Elena V. Romanova

Profiling metabolites and peptides in single cells

The intracellular levels and spatial localizations of metabolites and peptides reflect the state of a cell and its relationship to its surrounding environment. Moreover, the amounts and dynamics of metabolites and peptides are indicative of normal or pathological cellular conditions. Here we highlight established and evolving strategies for characterizing the metabolome and peptidome of single cells. Focused studies of the chemical composition of individual cells and functionally defined groups of cells promise to provide a greater understanding of cell fate, function and homeostatic balance. Single-cell bioanalytical microanalysis has also become increasingly valuable for examining cellular heterogeneity, particularly in the fields of neuroscience, stem cell biology and developmental biology.

Genome-Wide Analyses Reveal a Role for Peptide Hormones in Planarian Germline Development

Genomic/peptidomic analyses of the planarian Schmidtea mediterranea identifies >200 neuropeptides and uncovers a conserved neuropeptide required for proper maturation and maintenance of the reproductive system.

Distinguishing Endogenous d-Amino Acid-Containing Neuropeptides in Individual Neurons Using Tandem Mass Spectrometry

RNA-based protein synthesis produces L-amino acid-containing proteins and peptides. D-amino acid-containing peptides (DAACPs) can be generated from L-amino acid peptides via post-translational modification. In the nervous system, the conformational change of a single L-amino acid in a peptide to its D-form results in altered bioactivity, with some DAACPs having orders-of-magnitude enhanced efficacy. However, this modification is often overlooked when characterizing endogenous peptides. Here, with the use of matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry, neuropeptides that have the second residue isomerized to the D-isoform are distinguished from their L-epimers via differences in the relative amounts of specific fragment ions during tandem MS. With the appropriate fragment ions chosen, and in some cases with the use of metal adducts, epimer discrimination is optimized. Specifically, the cardioexcitatory peptide Asn-(D)Trp-Phe-amide (NdWFa) was assayed directly from neurons isolated from the sea slug Aplysia californica; the fraction of the peptide with the second residue (W) in the D- versus L-form was 90 ± 10%. We demonstrate that this approach is well suited for confirming DAACPs directly from cells and tissue, advancing our understanding of the l to d modification and the role it plays in cell-to-cell signaling.

Circadian Integration of Glutamatergic Signals by Little SAAS in Novel Suprachiasmatic Circuits

Background Neuropeptides are critical integrative elements within the central circadian clock in the suprachiasmatic nucleus (SCN), where they mediate both cell-to-cell synchronization and phase adjustments that cause light entrainment. Forward peptidomics identified little SAAS, derived from the proSAAS prohormone, among novel SCN peptides, but its role in the SCN is poorly understood. Methodology/Principal Findings Little SAAS localization and co-expression with established SCN neuropeptides were evaluated by immunohistochemistry using highly specific antisera and stereological analysis. Functional context was assessed relative to c-FOS induction in light-stimulated animals and on neuronal circadian rhythms in glutamate-stimulated brain slices. We found that little SAAS-expressing neurons comprise the third most abundant neuropeptidergic class (16.4%) with unusual functional circuit contexts. Little SAAS is localized within the densely retinorecipient central SCN of both rat and mouse, but not the retinohypothalamic tract (RHT). Some little SAAS colocalizes with vasoactive intestinal polypeptide (VIP) or gastrin-releasing peptide (GRP), known mediators of light signals, but not arginine vasopressin (AVP). Nearly 50% of little SAAS neurons express c-FOS in response to light exposure in early night. Blockade of signals that relay light information, via NMDA receptors or VIP- and GRP-cognate receptors, has no effect on phase delays of circadian rhythms induced by little SAAS. Conclusions/Significance Little SAAS relays signals downstream of light/glutamatergic signaling from eye to SCN, and independent of VIP and GRP action. These findings suggest that little SAAS forms a third SCN neuropeptidergic system, processing light information and activating phase-shifts within novel circuits of the central circadian clock.

Engineering the morphology and electrophysiological parameters of cultured neurons by microfluidic surface patterning

The ability to control the orientation, morphology, and electrophysiological characteristics of neurons in culture allows the construction of neural circuits with defined physiological properties. Using microfluidic protein deposition onto chemically modified glass, we achieve the controlled growth of Aplysia neurons on geometrical patterns of poly‐L‐lysine and collagen IV, surrounded by nonadhesive regions of bovine albumin. We investigate the parameters essential for forming functional neuronal networks, the morphology, biochemistry, and electrophysiology under engineered cell culture conditions. We demonstrate that not only the orientation of neurite extension but also the number of primary neurites originating from the cell soma, their length, and branching pattern depend on the spatial constraints presented by the size and shape of the adhesion region on the patterned substrate. In addition, the physicochemical properties of the support layer influence the electrical activity of the cultured neurons. Substrate‐dependent changes in the amplitude and in the dynamic parameters of the action potential cause decreased spike broadening in patterned neurons, which reflects changes in the number or functioning of active membrane ion channels. In contrast to morphology and electrophysiology, the neuropeptide content, as determined by mass spectrometry of individual patterned neurons, is not affected by the growth on patterned surfaces. Our results suggest that the morphological and electrophysiological parameters of neurons can be predictably altered/engineered by modulation of the chemical, physical, and topographical features of culture substrates. We also demonstrate that a full suite of techniques is required for functional characterization of neurons on engineered substrates.

Feedforward Compensation Mediated by the Central and Peripheral Actions of a Single Neuropeptide Discovered Using Representational Difference Analysis

Compensatory mechanisms are often used to achieve stability by reducing variance, which can be accomplished via negative feedback during homeostatic regulation. In principle, compensation can also be implemented through feedforward mechanisms where a regulator acts to offset the anticipated output variation; however, few such neural mechanisms have been demonstrated. We provide evidence that an Aplysia neuropeptide, identified using an enhanced representational difference analysis procedure, implements feedforward compensation within the feeding network. We named the novel peptide “allatotropin-related peptide” (ATRP) because of its similarity to insect allatotropin. Mass spectrometry confirmed the peptides identity, and in situ hybridization and immunostaining mapped its distribution in the Aplysia CNS. ATRP is present in the higher-order cerebral-buccal interneuron (CBI) CBI-4, but not in CBI-2. Previous work showed that CBI-4-elicited motor programs have a shorter protraction duration than those elicited by CBI-2. Here we show that ATRP shortens protraction duration of CBI-2-elicited ingestive programs, suggesting a contribution of ATRP to the parametric differences between CBI-4-evoked and CBI-2-evoked programs. Importantly, because Aplysia muscle contractions are a graded function of motoneuronal activity, one consequence of the shortening of protraction is that it can weaken protraction movements. However, this potential weakening is offset by feedforward compensatory actions exerted by ATRP. Centrally, ATRP increases the activity of protraction motoneurons. Moreover, ATRP is present in peripheral varicosities of protraction motoneurons and enhances peripheral motoneuron-elicited protraction muscle contractions. Therefore, feedforward compensatory mechanisms mediated by ATRP make it possible to generate a faster movement with an amplitude that is not greatly reduced, thereby producing stability.

Cerebrin prohormone processing, distribution and action in Aplysia californica

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17‐residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic purification guided by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F‐ and C‐clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral‐buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command‐like neuron CBI‐2, dramatically shortening the duration of the radula protraction in a concentration‐dependent manner, mimicking the motor‐pattern alterations observed in food induced arousal states. These findings suggest that cerebrin may contribute to food‐induced arousal in the animal. Cerebrin‐like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin‐like peptides may be widespread throughout gastropoda.

Distinct mechanisms produce functionally complementary actions of neuropeptides that are structurally related but derived from different precursors

Many bioactive neuropeptides containing RFamide at their C terminus have been described in both invertebrates and vertebrates. To obtain insight into the functional logic of RFamide signaling, we investigate it here in the feeding system of Aplysia. We focus on the expression, localization, and actions of two families of RFamide peptides, the FRFamides and FMRFamide, in the central neuronal circuitry and the peripheral musculature that generate the feeding movements. We describe the cloning of the FRFamide precursor protein and show that the FRFamides and FMRFamide are derived from different precursors. We map the expression of the FRFamide and FMRFamide precursors in the feeding circuitry using in situ hybridization and immunostaining and confirm proteolytic processing of the FRFamide precursor by mass spectrometry. We show that the two precursors are expressed in different populations of sensory neurons in the feeding system. In a representative feeding muscle, we demonstrate the presence of both FRFamides and FMRFamide and their release, probably from the processes of the sensory neurons in the muscle. Both centrally and in the periphery, the FRFamides and FMRFamide act in distinct ways, apparently through distinct mechanisms, and nevertheless, from an overall functional perspective, their actions are complementary. Together, the FRFamides and FMRFamide convert feeding motor programs from ingestive to egestive and depress feeding muscle contractions. We conclude that these structurally related peptides, although derived from different precursors, expressed in different neurons, and acting through different mechanisms, remain related to each other in the functional roles that they play in the system.

Classification of Large Cellular Populations and Discovery of Rare Cells Using Single Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Cell-to-cell variability and functional heterogeneity are integral features of multicellular organisms. Chemical classification of cells into cell type is important for understanding cellular specialization as well as organismal function and organization. Assays to elucidate these chemical variations are best performed with single cell samples because tissue homogenates average the biochemical composition of many different cells and oftentimes include extracellular components. Several single cell microanalysis techniques have been developed but tend to be low throughput or require preselection of molecular probes that limit the information obtained. Mass spectrometry (MS) is an untargeted, multiplexed, and sensitive analytical method that is well-suited for studying chemically complex individual cells that have low analyte content. In this work, populations of cells from the rat pituitary, the rat pancreatic islets of Langerhans, and from the Aplysia californica nervous system, are classified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) MS by their peptide content. Cells were dispersed onto a microscope slide to generate a sample where hundreds to thousands of cells were separately located. Optical imaging was used to determine the cell coordinates on the slide, and these locations were used to automate the MS measurements to targeted cells. Principal component analysis was used to classify cellular subpopulations. The method was modified to focus on the signals described by the lower principal components to explore rare cells having a unique peptide content. This approach efficiently uncovers and classifies cellular subtypes as well as discovers rare cells from large cellular populations.

MALDI mass spectrometric imaging using the stretched sample method to reveal neuropeptide distributions in Aplysia nervous tissue

Neuropeptides are a diverse set of complex cell-cell signaling molecules that modulate behavior, learning, and memory. Their spatially heterogeneous distributions, large number of post-translational modifications, and wide range of physiologically active concentrations make their characterization challenging. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging is well-suited to characterizing and mapping neuropeptides in the central nervous system. Because matrix application can cause peptide migration within tissue samples, application parameters for MALDI typically represent a compromise between attaining the highest signal quality and preserving native spatial distributions. The stretched sample approach minimizes this trade-off by fragmenting the tissue section into thousands of spatially isolated islands, each approximately 40 mum in size. This inhibits analyte migration between the pieces and, at the same time, reduces analyte-salt adduct formation. Here, we present methodological improvements that enable the imaging of stretched tissues and reveal neuropeptide distributions in nervous tissue from Aplysia californica. The distributions of known neuropeptides are shown to correspond with previous immunohistochemical results, demonstrating that the stretched imaging method is well-suited for working with easily redistributed molecules and heterogeneous tissues and reduces adducts from physiological salts.