Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics.

BACKGROUND: Rasal1 is a Ras GTPase-activating protein which contains C2 domains necessary for dynamic membrane association following intracellular calcium elevation. Membrane-bound Rasal1 inactivates Ras signaling through its RasGAP activity, and through such mechanisms has been implicated in regulating various cellular functions in the context of tumors. Although highly expressed in the brain, the contribution of Rasal1 to neuronal development and function has yet to be explored. RESULTS: We examined the contributions of Rasal1 to neuronal development in primary culture of hippocampal neurons through modulation of Rasal1 expression using molecular tools. Fixed and live cell imaging demonstrate diffuse expression of Rasal1 throughout the cell soma, dendrites and axon which localizes to the neuronal plasma membrane in response to intracellular calcium fluctuation. Pull-down and co-immunoprecipitation demonstrate direct interaction of Rasal1 with PKC, tubulin, and CaMKII. Consequently, Rasal1 is found to stabilize microtubules, through post-translational modification of tubulin, and accordingly inhibit dendritic outgrowth and branching. Through imaging, molecular, and electrophysiological techniques Rasal1 is shown to promote NMDA-mediated synaptic activity and CaMKII phosphorylation. CONCLUSIONS: Rasal1 functions in two separate roles in neuronal development; calcium regulated neurite outgrowth and the promotion of NMDA receptor-mediated postsynaptic events which may be mediated both by interaction with direct binding partners or calcium-dependent regulation of down-stream pathways. Importantly, the outlined molecular mechanisms of Rasal1 may contribute notably to normal neuronal development and synapse formation.

A Potential Role for the STXBP5-AS1 Gene in Adult ADHD Symptoms.

We aimed to detect Attention-deficit/hyperactivity (ADHD) risk-conferring genes in adults. In children, ADHD is characterized by age-inappropriate levels of inattention and/or hyperactivity-impulsivity and may persists into adulthood. Childhood and adulthood ADHD are heritable, and are thought to represent the clinical extreme of a continuous distribution of ADHD symptoms in the general population. We aimed to leverage the power of studies of quantitative ADHD symptoms in adults who were genotyped. Within the SAGA (Study of ADHD trait genetics in adults) consortium, we estimated the single nucleotide polymorphism (SNP)-based heritability of quantitative self-reported ADHD symptoms and carried out a genome-wide association meta-analysis in nine adult population-based and case-only cohorts of adults. A total of n = 14,689 individuals were included. In two of the SAGA cohorts we found a significant SNP-based heritability for self-rated ADHD symptom scores of respectively 15% (n = 3656) and 30% (n = 1841). The top hit of the genome-wide meta-analysis (SNP rs12661753; p-value = 3.02 × 10-7) was present in the long non-coding RNA gene STXBP5-AS1. This association was also observed in a meta-analysis of childhood ADHD symptom scores in eight population-based pediatric cohorts from the Early Genetics and Lifecourse Epidemiology (EAGLE) ADHD consortium (n = 14,776). Genome-wide meta-analysis of the SAGA and EAGLE data (n = 29,465) increased the strength of the association with the SNP rs12661753. In human HEK293 cells, expression of STXBP5-AS1 enhanced the expression of a reporter construct of STXBP5, a gene known to be involved in "SNAP" (Soluble NSF attachment protein) Receptor" (SNARE) complex formation. In mouse strains featuring different levels of impulsivity, transcript levels in the prefrontal cortex of the mouse ortholog Gm28905 strongly correlated negatively with motor impulsivity as measured in the five choice serial reaction time task (r2 = - 0.61; p = 0.004). Our results are consistent with an effect of the STXBP5-AS1 gene on ADHD symptom scores distribution and point to a possible biological mechanism, other than antisense RNA inhibition, involved in ADHD-related impulsivity levels.

Constitutive loss and acute pharmacological manipulation of ErbB4 signaling do not affect attention and inhibitory control in mice.

The receptor tyrosine kinase ErbB4 and its ligand trophic factors of the neuregulin (NRG) family have been associated with schizophrenia and other mental disorders in human genetic studies. In vivo studies in mice have shown how abnormal Nrg-ErbB4 signaling leads to deviant behaviors relevant to distinct aspects of schizophrenia, including hyperactivity, sensory gating deficits, working and spatial memory deficits and impaired social behavior. However, so far little is known on the role of ErbB4 in attention and inhibitory control, two aspects of executive functions that are impaired in schizophrenia. Here we investigated the effects of constitutive loss of ErbB4 in the central nervous system of mice on performance in a 5-choice serial reaction time task (5CSRTT) assessing attention and inhibitory control. In this task, ErbB4-/- mice did not show deficits in various parameters of attention, and premature responses as measure of inhibitory control. Nonetheless, ErbB4-/- mice recapitulated a specific set of behavioral phenotypes associated with schizophrenia, including a deficit in spatial learning and memory in the Barnes Maze and in contextual fear learning, and a trend for a deficit in sensorimotor gating. Furthermore, we investigated the effect of acute pharmacological inhibition of ErbB tyrosine kinase receptor using the pan-ErbB kinase inhibitor JNJ-28871063 (JNJ), in an automated version of the 5CSRTT. JNJ did not affect attention and inhibitory control. In conclusion, our data suggest no direct involvement of a classical Nrg-ErbB4 pathway in attention and inhibitory control in mice, while it confirms the involvement of this pathway in other domains relevant to schizophrenia.

Temporal profiling of depression vulnerability in a preclinical model of sustained depression.

Major Depression is a prevalent mental disorder that is characterized by negative mood and reduced motivation, and frequently results in social withdrawal and memory-related deficits. Repeated stressors, such as adverse life events, increase the risk for development of the disorder. Consequently, individual variability in stress response greatly weighs on depression-vulnerability and -resilience. Here, we employed the social defeat-induced persistent stress (SDPS) paradigm to identify depression-prone individuals and to examine the temporal development of depression in the months following exposure to brief defeat stress. Male Wistar rats were socially defeated (5 defeat episodes) and single-housed for a prolonged period of time (~24 weeks). We assessed the emergence of a sustained depressive-like state by repeatedly evaluating social motivation (social approach avoidance) and spatial memory (object place recognition) in SDPS rats during the isolation period. Individual variability in the effects of SDPS yielded two extreme subpopulations: an SDPS-prone group that showed gradual affective and cognitive deterioration in terms of social approach and memory retention, and a SDPS-resilient group that did not develop this phenotype. Notably, in SDPS-prone individuals, the affective deficits preceded later cognitive impairments, providing a novel temporal profile of the development of pathology in this preclinical model of sustained depression.

Evaluation of a chest radiograph reading and recording system for tuberculosis in a HIV-positive cohort.

AIM: To assess the impact of introducing a chest radiograph reading and recording system (CRRS) with a short training session, on the accuracy and inter-reader variability of tuberculosis (TB) interpretation of chest radiographs (CXRs) by a group of non-expert readers in a human immunodeficiency virus (HIV)-positive cohort. MATERIALS AND METHODS: A set of 139 CXRs was reviewed by a group of eight physicians pre- and post-intervention at two clinics in Shan State, Myanmar, providing HIV/TB diagnosis and treatment services. The results were compared against the consensus of expert radiologists for accuracy. RESULTS: Overall accuracy was similar pre- and post-intervention for most physicians with an average area under the receiver operating characteristic curve difference of 0.02 (95% confidence interval: -0.03, 0.07). The overall agreement among physicians was poor pre- and post-intervention (Fleiss κ=0.35 and κ=0.29 respectively). The assessment of agreement for specific disease patterns associated with active TB in HIV-infected patients showed that for intrinsically subtle findings, the agreement was generally poor but better for the more intrinsically obvious disease patterns: pleural effusion (Cohen's kappa range = 0.37-0.67) and milliary nodular pattern (Cohen's kappa range = 0.25-0.52). CONCLUSION: This study demonstrated limited impact of the introduction of a CRRS on CXR accuracy and agreement amongst non-expert readers. The role in which CXRs are used for TB diagnosis in a HIV-positive cohort in similar clinical contexts should be reviewed.

Synergistic targeting/prodrug strategies for intravesical drug delivery--lectin-modified PLGA microparticles enhance cytotoxicity of stearoyl gemcitabine by contact-dependent transfer.

The direct access to the urothelial tissue via intravesical therapy has emerged as a promising means for reducing the high recurrence rate of bladder cancer. However, few advanced delivery concepts have so far been evaluated to overcome critical inherent efficacy limitations imposed by short exposure times, low tissue permeability, and extensive washout. This study reports on a novel strategy to enhance gemcitabine treatment impact on urothelial cells by combining a pharmacologically advantageous prodrug approach with the pharmacokinetic benefits of a glycan-targeted carrier system. The conversion of gemcitabine to its 4-(N)-stearoyl derivative (GEM-C18) allowed for stable, homogeneous incorporation into PLGA microparticles (MP) without compromising intracellular drug activation. Fluorescence-labeled GEM-C18-PLGA-MP were surface-functionalized with wheat germ agglutinin (WGA) or human serum albumin (HSA) to assess in direct comparison the impact of biorecognitive interaction on binding rate and anchoring stability. MP adhesion on urothelial cells of non-malignant origin (SV-HUC-1), and low- (5637) or high-grade (HT-1376) carcinoma was correlated to the resultant antiproliferative and antimetabolic effect in BrdU and XTT assays. More extensive and durable binding of the WGA-GEM-C18-PLGA-MP induced a change in the pharmacological profile and substantially higher cytotoxicity, allowing for maximum response within the temporal restrictions of instillative administration (120 min). Mechanistically, a direct, contact-dependent transfer of stearoyl derivatives from the particle matrix to the urothelial membrane was found to account for this effect. With versatile options for future application, our results highlight the potential offered by the synergistic implementation of targeting/prodrug strategies in delivery systems tailored to the intravesical route.

A novel QTL underlying early-onset, low-frequency hearing loss in BXD recombinant inbred strains.

The DBA/2J inbred strain of mice has been used extensively in hearing research as it suffers from early-onset, progressive hearing loss. Initially, it mostly affects high frequencies, but already at 2-3 months hearing loss becomes broad. In search for hearing loss genes other than Cadherin 23 (otocadherin) and fascin-2, which make a large contribution to the high-frequency deficits, we used a large set of the genetic reference population of BXD recombinant inbred strains. For frequencies 4, 8, 16 and 32 kHz, auditory brainstem response hearing thresholds were longitudinally determined from 2-3 up to 12 weeks of age. Apart from a significant, broad quantitative trait locus (QTL) for high-frequency hearing loss on chromosome 11 containing the fascin-2 gene, we found a novel, small QTL for low-frequency hearing loss on chromosome 18, from hereon called ahl9. Real-time quantitative polymerase chain reaction of organs of Corti, isolated from a subset of strains, showed that a limited number of genes at the QTL were expressed in the organ of Corti. Of those genes, several showed significant expression differences based on the parental line contributing to the allele. Our results may aid in the future identification of genes involved in low-frequency, early-onset hearing loss.

Independent genetic loci for sensorimotor gating and attentional performance in BXD recombinant inbred strains.

A startle reflex in response to an intense acoustic stimulus is inhibited when a barely detectable pulse precedes the startle stimulus by 30-500 ms. It has been theorized that this phenomenon, named prepulse inhibition (PPI) of a startle response, is an automatic early-stage gating process contributing to the ability to focus attention. Deficits in PPI may therefore contribute to deficits in attentional processing. Both deficits are observed in schizophrenia spectrum disorders. Here, we investigated whether there is overlap in genetic control of PPI and attentional processing phenotypes in the panel of BXD recombinant inbred strains of mice. Using an individually titrated prepulse intensity to handle differences in perceived prepulse intensities among strains, we identified a significant quantitative trait locus (QTL) for PPI at the mid-distal end of chromosome 17. A measure of attentional processing in the five-choice serial reaction time task, response variability, mapped to a different locus on proximal-mid chromosome 16. In addition, the estimated genetic and environmental correlations between PPI and several attentional phenotypes were low and not significant. Taken together, the observation of separate genetic loci for PPI and attention and the absence of genetic and environmental correlations indicate that differences in sensorimotor gating do not contribute to differences in attentional performance. Therefore, it is worth pursuing the causative genes residing in both attention and PPI QTL, as these may contribute to separate molecular pathways implicated in neuropsychiatric diseases, such as schizophrenia.

Reduction in hippocampal neurogenesis after social defeat is long-lasting and responsive to late antidepressant treatment.

Major depressive disorder is a chronic disabling disease, often triggered and exacerbated by stressors of a social nature. Hippocampal volume reductions have been reported in depressed patients. In support of the neurogenesis theory of depression, in several stress-based animal models of depression, adult hippocampal neurogenesis was reduced and subsequently rescued by parallel antidepressant treatment. Here, we investigated whether repeated social defeat and subsequent individual housing for 3 months induces long-lasting changes in adult hippocampal neurogenesis in rats, and whether these can be normalized by late antidepressant treatment, as would match human depression. Neurogenesis was analysed by stereological quantification of the number of immature doublecortin (DCX)-immunopositive cells, in particular young (class I) and more mature (class II) DCX(+) cells, to distinguish differential effects of stress or drug treatment on these subpopulations. Using this social defeat paradigm, the total DCX(+) cell number was significantly reduced. This was most profound for older (class II) DCX(+) cells with long apical dendrites, whereas younger, class I cells remained unaffected. Treatment with the broad-acting tricyclic antidepressant imipramine, only during the last 3 weeks of the 3-month period after social defeat, completely restored the reduction in neurogenesis by increasing both class I and II DCX(+) cell populations. We conclude that despite the lack of elevated corticosterone plasma levels, neurogenesis is affected in a lasting manner by a decline in a distinct neuronal population of more mature newborn cells. Thus, the neurogenic deficit induced by this social defeat paradigm is long-lasting, but can still be normalized by late imipramine treatment.

Activity and impulsive action are controlled by different genetic and environmental factors.

Both impulsivity in operant tasks and locomotor activity in a novel open field are known to predict the development of addiction-related behavior in rodents. In this study, we investigated to what extent impulsivity in the five-choice serial reaction time task and various measures of novelty exploration are controlled by shared genetic and environmental factors in 12 different inbred mouse strains. No genetic correlation was observed between the level of impulsivity and levels of activity, a low correlation was observed with traditional measures of anxiety-like behavior (impulsive strains tend to be less anxious) and a highly significant correlation was found between impulsivity and specific aspects of movement. Furthermore, we found that impulsivity and all measures of novelty exploration were under control of different environmental factors. Interestingly, in the dorsal medial prefrontal cortex, a brain region involved in impulsivity and activity in novelty exploration tests; these behavioral measures correlated with the expression of different genes (respectively, Frzb, Snx5, BC056474 and the previously identified Glo1). Taken together, our study shows that impulsivity and activity in novelty exploration tests are genetically and environmentally distinct, suggesting that mouse models of these behaviors provide complementary insights into the development of substance abuse disorder.

Reduced expression of neuropeptide genes in a genome-wide screen of a secretion-deficient mouse.

Activity-dependent changes in synapses rely on functional changes in resident proteins and on gene expression. We addressed the relationship between synapse activity and the expression of synaptic genes by comparing RNA levels in the neocortex of normal mice versus secretion-deficient and therefore synaptically silent munc18-1 (mammalian homologue of Caenorhabditis elegans uncoordinated locomotion-18) null mutants, using microarray expression analysis, real-time quantitative PCR and northern blotting. We hypothesized that genes under the control of synaptic activity would be differentially expressed between mutants and controls. We found that few synaptic genes were differentially expressed. However, most neuropeptide genes with detectable expression on the microarray were differentially expressed, being expressed 3-20-fold higher in control cortex. Several other secreted proteins were also differentially expressed, but genes encoding their receptors and many other synaptic components were not. Differential expression was confirmed by real-time quantitative PCR analysis. In situ hybridization indicated that the difference in neuropeptide expression was uniform and not due to the loss of specific cells in the mutant. In primary sensory neurons, which do not depend on synaptic activity for their input, the differential expression of neuropeptides was not observed. These data argue against a general relationship between the activity of synapses and the expression of their resident proteins, but suggest a link between secretion and the expression of genes encoding the secreted products.

Bioflocculation of mesophilic and thermophilic activated sludge.

Thermophilic activated sludge treatment is often hampered by a turbid effluent. Reasons for this phenomenon are so far unknown. Here, the hypothesis of the temperature dependency of the hydrophobic interaction as a possible cause for diminished thermophilic activated sludge bioflocculation was tested. Adsorption of wastewater colloidal particles was monitored on different flat surfaces as a function of temperature. Adsorption on a hydrophobic surface varied with temperature between 20 and 60 degrees C and no upward or downward trend could be observed. This makes the hydrophobic interaction hypothesis unlikely in explaining the differences in mesophilic and thermophilic activated sludge bioflocculation. Both mesophilic and thermophilic biomass did not flocculate with wastewater colloidal particles under anaerobic conditions. Only in the presence of oxygen, with biologically active bacteria, the differences in bioflocculation behavior became evident. Bioflocculation was shown only to occur with the combination of wastewater and viable mesophilic biomass at 30 degrees C, in the presence of oxygen. Bioflocculation did not occur in case the biomass was inactivated or when oxygen was absent. Thermophilic activated sludge hardly showed any bioflocculation, also under mesophilic conditions. Despite the differences in bioflocculation behavior, sludge hydrophobicity and sludge zetapotentials were almost similar. Theoretical calculations using the DLVO (Derjaguin, Landau, Verweij and Overbeek) theory showed that flocculation is unlikely in all cases due to long-range electrostatic forces. These calculations, combined with the fact that bioflocculation actually did occur at 30 degrees C and the unlikelyness of the hydrophobic interaction, point in the direction of bacterial exo-polymers governing bridging flocculation. Polymer interactions are not included in the DLVO theory and may vary as a function of temperature.

GABAA receptor maturation in relation to eye opening in the rat visual cortex.

Changes in subunit composition of N-methyl-D-aspartate (NMDA) receptors have been reported to be affected by visual experience and may therefore form a major aspect of neuronal plasticity in the CNS during development. In contrast, putative alterations in the expression and functioning of the inhibitory GABAA receptor around eye opening have not been well defined yet. Here we describe the timing of changes in GABAA receptor subunit expression and the related synaptic functioning in the neonatal rat visual cortex and the influence of visual experience on this process. Quantitative analysis of all GABAA receptor subunit transcripts revealed a marked alpha3 to alpha1 subunit switch, in addition to a change in alpha4 and alpha5 expression. The changes were correlated with an acceleration of the decay of spontaneous inhibitory postsynaptic currents (sIPSCs). Both changes in receptor expression and synaptic functioning were initiated well before eye opening. Moreover, dark rearing could not prevent the robust upregulation of alpha1 or the change in sIPSC kinetics, indicating that this is not dependent of sensory (visual) input. Upon eye opening a positive correlation was observed between a faster decay of the sIPSCs and an increase in sIPSC frequency, which was absent in dark-reared animals. Thus, lack of extrinsic input to the cortex does not affect overall developmental regulation of synaptic functioning of GABAA receptors. However, we cannot exclude the possibility that visual experience is involved in proper shaping of the inhibitory network of the primary visual cortex.

Peptidergic modulation of male sexual behavior in Lymnaea stagnalis: structural and functional characterization of -FVamide neuropeptides.

In the simultaneous hermaphrodite snail Lymnaea stagnalis, copulation as a male is controlled by neurons that send axons to the male copulatory organs via a single penis nerve. Using direct mass spectrometry of a penis nerve sample, we show that one of the molecular ions has a mass corresponding to GAPRFVamide, previously identified from the buccal ganglia, and named Lymnaea inhibitory peptide (LIP). The identity of this peptide is confirmed by partial peptide purification from the penis nerve, followed by post source decay mass spectrometry. We cloned the LIP-encoding cDNA, which predicts a prohormone that gives rise to five copies of LIP (now re-named LIP A), two other -FVamide peptides (LIPs B and C), and five structurally unrelated peptides. The LIP gene is expressed in neurons of the right cerebral ventral lobe that send their axons into the penis nerve. We show that the LIP A peptide is present in these neurons and in the penis nerve, and confirmed the presence of LIP B and C in the penis nerve by post source decay mass spectrometry. Finally, we demonstrate that LIP A, B and C inhibit the contractions of the penis retractor muscle, thereby implicating their role in male copulation behavior.

Impaired dendritic spine maturation in GABAA receptor alpha1 subunit knock out mice.

In this study we investigated the functional implications of GABAA receptor alpha1 subunit deletion on dendritic arborization and spine maturation in the visual cortex. This subunit is normally strongly upregulated during early postnatal development. Our main finding is that mice lacking the GABAA receptor alpha1 subunit displayed an increased density of dendritic filopodia during the second and third postnatal weeks. However, there was a concomitant decreased density of mature mushroom-shaped spines, which became more pronounced in adults. In contrast, dendritic arborization was not altered in these mice. We propose that an increased efficacy of the inhibitory synaptic transmission in the alpha1 knock out mice may lead to an enhancement of the outgrowth of filopodia around eye opening, but to a failure in spine maturation at later stages.

Neuropeptide amidation: cloning of a bifunctional alpha-amidating enzyme from Aplysia.

One of the most common mechanisms of posttranslational modifications to generate biologically active (neuro)peptides is the process of peptide alpha-amidation. The only enzyme known to catalyze this important modification is peptidylglycine alpha-amidating monooxygenase (PAM): a (bifunctional) zymogen, giving rise to a monooxygenase (PHM) and a lyase (PAL). The highly peptidergic central nervous system and endocrine system of the marine mollusk Aplysia has homologs of various mammalian peptide processing enzymes, including furin, Afurin2, prohormone convertase 1 (PC1), PC2, carboxypeptidase E (CPE) and CPD. Previously, it has been shown that the abdominal ganglion of Aplysia, which contains approximately 800 peptidergic bag cell neurons, contains the highest specific alpha-amidating activity. We have identified and cloned multiple overlapping central nervous system and bag cell cDNAs that encode a predicted 748-residue protein that is a member of the PAM family. The protein sequence contains the contiguous sequence of the catalytic domains of PHM and PAL, clearly demonstrating the existence of bifunctional Aplysia PAM, the first invertebrate PAM zymogen with an organization similar to that in vertebrates. None of the characterized clones encoded the so-called exon A domain between the PHM and PAL domains. Furthermore, in a specific search by reverse transcription-polymerase chain reaction of RNA from multiple tissues we could only detect exon A-less transcripts. PAM expression was detected in the central nervous system, and in several endocrine and exocrine organs. Aplysia PAM is a candidate prohormone processing enzyme that plays an important role in the processing of Aplysia prohormones in the secretory pathway.

Family of prohormone convertases in Lymnaea: characterization of two alternatively spliced furin-like transcripts and cell-specific regulation of their expression.

The majority of neuropeptides in Lymnaea stagnalis are proteolytically processed from larger precursors at sites composed of single or multiple basic amino acid residues. Previous studies have identified several putative prohormone convertases in the brain of Lymnaea. To characterize the complete family, we undertook three independent approaches: reverse-transcribed polymerase chain reaction screening, and low-stringency cDNA and genomic library screenings. The central nervous system cDNA library screening yielded two cDNAs encoding Lfurin1 and its variant form, Lfurin1-X. Both proteins show the characteristic organization of (human) furin with a putative catalytic domain, a P domain, a Cys-rich domain, a transmembrane domain, and a cytoplasmic tail. Lfurin1 and Lfurin1-X are identical, apart from a putative alternatively spliced noncatalytic luminal protein domain, which is present exclusively in Lfurin1-X. In situ hybridization revealed that the Lfur1 gene is expressed throughout the Lymnaea brain, but that the level varies considerably from one neuron to another. Quantitative analysis of the expression level of the two alternatively spliced transcripts revealed that it is neuron type-specifically regulated. This probably indicates the functional importance of noncatalytic luminal protein domains in these enzymes. In addition, our findings suggest that apart from the identified convertases LPC2, Lfurin1/Lfurin1-X, and Lfurin2, additional prohormone convertase diversity is either not present or present only at low levels in the Lymnaea brain. Alternatively, additional prohormone convertases could exist with a lower degree of sequence conservation than the other Lymnaea prohormone convertase members. From our findings, it appears that the majority of prohormone processing in Lymnaea is carried out by the three thus far identified types of Kex2-related prohormone convertases despite the large number of neuropeptide precursors and diverse multiple basic cleavage sites hydrolyzed.

A molluscan peptide alpha-amidating enzyme precursor that generates five distinct enzymes.

Mechanisms underlying the specificity and efficiency of enzymes, which modify peptide messengers, especially with the variable requirements of synthesis in the neuronal secretory pathway, are poorly understood. Here, we examine the process of peptide alpha-amidation in individually identifiable Lymnaea neurons that synthesize multiple proproteins, yielding complex mixtures of structurally diverse peptide substrates. The alpha-amidation of these peptide substrates is efficiently controlled by a multifunctional Lymnaea peptidyl glycine alpha-amidating monooxygenase (LPAM), which contains four different copies of the rate-limiting Lymnaea peptidyl glycine alpha-hydroxylating monooxygenase (LPHM) and a single Lymnaea peptidyl alpha-hydroxyglycine alpha-amidating lyase. Endogenously, this zymogen is converted to yield a mixture of monofunctional isoenzymes. In vitro, each LPHM displays a unique combination of substrate affinity and reaction velocity, depending on the penultimate residue of the substrate. This suggests that the different isoenzymes are generated in order to efficiently amidate the many peptide substrates that are present in molluscan neurons. The cellular expression of the LPAM gene is restricted to neurons that synthesize amidated peptides, which underscores the critical importance of regulation of peptide alpha-amidation.

Direct mass spectrometric peptide profiling and sequencing of single neurons reveals differential peptide patterns in a small neuronal network.

Mass spectrometry (MS) was employed to detect and structurally characterize peptides in two functionally related neurons, named VD1 and RPD2, which form a network involved in the modulation of heartbeat in Lymnaea. Matrix-assisted laser desorption/ionization MS, directly applied to single neurons VD1 and RPD2, showed overlapping yet distinct mass profiles, with a subset of putative peptides specifically present in neuron VD1. Direct tandem MS of a single VD1 neuron revealed the primary structures of the VD1-specific peptides, which were identified as members of the family of small cardioactive peptides. Based on the tandem MS data, a degenerate oligonucleotide was made for use in a polymerase chain reaction strategy to isolate the cDNA encoding the precursor to the small cardioactive peptides from a brain-specific cDNA library. The calculated masses of the mature, posttranslationally modified peptides, as predicted from the corresponding cDNA, agreed with the measured masses of the actual peptides, as detected in single-cell MS analysis. In situ hybridization studies showed that the transcript encoding the precursor is present in VD1, but not in RPD2, thus corroborating the single-cell MS analysis. Finally, the small cardioactive peptides were shown to enhance the contractions of the auricle in vitro.

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.