Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain.

Uptake of L-glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Three transporters (VGLUT1-VGLUT3) are expressed in the mammalian CNS, with partial overlapping expression patterns, and VGLUT2 is the most abundantly expressed paralog in the thalamus, midbrain, and brainstem. Previous studies have shown that VGLUT1 is necessary for glutamatergic transmission in the hippocampus, but the role of VGLUT2 in excitatory transmission is unexplored in glutamatergic neurons and in vivo. We examined the electrophysiological and behavioral consequences of loss of either one or both alleles of VGLUT2. We show that targeted deletion of VGLUT2 in mice causes perinatal lethality and a 95% reduction in evoked glutamatergic responses in thalamic neurons, although hippocampal synapses function normally. Behavioral analysis of heterozygous VGLUT2 mice showed unchanged motor function, learning and memory, acute nociception, and inflammatory pain, but acquisition of neuropathic pain, maintenance of conditioned taste aversion, and defensive marble burying were all impaired. Reduction or loss of VGLUT2 in heterozygous and homozygous VGLUT2 knock-outs led to a graded reduction in the amplitude of the postsynaptic response to single-vesicle fusion in thalamic neurons, indicating that the vesicular VGLUT content is critically important for quantal size and demonstrating that VGLUT2-mediated reduction of excitatory drive affects specific forms of sensory processing.

Assay development for high throughput screening of p21(Waf1/Cip1) protein expression in intact cells using fluorometric microvolume assay technology.

The p21(Waf1/Cip1) protein represents a broad-acting cyclin-dependent kinase inhibitor that plays a key role in cell cycle regulation. Furthermore, p21(Waf1/Cip1) protein has been described as a direct participant in regulating genes involved in growth arrest, senescence and aging. In response to genotoxic insults (e.g., following chemotherapeutic treatment), p21(Waf1/Cip1) protein accumulates mainly through p53-mediated transcriptional activation and is also regulated at the post-transcriptional level. In tumor cells, p53 is frequently mutated leading to reduced p21(Waf1/Cip1) protein induction that may contribute to resistance to treatment by DNA-damaging agents. In order to identify compounds capable of restoring p21(Waf1/Cip1) protein level, we have developed a 96-multi-well plate-based high throughput screening assay in intact cells using the Applied Biosystems Fluorometric Microvolume Assay Technology (FMAT) macro-confocal system. Briefly, following incubation with test compounds, human MCF7 breast carcinoma cells were fixed and p21(Waf1/Cip1) protein was detected using anti-p21(Waf1/Cip1) monoclonal antibody and anti-mouse IgG conjugated to the red fluorescent dye Alexafluor 633. FMAT provides a set of raw images at a high magnification, in which fluorescence concentrated in a cell is detected as a specific signal. The mean fluorescence of a population of cells is calculated independently of the number of cells as with a classical FACS analysis. This is of particular interest for screening anticancer drugs that may affect cell number and therefore may impact on the readout. This assay was validated using reference compounds such as camptothecin and actinomycin D, known inducers of p21(Waf1/Cip1) protein.

Kirkinine, a new daphnane orthoester with potent neurotrophic activity from Synaptolepis kirkii.

The bioassay-guided fractionation of a dichloromethane extract from the roots of Synaptolepis kirkii using neuronal viability as a model allowed the isolation of the new daphnane orthoester kirkinine (1a) as a powerful neurotrophic constituent.

Mammalian GFRalpha -4, a divergent member of the GFRalpha family of coreceptors for glial cell line-derived neurotrophic factor family ligands, is a receptor for the neurotrophic factor persephin.

Four members of the glial cell line-derived neurotrophic factor family have been identified (GDNF, neurturin, persephin, and enovin/artemin). They bind to a specific membrane-anchored GDNF family receptor as follows: GFRalpha-1 for GDNF, GFRalpha-2 for neurturin, GFRalpha-3 for enovin/artemin, and (chicken) GFRalpha-4 for persephin. Subsequent signaling occurs through activation of a common transmembrane tyrosine kinase, cRET. GFRalpha-4, the coreceptor for persephin, was previously identified in chicken only. We describe the cloning and characterization of a mammalian persephin receptor GFRalpha-4. The novel GFRalpha receptor is substantially different in sequence from all known GFRalphas, including chicken GFRalpha-4, and lacks the first cysteine-rich domain present in all previously characterized GFRalphas. At least two different GFRalpha-4 splice variants exist in rat tissues, differing at their respective COOH termini. GFRalpha-4 mRNA is expressed at low levels in different brain areas in the adult as well as in some peripheral tissues including testis and heart. Recombinant rat GFRalpha-4 variants were expressed in mammalian cells and shown to be at least partially secreted from the cells. Persephin binds specifically and with high affinity (K(D) = 6 nm) to the rat GFRalpha-4 receptor, but no cRET activation could be demonstrated. Although the newly characterized mammalian GFRalpha-4 receptor is structurally divergent from previously characterized GFRalpha family members, we suggest that it is a mammalian orthologue of the chicken persephin receptor. This discovery will allow a more detailed investigation of the biological targets of persephin action and its potential involvement in diseases of the nervous system.

Binding of GDNF and neurturin to human GDNF family receptor alpha 1 and 2. Influence of cRET and cooperative interactions.

The members of the glial cell line-derived neurotrophic factor (GDNF) family signal via binding to the glycosyl phosphatidylinositol-anchored membrane proteins, the GDNF family receptors alpha (GFRalpha), and activation of cRET. We performed a detailed analysis of the binding of GDNF and neurturin to their receptors and investigated the influence of cRET on the binding affinities. We show that the rate of dissociation of (125)I-GDNF from GFRalpha1 is increased in the presence of 50 nm GDNF, an effect that can be explained by the occurrence of negative cooperativity. Scatchard plots of the ligand concentration binding isotherms reveal a pronounced downward curvature at low (125)I-GDNF concentrations suggesting the presence of positive cooperativity. This effect is observed in the range of GDNF concentrations responsible for biological activity (1-20 pm) and may have an important role in cRET-independent signaling. A high affinity site with a K(D) of 11 pm for (125)I-GDNF is detected only when GFRalpha1 is co-expressed with cRET at a DNA ratio of 1:3. These results suggest an interaction of GFRalpha1 and cRET in the absence of GDNF and demonstrate that the high affinity binding can be measured only when cRET is present.

Enovin, a member of the glial cell-line-derived neurotrophic factor (GDNF) family with growth promoting activity on neuronal cells. Existence and tissue-specific expression of different splice variants.

Glial cell-line-derived neurotrophic factor (GDNF), neurturin and persephin are neurotrophic factors involved in neuroneal differentiation, development and maintenance. They act on different types of neuroneal cells and signal through a receptor complex composed of a specific ligand-binding subunit of the GDNF family receptor alpha (GFRalpha) family together with a common signaling partner, the cRET protein tyrosine kinase. We describe the molecular cloning, expression, chromosomal localization and functional characterization of enovin, a fourth GDNF family member almost identical to the recently described artemin. We show the occurence in most tissues of several differently spliced mRNA variants for enovin, of which only two are able to translate into functional enovin protein. Some tissues seem to express only nonfunctional transcripts. These observations may underlie a complex transcriptional regulation pattern. Enovin mRNA expression is detectable in all adult and fetal human tissues examined, but expression levels are highest in peripheral tissues including prostate, placenta, pancreas, heart and kidney. This tissue distribution pattern is in accordance with that of GFRalpha-3, which here is shown to be the preferred ligand-binding receptor for enovin (Kd = 3.1 nM). The human enovin gene is localized on chromosome 1, region p31.3-p32. In vitro, enovin stimulates neurite outgrowth and counteracts taxol-induced neurotoxicity in staurosporine-differentiated SH-SY5Y human neuroblastoma cells. The peripheral expression pattern of enovin and its receptor together with its effects on neuroneal cells suggest that enovin might be useful for the treatment of neurodegenerative diseases in general and peripheral neuropathies in particular.

Molecular cloning, expression and tissue distribution of glial-cell-line-derived neurotrophic factor family receptor alpha-3 (GFRalpha-3).

Neurturin and glial-cell-line-derived neurotrophic factor (GDNF) promote the survival and maintenance of different types of neuronal cells and signal through a receptor complex composed of a ligand binding subunit, either GDNF family receptor alpha-1 (GFRalpha-1) or alpha-2 (GFRalpha-2), together with the cRET membrane-bound protein tyrosine kinase. We have cloned GFRalpha-3, a novel receptor belonging to the GFRalpha family, that is 35% identical by amino acid sequence to both GFRalpha-1 and GFRalpha-2. GFRalpha-3 is a protein composed of 400 amino acid residues with three potential N-linked glycosylation sites together with the features characteristic of a glycosyl-phosphatidylinositol-anchored membrane protein. The heterologous expression of a FLAG-tagged GFRalpha-3 in human embryonic kidney cells showed that the protein is bound to the cell surface via a glycosyl-PtdIns anchor and is glycosylated, with different glycoforms migrating on SDS/PAGE with apparent molecular masses ranging over 43-62 kDa. The gene for GFRalpha-3 was mapped to human chromosome 5 in a region (q31.1-q31.3) where several disease loci, growth factor and growth factor receptor genes have been localized. Using northern blot analysis or reverse-transcription PCR, GFRalpha-3 was shown to be expressed within the nervous system predominantly in the cerebellum and the spinal cord while in peripheral tissues GFRalpha-3 was found to be expressed mostly in the colon, small intestine, pancreas, heart, testis and prostate. Using a GFRalpha-3-specific [35S]cRNA[gammaS] probe, in situ hybridization histochemistry experiments confirmed the expression in the cerebellum.

An investigation into the role of N-glycosylation in the functional expression of a recombinant heteromeric NMDA receptor.

The effect of N-glycosylation on the assembly of N-methyl-D-aspartate (NMDA) heteromeric cloned receptors was studied. Thus human embryonic kidney (HEK) 293 cells were cotransfected with N-methyl-D-aspartate R1 (NR1) and N-methyl-D-aspartate R2A (NR2A) clones and the cells grown post-transfection in the presence of tunicamycin (TM). TM treatment resulted in a decrease of the NR1 subunit with M(r) 117 000 with a concomitant increase in a M(r) 97 000 immunoreactive species previously identified as the non-N-glycosylated NR1 subunit. In parallel, TM caused a dose-dependent inhibition of [3H]MK801 binding to the expressed receptor which was a result of an approximate four-fold reduction in the Dissociation Constant (KD) but with no change in the number of binding sites (Bmax). NMDA receptor cell surface expression was unchanged following TM treatment but it did result in a decrease in the percentage cell death post-transfection compared to control samples. The removal of TM from the cell culture media resulted in a return to the control KD value for [3H]MK801 binding and partial reglycosylation of newly synthesized NR1 subunit. These results demonstrate that N-glycosylation is requisite for the efficient expression of functional NR1/NR2A receptors. Furthermore, they suggest that N-glycosylation may be important for the correct formation of the channel domain of the NR1/NR2A receptor.

Molecular characterization of N-methyl-D-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule.

The N-methyl-D-aspartate R1 (NMDA R1), NMDA R2A, and NMDA R2C subunits were expressed transiently in double or triple combinations in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult mouse forebrain and cerebellum. Under conditions established for maximal expression, cotransfection of the NMDA R1 and R2C subunits yielded a protein detected immunologically with a molecular size of 780,000-850,000 daltons. No cell death was observed in the transfected cells, and the KD for [3H]MK801 binding to the NMDA R1/R2C receptor was 346 +/- 158 nM. This was in contrast to a value of KD = 22 +/- 9 nM found for native cerebellar receptors. Co-transfection with NMDA R1/R2A/R2C subunits with a DNA ratio, 1:3:3, resulted in the expression of a protein with a size similar to the NMDA R1/R2C combination, but the affinity of [3H]MK801 was now 22 +/- 5 nM, and the percentage cell death post-transfection was 89 +/- 17%. Immunoprecipitation assays of detergent-solubilized transfected cells with NMDA R1 subunit-specific antibodies co-precipitated the NMDA R2A and NMDA R2C subunits in 1/2A and 1/2C transfections, respectively. Similarly, immunoprecipitations with either NMDA R1 or NMDA R2C subunit-specific antibodies co-precipitated the NMDA R2A subunit in the R1/2A/2C triple transfections. These results show that the three NMDA receptor subunit types can co-assemble following their co-expression in mammalian cells with a pharmacological profile that is similar to that found for adult cerebellar NMDA receptors.

Single channel properties of cloned NMDA receptors in a human cell line: comparison with results from Xenopus oocytes.

1. Human embryonic kidney (HEK) 293 cells were transiently transfected with cDNAs encoding the NR1a-NMDA epsilon 1[NR2A] subunit combination of the NMDA receptor. Single channel behaviour was recorded from outside-out membrane patches, with the aim of comparing the results with those, recorded under the same conditions, from Xenopus oocytes injected with messenger RNA coding for the NR1a-NR2A combination. 2. Single channels in HEK 293 cells showed a main conductance level of 51.4 +/- 2.4 pS, compared with 50.1 +/- 1.4 pS for channels in oocytes. A subconductance level of 38.1 +/- 2.1 pS was found in HEK 293 cells, compared with 38.3 +/- 1.3 pS in oocytes. The frequencies of transitions between the shut and the two conductance levels were also very similar. 3. Distributions of shut times could be fitted with five exponential components. In HEK 293 cells the first three of these components had time constants of 39 +/- 4 microseconds, 0.54 +/- 0.04 ms and 9.94 +/- 1.3 ms; in oocytes the values were 69 +/- 35 microseconds, 0.54 +/- 0.15 ms and 6.53 +/- 4.6 ms, respectively. The relative areas of the components were also similar in the two systems. 4. The distribution of all apparent open times for the sublevels was fitted with two exponential components giving time constants of 0.18 +/- 0.02 ms and 1.31 +/- 0.17 ms (for HEK cells) or of 0.31 +/- 0.36 ms and 1.31 +/- 1.1 ms (for oocytes).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of NMDAR1-1a (N598Q)/NMDAR2A receptors results in decreased cell mortality.

Transient expression of wild-type N-methyl-D-aspartate, NMDAR1-1a/NMDAR2A heteromeric receptors, in mammalian cells yields cell death which was prevented by the inclusion of NMDA receptor antagonists in the cell culture media post-transfection. Transient expression of mutant NMDAR1-1a (N598Q)/NMDAR2A receptors resulted in a significant decrease in the percentage of cell death post-transfection. This mutation has been shown to reduce the Ca2+ permeability of cloned NMDA receptors. Thus these results provide indirect evidence for cell death via an NMDA receptor, Ca(2+)-mediated mechanism.

Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterization.

The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [3H]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180,000, 122,000, 97,000 and 54,000 in rat brain, but only a single band of M(r) 180,000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165,000-M(r) immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [3H]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity studies.

Immunological detection of the NMDAR1 glutamate receptor subunit expressed in embryonic kidney 293 cells and in rat brain.

The rat NMDAR1 (N-methyl-D-aspartate receptor) was expressed transiently in human embryonic kidney cells. Transfected cell homogenates showed saturable [3H]MK-801 binding activity that was best fit by a single high-affinity site with a KD of 9 nM and a Bmax of 113 fmol of binding sites/mg of protein. Antibodies raised against the peptide sequence NMDAR1 (929-938) coupled to keyhole limpet haemocyanin specifically recognised a single band with M(r) 117,000 in immunoblots from adult rat brain. In the transfected cells, the antibody recognised two bands: one with M(r) 117,000, which was coincident with that from brain membranes, and one with M(r) 97,000, which was identified as nonglycosylated NMDAR1 subunit. These results identify the NMDAR1 of rat brain and further show that the homooligomer binds MK-801, albeit at low efficiency.

Species dependent effects of dihydroergosine on [3H]TBOB binding to membranes from the human, rat, bovine and mouse brain.

Dihydroergosine enhanced [3H]TBOB binding to the crude synaptosomal membranes prepared from the whole rat brain and human frontal cortex. Higher concentrations of the same drug inhibited [3H]TBOB binding in the preparations obtained from the whole mouse brain and bovine frontal cortex. Bicuculline-induced enhancement and GABA- or diazepam-induced inhibition of [3H]TBOB binding were similar in the four species examined. The results indicate that dihydroergosine modulates species-dependently GABA/benzodiazepine receptors.