The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling

Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX3C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51. Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein. However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings. These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology. Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET). Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate. The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A⋅T base pairs for recognition of homology. We conclude that Dmc1, like human Rad51 and E. coli RecA protein, promotes homologous pairing and strand exchange by a “synaptic pathway” involving a three-stranded nucleoprotein intermediate, rather than by a “helicase pathway” involving the separation and reannealing of DNA strands.

Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells.

Hippocampal pyramidal cells, receiving domain specific GABAergic inputs, express up to 10 different subunits of the gamma-aminobutyric acid type A (GABAA) receptor, but only 3 different subunits are needed to form a functional pentameric channel. We have tested the hypothesis that some subunits are selectively located at subsets of GABAergic synapses. The alpha 1 subunit has been found in most GABAergic synapses on all postsynaptic domains of pyramidal cells. In contrast, the alpha 2 subunit was located only in a subset of synapses on the somata and dendrites, but in most synapses on axon initial segments innervated by axo-axonic cells. The results demonstrate that molecular specialization in the composition of postsynaptic GABAA receptor subunits parallels GABAergic cell specialization in targeting synapses to a specific domain of postsynaptic cortical neurons.

Cooperative interactions in the induction of long-term potentiation and depression of synaptic excitation between hippocampal CA3-CA1 cell pairs in vitro.

The requirement for cooperative interactions between multiple synaptic inputs in the induction of long-term potentiation (LTP) and long-term depression (LTD) has been tested at Schaffer collateral synapses with paired recordings from monosynaptically coupled CA3-CA1 cell pairs in rat hippocampal slice cultures. Tetanization of single presynaptic neurons at 50 Hz (repeated 5-7 times for 300-500 ms each) induced only a transient potentiation (< 3 min) of excitatory postsynaptic potentials (EPSPs). Persistent potentiation (> 15 min) was induced only when single presynaptic action potentials were synchronously paired with directly induced postsynaptic depolarizing pulses (repeated 50-100 times). Tetanus-induced potentiation of extracellularly evoked EPSPs lasting > 4 min could only be obtained if the EPSP was > 4 mV. Because unitary EPSP amplitudes average approximately 1 mV, we conclude that high-frequency discharge must occur synchronously] in 4-5 CA3 cells for LTP to be induced in a common postsynaptic CA1 cell. Asynchronous pairing of presynaptic action potentials with postsynaptic depolarizing current pulses (preceding each EPSP by 800 ms) depressed both naive and previously potentiated unitary EPSPs. Likewise, homosynaptic LTD of unitary EPSPs was induced when the presynaptic cell was tetanized at 3 Hz for 3 min, regardless of their amplitude (0.3-3.2 mV). Homosynaptic LTD of extracellularly evoked Schaffer collateral EPSPs < 4 mV could be induced if no inhibitory postsynaptic potential was apparent, but was prevented by eliciting a large inhibitory postsynaptic potential or by injection of hyperpolarizing current in the postsynaptic cell. We conclude that cooperative interactions among multiple excitatory inputs are not required for induction of homosynaptic LTD of unitary EPSPs.

Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling.

During receptor mediated endocytosis, at least a fraction of recycling cargo typically accumulates in a pericentriolar cluster of tubules and vesicles. However, it is not clear if these endosomal structures are biochemically distinct from the early endosomes from which they are derived. To better characterize this pericentriolar endosome population, we determined the distribution of two endogenous proteins known to be functionally involved in receptor recycling [Rab4, cellubrevin (Cbvn)] relative to the distribution of a recycling ligand [transferrin (Tfn)] as it traversed the endocytic pathway. Shortly after internalization, Tfn entered a population of early endosomes that contained both Rab4 and Cbvn, demonstrated by triple label immunofluorescence confocal microscopy. Tfn then accumulated in the pericentriolar cluster of recycling vesicles (RVs). However, although these pericentriolar endosomes contained Cbvn, they were strikingly depleted of Rab4. The ability of internalized Tfn to reach the Rab4-negative population was not blocked by nocodazole, although the characteristic pericentriolar location of the population was not maintained in the absence of microtubules. Similarly, Rab4-positive and -negative populations remained distinct in cells treated with brefeldin A, with only Rab4-positive elements exhibiting the extended tubular morphology induced by the drug. Thus, at least with respect to Rab4 distribution, the pathway of Tfn receptor recycling consists of at least two biochemically and functionally distinct populations of endosomes, a Rab4-positive population of early endosomes to which incoming Tfn is initially delivered and a Rab4-negative population of recycling vesicles that transiently accumulates Tfn on its route back to the plasma membrane.

Operant conditioning of H-reflex changes synaptic terminals on primate motoneurons.

Operant conditioning of the primate triceps surae H-reflex, the electrical analog of the spinal stretch reflex, creates a memory trace that includes changes in the spinal cord. To define the morphological correlates of this plasticity, we analyzed the synaptic terminal coverage of triceps surae motoneurons from animals in which the triceps surae H-reflex in one leg had been increased (HRup mode) or decreased (HRdown mode) by conditioning and compared them to each other and to motoneurons from unconditioned animals. Motoneurons were labeled by intramuscular injection of cholera toxin-horseradish peroxidase. A total of 5055 terminals on the cell bodies and proximal dendrites of 114 motoneurons from 14 animals were studied by electron microscopy. Significant differences were found between HRup and HRdown animals and between HRup and naive (i.e., unconditioned) animals. F terminals (i.e., putative inhibitory terminals) were smaller and their active zone coverage on the cell body was lower on motoneurons from the conditioned side of HRup animals than on motoneurons from the conditioned side of HRdown animals. C terminals (i.e., terminals associated with postsynaptic cisterns and rough endoplasmic reticulum) were smaller and the number of C terminals in each C complex (i.e., a group of contiguous C terminals) was larger on motoneurons from the conditioned side of HRup animals than on motoneurons either from the conditioned side of HRdown animals or from naive animals. Because the treatment of HRup and HRdown animals differed only in the reward contingency, the results imply that the two contingencies had different effects on motoneuron synaptic terminals. In combination with other recent data, they show that H-reflex conditioning produces a complex pattern of spinal cord plasticity that includes changes in motoneuron physiological properties as well as in synaptic terminals. Further delineation of this pattern should reveal the contribution of the structural changes described here to the learned change in behavior.

Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex.

The primate temporal cortex has been demonstrated to play an important role in visual memory and pattern recognition. It is of particular interest to investigate whether activity-dependent modification of synaptic efficacy, a presumptive mechanism for learning and memory, is present in this cortical region. Here we address this issue by examining the induction of synaptic plasticity in surgically resected human inferior and middle temporal cortex. The results show that synaptic strength in the human temporal cortex could undergo bidirectional modifications, depending on the pattern of conditioning stimulation. High frequency stimulation (100 or 40 Hz) in layer IV induced long-term potentiation (LTP) of both intracellular excitatory postsynaptic potentials and evoked field potentials in layers II/III. The LTP induced by 100 Hz tetanus was blocked by 50-100 microM DL-2-amino-5-phosphonovaleric acid, suggesting that N-methyl-D-aspartate receptors were responsible for its induction. Long-term depression (LTD) was elicited by prolonged low frequency stimulation (1 Hz, 15 min). It was reduced, but not completely blocked, by DL-2-amino-5-phosphonovaleric acid, implying that some other mechanisms in addition to N-methyl-DL-aspartate receptors were involved in LTD induction. LTD was input-specific, i.e., low frequency stimulation of one pathway produced LTD of synaptic transmission in that pathway only. Finally, the LTP and LTD could reverse each other, suggesting that they can act cooperatively to modify the functional state of cortical network. These results suggest that LTP and LTD are possible mechanisms for the visual memory and pattern recognition functions performed in the human temporal cortex.

A presynaptic locus for long-term potentiation of elementary synaptic transmission at mossy fiber synapses in culture.

The complex circuitry of the CA3 region and the abundance of collateral connections has made it difficult to study the mossy fiber pathway in hippocampal slices and therefore to establish the site of expression of long-term potentiation at these synapses. Using a novel cell culture system, we have produced long-term potentiation of the elementary synaptic connections on single CA3 pyramidal neurons following tetanic stimulation of individual dentate gyrus granule cells. As is the case for the hippocampal slice, this potentiation was independent of N-methyl-D-aspartate receptor activation, was simulated by application of forskolin, and its induction did not require any modulatory input. The increase in synaptic strength was accompanied by a reduction in the number of failures of transmission and by an increase in the coefficient of variation of the responses and was prevented by presynaptic injection of an inhibitor of protein kinase A. These findings show that mossy fiber long-term potentiation has a presynaptic locus and that its expression is dependent on protein kinase A.

Aromatic amino acid transamination and methionine recycling in trypanosomatids.

Although trypanosomatids are known to rapidly transaminate exogenous aromatic amino acids in vitro and in vivo, the physiological significance of this reaction is not understood. In postmitochondrial supernatants prepared from Trypanosoma brucei brucei and Crithidia fasciculata, we have found that aromatic amino acids were the preferred amino donors for the transamination of alpha-ketomethiobutyrate to methionine. Intact C. fasciculata grown in the presence of [15N]tyrosine were found to contain detectable [15N]methionine, demonstrating that this reaction occurs in situ in viable cells. This process is the final step in the recycling of methionine from methylthioadenosine, a product of decarboxylated S-adenosylmethionine from the polyamine synthetic pathway. Mammalian liver, in contrast, preferentially used glutamine for this reaction and utilized a narrower range of amino donors than seen with the trypanosomatids. Studies with methylthioadenosine showed that this compound was readily converted to methionine, demonstrating a fully functional methionine-recycling pathway in trypanosomatids.

Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles.

Immunohistochemical visualization of the rat vesicular acetylcholine transporter (VAChT) in cholinergic neurons and nerve terminals has been compared to that for choline acetyltransferase (ChAT), heretofore the most specific marker for cholinergic neurons. VAChT-positive cell bodies were visualized in cerebral cortex, basal forebrain, medial habenula, striatum, brain stem, and spinal cord by using a polyclonal anti-VAChT antiserum. VAChT-immuno-reactive fibers and terminals were also visualized in these regions and in hippocampus, at neuromuscular junctions within skeletal muscle, and in sympathetic and parasympathetic autonomic ganglia and target tissues. Cholinergic nerve terminals contain more VAChT than ChAT immunoreactivity after routine fixation, consistent with a concentration of VAChT within terminal neuronal arborizations in which secretory vesicles are clustered. These include VAChT-positive terminals of the median eminence or the hypothalamus, not observed with ChAT antiserum after routine fixation. Subcellular localization of VAChT in specific organelles in neuronal cells was examined by immunoelectron microscopy in a rat neuronal cell line (PC 12-c4) expressing VAChT as well as the endocrine and neuronal forms of the vesicular monoamine transporters (VMAT1 and VMAT2). VAChT is targeted to small synaptic vesicles, while VMAT1 is found mainly but not exclusively on large dense-core vesicles. VMAT2 is found on large dense-core vesicles but not on the small synaptic vesicles that contain VAChT in PC12-c4 cells, despite the presence of VMAT2 immunoreactivity in central and peripheral nerve terminals known to contain monoamines in small synaptic vesicles. Thus, VAChT and VMAT2 may be specific markers for "cholinergic" and "adrenergic" small synaptic vesicles, with the latter not expressed in nonstimulated neuronally differentiated PC12-c4 cells.

Low-calcium-induced enhancement of chemical synaptic transmission from photoreceptors to horizontal cells in the vertebrate retina.

According to the classical calcium hypothesis of synaptic transmission, the release of neurotransmitter from presynaptic terminals occurs through an exocytotic process triggered by depolarization-induced presynaptic calcium influx. However, evidence has been accumulating in the last two decades indicating that, in many preparations, synaptic transmitter release can persist or even increase when calcium is omitted from the perfusing saline, leading to the notion of a "calcium-independent release" mechanism. Our study shows that the enhancement of synaptic transmission between photoreceptors and horizontal cells of the vertebrate retina induced by low-calcium media is caused by an increase of calcium influx into presynaptic terminals. This paradoxical effect is accounted for by modifications of surface potential on the photoreceptor membrane. Since lowering extracellular calcium concentration may likewise enhance calcium influx into other nerve cells, other experimental observations of "calcium-independent" release may be reaccommodated within the framework of the classical calcium hypothesis without invoking unconventional processes.

Mice deficient for prion protein exhibit normal neuronal excitability and synaptic transmission in the hippocampus.

We recorded in the CA1 region from hippocampal slices of prion protein (PrP) gene knockout mice to investigate whether the loss of the normal form of prion protein (PrPC) affects neuronal excitability as well as synaptic transmission in the central nervous system. No deficit in synaptic inhibition was found using field potential recordings because (i) responses induced by stimulation in stratum radiatum consisted of a single population spike in PrP gene knockout mice similar to that recorded from control mice and (ii) the plot of field excitatory postsynaptic potential slope versus the population spike amplitude showed no difference between the two groups of mice. Intracellular recordings also failed to detect any difference in cell excitability and the reversal potential for inhibitory postsynaptic potentials. Analysis of the kinetics of inhibitory postsynaptic current revealed no modification. Finally, we examined whether synaptic plasticity was altered and found no difference in long-term potentiation between control and PrP gene knockout mice. On the basis of our findings, we propose that the loss of the normal form of prion protein does not alter the physiology of the CA1 region of the hippocampus.

Short-term synaptic enhancement and long-term potentiation in neocortex.

Repetitive stimuli reliably induce long-term potentiation (LTP) of synapses in the upper layers of the granular somatosensory cortex but not the agranular motor cortex of rats. Herein we examine, in these same cortical areas, short-term changes in synaptic strength that occur during the LTP induction period. theta-Burst stimulation produced a strong short-term enhancement of synapses in the granular area but only weak enhancement in the agranular area. The magnitude of enhancement during stimulation was strongly correlated with the magnitude of LTP subsequently expressed. Short-term enhancement was abolished by an antagonist of N-methyl-D-aspartate (NMDA) receptors but remained in the presence of a non-NMDA receptor antagonist. Inhibitory postsynaptic potentials of the granular and agranular areas displayed similar frequency sensitivity, but the frequency sensitivity of NMDA receptor-dependent excitatory postsynaptic potentials differed significantly between areas. We propose that pathway-specific differences in short-term enhancement are due to variations in the frequency dependence of NMDA currents; different capacities for short-term enhancement may explain why repetitive stimulation more readily induces LTP in the somatosensory cortex than in the motor cortex.

Calcium/calmodulin-dependent kinase II and long-term potentiation enhance synaptic transmission by the same mechanism.

Ca(2+)-sensitive kinases are thought to play a role in long-term potentiation (LTP). To test the involvement of Ca2+/calmodulin-dependent kinase II (CaM-K II), truncated, constitutively active form of this kinase was directly injected into CA1 hippocampal pyramidal cells. Inclusion of CaM-K II in the recording pipette resulted in a gradual increase in the size of excitatory postsynaptic currents (EPSCs). No change in evoked responses occurred when the pipette contained heat-inactivated kinase. The effects of CaM-K II mimicked several features of LTP in that it caused a decreased incidence of synaptic failures, an increase in the size of spontaneous EPSCs, and an increase in the amplitude of responses to iontophoretically applied alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. To determine whether the CaM-K II-induced enhancement and LTP share a common mechanism, occlusion experiments were carried out. The enhancing action of CaM-K II was greatly diminished by prior induction of LTP. In addition, following the increase in synaptic strength by CaM-K II, tetanic stimulation failed to evoke LTP. These findings indicate that CaM-K II alone is sufficient to augment synaptic strength and that this enhancement shares the same underlying mechanism as the enhancement observed with LTP.

Identification of a DNA element determining synaptic expression of the mouse acetylcholine receptor delta-subunit gene.

mRNAs for acetylcholine receptor genes are highly concentrated in the endplate region of adult skeletal muscle largely as a result of a transcription restricted to the subneural nuclei. To identify the regulatory elements involved, we employed a DNA injection of a plasmid containing a fragment of the acetylcholine receptor delta-subunit gene promoter (positions -839 to +45) linked to the reporter gene lacZ with a nuclear localization signal. Injection of the wild-type construct into mouse leg muscles yielded preferential expression of the reporter gene in the synaptic region. Analysis of various mutant promoters resulted in the identification of a DNA element (positions -60 to -49), referred to as the N box, that plays a critical role in subneural expression. Disruption of this 12-bp element in the context of a mouse delta-subunit promoter from positions -839 to +45 gives widespread expression of the reporter gene throughout the entire muscle fiber, indicating that this element is a silencer that represses delta-subunit gene transcription in extrajunctional areas. On the other hand, this element inserted upstream of a heterologous basal promoter preferentially enhances expression in the endplate region. This element therefore regulates the restricted expression of the delta-subunit gene both as an enhancer at the endplate level and as a silencer in extrajunctional areas. Furthermore, gel-shift experiments with mouse muscle extracts reveal an activity that specifically binds the 6-bp sequence TTCCGG of this element, suggesting that a transcription factor(s) controls the expression of the delta-subunit gene via this element.