Structural domains of IS10 transposase and reconstitution of transposition activity from proteolytic fragments lacking an interdomain linker.

All of the DNA cleavage and strand transfer events required for transposition of insertion sequence IS10 are carried out by a 46-kDa IS10-encoded transposase protein. Limited proteolysis demonstrates that transposase has two principal structural domains, a 28-kDa N-terminal domain (N alpha beta; aa 1-246) and a 17-kDa C-terminal domain (C; aa 256-402). The two domains are connected by a 1-kDa proteolytic-sensitive linker region (aa 247-255). The N-terminal domain N alpha beta can be further subdivided into domains N alpha and N beta by a weaker protease-sensitive site located 6 kDa (53 aa) from the N terminus. The N beta and N alpha beta fragments are capable of nonspecific DNA binding as determined by Southwestern blot analysis. None of the fragments alone is capable of carrying out the first step of transposition, assembly of a synaptic complex containing a pair of transposon ends. Remarkably, complete transposition activity can be reconstituted by mixing fragment N alpha beta and fragment C, with or without the intervening linker region. We infer that the structural integrity of transposase during the transitions involved in the chemical steps of the transposition reaction is maintained independent of the linker, presumably by direct contacts between and among the principal domains. Reconstitution of activity in the absence of the linker region is puzzling, however, because mutations that block strand transfer or affect insertion specificity alter linker region residues. Additional reconstitution experiments demonstrate that the N alpha region is dispensable for formation of a synaptic complex but is required for complexes to undergo cleavage.

Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase receptors.

Although neurotrophins are primarily associated with long-term effects on neuronal survival and differentiation, recent studies have shown that acute changes in synaptic transmission can also be produced. In the hippocampus, an area critically involved in learning and memory, we have found that brain-derived neurotrophic factor (BDNF) rapidly enhanced synaptic efficacy through a previously unreported mechanism--increased postsynaptic responsiveness via a phosphorylation-dependent pathway. Within minutes of BDNF application to cultured hippocampal neurons, spontaneous firing rate was dramatically increased, as were the frequency and amplitude of excitatory postsynaptic currents. The increased frequency of postsynaptic currents resulted from the change in presynaptic firing. However, the increased amplitude was postsynaptic in origin because it was selectively blocked by intracellular injection of the tyrosine kinase receptor (Ntrk2/TrkB) inhibitor K-252a and potentiated by injection of the phosphatase inhibitor okadaic acid. These results suggest a role for BDNF in the modulation of synaptic transmission in the hippocampus.

Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules.

We have investigated the relationship of the so-called small dense core vesicle (SDCV), the major catecholamine-containing neurosecretory vesicle of sympathetic neurons, to synaptic vesicles containing classic neurotransmitters and secretory granules containing neuropeptides. SDCVs contain membrane proteins characteristic of synaptic vesicles such as synaptophysin and synaptoporin. However, SDCVs also contain membrane proteins characteristic of certain secretory granules like the vesicular monoamine transporter and the membrane-bound form of dopamine beta-hydroxylase. In neurites of sympathetic neurons, synaptophysin and dopamine beta-hydroxylase are found in distinct vesicles, consistent with their transport from the trans-Golgi network to the site of SDCV formation in constitutive secretory vesicles and secretory granules, respectively. Hence, SDCVs constitute a distinct type of neurosecretory vesicle that is a hybrid of the synaptic vesicle and the secretory granule membranes and that originates from the contribution of both the constitutive and the regulated pathway of protein secretion.

Presynaptic modulation of cortical synaptic activity by calcineurin.

Synaptic plasticity is modulated by Ca(2+)-induced alterations in the balance between phosphorylation and dephosphorylation. Recent evidence suggests that calcineurin, the Ca(2+)-calmodulin-dependent phosphatase (2B), modulates the activity of postsynaptic glutamate receptors. However, in rat cortex, calcineurin is enriched mainly in presynaptic, not postsynaptic, fractions. To determine if calcineurin modulates glutamatergic neurotransmission through a presynaptic mechanism, we used whole-cell patch clamp experiments to test effects of two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, on synaptic activity in fetal rat cortical neurons. The rate of spontaneous action-potential firing was markedly increased by either CsA or FK506 but was unaffected by rapamycin, a structural analog of FK506 which has no effect on calcineurin. In voltage-clamp experiments, CsA increased the rate but not the amplitude of glutamate receptor-mediated, excitatory postsynaptic currents, suggesting an increased rate of glutamate release. CsA had no effect on the amplitude of currents evoked by brief bath application of selective glutamate receptor agonists, providing further evidence for a pre- rather than postsynaptic site of action. In conclusion, these data indicate that calcineurin modulates glutamatergic neurotransmission in rat cortical neurons through a presynaptic mechanism.

Somatodendritic expression of an immediate early gene is regulated by synaptic activity.

Trans-synaptic activation of gene expression is linked to long-term plastic adaptations in the nervous system. To examine the molecular program induced by synaptic activity, we have employed molecular cloning techniques to identify an immediate early gene that is rapidly induced in the brain. We here report the entire nucleotide sequence of the cDNA, which encodes an open reading frame of 396 amino acids. Within the hippocampus, constitutive expression was low. Basal levels of expression in the cortex were high but can be markedly reduced by blockade of N-methyl-D-aspartate receptors. By contrast, synaptic activity induced by convulsive seizures increased mRNA levels in neurons of the cortex and hippocampus. High-frequency stimulation of the perforant path resulted in long-term potentiation and a spatially confined dramatic increase in the level of mRNA in the granule cells of the ipsilateral dentate gyrus. Transcripts were localized to the soma and to the dendrites of the granule cells. The dendritic localization of the transcripts offers the potential for local synthesis of the protein at activated postsynaptic sites and may underlie synapse-specific modifications during long-term plastic events.

Redistribution of synaptic vesicles and their proteins in temperature-sensitive shibire(ts1) mutant Drosophila.

From an extract of Drosophila melanogaster head homogenates, a membrane fraction can be isolated that has the same sedimentation properties as vertebrate synaptic vesicles and contains Drosophila synaptotagmin. The fraction disappears from homogenates of temperature-sensitive (ts) mutant shibire(ts1) (shi(ts1)) flies paralyzed by exposure to non-permissive temperatures, and reappears on return to permissive temperatures. Since reversible, temperature-dependent depletion of synaptic vesicles is known to occur in shibire(ts1) flies, we conclude that the fraction we have identified contains synaptic vesicles. We have examined the fate of synaptic vesicle membrane proteins in shibire flies at nonpermissive temperatures and found that all of these vesicle antigens are transferred to rapidly sedimenting membranes and codistribute with a plasma membrane marker by both glycerol velocity and metrizamide density sedimentation and by confocal microscopy. Three criteria were used to establish that other neuron-specific antigens--neuronal synaptobrevin and cysteine-string proteins--are legitimate components of synaptic vesicles: cosedimentation with Drosophila synaptotagmin, immunoadsorption, and disappearance of these antigens from the vesicle fractions in paralyzed shibire flies.

Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures.

Voltage- and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole-cell current and voltage clamp techniques. LHRH neurons were maintained in explant cultures derived from olfactory pit regions of embryonic mice. Cells were marked intracellularly with Lucifer yellow following recording. Sixty-two cells were unequivocally identified as LHRH neurons by Lucifer yellow and LHRH immunocytochemistry. The cultured LHRH neurons had resting potentials around -50 mV, exhibited spontaneous discharges generated by intrinsic and/or synaptic activities and contained a time-dependent inward rectifier (Iir). Voltage clamp analysis of ionic currents in the LHRH neuron soma revealed a tetrodotoxin-sensitive Na+ current (INa) and two major types of K+ currents, a transient current (IA), a delayed rectifier current (IK) and low- and high-voltage-activated Ca2+ currents. Spontaneous depolarizing synaptic potentials and depolarizations induced by direct application of gamma-aminobutyrate were both inhibited by picrotoxin or bicuculline, demonstrating the presence of functional gamma-aminobutyrate type A synapses on these neurons. Responses to glutamate were found in LHRH neurons in older cultures. Thus, embryonic LHRH neurons not yet positioned in their postnatal environment in the forebrain contained a highly differentiated repertoire of voltage- and ligand-gated channels.

D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release.

Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the cerebral cortex, hippocampus, anterior olfactory nucleus, olfactory tubercle, and amygdala. Localizations of D-serine immunoreactivity correlate closely with those of D-serine binding to the glycine modulatory site of the N-methyl-D-aspartate (NMDA) receptor as visualized by autoradiography and are inversely correlated to the presence of D-amino acid oxidase. D-Serine is enriched in process-bearing glial cells in neuropil with the morphology of protoplasmic astrocytes. In glial cultures of rat cerebral cortex, D-serine is enriched in type 2 astrocytes. The release of D-serine from these cultures is stimulated by agonists of non-NMDA glutamate receptors, suggesting a mechanism by which astrocyte-derived D-serine could modulate neurotransmission. D-Serine appears to be the endogenous ligand for the glycine site of NMDA receptors.

Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin.

In natural streptavidin, tryptophan 120 of each subunit makes contacts with the biotin bound by an adjacent subunit through the dimer-dimer interface. To understand quantitatively the role of tryptophan 120 and its intersubunit communication in the properties of streptavidin, a streptavidin mutant in which tryptophan 120 is converted to phenylalanine was produced and characterized. The streptavidin mutant forms a tetrameric molecule and binds one biotin per subunit, as does natural streptavidin, indicating that the mutation of tryptophan 120 to phenylalanine has no significant effect on the basic properties of streptavidin. However, its biotin-binding affinity was reduced substantially, to approximately 10(8) M-1, indicating that the contact made by tryptophan 120 to biotin has a considerable contribution to the extremely tight biotin binding by streptavidin. The mutant retained bound biotin over a wide pH range or with the addition of urea up to 6 M at neutral pH. However, bound biotin was efficiently released by the addition of excess free biotin due, presumably, to exchange reactions. Electrophoretic analysis revealed that the intersubunit contact made by tryptophan 120 to biotin through the dimer-dimer interface is the major interaction responsible for the biotin-induced, tighter subunit association of streptavidin. In addition, the mutant has weaker subunit association than natural streptavidin even in the absence of biotin, indicating that tryptophan 120 also contributes to the subunit association of tetramers in the absence of biotin.

Refinement of odor molecule tuning by dendrodendritic synaptic inhibition in the olfactory bulb.

Mitral/tufted cells (M/T cells) and granule cells form reciprocal dendrodendritic synapses in the main olfactory bulb; the granule cell is excited by glutamate from the M/T cell and in turn inhibits M/T cells by gamma-aminobutyrate. The trans-synaptically excited granule cell is thought to induce lateral inhibition in neighboring M/T cells and to refine olfactory information. It remains, however, elusive how significantly and specifically this synaptic regulation contributes to the discrimination of different olfactory stimuli. This investigation concerns the mechanism of olfactory discrimination by single unit recordings of responses to a series of normal aliphatic aldehydes from individual rabbit M/T cells. This analysis revealed that inhibitory responses are evoked in a M/T cell by a defined subset of odor molecules with structures closely related to the excitatory odor molecules. Furthermore, blockade of the reciprocal synaptic transmission by the glutamate receptor antagonist or the gamma-aminobutyrate receptor antagonist markedly suppressed the odor-evoked inhibition, indicating that the inhibitory responses are evoked by lateral inhibition via the reciprocal synaptic transmission. The synaptic regulation in the olfactory bulb thus greatly enhances the tuning specificity of odor responses and would contribute to discrimination of olfactory information.

Acute Synaptic Activity Causes Differential miRNA Expression in The Drosophila melanogaster Larval Central Nervous System

The primary goal of this thesis was to determine if spaced synaptic stimulation induced the differential expression of microRNAs (miRNAs) in the Drosophila melanogaster central nervous system (CNS). Prior to attaining this goal, we needed to identify and validate a spaced stimulation paradigm that could induce the formation of new synaptic growth at a model synapse, the larval neuromuscular junction (NMJ). Both Channelrhodopsin- and high potassium-based stimulation paradigms adapted from (Ataman, et al. 2008) were tested. Once validation of these paradigms was complete, we sought to characterize the miRNA expression profile of the larval CNS by miRNA array. Following attainment of these data, we used quantitative real-time PCR (RT-qPCR) to determine if acute synaptic stimulation caused the differential expression of neuronal miRNAs. We found that upon high potassium spaced training in a wild type (Canton S) genotype, 5 miRNAs showed significant differential expression when normalized to a validated reference gene, the U1 snRNA. Moreover, absolute quantification of our RT-qPCR study implicated one miRNA: miR-958 as being significantly regulated by activity. Investigation into potential targets for miR-958 revealed it to be a potential regular of Dlar, a protein tyrosine phosphatase implicated in synapse development. This investigation provides the foundation to directly test our underlying hypothesis that, following spaced training, differential expression of miRNAs alters the translation of proteins required to induce and maintain these structural changes at the synapse.

Activity-Regulated microRNAs: Modulators of Synaptic Growth at the Drosophila Neuromuscular Junction

It is well established that long-term changes in synaptic structure and function are mediated by rapid activity-dependent gene transcription and new protein synthesis. A growing body of evidence supports the involvement of the microRNA (miRNA) pathway in these processes. We have used the Drosophila neuromuscular junction (NMJ) as a model synapse to characterize activity-regulated miRNAs and their important mRNA targets. Here, we have identified five neuronal miRNAs (miRs-1, -8, -289, -314, and -958) that are significantly downregulated in response to neuronal activity. Furthermore we have discovered that neuronal misexpression of three of these miRNAs (miR-8, -289, and -958) is capable of suppressing new synaptic growth in response to activity suggesting that these miRNAs control the translation of biologically relevant target mRNAs. Putative targets of the activity-regulated miRNAs-8 and -289 are significantly enriched in clusters mapping to functional processes including axon development, pathfinding, and axon growth. We demonstrate that activity-regulated miR-8 regulates the 3'UTR of wingless, a presynaptic regulatory protein involved in the process of activity-dependent axon terminal growth. Additionally, we show that the 3'UTR of the protein tyrosine phosophatase leukocyte antengen related (lar), a protein required for axon guidance and synaptic growth, is regulated by activity-regulated miRNAs-8, -289, and -958 in vitro. Both wg and lar were identified as relevant putative targets for co-regulation based through our functional cluster analysis. One putative target of miR-289 is the Ca2+/calmodulin-dependent protein kinase II (CamKII). While CamKII is not predicted as a target for co-regulation by multiple activity-regulated miRNAs we identified it as an especially pertinent target for analysis in our system for two reasons. First, CamKII has an extremely well characterized role in postsynaptic plasticity, but its presynaptic role is less well characterized and bears further analysis. Second, local translation of CamKII mRNA is regulated in part by the miRNA pathway in an activity-dependent manner in dendrites. We find that the CamKII 3'UTR is regulated by miR-289 in-vitro and this regulation is alleviated by mutating the `seed region' of the miR-289 binding site within the CamKII 3'UTR. Furthermore, we demonstrate a requirement for local translation of CamKII in motoneurons in the process of activity-regulated axon terminal growth.

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.

Immunohistochemical evidence of synaptic retraction, cytoarchitectural remodeling, and cell death in the inner retina of the rat model of Oygen-Induced Retinopathy (OIR)

Purpose. Postnatal exposure to hyperoxia destroys the plexiform layers of the neonatal rat retina, resulting in significant electroretinographic anomalies. The purpose of this study was to identify the mechanisms at the origin of this loss. Methods. Sprague-Dawley (SD) and Long Evans (LE) rats were exposed to hyperoxia from birth to postnatal day (P) 6 or P14 and from P6 to P14, after which rats were euthanatized at P6, P14, or P60. Results. At P60, synaptophysin staining confirmed the lack of functional synaptic terminals in SD (outer plexiform layer [OPL]) and LE (OPL and inner plexiform layer [IPL]) rats. Uneven staining of ON-bipolar cell terminals with mGluR6 suggests that their loss could play a role in OPL thinning. Protein kinase C(PKC)-α and recoverin (rod and cone ON-bipolar cells, respectively) showed a lack of dendritic terminals in the OPL with disorganized axonal projections in the IPL. Although photoreceptor nuclei appeared intact, a decrease in bassoon staining (synaptic ribbon terminals) suggests limited communication to the inner retina. Findings were significantly more pronounced in LE rats. An increase in TUNEL-positive cells was observed in LE (inner nuclear layer [INL] and outer nuclear layer [ONL]) and SD (INL) rats after P0 to P14 exposure (425.3%, 102.2%, and 146.3% greater than control, respectively [P < 0.05]). Conclusions. Results suggest that cell death and synaptic retraction are at the root of OPL thinning. Increased TUNEL-positive cells in the INL confirm that cells die, at least in part, because of apoptosis. These findings propose a previously undescribed mechanism of cell death and synaptic retraction that are likely at the origin of the functional consequences of hyperoxia.