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R Sprengel

Publications and source records attributed to R Sprengel.

At least 37 records · Page 2Linked to original sources
PubMed2000

Developmental profile of kainate receptor subunit KA1 revealed by Cre expression in YAC transgenic mice.

To determine the spatio-temporal expression in brain of the high-affinity kainate receptor subunit KA1, we generated transgenic mice expressing Cre recombinase from the KA1 gene on a chromosomally integrated 550 kb yeast artificial chromosome (YAC). Activity of the KA1 gene promoter during brain development was visualized by Cre immunohistochemistry, and by X-gal staining of beta-galactosidase induced by Cre recombinase in double transgenic KA1-Cre/lacZ indicator mice. During early brain development, expression from the YAC-carried KA1-Cre transgene was observed in all major brain areas, predicting a function for KA1 in the developing central nervous system. In the adult brain, KA1-Cre transgene expression was restricted mainly to hippocampal CA3 pyramidal and dentate gyrus granule cells, an adult expression pattern characteristic for the endogenous KA1 alleles. KA1-Cre transgenic mice may help in elucidating the role of floxed genes ablated in vivo in KA1 expressing neurons.

Aging↗
PubMed2000

Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2.

RNA editing by site-selective deamination of adenosine to inosine alters codons and splicing in nuclear transcripts, and therefore protein function. ADAR2 (refs 7, 8) is a candidate mammalian editing enzyme that is widely expressed in brain and other tissues, but its RNA substrates are unknown. Here we have studied ADAR2-mediated RNA editing by generating mice that are homozygous for a targeted functional null allele. Editing in ADAR2-/- mice was substantially reduced at most of 25 positions in diverse transcripts; the mutant mice became prone to seizures and died young. The impaired phenotype appeared to result entirely from a single underedited position, as it reverted to normal when both alleles for the underedited transcript were substituted with alleles encoding the edited version exonically. The critical position specifies an ion channel determinant, the Q/R site, in AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor GluR-B pre-messenger RNA. We conclude that this transcript is the physiologically most important substrate of ADAR2.

Adenosine Deaminase↗
PubMed2000

Dysfunctions in mice by NMDA receptor point mutations NR1(N598Q) and NR1(N598R).

NMDA receptors in mice were mutated by gene targeting to substitute asparagine (N) in position 598 of the NR1 subunit to glutamine (Q) or arginine (R). Animals expressing exclusively the mutated NR1 alleles, NR1(Q/Q) and NR1(-/R) mice, developed a perinatally lethal phenotype mainly characterized by respiratory failure. The dysfunctions were partially rescued in heterozygous mice by the presence of pure wild-type receptors. Thus, NR1(+/Q) mice exhibited reduced life expectancy, with females being impaired in nurturing; NR1(+/R) mice displayed signs of underdevelopment such as growth retardation and impaired righting reflex, and died before weaning. We analyzed the key properties of NMDA receptors, high Ca(2+) permeability, and voltage-dependent Mg(2+) block, in the mutant mice. Comparison of the complex physiological and phenotypical changes observed in the different mutants indicates that properties controlled by NR1 subunit residue N598 are important for autonomic brain functions at birth and during postnatal development. We conclude that disturbed NMDA receptor signaling mediates a variety of neurological phenotypes.

Alleles↗
PubMed1999

Importance of AMPA receptors for hippocampal synaptic plasticity but not for spatial learning.

Gene-targeted mice lacking the L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR-A exhibited normal development, life expectancy, and fine structure of neuronal dendrites and synapses. In hippocampal CA1 pyramidal neurons, GluR-A-/- mice showed a reduction in functional AMPA receptors, with the remaining receptors preferentially targeted to synapses. Thus, the CA1 soma-patch currents were strongly reduced, but glutamatergic synaptic currents were unaltered; and evoked dendritic and spinous Ca2+ transients, Ca2+-dependent gene activation, and hippocampal field potentials were as in the wild type. In adult GluR-A-/- mice, associative long-term potentiation (LTP) was absent in CA3 to CA1 synapses, but spatial learning in the water maze was not impaired. The results suggest that CA1 hippocampal LTP is controlled by the number or subunit composition of AMPA receptors and show a dichotomy between LTP in CA1 and acquisition of spatial memory.

Action Potentials↗
PubMed1999

Studies on conditional gene expression in the brain.

This manuscript summarizes our recent attempts to regulate in vitro and in vivo the expression of genes encoding components and regulators of the postsynaptic machinery along with marker genes such as lacZ and GFP. In particular, we studied tTA-dependent regulation and utilized Cre in combination with reversible silencing by intron engineering of dominant negative alleles. We further present a "knockin" approach for on-site artificial regulation of chromosomal genes.

Animals↗
PubMed1999

Mice with genetically modified NMDA and AMPA receptors.

This manuscript summarizes mouse mutants for ionotropic glutamate receptors that were generated by different laboratories to analyze the function of the NMDA and AMPA receptors in the mouse. Thus, NMDA receptor mutant mice that were generated by the "knock-in" technology demonstrate that the NR1 and the NR2B subunits participate in the formation of NMDA receptors that are involved in vital functions like breathing and suckling of a newborn mouse. Mice that lack NR2A, -2C, and -2D subunits were described to be viable and have been used to study the role of NMDA receptors in adult mice. The depletion of the GluR-B subunit revealed an NMDA receptor-independent form of long-term potentiation (LTP). This AMPA receptor-mediated LTP at CA3/CA1 synapses was also observed in mice that carry an editing-deficient GluR-B allele even though these mice die prematurely after heavy epileptic seizures. In other mutants, the intracellular COOH-terminal domain of the NMDA receptor was truncated; and when compared to NMDA receptor "knock-out" mice, a functional knock-out of the NMDA receptor was observed. However, in the synapses of NR2AC/AC mutants, gatable NMDA receptors were synaptically activated, indicating that the knock-out phenotypes mediated by the COOH-terminally truncated NMDA receptors appear to reflect defective intracellular signaling.

Animals↗
PubMed1999

GABA- and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice.

Mice were generated expressing green fluorescent protein (GFP) under the control of the gonadotropin-releasing hormone (GnRH) promoter. Green fluorescence was observed in, and restricted to, GnRH-immunopositive neuronal somata in the olfactory bulb, ganglion terminale, septal nuclei, diagonal band of Broca (DBB), preoptic area (POA), and caudal hypothalamus, as well as GnRH neuronal dendrites and axons, including axon terminals in the median eminence and organum vasculosum of the lamina terminalis (OVLT). Whole-cell recordings from GFP-expressing GnRH neurons in the OVLT-POA-DBB region revealed a firing pattern among GFP-expressing GnRH neurons distinct from that of nonfluorescent neurons. Nucleated patches of GFP-expressing GnRH neurons exhibited pronounced responses to fast application of GABA and smaller responses to L-glutamate and AMPA. One-fifth of the nucleated patches responded to NMDA. The GABA-A, AMPA, and NMDA receptor channels on GnRH neurons mediating these responses may play a role in the modulation of GnRH secretory oscillations.

Action Potentials↗
PubMed1999

Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B.

We generated mouse mutants with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at different levels and deficient in Q/R-site editing. All mutant lines had increased AMPAR calcium permeabilities in pyramidal neurons, and one showed elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx induced NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium-triggered neuronal death was not observed, but mutants had mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlated with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons.

Alleles↗
PubMed1998

The AMPA receptor subunit GluR-B in its Q/R site-unedited form is not essential for brain development and function.

Calcium permeability of L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in excitatory neurons of the mammalian brain is prevented by coassembly of the GluR-B subunit, which carries an arginine (R) residue at a critical site of the channel pore. The codon for this arginine is created by site-selective adenosine deamination of an exonic glutamine (Q) codon at the pre-mRNA level. Thus, central neurons can potentially control the calcium permeability of AMPARs by the level of GluR-B gene expression as well as by the extent of Q/R-site editing, which in postnatal brain, positions the R codon into >99% of GluR-B mRNA. To study whether the small amount of unedited GluR-B is of functional relevance, we have generated mice carrying GluR-B alleles with an exonic arginine codon. We report that these mutants manifest no obvious deficiencies, indicating that AMPAR-mediated calcium influx into central neurons can be solely regulated by the levels of Q/R site-edited GluR-B relative to other AMPAR subunits. Notably, a targeted GluR-B gene mutant with 30% reduced GluR-B levels had 2-fold higher AMPAR-mediated calcium permeability in hippocampal pyramidal cells with no sign of cytotoxicity. This constitutes proof in vivo that elevated calcium influx through AMPARs need not generate pathophysiological consequences.

Animals↗
PubMed1998

Stable transfection of the rat follicle-stimulating hormone receptor complementary DNA into an immortalized murine Sertoli cell line.

A plasmid expressing the rat FSH receptor (R) cDNA under the Simian virus (SV) 40 promoter/enhancer was stably transfected into a mouse Sertoli cell (SC) line (MSC-1) established from transgenic mice carrying a fusion gene of the human anti-Müllerian hormone (AMH) promoter sequences linked to the SV40 T-antigen gene (Peschon et al., 1992). The original cell line has numerous SC characteristics, but it was reported not to express the inhibin-alpha and follicle-stimulating hormone (FSH)R genes. The new FSHR expressing cell line possessed approximately 2000 per cell with equilibrium association constant (Ka) of 1.5 x 10(9) l/mol. In Northern blots, an FSHR mRNA species of 2.6 kb was found. The cells responded to recombinant human FSH (recFSH) and pertussis toxin (PT) with stimulated cAMP production. Moreover, PT enhanced the FSH-stimulated cAMP production in these cells, indicating the presence of a functional Gi protein. 12-O-tetradecanoylphorbol-13-acetate (TPA) suppressed the FSH-stimulated cAMP production of the cells, which effect was similar to that observed previously upon protein kinase C (PKC) activation in rat seminiferous tubules in vitro. Hence, the FSHR signalling, and its modulatory pathways, were intact in the FSHR expressing MSC-1 cell line. RT-PCR with inhibin-alpha specific oligonucleotide primers. followed by Southern hybridization, indicated that, unlike previously shown, the original and the FSHR expressing MSC-1 cells do express the inhibin alpha gene. FSH stimulation of the cells decreased their proliferation and, unexpectedly, the inhibin-alpha mRNA levels. The cells have functional features both from neonatal and mature SC. A feature of the former cells is the lack of FSH-stimulated up-regulation of inhibin-alpha expression; in fact FSH decreased this message. The antiproliferative, and apparently differentiating, effect of FSH on these cells resembled mature SC functions. Since adult SC do not proliferate in vitro, the new FSHR expressing and proliferating cell line provides a useful in vitro model for studying some facets of SC functions, though keeping in mind that these transformed cells do not behave identically with adult SC in vivo. The constitutive expression of FSHR in these cells allows the study of posttranscriptional events in the FSHR regulation.

Animals↗
PubMed1998

Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo.

NMDA receptors, a class of glutamate-gated cation channels with high Ca2+ conductance, mediate fast transmission and plasticity of central excitatory synapses. We show here that gene-targeted mice expressing NMDA receptors without the large intracellular C-terminal domain of any one of three NR2 subunits phenotypically resemble mice made deficient in that particular subunit. Mice expressing the NR2B subunit in a C-terminally truncated form (NR2B(deltaC/deltaC) mice) die perinatally. NR2A(deltaC/deltaC) mice are viable but exhibit impaired synaptic plasticity and contextual memory. These and NR2C(deltaC/deltaC) mice display deficits in motor coordination. C-terminal truncation of NR2 subunits does not interfere with the formation of gateable receptor channels that can be synaptically activated. Thus, the phenotypes of our mutants appear to reflect defective intracellular signaling.

Amino Acid Sequence↗
PubMed1998

RNA editing of brain glutamate receptor channels: mechanism and physiology.

Glutamate-gated cation selective channels mediate fast excitatory neurotransmission in the mammalian brain. Functionally critical channel positions contain amino acid residues not predicted from the exonic sequence for the channel subunits. The codons for these residues are created in the respective primary gene transcripts by the site selective deamination of adenosine to inosine. This type of RNA editing requires a short double-stranded RNA structure formed by the exonic sequence around the adenosine targeted for deamination with a complementary sequence in the downstream intron and hence, it precedes splicing. Candidate enzymes for nuclear transcript editing currently comprise three molecularly cloned mammalian RNA-dependent adenosine deaminases. Two of these are expressed in most body tissues, perhaps indicating that adenosine deamination in transcripts is more global than has been recognized. Indeed, numerous mRNAs in different tissues may contain inosine residues and encode proteins with amino acid substitutions and different properties relative to the exonically encoded forms. If so, RNA editing by adenosine deamination may significantly enlarge the functional repertoire of the mammalian genome.

Amino Acid Sequence↗
PubMed1997

Tissue specific control regions of the N-methyl-D-aspartate receptor subunit NR2C promoter.

In the mouse brain, the N-methyl-D-aspartate receptor subunit NR2C (epsilon-3) is mainly detected in the cerebellar granule cells starting from the second week of postnatal life. In order to improve our understanding of molecular mechanisms of this neuron-specific, spatial and temporal gene expression, different promoter fragments were used to control indicator genes in nondifferentiated rat pheochromocytoma (PC12) cells, in human embryonal kidney (HEK293) cells and in transgenic mice. A 400 bp NR2C promoter region upstream of the transcriptional start site was identified as a basal promoter that was negatively regulated possibly by a neuron restrictive silencer element (NRSE) that is localized 664 base pairs downstream from the transcriptional start sites.

Animals↗
PubMed1996

RED2, a brain-specific member of the RNA-specific adenosine deaminase family.

The mammalian RNA-specific adenosine deaminases DRADA/dsRAD (alias ADAR) and RED1 (alias ADARB1) have been implicated in the site-selective editing of brain-expressed pre-mRNAs for glutamate receptor subunits and of antigenomic RNA of hepatitis delta virus. These enzymes are expressed in many if not all tissues, predicting an as yet unappreciated significance for adenosine deamination-mediated recoding of gene transcripts in the mammalian organism. We now report the molecular cloning of cDNA for RED2 (alias ADARB2), a third member of the RNA-specific adenosine deaminase family in the rodent. RED2 is closely sequence-related to RED1 but appears to be expressed only in the brain, where expression is widespread reaching highest levels in olfactory bulb and thalamus. RED2 further differs from RED1 in having a 54-residue amino-terminal extension which includes an arginine-rich motif. Different from DRADA and RED1, recombinantly expressed RED2 did not deaminate adenosines in extended synthetic dsRNA or in GluR-B pre-mRNA. However, a chimera of RED1 and RED2 edited the GluR-B Q/R and R/G sites with moderate efficiency. Our data suggest that RED2 may edit brain-specific transcripts with distinct structural features.

Adenosine Deaminase↗
PubMed1996

Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse.

Targeting vectors for embryonic stem (ES) cells typically contain a mouse gene segment of >7 kb with the neo gene inserted for positive selection of the targeting event. More complex targeting vectors carry additional genetic elements (e.g. lacZ, loxP, point mutations). Here we use homologous recombination in yeast to construct targeting vectors for the incorporation of genetic elements (GEs) into mouse genes. The precise insertion of GEs into any position of a mouse gene segment cloned in an Escherichia coli/yeast shuttle vector is directed by short recombinogenic arms (RAs) flanking the GEs. In this way, complex targeting vectors can be engineered with considerable ease and speed, obviating extensive gene mapping in search for suitable restriction sites.

Animals↗
PubMed1996

Q/R site editing in kainate receptor GluR5 and GluR6 pre-mRNAs requires distant intronic sequences.

RNA editing by adenosine deamination in brain-expressed pre-mRNAs for glutamate receptor (GluR) subunits alters gene-specified codons for functionally critical positions, such as the channel's Q/R site. We show by transcript analysis of minigenes transiently expressed in PC-12 cells that, in contrast to GluR-B pre-mRNA, where the two editing sites (Q/R and R/G) require base pairing with nearby intronic editing site complementary sequences (ECSs), editing in GluR5 and GluR6 pre-mRNAs recruits an ECS located as far as 1900 nucleotides distal to the Q/R site. The exon-intron duplex structure of the GluR5 and GluR6 pre-mRNAs appears to be a substrate of double-stranded RNA-specific adenosine deaminase. This enzyme when coexpressed in HEK 293 cells preferentially targets the adenosine of the Q/R site and of an unpaired position in the ECS which is highly edited in brain.

Adenosine↗
PubMed1996

A mammalian RNA editing enzyme.

Editing of RNA by site-selective adenosine deamination alters codons in brain-expressed pre-messenger RNAs for glutamate receptor (GluR) subunits including a codon for a channel determinant (Q/R site) in GluR-B, which controls the Ca2+ permeability of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. Editing of GluR pre-mRNAs requires a double-stranded RNA (dsRNA) structure formed by exonic and intronic sequences and is catalysed by an unknown dsRNA adenosine deaminase. Here we report the cloning of complementary DNA for RED1, a dsRNA adenosine deaminase expressed in brain and peripheral tissues that efficiently edits the Q/R site in GluR-B pre-mRNA in vitro. This site is poorly edited by DRADA, which is distantly sequence-related to RED1. Both deaminases edit the R/G site in GluR-B pre-mRNA, indicating that members of an emerging gene family catalyse adenosine deamination in nuclear transcripts with distinct but overlapping substrate specificities.

Adenosine Deaminase↗