TAAR1-Dependent and -Independent Actions of Tyramine in Interaction With Glutamate Underlie Central Effects of Monoamine Oxidase Inhibition.

BACKGROUND: Monoamine oxidase inhibitors (MAOIs) exert therapeutic actions by elevating extracellular levels of monoamines in the brain. Irreversible MAOIs cause serious hypertensive crises owing to peripheral accumulation of tyramine, but the role of tyramine in the central effects of MAOIs remains elusive, an issue addressed herein. To achieve robust inhibition of MAOA/B, the clinically used antidepressant tranylcypromine (TCP) was employed. METHODS: Behavioral, histological, mass spectrometry imaging, and biosensor-mediated measures of glutamate were conducted with MAOIs in wild-type and TAAR1-knockout (KO) mice. RESULTS: Both antidepressant and locomotion responses to TCP were enhanced in TAAR1-KO mice. A recently developed fluoromethylpyridinium-based mass spectrometry imaging method revealed robust accumulation of striatal tyramine on TCP administration. Furthermore, tyramine accumulation was higher in TAAR1-KO versus wild-type mice, suggesting a negative feedback mechanism for TAAR1 in sensing tyramine levels. Combined histoenzymological and immunohistological studies revealed hitherto unknown TAAR1 localization in brain areas projecting to the substantia nigra/ventral tegmental area. Using an enzyme-based biosensor technology, we found that both TCP and tyramine reduced glutamate release in the substantia nigra in wild-type but not in TAAR1-KO mice. Moreover, glutamate measures in freely moving animals treated with TCP demonstrated that TAAR1 prevents glutamate accumulation in the substantia nigra during hyperlocomotive states. CONCLUSIONS: These observations suggest that tyramine, in interaction with glutamate, is involved in centrally mediated behavioral, transcriptional, and neurochemical effects of MAOIs.

Comprehensive mapping of neurotransmitter networks by MALDI-MS imaging.

We present a mass spectrometry imaging (MSI) approach for the comprehensive mapping of neurotransmitter networks in specific brain regions. Our fluoromethylpyridinium-based reactive matrices facilitate the covalent charge-tagging of molecules containing phenolic hydroxyl and/or primary or secondary amine groups, including dopaminergic and serotonergic neurotransmitters and their associated metabolites. These matrices improved the matrix-assisted laser desorption/ionization (MALDI)-MSI detection limit toward low-abundance neurotransmitters and facilitated the simultaneous imaging of neurotransmitters in fine structures of the brain at a lateral resolution of 10 µm. We demonstrate strategies for the identification of unknown molecular species using the innate chemoselectivity of the reactive matrices and the unique isotopic pattern of a brominated reactive matrix. We illustrate the capabilities of the developed method on Parkinsonian brain samples from human post-mortem tissue and animal models. The direct imaging of neurotransmitter systems provides a method for exploring how various neurological diseases affect specific brain regions through neurotransmitter modulation.

α-synuclein-lipoprotein interactions and elevated ApoE level in cerebrospinal fluid from Parkinson's disease patients.

The progressive accumulation, aggregation, and spread of α-synuclein (αSN) are common hallmarks of Parkinson's disease (PD) pathology. Moreover, numerous proteins interact with αSN species, influencing its toxicity in the brain. In the present study, we extended analyses of αSN-interacting proteins to cerebrospinal fluid (CSF). Using coimmunoprecipitation, followed by mass spectrometry, we found that αSN colocalize with apolipoproteins on lipoprotein vesicles. We confirmed these interactions using several methods, including the enrichment of lipoproteins with a recombinant αSN, and the subsequent uptake of prepared vesicles by human dopaminergic neuronal-like cells. Further, we report an increased level of ApoE in CSF from early PD patients compared with matched controls in 3 independent cohorts. Moreover, in contrast to controls, we observed the presence of ApoE-positive neuromelanin-containing dopaminergic neurons in substantia nigra of PD patients. In conclusion, the cooccurrence of αSN on lipoprotein vesicles, and their uptake by dopaminergic neurons along with an increase of ApoE in early PD, proposes a mechanism(s) for αSN spreading in the extracellular milieu of PD.

Molecular imaging identifies age-related attenuation of acetylcholine in retrosplenial cortex in response to acetylcholinesterase inhibition.

The neurotransmitter of the cholinergic system, acetylcholine plays a major role in the brain's cognitive function and is involved in neurodegenerative disorders. Here, we present age-related alterations of acetylcholine levels after administration of the acetylcholinesterase inhibitor drug tacrine in normal mice. Using a quantitative, robust and molecular-specific mass spectrometry imaging method we found that tacrine administration significantly raised acetylcholine levels in most areas of sectioned mice brains, inter alia the striatum, hippocampus and cortical areas. However, acetylcholine levels in retrosplenial cortex were significantly lower in 14-month-old than in 12-week-old animals following its administration, indicating that normal aging affects the cholinergic system's responsivity. This small brain region is interconnected with an array of brain networks and is involved in numerous cognitive tasks. Simultaneous visualization of distributions of tacrine and its hydroxylated metabolites in the brain revealed a significant decrease in levels of the metabolites in the 14-month-old mice. The results highlight strengths of the imaging technique to simultaneously investigate multiple molecular species and the drug-target effects in specific regions of the brain. The proposed approach has high potential in studies of neuropathological conditions and responses to neuroactive treatments.

How Does the Sweet Violet (Viola odorata L.) Fight Pathogens and Pests - Cyclotides as a Comprehensive Plant Host Defense System.

Cyclotides are cyclic plant polypeptides of 27-37 amino acid residues. They have been extensively studied in bioengineering and drug development contexts. However, less is known about the relevance of cyclotides for the plants producing them. The anti-insect larvae effects of kB1 and antibacterial activity of cyO2 suggest that cyclotides are a part of plant host defense. The sweet violet (Viola odorata L.) produces a wide array of cyclotides, including kB1 (kalata B1) and cyO2 (cycloviolacin O2), with distinct presumed biological roles. Here, we evaluate V. odorata cyclotides' potency against plant pathogens and their mode of action using bioassays, liposome experiments and immunogold labeling for transmission electron microscopy (TEM). We explore the link between the biological activity and distribution in plant generative, vegetative tissues and seeds, depicted by immunohistochemistry and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Cyclotides cyO2, cyO3, cyO13, and cyO19 are shown to have potent activity against model fungal plant pathogens (Fusarium oxysporum, F. graminearum, F. culmorum, Mycosphaerella fragariae, Botrytis cinerea) and fungi isolated from violets (Colletotrichum utrechtense and Alternaria alternata), with minimal inhibitory concentrations (MICs) ranging from 0.8 µM to 25 µM. Inhibition of phytopathogenic bacteria - Pseudomonas syringae pv. syringae, Dickeya dadantii and Pectobacterium atrosepticum - is also observed with MIC = 25-100 µM. A membrane-disrupting antifungal mode of action is shown. Finding cyO2 inside the fungal spore cells in TEM images may indicate that other, intracellular targets may be involved in the mechanism of toxicity. Fungi can not break down cyclotides in the course of days. varv A (kalata S) and kB1 show little potency against pathogenic fungi when compared with the tested cycloviolacins. cyO2, cyO3, cyO19 and kB1 are differentially distributed and found in tissues vulnerable to pathogen (epidermis, rizodermis, vascular bundles, protodermis, procambium, ovary walls, outer integuments) and pest (ground tissues of leaf and petiole) attacks, respectively, indicating a link between the cyclotides' sites of accumulation and biological role. Cyclotides emerge as a comprehensive defense system in V. odorata, in which different types of peptides have specific targets that determine their distribution in plant tissues.

Striatal Tyrosine Hydroxylase Is Stimulated via TAAR1 by 3-Iodothyronamine, But Not by Tyramine or ß-Phenylethylamine.

The trace amine-associated receptor 1 (TAAR1) is expressed by dopaminergic neurons, but the precise influence of trace amines upon their functional activity remains to be fully characterized. Here, we examined the regulation of tyrosine hydroxylase (TH) by tyramine and beta-phenylethylamine (ß-PEA) compared to 3-iodothyronamine (T1AM). Immunoblotting and amperometry were performed in dorsal striatal slices from wild-type (WT) and TAAR1 knockout (KO) mice. T1AM increased TH phosphorylation at both Ser19 and Ser40, actions that should promote functional activity of TH. Indeed, HPLC data revealed higher rates of L-dihydroxyphenylalanine (DOPA) accumulation in WT animals treated with T1AM after the administration of a DOPA decarboxylase inhibitor. These effects were abolished both in TAAR1 KO mice and by the TAAR1 antagonist, EPPTB. Further, they were specific inasmuch as Ser845 phosphorylation of the post-synaptic GluA1 AMPAR subunit was unaffected. The effects of T1AM on TH phosphorylation at both Ser19 (CamKII-targeted), and Ser40 (PKA-phosphorylated) were inhibited by KN-92 and H-89, inhibitors of CamKII and PKA respectively. Conversely, there was no effect of an EPAC analog, 8-CPT-2Me-cAMP, on TH phosphorylation. In line with these data, T1AM increased evoked striatal dopamine release in TAAR1 WT mice, an action blunted in TAAR1 KO mice and by EPPTB. Mass spectrometry imaging revealed no endogenous T1AM in the brain, but detected T1AM in several brain areas upon systemic administration in both WT and TAAR1 KO mice. In contrast to T1AM, tyramine decreased the phosphorylation of Ser40-TH, while increasing Ser845-GluA1 phosphorylation, actions that were not blocked in TAAR1 KO mice. Likewise, ß-PEA reduced Ser40-TH and tended to promote Ser845-GluA1 phosphorylation. The D1 receptor antagonist SCH23390 blocked tyramine-induced Ser845-GluA1 phosphorylation, but had no effect on tyramine- or ß-PEA-induced Ser40-TH phosphorylation. In conclusion, by intracellular cascades involving CaMKII and PKA, T1AM, but not tyramine and ß-PEA, acts via TAAR1 to promote the phosphorylation and functional activity of TH in the dorsal striatum, supporting a modulatory influence on dopamine transmission.

Peptide ion channel toxins from the bootlace worm, the longest animal on Earth.

Polypeptides from animal venoms have found important uses as drugs, pharmacological tools, and within biotechnological and agricultural applications. We here report a novel family of cystine knot peptides from nemertean worms, with potent activity on voltage-gated sodium channels. These toxins, named the α-nemertides, were discovered in the epidermal mucus of Lineus longissimus, the 'bootlace worm' known as the longest animal on earth. The most abundant peptide, the 31-residue long α-1, was isolated, synthesized, and its 3D NMR structure determined. Transcriptome analysis including 17 species revealed eight α-nemertides, mainly distributed in the genus Lineus. α-1 caused paralysis and death in green crabs (Carcinus maenas) at 1 µg/kg (~300 pmol/kg). It showed profound effect on invertebrate voltage-gated sodium channels (e.g. Blattella germanica Nav1) at low nanomolar concentrations. Strong selectivity for insect over human sodium channels indicates that α-nemertides can be promising candidates for development of bioinsecticidal agents.

A Space Efficient Direct Access Data Compression Approach for Mass Spectrometry Imaging.

Advances in mass spectrometry imaging that improve both spatial and mass resolution are resulting in increasingly larger data files that are difficult to handle with current software. We have developed a novel near-lossless compression method with data entropy reduction that reduces the file size significantly. The reduction in data size can be set at four different levels (coarse, medium, fine, and superfine) prior to running the data compression. This can be applied to spectra or spectrum-by-spectrum, or it can be applied to transpose arrays or array-by-array, to efficiently read the data without decompressing the whole data set. The results show that a compression ratio of up to 5.9:1 was achieved for data from commercial mass spectrometry software programs and 55:1 for data from our in-house developed msIQuant program. Comparing the average signals from regions of interest, the maximum deviation was 0.2% between compressed and uncompressed data sets with coarse accuracy for the data entropy reduction. In addition, when accessing the compressed data by selecting a random m/ z value using msIQuant, the time to update an image on the computer screen was only slightly increased from 92 (±32) ms (uncompressed) to 114 (±13) ms (compressed). Furthermore, the compressed data can be stored on readily accessible servers for data evaluation without further data reprocessing. We have developed a space efficient, direct access data compression algorithm for mass spectrometry imaging, which can be used for various data-demanding mass spectrometry imaging applications.

A mass spectrometry imaging approach for investigating how drug-drug interactions influence drug blood-brain barrier permeability.

There is a high need to develop quantitative imaging methods capable of providing detailed brain localization information of several molecular species simultaneously. In addition, extensive information on the effect of the blood-brain barrier on the penetration, distribution and efficacy of neuroactive compounds is required. Thus, we have developed a mass spectrometry imaging method to visualize and quantify the brain distribution of drugs with varying blood-brain barrier permeability. With this approach, we were able to determine blood-brain barrier transport of different drugs and define the drug distribution in very small brain structures (e.g., choroid plexus) due to the high spatial resolution provided. Simultaneously, we investigated the effect of drug-drug interactions by inhibiting the membrane transporter multidrug resistance 1 protein. We propose that the described approach can serve as a valuable analytical tool during the development of neuroactive drugs, as it can provide physiologically relevant information often neglected by traditional imaging technologies.

Direct imaging of elemental distributions in tissue sections by laser ablation mass spectrometry.

We present a strategy for imaging of elements in biological tissues using laser ablation (LA) mass spectrometry (MS), which was compared to laser ablation inductively coupled plasma (LA-ICP) MS. Both methods were adopted for quantitative imaging of elements in mouse kidney, as well as traumatic brain injury model tissue sections. MS imaging (MSI) employing LA provides quantitative data by comparing signal abundances of sodium from tissues to those obtained by imaging quantitation calibration standards of the target element applied to adjacent control tissue sections. LA-ICP MSI provided quantitative data for several essential elements in both brain and kidney tissue sections using a dried-droplet approach. Both methods were used to image a rat model of traumatic brain injury, revealing accumulations of sodium and calcium in the impact area and its peripheral regions. LA MSI is shown to be a viable option for quantitative imaging of specific elements in biological tissue sections.

Simultaneous imaging of multiple neurotransmitters and neuroactive substances in the brain by desorption electrospray ionization mass spectrometry.

With neurological processes involving multiple neurotransmitters and neuromodulators, it is important to have the ability to directly map and quantify multiple signaling molecules simultaneously in a single analysis. By utilizing a molecular-specific approach, namely desorption electrospray ionization mass spectrometry imaging (DESI-MSI), we demonstrated that the technique can be used to image multiple neurotransmitters and their metabolites (dopamine, dihydroxyphenylacetic acid, 3-methoxytyramine, serotonin, glutamate, glutamine, aspartate, γ-aminobutyric acid, adenosine) as well as neuroactive drugs (amphetamine, sibutramine, fluvoxamine) and drug metabolites in situ directly in brain tissue sections. The use of both positive and negative ionization modes increased the number of identified molecular targets. Chemical derivatization by charge-tagging the primary amines of molecules significantly increased the sensitivity, enabling the detection of low abundant neurotransmitters and other neuroactive substances previously undetectable by MSI. The sensitivity of the imaging approach of neurochemicals has a great potential in many diverse applications in fields such as neuroscience, pharmacology, drug discovery, neurochemistry, and medicine.

msIQuant--Quantitation Software for Mass Spectrometry Imaging Enabling Fast Access, Visualization, and Analysis of Large Data Sets.

This paper presents msIQuant, a novel instrument- and manufacturer-independent quantitative mass spectrometry imaging software suite that uses the standardized open access data format imzML. Its data processing structure enables rapid image display and the analysis of very large data sets (>50 GB) without any data reduction. In addition, msIQuant provides many tools for image visualization including multiple interpolation methods, low intensity transparency display, and image fusion. It also has a quantitation function that automatically generates calibration standard curves from series of standards that can be used to determine the concentrations of specific analytes. Regions-of-interest in a tissue section can be analyzed based on a number of quantities including the number of pixels, average intensity, standard deviation of intensity, and median and quartile intensities. Moreover, the suite's export functions enable simplified postprocessing of data and report creation. We demonstrate its potential through several applications including the quantitation of small molecules such as drugs and neurotransmitters. The msIQuant suite is a powerful tool for accessing and evaluating very large data sets, quantifying drugs and endogenous compounds in tissue areas of interest, and for processing mass spectra and images.

An introduction to MS imaging in drug discovery and development.

A vital process in drug discovery and development is to assess the absorption, distribution, metabolism, excretion and toxicology of potentially therapeutic compounds in the body. The potential utility of MS imaging has been demonstrated in many studies focusing on molecules including peptides, proteins and lipids. However, MS imaging also permits the direct analysis of drugs and drug metabolites in tissue samples without requiring the use of target-specific labels or reagents. Here, a brief technical description of the technique is presented along with examples of its usefulness at different stages of the drug discovery and development process including absorption, distribution, metabolism, excretion and toxicology, and blood-brain barrier drug penetration investigations.

Pyrylium Salts as Reactive Matrices for MALDI-MS Imaging of Biologically Active Primary Amines.

Many neuroactive substances, including endogenous biomolecules, environmental compounds, and pharmaceuticals possess primary amine functional groups. Among these are catecholamine neurotransmitters (e.g., dopamine), many substituted phenethylamines (e.g., amphetamine), as well as amino acids and neuropeptides. In most cases, mass spectrometric (ESI and MALDI) analyses of trace amounts of such compounds are challenging because of their poor ionization properties. We present a method for chemical derivatization of primary amines by reaction with pyrylium salts that facilitates their detection by MALDI-MS and enables the imaging of primary amines in brain tissue sections. A screen of pyrylium salts revealed that the 2,4-diphenyl-pyranylium ion efficiently derivatizes primary amines and can be used as a reactive MALDI-MS matrix that induces both derivatization and desorption. MALDI-MS imaging with such matrix was used to map the localization of dopamine and amphetamine in brain tissue sections and to quantitatively map the distribution of the neurotoxin ß-N-methylamino-L-alanine.

Direct targeted quantitative molecular imaging of neurotransmitters in brain tissue sections.

Current neuroimaging techniques have very limited abilities to directly identify and quantify neurotransmitters from brain sections. We have developed a molecular-specific approach for the simultaneous imaging and quantitation of multiple neurotransmitters, precursors, and metabolites, such as tyrosine, tryptamine, tyramine, phenethylamine, dopamine, 3-methoxytyramine, serotonin, GABA, glutamate, acetylcholine, and L-alpha-glycerylphosphorylcholine, in histological tissue sections at high spatial resolutions. The method is employed to directly measure changes in the absolute and relative levels of neurotransmitters in specific brain structures in animal disease models and in response to drug treatments, demonstrating the power of mass spectrometry imaging in neuroscience.

Mass spectrometry imaging, an emerging technology in neuropsychopharmacology.

Mass spectrometry imaging is a powerful tool for directly determining the distribution of proteins, peptides, lipids, neurotransmitters, metabolites and drugs in neural tissue sections in situ. Molecule-specific imaging can be achieved using various ionization techniques that are suited to different applications but which all yield data with high mass accuracies and spatial resolutions. The ability to simultaneously obtain images showing the distributions of chemical species ranging from metal ions to macromolecules makes it possible to explore the chemical organization of a sample and to correlate the results obtained with specific anatomical features. The imaging of biomolecules has provided new insights into multiple neurological diseases, including Parkinson's and Alzheimer's disease. Mass spectrometry imaging can also be used in conjunction with other imaging techniques in order to identify correlations between changes in the distribution of important chemical species and other changes in the properties of the tissue. Here we review the applications of mass spectrometry imaging in neuroscience research and discuss its potential. The results presented demonstrate that mass spectrometry imaging is a useful experimental method with diverse applications in neuroscience.

Deuterated matrix-assisted laser desorption ionization matrix uncovers masked mass spectrometry imaging signals of small molecules.

D(4)-α-Cyano-4-hydroxycinnamic acid (D(4)-CHCA) has been synthesized for use as a matrix for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and MALDI-MS imaging (MSI) of small molecule drugs and endogenous compounds. MALDI-MS analysis of small molecules has historically been hindered by interference from matrix ion clusters and fragment peaks that mask signals of low molecular weight compounds of interest. By using D(4)-CHCA, the cluster and fragment peaks of CHCA, the most common matrix for analysis of small molecules, are shifted by + 4, + 8 and + 12 Da, which expose signals across areas of the previously concealed low mass range. Here, obscured MALDI-MS signals of a synthetic small molecule pharmaceutical, a naturally occurring isoquinoline alkaloid, and endogenous compounds including the neurotransmitter acetylcholine have been unmasked and imaged directly from biological tissue sections.

Novel mass spectrometry imaging software assisting labeled normalization and quantitation of drugs and neuropeptides directly in tissue sections.

MALDI MS imaging has been extensively used to produce qualitative distribution maps of proteins, peptides, lipids, small molecule pharmaceuticals and their metabolites directly in biological tissue sections. There is growing demand to quantify the amount of target compounds in the tissue sections of different organs. We present a novel MS imaging software including protocol for the quantitation of drugs, and for the first time, an endogenous neuropeptide directly in tissue sections. After selecting regions of interest on the tissue section, data is read and processed by the software using several available methods for baseline corrections, subtractions, denoising, smoothing, recalibration and normalization. The concentrations of in vivo administered drugs or endogenous compounds are then determined semi-automatically using either external standard curves, or by using labeled compounds, i.e., isotope labeled analogs as standards. As model systems, we have quantified the distribution of imipramine and tiotropium in the brain and lung of dosed rats. Substance P was quantified in different mouse brain structures, which correlated well with previously reported peptide levels. Our approach facilitates quantitative data processing and labeled standards provide better reproducibility and may be considered as an efficient tool to quantify drugs and endogenous compounds in tissue regions of interest.

Controlled-pH tissue cleanup protocol for signal enhancement of small molecule drugs analyzed by MALDI-MS imaging.

The limit of detection of low-molecular weight compounds in tissue sections, analyzed by matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), was significantly improved by employing sample washing using a pH-controlled buffer solution. The pH of the washing solutions were set at values whereby the target analytes would have low solubility. Washing the tissue sections in the buffered solution resulted in removal of endogenous soluble ionization-suppressing compounds and salts, while the target compound remained in situ with minor or no delocalization during the buffered washing procedure. Two pharmaceutical compounds (cimetidine and imipramine) and one new protease inhibitor compound were successfully used to evaluate the feasibility of the pH-controlled tissue washing protocol for MALDI-MSI. Enhancement in signal-to-noise ratio was achieved by a factor of up to 10.