AS601245, a c-Jun NH2-terminal kinase (JNK) inhibitor, reduces axon/dendrite damage and cognitive deficits after global cerebral ischaemia in gerbils.

BACKGROUND AND PURPOSE: Based on their proven ability, in animal models of stroke, to reduce damage to brain grey matter, many drugs have been tested in clinical trials but without success. Failure to save axons from injury and to protect functional outcome has been proposed as the major reason for this lack of success. We have previously demonstrated in two rodent models of cerebral ischaemia, that AS601245 (1,3-benzothiazol-2-yl (2-([2-(3-pyridinyl) ethyl] amino)-4 pyrimidinyl) acetonitrile), an inhibitor of the c-Jun NH(2)-terminal kinase (JNK), has neuroprotective properties. The aim of the present study was to further investigate if AS601245 in addition to its ability to protect neurons also could protect neurites and preserve memory after cerebral ischaemia, in gerbils. EXPERIMENTAL APPROACH: Using immunohistochemical techniques and a behavioural test, we studied the effect of the compound AS601245 on neurodegeneration and cognitive deficits after global cerebral ischaemia in gerbils. KEY RESULTS: At a dose of 80 mg kg(-1), i.p., AS601245 reduced damage to neurites by 67% (P<0.001 versus controls) and activation of astrocytes by 84% (P<0.001 versus controls). In addition, AS601245 (80 mg kg(-1), i.p.) prevented ischaemia-induced impairment of memory in the inhibitory avoidance task model. CONCLUSIONS AND IMPLICATIONS: The present results suggest that AS601245 reduced damage to neurites and decreased astrogliosis following global ischaemia and also improved long-term memory, supporting JNK inhibition as a promising therapeutic strategy for ischaemic insults to the CNS.

Apolipoprotein E expression by neurons surviving excitotoxic stress.

In the adult brain, apolipoprotein E (apoE) mRNA is thought to be expressed by nonneuronal cells. Yet, when a brain damage has occurred, the protein is found in neurons. We have studied apoE expression following systemic kainic acid (KA), injected in rats to induce hippocampal neurodegeneration. We describe two effects. First, a moderate increase of apoE levels in astrocytes. Second, and unexpected, a very strong increase of apoE mRNA levels in clusters of CA1 and CA3 pyramidal neurons. Neuronal identity of these cells is supported by a series of observations. First, apoE hybridization signals were found in cells with morphological characteristics of pyramidal neurons. Second, the cells were positive for the neuronal marker MAP2. Third, the cells were negative for the astrocytic marker GFAP and for the microglia marker OX42. Fourth, the same distribution pattern was found with probes hybridizing to c-fos, a transcription factor transiently expressed in neurons under stress. At 48 and 72 h following KA, most of the excitotoxic cell death had already occurred. Since no morphological signs of programmed cell death were observed in apoE-positive pyramidal neurons, we suggest that expression of apoE by neurons may be part of a rescue program to counteract neurodegeneration.

Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase.

MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.

The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate binding and enzymatic specificity.

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.

Induction of the mitogen-activated protein kinase phosphatase MKP3 by nerve growth factor in differentiating PC12.

In PC12 sympathetic neurons activation and nuclear translocation of ERK family MAP kinases plays an essential role in processes underlying nerve growth factor (NGF)-dependent differentiation. We have recently cloned MKP-3 as a novel dual specificity phosphatase displaying selectivity towards inactivation of the ERK1 and ERK2 MAP kinases. Here we report that in PC12 cells, MKP-3 undergoes powerful and specific up-regulation by NGF while a number of mitogens and cellular stresses are ineffective. NGF-stimulated MKP-3 expression appears after 1 h, is maximal at 3 h, and is sustained for 5 days. This coincides with a critical period of neurite outgrowth and terminal differentiation. Consistent with a role mediating inhibition of PC12 cell MAP kinases, NGF-stimulated ERK2 activation was suppressed considerably following pretreatment with fibroblast growth factor and 9-cis-retinal, two additional differentiation factors found to induce powerfully MKP-3 expression. Given the clear cytosolic localization of MKP3 in PC12 cells and sympathetic neurons, these results suggest a critical role for inactivating ERK MAP kinases in non-nuclear compartments during essential stages of NGF-mediated PC12 differentiation.

Regulated expression of dual specificity protein phosphatases in rat brain.

Activated mitogen-activated protein (MAP) kinases play an essential role controlling many neuronal functions. Dual specificity protein phosphatases (DS-PTPs) elicit selective inactivation of MAP kinases and are under tight transcriptional control. We have studied expression of four DS-PTPs (MKP-1, MKP-X, MKP-3 and B23) in rat brain and examined changes during post-natal development and following kainic acid induced seizure activity. In normal adult brain these DS-PTPs exhibit a strikingly different expression pattern. Only MKP-1 was regulated during development with levels increased transiently (P15-P21) within the thalamus and somatosensory cortex. Following kainate treatment, MKP-1, MKP-3 and B23 all exhibit striking changes in expression within hippocampal subfields CA1-3 and dentate gyrus. Regulated transcription of DS-PTPs may play a critical role controlling MAP kinase dependent processes including synaptic remodeling and neuronal death.

Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1.

We have studied the phosphorylation of the Bcl-2 family of proteins by different mitogen-activated protein (MAP) kinases. Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKbeta, and this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKbeta and the constitutive Rac1 mutant G12V. This is seen by both 32PO4 labeling and the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKbeta activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A, S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKbeta phosphorylates identical sites in vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses.

Induction of the dual specificity phosphatase PAC1 in rat brain following seizure activity.

Recurrent seizure activity leads to delayed neuronal death as well as to inflammatory responses involving microglia in hippocampal subfields CA1, CA3 and CA4. Since mitogen activated protein (MAP) kinases control neuronal apoptosis and trigger generation of inflammatory cytokines, their activation state could determine seizure-related brain damage. PAC1 is a dual specificity protein phosphatase inactivating MAP kinases which we have found to be undetectable in normal brain. Despite this, kainic acid-induced seizure activity lead to rapid (approximately 3 h) but transient appearance of PAC1 mRNA in granule cells of the dentate gyrus as well as in pyramidal CA1 neurons. This pattern changed with time and after 2-3 days PAC1 was induced in dying CA1 and CA3 neurons. At this time PAC1 mRNA was also expressed in white matter microglia as well as in microglia invading the damaged hippocampus. PAC1 may play an important role controlling MAP kinase involvement in both neuronal death and neuro-inflammation following excitotoxic damage.

Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase, MKP-4.

Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli. Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues. By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4). The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX3AYLM (where X is any amino acid) conserved in dual specificity phosphatases. MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2. When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK. This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK). Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver. Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells. Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28. The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression.

The dual specificity phosphatases M3/6 and MKP-3 are highly selective for inactivation of distinct mitogen-activated protein kinases.

The mitogen-activated protein (MAP) kinase family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) as structurally and functionally distinct enzyme classes. Here we describe two new dual specificity phosphatases of the CL100/MKP-1 family that are selective for inactivating ERK or JNK/SAPK and p38 MAP kinases when expressed in COS-7 cells. M3/6 is the first phosphatase of this family to display highly specific inactivation of JNK/SAPK and p38 MAP kinases. Although stress-induced activation of p54 SAPKbeta, p46 SAPKgamma (JNK1) or p38 MAP kinases is abolished upon co-transfection with increasing amounts of M3/6 plasmid, epidermal growth factor-stimulated ERK1 is remarkably insensitive even to the highest levels of M3/6 expression obtained. In contrast to M3/6, the dual specificity phosphatase MKP-3 is selective for inactivation of ERK family MAP kinases. Low level expression of MKP-3 blocks totally epidermal growth factor-stimulated ERK1, whereas stress-induced activation of p54 SAPKbeta and p38 MAP kinases is inhibited only partially under identical conditions. Selective regulation by M3/6 and MKP-3 was also observed upon chronic MAP kinase activation by constitutive p21(ras) GTPases. Hence, although M3/6 expression effectively blocked p54 SAPKbeta activation by p21(rac) (G12V), ERK1 activated by p21(ras) (G12V) was insensitive to this phosphatase. ERK1 activation by oncogenic p21(ras) was, however, blocked totally by co-expression of MKP-3. This is the first report demonstrating reciprocally selective inhibition of different MAP kinases by two distinct dual specificity phosphatases.

Developmental and plasticity-related differential expression of two SNAP-25 isoforms in the rat brain.

In this article we study the relationship between the expression pattern of two recently identified isoforms of the 25-kD synaptosomal-associated protein (SNAP-25a and SNAP-25b) and the morphological changes inherent to neuronal plasticity during development and kainic acid treatment. SNAP-25 has been involved in vescicle fusion in the nerve terminal, and most likely participates in different membrane fusion-related processes, such as those involved in neurotransmitter release and axonal growth. In the adult brain, SNAP-25b expression exceeded SNAP-25a in distribution and intensity, being present in most brain structures . Moderate or high levels of SNAP-25a hybridization signal were found in neurons of the olfactory bulb, the layer Va of the frontal and parietal cortices, the piriform cortex, the subiculum and the hippocampal CA4 field, the substantia nigra/pars compacta, and the pineal gland, partially overlapping SNAP-25b mRNA distribution. In restricted regions of cerebral cortex, thalamus, mammillary bodies, substantia nigra, and pineal glands the two isoforms were distributed in reciprocal fashion. During development SNAP-25a mRNA was the predominant isoform, whereas SNAP-25b expression increased postnatally. The early expression of SNAP-25a in the embryo and the decrease after P21 is suggestive of a potential involvement of this isoform in axonal growth and/or synaptogenesis. This conclusion is indirectly supported by the observation that SNAP-25a mRNA, but not SNAP-25b mRNA, was upregulated in the granule cells of the adult dentate gyrus 48 hours after kainate-induced neurotoxic damage of the hippocampal CA3-CA4 regions. Increase of SNAP-25 immunoreactivity was observed as early as 4 days after kainate injection within the mossy fiber terminals of the CA3 region, and in the newly formed mossy fiber aberrant terminals of the supragranular layer. These data suggest an isoform-specific role of SNAP-25 in neural plasticity.

MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase.

MKP-1 (also known as CL100, 3CH134, Erp, and hVH-1) exemplifies a class of dual-specificity phosphatase able to reverse the activation of mitogen-activated protein (MAP) kinase family members by dephosphorylating critical tyrosine and threonine residues. We now report the cloning of MKP-3, a novel protein phosphatase that also suppresses MAP kinase activation state. The deduced amino acid sequence of MKP-3 is 36% identical to MKP-1 and contains the characteristic extended active-site sequence motif VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid) as well as two N-terminal CH2 domains displaying homology to the cell cycle regulator Cdc25 phosphatase. When expressed in COS-7 cells, MKP-3 blocks both the phosphorylation and enzymatic activation of ERK2 by mitogens. Northern analysis reveals a single mRNA species of 2.7 kilobases with an expression pattern distinct from other dual-specificity phosphatases. MKP-3 is expressed in lung, heart, brain, and kidney, but not significantly in skeletal muscle or testis. In situ hybridization studies of MKP-3 in brain reveal enrichment within the CA1, CA3, and CA4 layers of the hippocampus. Metrazole-stimulated seizure activity triggers rapid (<1 h) but transient up-regulation of MKP-3 mRNA in the cortex, piriform cortex, and some amygdala nuclei. Metrazole stimulated similar regional up-regulation of MKP-1, although this was additionally induced within the thalamus. MKP-3 mRNA also undergoes powerful induction in PC12 cells after 3 h of nerve growth factor treatment. This response appears specific insofar as epidermal growth factor and dibutyryl cyclic AMP fail to induce significant MKP-3 expression. Subcellular localization of epitope-tagged MKP-3 in sympathetic neurons reveals expression in the cytosol with exclusion from the nucleus. Together, these observations indicate that MKP-3 is a novel dual-specificity phosphatase that displays a distinct tissue distribution, subcellular localization, and regulated expression, suggesting a unique function in controlling MAP kinase family members. Identification of a second partial cDNA clone (MKP-X) encoding the C-terminal 280 amino acids of an additional phosphatase that is 76% identical to MKP-3 suggests the existence of a distinct structurally homologous subfamily of MAP kinase phosphatases.

The mouse 5-hydroxytryptamine1B receptor is localized predominantly on axon terminals.

The 5-hydroxytryptamine1B receptor is a serotonin receptor subtype which is expressed predominantly in the basal ganglia. It has been suggested to play a role in movement and appetite control as well as in certain pathological states such as migraine. The recent cloning of the 5-hydroxytryptamine1B gene as well as the discovery of a radioligand that labels in rodents 5-hydroxytryptamine1B and possibly 5-hydroxytryptamine1D alpha receptors (S-CM-G[125I]TNH2) allowed us to compare the distribution of the messenger RNA and of the protein in mouse brain sections. A high 5-hydroxytryptamine1B messenger RNA level is found in the caudate-putamen in medium spiny neurons that project to the globus pallidus and the substantia nigra. In contrast, no messenger RNA is expressed in the globus pallidus and substantia nigra although these structures reveal the highest level of 5-hydroxytryptamine1B binding sites. In the hippocampus, 5-hydroxytryptamine1B messenger RNA is localized in the cell bodies of pyramidal cells of the CA1 field while the protein is found predominantly in the dorsal subiculum, a projection zone for the CA1 pyramidal neurons. In the cerebellum, 5-hydroxytryptamine1B messenger RNA is expressed in the Purkinje cells, which display no receptor binding sites. Conversely, moderate binding is found in the deep nuclei of the cerebellum, the main projection zone of the Purkinje cells. 5-Hydroxytryptamine1B sites are also detected in the superficial gray layer of the superior colliculus and the lateral geniculate nucleus, brain regions containing the terminals of retinal ganglion cells. The soma of these ganglion cells express high levels of 5-hydroxytryptamine1B messenger RNA while no 5-hydroxytryptamine1B binding sites were found in the retina. This study demonstrates that the main brain regions, expressing 5-hydroxytrypamine1B messenger RNA contain low densities of 5-hydroxytryptamine1B binding sites. Conversely, the major projection areas of these anatomical structures do not express detectable levels of 5-hydroxytryptamine1B messenger RNA, but present a high density of binding sites. In addition, our data suggest that the distribution of the 5-hydroxytryptamine1D alpha binding sites is different from that of the 5-hydroxytryptamine1D alpha messenger RNA. These results together with previous lesion studies, indicate that the 5-hydroxytryptamine1B and possibly the 5-hydroxytryptamine1D alpha receptors are localized predominantly on axon terminals, while their expression is low or absent at the somatodendritic level. The 5-hydroxytryptamine1D alpha proteins might therefore contain an addressing signal allowing their transport toward nerve endings.(ABSTRACT TRUNCATED AT 400 WORDS)

Mouse 5-hydroxytryptamine5A and 5-hydroxytryptamine5B receptors define a new family of serotonin receptors: cloning, functional expression, and chromosomal localization.

Serotonin [5-hydroxytryptamine (5-HT)] is a neuromodulator that mediates a wide range of physiological functions by activating multiple receptors. Using a strategy based on amino acid sequence homology between 5-HT receptors that interact with guanine nucleotide-binding proteins, we have isolated from a mouse brain library a cDNA encoding a new serotonin receptor. Amino acid sequence comparisons revealed that this receptor was a close relative of the previously identified 5-HT5 receptor but was distant from all other 5-HT receptor subtypes; we therefore named it 5-HT5B. When expressed in COS-7 cells, the 5-HT5B receptor displayed a high affinity for the serotonergic radioligand 125I-lysergic acid diethylamide. Its pharmacological profile was distinct from that of all classic 5-HT receptor subtypes. However, the high affinity of the 5-HT5B receptor for 5-carboxamidotryptamine and its low affinity for sumatriptan indicated that it might correspond to recently described 5-HT1D-like binding sites that were labeled with [3H]5-carboxamidotryptamine and insensitive to sumatriptan. In situ hybridization experiments revealed that the 5-HT5B mRNA was expressed predominantly in the habenula and in the CA1 field of the hippocampus. We also determined the chromosomal localization of the 5-HT5A and 5-HT5B genes and of their human counterparts. The 5-HT5A gene colocalized with the mouse mutation reeler and the human mutation holoprosencephaly type 3, which both result in abnormal brain development, raising the possibility that the 5-HT5A receptor plays a role in brain development.

Gene in the region of the Friedreich ataxia locus encodes a putative transmembrane protein expressed in the nervous system.

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disorder that affects the cerebellum, spinal cord, and peripheral nerves. The FRDA gene was localized in 9q13-q21 within 0.7 centimorgan of the D9S5 and D9S15 loci. One recently reported recombination event and haplotype analysis in a population with a founder effect suggested that the FRDA locus is on the D9S5 side. Using a conserved probe from the D9S5 locus, we have now identified an approximately 7-kilobase (kb) transcript and report cloning of its cDNA. The corresponding gene, X11, extends at least 80 kb in a direction opposite D9S15. The gene is expressed in the brain, including the cerebellum, but is not detectable in several nonneuronal tissues and cell lines. In situ hybridization of adult mouse brain sections showed prominant expression in the granular layer of the cerebellum. Expression was also found in the spinal cord. The cDNA contains an open reading frame encoding a 708-amino acid sequence that shows no significant similarity to other known proteins but contains a unique, 24-residue-long, putative transmembrane segment. On the basis of its genomic localization and its neuronal site of expression, particularly in the cerebellum, this "pioneer" gene represents a candidate for FRDA. Direct evidence of its involvement in FRDA will require a search for causative point mutations in patients.

The mouse 5HT5 receptor reveals a remarkable heterogeneity within the 5HT1D receptor family.

Serotonin (5-HT) is a neuromodulator that mediates a wide range of physiological functions by activating multiple receptors. Using a strategy based on amino acid sequence homology between 5-HT receptors that interact with G proteins, we have isolated a cDNA encoding a new serotonin receptor from a mouse brain library. Amino acid sequence comparisons revealed that this receptor was a distant relative of all previously identified 5-HT receptors; we therefore named it 5HT5. When expressed in Cos-7 cells and NIH-3T3 cells, the 5HT5 receptor displayed a high affinity for the serotonergic radioligand [125I]LSD. Surprisingly, its pharmacological profile resembled that of the 5HT1D receptor, which is a 5-HT receptor subtype which has been shown to inhibit adenylate cyclase and which is predominantly expressed in basal ganglia. However, unlike 5HT1D receptors, the 5HT5 receptor did not inhibit adenylate cyclase and its mRNA was not found in basal ganglia. On the contrary, in situ hybridization experiments revealed that the 5HT5 mRNA was expressed predominantly in cerebral cortex, hippocampus, habenula, olfactory bulb and granular layer of the cerebellum. Our results therefore demonstrate that the 5HT1D receptors constitute a heterogeneous family of receptors with distinct intracellular signalling properties and expression patterns.

Isolation of a mouse "5HT1E-like" serotonin receptor expressed predominantly in hippocampus.

Using a strategy based on amino acid sequence homology between 5-hydroxytryptamine (5-HT) receptors that interact with G proteins, we have isolated from a mouse brain library a cDNA encoding a new serotonin receptor, the 5HT1E beta receptor. Amino acid sequence comparisons revealed that its closest relatives were the recently characterized 5HT1E receptor (S31) and the 5HT1B and 5HT1D receptors. When expressed transiently in Cos-7 cells, the 5HT1E beta receptor displayed a high affinity for the nonspecific serotonergic radioligand 2-[125I]iodolysergic acid diethylamide (Kd = 980 pM). The pharmacological profile of the 5HT1E beta receptor resembled that of previously reported 5HT1E sites that have a low affinity for 5-carboxamidotryptamine and that have been found in human and rat brain. When stably expressed in NIH-3T3 cells, the 5HT1E beta receptor was negatively coupled to adenylate cyclase. In situ hybridization experiments revealed that the 5HT1E beta transcripts were detected only in the CA1, CA2, and CA3 layers of the hippocampus. Our results therefore demonstrate that the 5HT1E receptors constitute a heterogeneous family of receptors.

Mouse 5HT1B serotonin receptor: cloning, functional expression, and localization in motor control centers.

Serotonin is a neuromodulator that mediates a wide range of effects by interacting with multiple receptors. Using a strategy based on nucleotide sequence homology between genes encoding receptors that interact with guanine nucleotide-binding proteins, we have isolated a mouse gene encoding an additional serotonin receptor. When expressed in cultured cells, it displayed the pharmacological profile and coupling with adenylate cyclase characteristic of the 5HT1B receptor subtype. In NIH 3T3 cells expressing this receptor, serotonin induced a decrease in forskolin-stimulated cAMP levels. This effect was blocked by pertussis toxin, indicating that the 5HT1B receptor interacts with a pertussis toxin-sensitive guanine nucleotide-binding protein. To obtain clues as to the possible function of the 5HT1B receptor, we have analyzed its pattern of expression in the adult mouse brain by in situ hybridization. Our results, together with previous autoradiographic studies, suggest that the 5HT1B receptors are localized presynaptically on the terminals of striatal neurons and Purkinje cells and that they might modulate the release of neurotransmitters such as gamma-aminobutyric acid. The predominant expression of the 5HT1B receptor in the striatum and cerebellum points to an involvement of this receptor in motor control.

A family of Drosophila serotonin receptors with distinct intracellular signalling properties and expression patterns.

Biogenic amines such as serotonin elicit or modulate a wide range of behaviours by interacting with multiple receptor subtypes. We have isolated cDNA clones encoding three distinct Drosophila serotonin receptors which belong to the G protein-coupled receptor family. When expressed in mammalian cells, these receptors activate different intracellular effector systems. The 5HT-dro1 receptor stimulates adenylate cyclase while the 5HT-dro2A and the 5HT-dro2B receptors inhibit adenylate cyclase and activate phospholipase C. Expression of all three receptors starts in late embryos and is restricted to distinct populations of cells in the central nervous system. The 5HT-dro2A receptor is predominantly expressed in midline motor neurons (VUM neurons) that innervate larval muscles thus suggesting a role for this receptor in motor control.

Genetic and developmental analysis of irreC, a genetic function required for optic chiasm formation in Drosophila.

Irregular chiasm C (irreC) is an X-linked genetic function necessary for the correct projection of visual fibers in the optic chiasms of Drosophila optic ganglia. In addition to a severe disorganization of the inner optic chiasm irreC mutants display a subtle phenotype in the outer optic chiasm, in which some bundles of axons that leave the posterior equatorial part of the lamina on their way to the anterior medulla take a long detour before eventually finding their specific targets in the medulla neuropile. Deletion and recombination mapping of two irreC alleles (one P-element induced, the other associated with an inversion) have yielded a precise cytogenetic location in 3C4-5. A complex complementation pattern between roughest (rst) and irreC alleles indicates that both genetic functions are structurally and/or functionally closely interrelated. Flies in which the irreC locus is completely deleted by overlapping deficiencies are viable and their defects in the optic chiasms are similar to those seen in the two alleles. The defects in the outer and inner optic chiasms are not epigenetically connected and mosaic analyses have shown them to be independent from the genotype of the compound eye. Although the larval visual nerve looks normal, we have found that in the optic lobes of irreC mutants a group of early differentiating larval neurons is misplaced, suggesting a pioneering function of these cells during organization of the outer optic chiasms.