K.W. Li

Rapid Communication: Neuropeptide Expression and Processing as Revealed by Direct Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Single Neurons

Abstract: Neuropeptides were directly detected in single identified neurons and the neurohemal area of peptidergic (neuroendocrine) systems in the Lymnaea brain by using matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS). The samples were placed in matrix solution and ruptured to allow mixing of cell contents with the matrix solution. After formation of matrix crystals, the analytes were analyzed by MALDI‐MS. It was surprising that clean mass spectra were produced, displaying extreme sensitivity of detection. In one of the neuroendocrine systems studied, we could demonstrate for the first time, by comparing the peptide patterns of soma and of neurohemal axon terminals, that processing of the complex prohormone expressed in this system occurs entirely in the soma. In the other system studied, novel peptides could be detected in addition to peptides previously identified by conventional molecular biological and peptide chemical methods. Thus, complex peptide processing and expression patterns could be predicted that were not detected in earlier studies using conventional methods. As the first MALDI‐ MS study of direct peptide fingerprinting in the single neuron these experients demonstrate that MALDI‐MS forms a new and valuable approach to the study of the synthesis and expression of bioactive peptides, with potential application to single‐cell studies in vertebrates, including humans.

Towards Understanding the Role of Insulin in the Brain: Lessons from Insulin-related Signaling Systems in the Invertebrate Brain

Insulin is a molecule that has played a key role in several of the most important landmarks in medical and biological research. It is one of the most extensively studied protein hormones, and its structure and function have been elucidated in many vertebrate species, ranging from man to hagfish and turkey. The structure, function as well as tissue of synthesis of vertebrate insulins are strictly conserved. The structural identification of insulin-related peptides from invertebrates has disrupted the picture of an evolutionary stable peptide hormone. Insulin-related peptides in molluscs and insects turned out to be a structurally diverse group encoded by large multi-gene families that are uniquely expressed in the brain and serve functions different from vertebrate insulin. In this review, we discuss invertebrate insulins in detail. We examine how these peptides relate to the model role that vertebrate insulin has played over the years; however, more importantly, we discuss several unique principles that can be learned from them. We show how diversity of these peptides is generated at the genetic level and how the structural diversity of the peptides is linked to the exclusive presence of a single type of neuronal insulin receptor-related receptor. We also discuss the fact that the invertebrate peptides, in addition to a hormonal role, may also act in a synaptic and/or nonsynaptic fashion as transmitters/neuromodulators on neurons in the brain. It can be expected that the use of well-defined neuronal preparations in invertebrates may lead to a further understanding of these novel functions and may act as guide preparations for a possible role of insulin and its relatives in the vertebrate brain.

Structural and functional characterization of neuropeptides involved in the control of male mating behavior of Lymnaea stagnalis

Mating as a male in the simultaneous hermaphrodite freshwater snail, Lymnaea stagnalis, comprises a series of complex behaviors that are a prelude to copulation. Copulatory behavior itself is assumed to be controlled by various types of peptidergic neurons as well as serotonergic cells. Here we report the primary structure of two peptides that were extracted from a cluster of neurons that innervates the penial complex and that is located in the anterior lobe of the right cerebral ganglion. The sequences of the peptides were determined as: Ala-Pro-Gly-Trp-amide and Ser-Gly-Ser-Asp-Tyr-Cys-Glu-Thr-Leu-Lys-Glu-Val-Ala-Asp-Glu-Tyr-Ile-Leu- Leu- Ser-Tyr-Lys-Ile-Glu-Glu-Gln-Arg-Ala-Ala-Asp-Cys-Gly-Gly-Glu-Pro-Pro-Asn- Ser- Gln(amide), respectively. The longer peptide is a homodimer. Both peptides are processed from the recently identified Ala-Pro-Gly-Trp-amide prohormone, which is expressed in the neurons of the anterior lobe of the right cerebral ganglion. Ala-Pro-Gly-Trp-amide could also be recovered from the penial complex. This peptide, when applied in vitro, inhibits the contractions of the penis retractor muscles evoked by serotonin in a dose-dependent fashion.

Protein synthesis in synaptosomes: a proteomics analysis

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre‐synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [35S]methionine and applied two‐dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two‐dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver‐stained gels that were run in parallel. In‐gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear‐encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre‐synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.

Co-localized neuropeptides conopressin and Ala-Pro-Gly-Trp-NH2 have antagonistic effects on the vas deferens of Lymnaea

We examined functional aspects of co-localization of neuropeptides involved in the regulation of male copulation behaviour in the simultaneous hermaphrodite snail Lymnaea stagnalis. The copulation behaviour is controlled by several types of peptidergic neurons that include a cluster of neurons in the anterior lobe of the right cerebral ganglion. All anterior lobe neurons express the gene encoding Ala-Pro-Gly-Trp-NH2 (APGWamide), and a subset of neurons also express the vasopressin-related conopressin gene. Immunocytochemical and peptide chemical experiments show that both APGWamide and conopressin are transported to the penis complex and the vas deferens via the penis nerve. Co-localization of the two peptides was also observed in some, but not all, axon bundles that run along the vas deferens. APGWamide and conopressin were structurally identified from the penis complex with vas deferens. Conopressin excites the vas deferens in vitro, whereas APGWamide inhibits the excitatory effects of conopressin, both in a dose-dependent fashion. We propose that the antagonistic effects of these peptides on the vas deferens underlie its peristalsis. Thus, these peptides play an important role in the control of ejaculation of semen during copulation.

A comparison of the synaptic proteome in human chronic schizophrenia and rat ketamine psychosis suggest that prohibitin is involved in the synaptic pathology of schizophrenia

Many studies in recent years suggest that schizophrenia is a synaptic disease that crucially involves a hypofunction of N-methyl-D-aspartate receptor-mediated signaling. However, at present it is unclear how these pathological processes are reflected in the protein content of the synapse. We have employed two-dimensional gel electrophoresis in conjunction with mass spectrometry to characterize and compare the synaptic proteomes of the human left dorsolateral prefrontal cortex in chronic schizophrenia and of the cerebral cortex of rats treated subchronically with ketamine. We found consistent changes in the synaptic proteomes of human schizophrenics and in rats with induced ketamine psychosis compared to controls. However, commonly regulated proteins between both groups were very limited and only prohibitin was found upregulated in both chronic schizophrenia and the rat ketamine model. Prohibitin, however, could be a new potential marker for the synaptic pathology of schizophrenia and might be causally involved in the disease process.

MICAL-1 is a negative regulator of MST-NDR kinase signaling and apoptosis.

ABSTRACT MICALs (molecules interacting with CasL) are atypical multidomain flavoenzymes with diverse cellular functions. The molecular pathways employed by MICAL proteins to exert their cellular effects remain largely uncharacterized. Via an unbiased proteomics approach, we identify MICAL-1 as a binding partner of NDR (nuclear Dbf2-related) kinases. NDR1/2 kinases are known to mediate apoptosis downstream of the mammalian Ste-20-like kinase MST1, and ablation of NDR1 in mice predisposes the mice to cancer as a result of compromised apoptosis. MST1 phosphorylates NDR1/2 kinases at their hydrophobic motif, thereby facilitating full NDR kinase activity and function. However, if and how this key phosphorylation event is regulated are unknown. Here we show that MICAL-1 interacts with the hydrophobic motif of NDR1/2 and that overexpression or knockdown of MICAL-1 reduces or augments NDR kinase activation or activity, respectively. Surprisingly, MICAL-1 is a phosphoprotein but not an NDR or MST1 substrate. Rather, MICAL-1 competes with MST1 for NDR binding and thereby antagonizes MST1-induced NDR activation. In line with this inhibitory effect, overexpression or knockdown of MICAL-1 inhibits or enhances, respectively, NDR-dependent proapoptotic signaling induced by extrinsic stimuli. Our findings unveil a previously unknown biological role for MICAL-1 in apoptosis and define a novel negative regulatory mechanism of MST-NDR signaling.

Structural identification, neuronal synthesis, and role in male copulation of myomodulin-A of Lymnaea: a study involving direct peptide profiling of nervous tissue by mass spectrometry.

We used a strategy combining immunodetection, peptide chemistry, and a novel method, direct peptide fingerprinting of neurons and small pieces of nerve by using matrix-assisted laser desorption ionization mass spectrometry, to structurally identify and localize the neuropeptide myomodulin-A in the mollusc, Lymnaea stagnalis. Lymnaea myomodulin appeared to be identical to Aplysia myomodulin-A and is produced by many central neurons, including neurons located in the ventral lobe of the right cerebral ganglion that innervate the penis complex via the penis nerve. Myomodulin-A could also be characterized from the penis complex, and physiological concentrations of the peptide enhanced the relaxation rate of electrically induced contractions of the penis retractor muscle in vitro in a dose-dependent fashion.

LFRFamides: a novel family of parasitation-induced -RFamide neuropeptides that inhibit the activity of neuroendocrine cells in Lymnaea stagnalis

We report the characterization of a cDNA encoding a novel ‐RFamide neuropeptide precursor that is up‐regulated during parasitation in the snail Lymnaea stagnalis. Processing of this precursor yields five structurally related neuropeptides, all but one ending with the C‐terminal sequence ‐LFRFamide, as was confirmed by direct mass spectrometry of brain tissue. The LFRFamide gene is expressed in a small cluster of neurons in each buccal ganglion, three small clusters in each cerebral ganglion, and one cluster in each lateral lobe of the cerebral ganglia. Application of two of the LFRFamide peptides to neuroendocrine cells that control either growth and metabolism or reproduction induced similar hyperpolarizing K+‐currents, and inhibited electrical activity. We conclude that up‐regulation of inhibitory LFRFamide neuropeptides during parasitation probably reflects an evolutionary adaptation that allows endoparasites to suppress host metabolism and reproduction in order to fully exploit host energy recourses.

Peptidomics of a single identified neuron reveals diversity of multiple neuropeptides with convergent actions on cellular excitability

In contrast to classical transmitters, the detailed structures and cellular and synaptic actions of neuropeptides are less well described. Peptide mass profiling of single identified neurons of the mollusc Lymnaea stagnalis indicated the presence of 17 abundant neuropeptides in the cardiorespiratory neuron, visceral dorsal 1 (VD1), and a subset of 14 peptides in its electrically coupled counterpart, right parietal dorsal 2. Altogether, based on this and previous work, we showed that the high number of peptides arises from the expression and processing of four distinct peptide precursor proteins, including a novel one. Second, we established a variety of posttranslational modifications of the generated peptides, including phosphorylation, disulphide linkage, glycosylation, hydroxylation, N-terminal pyroglutamylation, and C-terminal amidation. Specific synapses between VD1 and its muscle targets were formed, and their synaptic physiology was investigated. Whole-cell voltage-clamp analysis of dissociated heart muscle cells revealed, as tested for a selection of representative family members and their modifications, that the peptides of VD1 exhibit convergent activation of a high-voltage-activated Ca current. Moreover, the differentially glycosylated and hydroxylated α2 peptides were more potent than the unmodified α2 peptide in enhancing these currents. Together, this study is the first to demonstrate that single neurons exhibit such a complex pattern of peptide gene expression, precursor processing, and differential peptide modifications along with a remarkable degree of convergence of neuromodulatory actions. This study thus underscores the importance of a detailed mass spectrometric analysis of neuronal peptide content and peptide modifications related to neuromodulatory function.