Parichehr Malherbe

In situ hybridization histochemistry reveals a diversity of GABAA receptor subunit mRNAs in neurons of the rat spinal cord and dorsal root ganglia

The distribution and relative abundance of gene transcripts for diverse GABAA receptor subunits (alpha 1-3,5, beta 1-3, gamma 2) in neurons of the rat cervical spinal cord and dorsal root ganglia were determined by in situ hybridization histochemistry using 35S-labeled 60mer oligonucleotide probes. The receptor proteins (mapped by benzodiazepine receptor radioautography and immunohistochemistry with [3H]flumazenil and a monoclonal antibody for the beta 2 + beta 3 subunits, respectively) were most abundant in the dorsal horn (layers II and III) and in layer X around the central canal. Although diverse receptor subunit mRNAs were detected (to varying degrees) in neurons throughout layers II-X of the spinal cord, motoneurons in layer IX were particularly strongly labeled. The gamma 2 mRNA was the most ubiquitous and abundant of the subunit variants investigated. The labeling of motoneurons in layer IX was particularly strong for alpha 2, moderate for beta 3 and gamma 2 and extremely weak for alpha 1 and alpha 3. In layers VII, VIII and X the beta 3 and gamma 2 transcripts were moderately expressed whereas the alpha 1 and beta 2 transcript levels differed markedly among the cells of these layers. Although the mRNAs of all subunit variants could be detected in layers IV-VI, only alpha 3, alpha 5, beta 3 and gamma 2 hybridization signals were observed in layers II and III. In the dorsal root ganglia, whereas alpha 2 transcripts were abundant in virtually all large sensory neurons and to a much lower degree in the small diameter cells, gamma 2 transcripts were confined to a subpopulation of large and small neurons. Furthermore, beta 2 and alpha 1 transcripts were even more restricted in their distribution. The findings provided a basis for the mediation of synaptic inhibition in the spinal cord by diverse GABAA receptors and further strong evidence for the long-established view that presynaptic inhibition of inter- and motoneurons, via axoaxonic synapses between GABAergic interneurons and primary afferent terminals, is mediated by GABAA receptors. The physiological roles and pharmacological implications of this receptor diversity have yet to be determined.

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABAA receptors in neuronal populations within the olfactory bulb.

Molecular neuroanatomy of human monoamine oxidases A and B revealed by quantitative enzyme radioautography and in situ hybridization histochemistry

Monoamine oxidases are key enzymes in the metabolism of amine neurotransmitters and neuromodulators and are targets for drug therapy in depression, Parkinsons and Alzheimers diseases. Knowledge of their distribution in the brain is essential to understand their physiological role. To study the regional distribution and abundance of monoamine oxidases A and B in human brain, pituitary and superior cervical ganglion, we used quantitative enzyme radioautography with radioligands [3H]Ro41-1049 and [3H]lazabemide, respectively. Furthermore, 35S-labelled oligonucleotides complementary to isoenzyme messengerRNAs were used to map the cellular location of the respective transcripts in adjacent sections by in situ hybridization histochemistry. A markedly different pattern of distribution of the isoenzymes was observed. Highest levels of monoamine oxidase A were measured in the superior cervical ganglion, locus coeruleus, interpeduncular nucleus and ventromedial hypothalamic nucleus. The corresponding messengerRNA was detected only in the noradrenergic neurons of the superior cervical ganglion and locus coeruleus. In contrast to rat brain, monoamine oxidase B was much more abundant in most human brain regions investigated. Highest levels were measured in the ependyma of ventricles, stria terminalis and in individual hypothalamic neurons. Monoamine oxidase B transcripts were detected in serotoninergic raphe neurons, histaminergic hypothalamic neurons and in dentate gyrus granule cells of the hippocampal formation. We conclude that [3H]Ro41-1049 and [3H]azabemide are extremely useful radioligands for high-resolution analyses of the abundance and distribution of catalytic sites of monoamine oxidases A and B, respectively, in human brain sections. From levels of messenger RNA detected, the cellular sites of synthesis of the isoenzymes are the noradrenergic neurons of the locus coeruleus (for monoamine oxidase A) and the serotoninergic and histaminergic neurons of the raphe and posterior hypothalamus, respectively (for monoamine oxidase B). The combination of quantitative enzyme radioautography with in situ hybridization histochemistry is a useful approach to study, with high resolution, both the physiology and pathophysiology of monoamine oxidases in human brain.

The γ3-subunit of the GABAA-receptor confers sensitivity to benzodiazepine receptor ligands

The γ3‐subunit of the GABAA‐receptor in rat brain has been identified by molecular cloning. When co‐expressed with the α5‐ and β2‐subunits in transfected cells a high potency for GABA (K a = 4.9 ± 1.2 μM; and a strong cooperativity in gating the channel (H = 1.9 ± 0.2) was observed. The GABA response was potentiated in the presence of flunitrazepam reduced by βCCM. An analogous bi‐directional modulation of the GABA response was observed with diazepam and DMCM as tested with the subunit combinations α1β2γ3 and α3β2γ3 expressed in Xenopus oocytes. Since the benzodiazepine receptor ligands were virtually inactive in the absence of the γ3‐subunit, as tested with the α3β2‐ and α5β2‐subunit combinations, the γ3‐subunit is a prerequisite for the benzodiazepine receptor sensitivity of the expressed GABAA‐receptors. The γ3‐subunit could functionally replace the γ2‐subunit with regard to the bi‐directional allosteric drug modulation.

Localization of GABAA receptor subunit mRNAs in the rat locus coeruleus.

N-Methyl-D-aspartate (NMDA)-activated ionotropic glutamate receptors in the CNS are thought to play a crucial role in cognitive processes, neurological disorders as well as in progressive neurodegenerative diseases. In spite of the overwhelming evidence for the existence of structurally different subunits of NMDA receptors in the CNS, the functional relevance of this heterogeneity is still poorly understood. A first step in this direction is to demonstrate the receptor composition in well-characterized transmitter-specific neuronal populations, such as the noradrenergic neurons of the rat locus coeruleus (LC). LC neurons may play a key role in the regulation of vigilance, attention, learning and memory, as well as anxiety and are affected in neurodegenerative disorders. In this study we examined, by means of in situ hybridization with 35S-labelled oligodeoxynucleotide probes, the distribution of mRNAs encoding the splice variants of the NMDAR1 subunit as well as four NMDAR2 subunits (A-D) in the rat LC. Identified neurons express mRNAs encoding several NMDAR1 subunit isoforms (4a, 2a > 2b, 4b) as well as NMDAR2 subunits (2B > 2D), whereas other transcripts (1a,1b,3a,3b,2A,2C) were not detected. These findings suggest that NMDA receptors in the LC are composed of unique combination(s) of subunits, e.g. 4a-2B, of as yet unknown stoichiometry. Whether the identification of this potential drug target can be exploited, e.g. in the development of new anxiolytics, antidepressants, or neuroprotective agents, awaits further investigations.

Biochemical and behavioural characterization of EMPA, a novel high‐affinity, selective antagonist for the OX2 receptor

Background and purpose:  The OX2 receptor is a G‐protein‐coupled receptor that is abundantly found in the tuberomammillary nucleus, an important site for the regulation of the sleep‐wake state. Herein, we describe the in vitro and in vivo properties of a selective OX2 receptor antagonist, N‐ethyl‐2‐[(6‐methoxy‐pyridin‐3‐yl)‐(toluene‐2‐sulphonyl)‐amino]‐N‐pyridin‐3‐ylmethyl‐acetamide (EMPA).

Alternatively spliced isoforms of the N-methyl-d-aspartate receptor subunit 1 are differentially distributed within the rat spinal cord

N-Methyl-D-aspartate-activated ionotropic glutamate receptors play a crucial role in synaptic transmission in the spinal cord. Molecular cloning has identified two polymorphic subunits--N-methyl-D-aspartate receptor subunits 1 and 2--the products of alternative splicing (subunit 1a-4b) or of different genes (subunit 2 A-D). While the distribution of N-methyl-D-aspartate receptor subunit 1 splice variants is unknown in the spinal cord, that of subunit 2 appears controversial. We examined, by means of in situ hybridization, the distribution of messenger RNAs encoded by these genes in rat cervical spinal cord. Most neurons throughout all the laminae express predominantly type b variants of subunit 1 (dorsal horn: 3b; ventral horn: 4b) and the 2A subunit, although some neurons in laminae 2 and 9 also express subunit 2B. Our findings demonstrate that subunit 1 splice variants are differentially distributed in the rat cervical cord and, since they fall into two physiologically and pharmacologically distinct groups, may reveal the distribution of antagonist- and agonist-preferring N-methyl-D-aspartate receptor subclasses. They also indicate the co-distribution of receptor subunits 1 and 2, suggesting the existence of heteromeric N-methyl-D-aspartate receptor complexes. Thus, in the spinal cord, different combinations of subunit 1 isoforms as well as subunit 2 may form N-methyl-D-aspartate receptors with different physiological and pharmacological properties. If this structural diversity of presumptive N-methyl-D-aspartate receptors exists in human spinal cord, it might identify potential targets for drug therapy.

Expression of functional membrane-bound and soluble catechol-O-methyltransferase in Escherichia coli and a mammalian cell line.

Abstract: Human catechol‐O‐methyltransferase (hCOMT) cDNA was used to express the recombinant hCOMT enzyme in sufficient quantities in prokaryotic as well as in eukaryotic cells to allow kinetic studies. When human membrane‐bound catechol‐O‐methyltransferase (MB‐COMT; amino acids 1‐271) and the soluble catechol‐O‐methyltransferase COMT (S‐COMT; Δ membrane anchor hCOMT; amino acids 27‐271), with the latter lacking the first 26 hydrophobic amino acids, were expressed in Escherichia coli, a relatively high‐level synthesis of catalytically active enzymes was obtained. Insertion of the human MB‐COMT‐coding sequence into an eukaryotic expression vector under transcriptional control of the cytomegalovirus (CMV) promoter and enhancer yielded large quantities of hCOMT in human kidney 293 cells. Subcellular fractionation of 293 cells transfected with pBC12/CMV‐hCOMT showed hCOMT to be located predominantly in the membrane fraction. The catechol‐O‐methyltransferase (COMT) activity was measured in cytosolic and membrane fractions at 37°C, giving values of 33 and 114 units/mg of protein, respectively (1 unit produces 1 nmol of guaiacol/h). Km values were 10 μM for MB‐COMT and 108 μM for S‐COMT, indicating that recombinant MB‐COMT exhibits a higher affinity for catechol as the substrate than the soluble form. RNA blot analysis of human hepatome cells (Hep G2), kidney, liver, and fetal brain revealed only one species of hCOMT mRNA of ∼ 1.4 kb. Its level in these various tissues was similar to those of COMT protein in each tissue. Using the polymerase chain reaction (PCR) with primers surrounding the putative membrane anchor region, we have clearly identified a single‐size PCR product generated from hCOMT mRNA of various human tissues. Hence, the two forms of the enzyme cannot be the products of an alternative splicing of transcripts. We suggest that S‐COMT is generated by proteolytic cleavage between the NH2‐terminal membrane anchor and the catalytic domain of the membrane‐bound form. Lack of the N‐terminal fragments reduces the catalytic activity of the enzyme.

Characterisation of wild-type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line

Monoamine oxidase (MAO)‐A and MAO‐B are FAD‐containing mitochondrial enzymes which catabolize biogenic and xenobiotic amines. The N‐terminal regions of both forms of MAO contain an ADP‐binding consensus sequence found in several dinucleotide‐dependent enzymes, but otherwise show remarkable sequence differences. In order to investigate whether the N‐terminal region of MAOs participates in the different catalytic properties and inhibitor specificities exhibited by MAO‐A and MAO‐B, we constructed chimeric A/B forms and expressed them in a human embryonic kidney cell line (293 cells). The MAO‐A chimeric form containing the N‐terminus (36 amino acids) of MAO‐B and the B chimera having the first 45 amino acid sequence of MAO‐A were both catalytically active. Compared to the respective wild‐type form, they did not show any significant difference in their catalytic properties (K m, k cat) towards the substrates tested or in their sensitivity towards inhibitors. This indicates that the N‐terminal region of the two isoenzymes is not involved in the different specificities of MAO‐A and MAO‐B. Substitution of Cys‐397 of MAO‐B, i.e. the residue covalently anchoring FAD, with an Ala or a His residue resulted in the total loss of enzymatic activity, suggesting that the covalent coupling of FAD to MAO occurs specifically at the ‐SH group of cysteine.

Lack of β-Amyloidosis in transgenic mice expressing low levels of familial Alzheimer's disease missense mutations

Point mutations within the beta-amyloid precusor protein (beta-APP) gene known to segregate with Alzheimers disease in certain families were introduced into human beta-APP cDNAs and expressed under the control of a neuron-specific enolase (NSE) promoter in mice. The transgenic animals exhibited transgene expression predominantly in neocortex and hippocampus where the levels were maximally 1.3-fold of those of wild-type mouse beta-APP. Quantitative immunoblot analysis in homozygous mice carrying different missense mutations showed slightly increased alpha-secretory processing. In V7171 mice compared to nontransgenic mice there was more alpha-secretory beta-APP (beta-APPsec) in cortex/hippocampus, less in cerebellum, and no difference in midbrain/brain stem. In none of the transgenic animals tested was a 4 kDa amyloid fragment detected by Western blotting of brain extracts, immunohistochemistry, or by 125I-A beta-binding onto brain sections. No glial reaction was observed. Behavioral analysis of mice carrying the V7171 mutation showed no appreciable deficit in comparison to wild-type mice. Together, these data suggest that low levels of expression of mutated beta-APP in 10-12-month-old transgenic mouse brains result in slightly more beta-APPsec, and are insufficient to induce amyloidogenic processing and AD-like pathology.