Quantitative Characterization of the Neuropeptide Level Changes in Dorsal Horn and Dorsal Root Ganglia Regions of the Murine Itch Models.

Chronic itch can be extremely devastating and, in many cases, difficult to treat. One challenge in treating itch disorders is the limited understanding of the multitude of chemical players involved in the communication of itch sensation from the peripheral to the central nervous system. Neuropeptides are intercellular signaling molecules that are known to be involved in the transmission of itch signals from primary afferent neurons, which detect itch in the skin, to higher-order circuits in the spinal cord and brain. To investigate the role of neuropeptides in transmitting itch signals, we generated two mouse models of chronic itch-Acetone-Ether-Water (AEW, dry skin) and calcipotriol (MC903, atopic dermatitis). For peptide identification and quantitation, we analyzed the peptide content of dorsal root ganglia (DRG) and dorsal horn (DH) tissues from chronically itchy mice using liquid chromatography coupled to tandem mass spectrometry. De novo-assisted database searching facilitated the identification and quantitation of 335 peptides for DH MC903, 318 for DH AEW, 266 for DRG MC903, and 271 for DRG AEW. Of these quantifiable peptides, we detected 30 that were differentially regulated in the tested models, after accounting for multiple testing correction (q ≤ 0.1). These include several peptide candidates derived from neuropeptide precursors, such as proSAAS, protachykinin-1, proenkephalin, and calcitonin gene-related peptide, some of them previously linked to itch. The peptides identified in this study may help elucidate our understanding about these debilitating disorders. Data are available via ProteomeXchange with identifier PXD015949.

Optically Guided Single Cell Mass Spectrometry of Rat Dorsal Root Ganglia to Profile Lipids, Peptides and Proteins.

The mammalian dorsal root ganglia (DRG) are located on the dorsal roots of the spinal nerves and contain cell bodies of primary sensory neurons. DRG cells have been classified into subpopulations based on their size, morphology, intracellular markers, response to stimuli, and neuropeptides. To understand the connections between DRG chemical heterogeneity and cellular function, we performed optically guided, high-throughput single cell profiling using sequential matrix-assisted laser desorption/ionization mass spectrometry (MS) to detect lipids, peptides, and several proteins in individual DRG cells. Statistical analysis of the resulting mass spectra allows stratification of the DRG population according to cellular morphology and, presumably, major cell types. A subpopulation of small cells contained myelin proteins, which are abundant in Schwann cells, and mass spectra of several larger cells contained peaks matching neurofilament, vimentin, myelin basic protein S, and thymosin beta proteins. Of the over 1000 cells analyzed, approximately 78 % produced putative peptide-rich spectra, allowing the population to be classified into three distinct cell types. Two signals with m/z 4404 and 5487 were exclusively observed in a cell type, but could not be matched to results of our previous liquid chromatography-MS analyses.

Mass spectrometry-based characterization of endogenous peptides and metabolites in small volume samples.

Technologies to assay single cells and their extracellular microenvironments are valuable in elucidating biological function, but there are challenges. Sample volumes are low, the physicochemical parameters of the analytes vary widely, and the cellular environment is chemically complex. In addition, the inherent difficulty of isolating individual cells and handling small volume samples complicates many experimental protocols. Here we highlight a number of mass spectrometry (MS)-based measurement approaches for characterizing the chemical content of small volume analytes, with a focus on methods used to detect intracellular and extracellular metabolites and peptides from samples as small as individual cells. MS has become one of the most effective means for analyzing small biological samples due to its high sensitivity, low analyte consumption, compatibility with a wide array of sampling approaches, and ability to detect a large number of analytes with different properties without preselection. Having access to a flexible portfolio of MS-based methods allows quantitative, qualitative, untargeted, targeted, multiplexed, and spatially resolved investigations of single cells and their similarly scaled extracellular environments. Combining MS with on-line and off-line sample conditioning tools, such as microfluidic and capillary electrophoresis systems, significantly increases the analytical coverage of the sample's metabolome and peptidome, and improves individual analyte characterization/identification. Small volume assays help to reveal the causes and manifestations of biological and pathological variability, as well as the functional heterogeneity of individual cells within their microenvironments and within cellular populations. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

Mitogen-activated protein kinase phosphatase-1 promotes neovascularization and angiogenic gene expression.

OBJECTIVE: Angiogenesis is the formation of new blood vessels through endothelial cell sprouting. This process requires the mitogen-activated protein kinases, signaling molecules that are negatively regulated by the mitogen-activated protein kinase phosphatase-1 (MKP-1). The purpose of this study was to evaluate the role of MKP-1 in neovascularization in vivo and identify associated mechanisms in endothelial cells. APPROACH AND RESULTS: We used murine hindlimb ischemia as a model system to evaluate the role of MKP-1 in angiogenic growth, remodeling, and arteriogenesis in vivo. Genomic deletion of MKP-1 blunted angiogenesis in the distal hindlimb and microvascular arteriogenesis in the proximal hindlimb. In vitro, endothelial MKP-1 depletion/deletion abrogated vascular endothelial growth factor-induced migration and tube formation, and reduced proliferation. These observations establish MKP-1 as a positive mediator of angiogenesis and contrast with the canonical function of MKP-1 as a mitogen-activated protein kinase phosphatase, implying an alternative mechanism for MKP-1-mediated angiogenesis. Cloning and sequencing of MKP-1-bound chromatin identified localization of MKP-1 to exonic DNA of the angiogenic chemokine fractalkine, and MKP-1 depletion reduced histone H3 serine 10 dephosphorylation on this DNA locus and blocked fractalkine expression. In vivo, MKP-1 deletion abrogated ischemia-induced fractalkine expression and macrophage and T-lymphocyte infiltration in distal hindlimbs, whereas fractalkine delivery to ischemic hindlimbs rescued the effect of MKP-1 deletion on neovascular hindlimb recovery. CONCLUSIONS: MKP-1 promoted angiogenic and arteriogenic neovascular growth, potentially through dephosphorylation of histone H3 serine 10 on coding-region DNA to control transcription of angiogenic genes, such as fractalkine. These observations reveal a novel function for MKP-1 and identify MKP-1 as a potential therapeutic target.

Integrating Mass Spectrometry with Microphysiological Systems for Improved Neurochemical Studies.

Microphysiological systems, often referred to as "organs-on-chips", are in vitro platforms designed to model the spatial, chemical, structural, and physiological elements of in vivo cellular environments. They enhance the evaluation of complex engineered biological systems and are a step between traditional cell culture and in vivo experimentation. As neurochemists and measurement scientists studying the molecules involved in intercellular communication in the nervous system, we focus here on recent advances in neuroscience using microneurological systems and their potential to interface with mass spectrometry. We discuss a number of examples - microfluidic devices, spheroid cultures, hydrogels, scaffolds, and fibers - highlighting those that would benefit from mass spectrometric technologies to obtain improved chemical information.

3D-Printed pHEMA Materials for Topographical and Biochemical Modulation of Dorsal Root Ganglion Cell Response.

Understanding and controlling the interactions occurring between cells and engineered materials are central challenges toward progress in the development of biomedical devices. In this work, we describe materials for direct ink writing (DIW), an extrusion-based type of 3D printing, that embed a custom synthetic protein (RGD-PDL) within the microfilaments of 3D-hydrogel scaffolds to modify these interactions and differentially direct tissue-level organization of complex cell populations in vitro. The RGD-PDL is synthesized by modifying poly-d-lysine (PDL) to varying extents with peptides containing the integrin-binding motif Arg-Gly-Asp (RGD). Compositional gradients of the RGD-PDL presented by both patterned and thin-film poly(2-hydroxyethyl) methacrylate (pHEMA) substrates allow the patterning of cell-growth compliance in a grayscale form. The surface chemistry-dependent guidance of cell growth on the RGD-PDL-modified pHEMA materials is demonstrated using a model NIH-3T3 fibroblast cell line. The formation of a more complex cellular system-organotypic primary murine dorsal root ganglion (DRG)-in culture is also achieved on these scaffolds, where distinctive forms of cell growth and migration guidance are seen depending on their RGD-PDL content and topography. This experimental platform for the study of physicochemical factors on the formation and the reorganization of organotypic cultures offers useful capabilities for studies in tissue engineering, regenerative medicine, and diagnostics.

Peptidomics and Secretomics of the Mammalian Peripheral Sensory-Motor System.

The dorsal root ganglion (DRG) and its anatomically and functionally associated spinal nerve and ventral and dorsal roots are important components of the peripheral sensory-motor system in mammals. The cells within these structures use a number of peptides as intercellular signaling molecules. We performed a variety of mass spectrometry (MS)-based characterizations of peptides contained within and secreted from these structures, and from isolated and cultured DRG cells. Liquid chromatography-Fourier transform MS was utilized in DRG and nerve peptidome analysis. In total, 2724 peptides from 296 proteins were identified in tissue extracts. Neuropeptides are among those detected, including calcitonin gene-related peptide I, little SAAS, and known hemoglobin-derived peptides. Solid phase extraction combined with direct matrix-assisted laser desorption/ionization time-of-flight MS was employed to investigate the secretome of these structures. A number of peptides were detected in the releasate from semi-intact preparations of DRGs and associated nerves, including neurofilament- and myelin basic protein-related peptides. A smaller set of analytes was observed in releasates from cultured DRG neurons. The peptide signals observed in the releasates have been mass-matched to those characterized and identified in homogenates of entire DRGs and associated nerves. This data aids our understanding of the chemical composition of the mammalian peripheral sensory-motor system, which is involved in key physiological functions such as nociception, thermoreception, itch sensation, and proprioception.

HOXA9 methylation by PRMT5 is essential for endothelial cell expression of leukocyte adhesion molecules.

The induction of proinflammatory proteins in stimulated endothelial cells (EC) requires activation of multiple transcription programs. The homeobox transcription factor HOXA9 has an important regulatory role in cytokine induction of the EC-leukocyte adhesion molecules (ELAM) E-selectin and vascular cell adhesion molecule 1 (VCAM-1). However, the mechanism underlying stimulus-dependent activation of HOXA9 is completely unknown. Here, we elucidate the molecular mechanism of HOXA9 activation by tumor necrosis factor alpha (TNF-α) and show an unexpected requirement for arginine methylation by protein arginine methyltransferase 5 (PRMT5). PRMT5 was identified as a TNF-α-dependent binding partner of HOXA9 by mass spectrometry. Small interfering RNA (siRNA)-mediated depletion of PRMT5 abrogated stimulus-dependent HOXA9 methylation with concomitant loss in E-selectin or VCAM-1 induction. Chromatin immunoprecipitation analysis revealed that PRMT5 is recruited to the E-selectin promoter following transient HOXA9 binding to its cognate recognition sequence. PRMT5 induces symmetric dimethylation of Arg140 on HOXA9, an event essential for E-selectin induction. In summary, PRMT5 is a critical coactivator component in a newly defined, HOXA9-containing transcription complex. Moreover, stimulus-dependent methylation of HOXA9 is essential for ELAM expression during the EC inflammatory response.