconvolution procedure is critical for resolving modified peptides with relatively small precursor mass shifts that cause the same peptide sequence in modified and unmodified forms to theoretically cofragment in the same raw MS/MS spectra. The μDIA workflow is implemented in the PROTALIZER software tool which fully automates tandem mass spectral deconvolution, queries every peptide with a library-free search algorithm against a user-defined protein database, and confidently identifies multiple peptides in a single tandem mass spectrum. We also benchmarked μDIA against DDA using a 90 min gradient analysis of HeLa and Escherichia coli peptides that were mixed in predefined quantitative ratios, and our results showed μDIA provided 24% more true positives at the same false positive rate.

Background Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MeCP2. Much of our understanding of MeCP2 function is derived from transcriptomic studies with the general assumption that alterations in the transcriptome correlate with proteomic changes. Advances in mass spectrometry-based proteomics have facilitated recent interest in the examination of global protein expression to better understand the biology between transcriptional and translational regulation. Methods We therefore performed the first comprehensive transcriptome-proteome comparison in a RTT mouse model to elucidate RTT pathophysiology, identify potential therapeutic targets, and further our understanding of MeCP2 function. The whole cortex of wild-type and symptomatic RTT male littermates (n = 4 per genotype) were analyzed using RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Ingenuity® Pathway Analysis was used to identify significantly affected pathways in the transcriptomic and proteomic data sets. Results Our results indicate these two “omics” data sets supplement one another. In addition to confirming previous works regarding mRNA expression in Mecp2-deficient animals, the current study identified hundreds of novel protein targets. Several selected protein targets were validated by Western blot analysis. These data indicate RNA metabolism, proteostasis, monoamine metabolism, and cholesterol synthesis are disrupted in the RTT proteome. Hits common to both data sets indicate disrupted cellular metabolism, calcium signaling, protein stability, DNA binding, and cytoskeletal cell structure. Finally, in addition to confirming disrupted pathways and identifying novel hits in neuronal structure and synaptic transmission, our data indicate aberrant myelination, inflammation, and vascular disruption. Intriguingly, there is no evidence of reactive gliosis, but instead, gene, protein, and pathway analysis suggest astrocytic maturation and morphological deficits. Conclusions This comparative omics analysis supports previous works indicating widespread CNS dysfunction and may serve as a valuable resource for those interested in cellular dysfunction in RTT. Electronic supplementary material The online version of this article (10.1186/s13229-017-0174-4) contains supplementary material, which is available to authorized users.

Data-independent acquisition (DIA)-based proteomics has become increasingly complicated in recent years because of the vast number of workflows described, coupled with a lack of studies indicating a rational framework for selecting effective settings to use. To address this issue and provide a resource for the proteomics community, we compared 12 DIA methods that assay tryptic peptides using various mass-isolation windows. Our findings indicate that the most sensitive single injection LC-DIA method uses 6 m/z isolation windows to analyze the densely populated tryptic peptide range from 450 to 730 m/z, which allowed quantification of 4465 Escherichia coli peptides. In contrast, using the sequential windowed acquisition of all theoretical fragment-ions (SWATH) approach with 26 m/z isolation windows across the entire 400-1200 m/z range, allowed quantification of only 3309 peptides. This reduced sensitivity with 26 m/z windows is caused by an increase in co-eluting compounds with similar precursor values detected in the same tandem MS spectra, which lowers the signal-to-noise of peptide fragment-ion chromatograms and reduces the amount of low abundance peptides that can be quantified from 410 to 920 m/z. Above 920 m/z, more peptides were quantified with 26 m/z windows because of substantial peptide (13) C isotope distributions that parse peptide ions into separate isolation windows. Because reproducible quantification has been a long-standing aim of quantitative proteomics, and is a so-called trait of DIA, we sought to determine whether precursor-level chromatograms used in some methods rather than their fragment-level counterparts have similar precision. Our data show that extracted fragment-ion chromatograms are the reason DIA provides superior reproducibility. Copyright © 2015 John WileySons, Ltd.

Over the past decade, growing evidence has emerged, suggesting that protein deimination is increased in human neurodegenerative disorders such as Alzheimer's disease, Creutzfeldt–Jakob disease, and Parkinson's disease. As an additional tool to identify affected proteins, some of these experiments utilized the F95 monoclonal antibody, which can theoretically recognize any protein in which arginine amino acids have been transformed to citrullines. This chapter outlines previous studies of brain protein deimination in these aforementioned maladies using F95 as well as ongoing unpublished studies of other neurodegenerative disorders such as Alexander's disease, amyotrophic lateral sclerosis, diffuse Lewy body disease, some primary astrocytic neoplasms, and normal brain aging. In addition, some data associated with the animal models of these conditions are also presented. Collectively, most of these conditions support the trending concept that neurodegeneration, for whatever reason, is accompanied by amplified citrullination, although the proteins affected and pattern of increased deimination in the central nervous system may differ in different disease states.

We investigated Rorschach responses associated with narcissism and hysteria in a group of antisocial personality disordered offenders. The Rorschach protocols of 42 subjects who met the criteria from the Diagnostic and Statistical Manual of Mental Disorders (3rd ed., rev. [DSM-III-R]; American Psychiatric Association, 1987) for antisocial personality disorder were analyzed using Exner's (1986) criteria for pairs, reflections, and personal responses, and Gacono's (1988) criteria for the impressionistic response. Severe, or primary psychopaths (n = 21), scoring greater than or equal to 30 on the Hare (1980) Psychopathy Checklist (PCL), were compared to moderate, or secondary pscyhopaths (n = 21), scoring less than 30 on the PCL. The mean number of pair and impressionistic responses did not significantly differ for the two antisocial groups. The highly psychopathic group, however, did exhibit a significantly greater mean number of reflection and personal responses. We discuss pair and reflection responses and their relationship to narcissism in psychopathic disturbance. We recommend interpreting the personal response within the context of the psychopathic character and view personal responses as expressions of narcissism and omnipotence in highly psychopathic subjects. We also hypothesize that the impressionistic responses are indicative of primitive dissociative processes and hysteria in psychopathic subjects, and that their presence provides construct validity for the work of Guze (1976) and others who suggested an underlying histrionic dimension to psychopathy.