Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin.

Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT.

Characterization of the interaction between a novel convulsant agent, norbiphen, and GABA(A) and other ligand-gated ion channels.

A hybrid molecule composed of the antimicrobial, norfloxacin, linked to the non-steroidal anti-inflammatory drug (NSAID), biphenylacetic acid, which we have termed norbiphen, is a lethal convulsant in vivo and an antagonist of rodent GABA(A) receptors in vitro. In the present study, the selectivity, molecular site(s) and mechanism of action of this novel convulsant were investigated using electrophysiological techniques. Sub-maximal GABA-evoked currents recorded from rodent hippocampal neurons were reversibly inhibited by norbiphen (1 microM) to 5+/-2% of control whereas glutamate, NMDA and glycine activated responses were little or unaffected. Sub-maximal GABA-evoked currents recorded from oocytes expressing recombinant human alpha1beta2gamma2s or alpha1beta2 GABA(A) receptors were also reversibly inhibited by norbiphen (1-1000 nM) with an IC(50) (+/-s.e.m.) of 5.7+/-1 and 8.8+/-1 nM, respectively. Similarly, GABA currents recorded from alpha1beta1gamma2s, alpha1beta1 and beta2gamma2s receptors were inhibited with IC(50)s of 16.1+/-1, 18.8+/-1 and 4.2+/-1 nM, respectively. In contrast, norbiphen (100 nM) had little or no effect at rho1 GABA(C) homomers. At alpha1beta2gamma2s receptors, norbiphen had no affect on the GABA reversal potential, and inhibition was not voltage-dependent, suggesting that this compound does not act at the ion channel. The GABA concentration response curve was shifted in a competitive-like fashion by norbiphen (10-300 nM) and a Schild analysis of these data yielded a slope of 0.94+/-0.1 and a pA(2) of 7.77. Our data reveal a novel, selective and highly potent antagonist of GABA(A) receptors. Norbiphen should be a valuable agent in future studies of this receptor complex.

Inhibition of nicotinic acetylcholine receptors by bicuculline.

A study was made on the effects of bicuculline, the classical gamma-aminobutyric acid-A receptor antagonist, on heteromeric mouse muscle alphabetagammadelta, heteromeric neuronal rat alpha2beta4 and alpha4beta2 and homomeric human alpha7 nicotinic acetylcholine receptors (nAChRs), expressed in Xenopus oocytes. Bicuculline reduced the ACh-induced currents in a rapid and reversible way, with IC50 values of 34+/-1.5 microM for mouse muscle alphabetagammadelta and 12.4+/-0.7 and 18+/-1 microM for rat neuronal alpha2beta4 and alpha4beta2 nAChRs, respectively. Therefore, the three types of heteromeric receptors are inhibited by bicuculline but the neuronal alpha2beta4 and alpha4beta2 receptors were more sensitive than the muscle alphabetagammadelta receptor. The Hill coefficients for ACh-current inhibition were close to one for all types of receptors, suggesting a single site of action for bicuculline inhibition of nAChRs. Bicuculline shifted the ACh-dose-current response curve to the right and the maximal current was reduced, a reduction that for the heteromeric receptors was not overcome by high concentrations of ACh. The effect of bicuculline was examined at different membrane potentials, and the ACh-current-membrane potential relationships obtained indicate that the inhibition by bicuculline is voltage-dependent for muscle alphabetagammadelta and neuronal alpha2beta4 and alpha4beta2 nAChRs. All these results are consistent with the notion that bicuculline blocks the heteromeric muscle and neuronal nAChRs in a non-competitive way. Studies were also made on the wild type (wt alpha7) and mutant leu-to-threo (L248T) homomeric human neuronal alpha7-nAChRs. In sharp contrast to the heteromeric ACh receptors examined, bicuculline blocked in a competitive way the homomeric wt alpha7-nAChRs, as evidenced by a parallel shift of the bicuculline dose-ACh-current inhibition on raising the ACh concentration. Moreover, similar to the effects of serotonin on wt and mutant alpha7 ACh receptors, the mutation converted bicuculline from an antagonist into a competitive agonist. All this suggests that bicuculline may serve as a lead molecule to design new anticholinergic substances.

Functional and pharmacological properties of GABArho1delta51 receptors.

Gamma-aminobutyrate is the main inhibitory neurotransmitter in the vertebrate brain, and the gamma-aminobutyric acid (GABA) receptor subunit GABArho1delta51 is an alternatively spliced form of the GABArho1 receptor that was recently isolated from human retina cDNA libraries. The rho1delta51 receptor subunit lacks 17 amino acids in the extracellular N-terminal domain and, when expressed in Xenopus oocytes, forms functional homomeric GABA receptors. Unexpectedly, even after a such a big deletion, the fundamental properties of the deleted variant receptors are very similar to those of the complete GABArho1 receptors. For example, both types of receptors are bicuculline resistant, desensitize very little, and are negatively modulated by Zn2+ and positively modulated by La3+. In spite of such similarities, the GABArho1delta51 receptors are more sensitive to GABA, to the specific GABA(C) antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid and to Zn2+, than the complete GABArho1 receptors. The GABArho1delta51 receptors extend the variety of inhibitory receptors in the retina. Their functional significance still remains to be determined.

GABArho 1/GABAAalpha 1 receptor chimeras to study receptor desensitization.

gamma-Aminobutyrate type C (GABA(C)) receptors are ligand-gated ion channels that are expressed preponderantly in the vertebrate retina and are characterized, among other things, by a very low rate of desensitization and resistance to the specific GABA(A) antagonist bicuculline. To examine which structural elements determine the nondesensitizing character of the human homomeric rho1 receptor, we used a combination of gene chimeras and electrophysiology of receptors expressed in Xenopus oocytes. Two chimeric genes were constructed, made up of portions of the rho1-subunit and of the alpha1-subunit of the GABA(A) receptor. When expressed in Xenopus oocytes, one chimeric gene (rho1/alpha1) formed functional homooligomeric receptors that were fully resistant to bicuculline and were blocked by the specific GABA(C) antagonist (1,2,5, 6-tetrahydropyridine-4-yl)methylphosphinic acid and by zinc. Moreover, these chimeric receptors had a fast-desensitizing component, even faster than that of heterooligomeric GABA(A) receptors, in striking contrast to the almost nil desensitization of wild-type rho1 (wt rho1) receptors. To see whether the fast-desensitizing characteristic of the chimera was determined by the amino acids forming the ion channels, we replaced the second transmembrane segment (TM2) of rho1 by that of the alpha1-subunit of GABA(A). Although the alpha1-subunit forms fast-desensitizing receptors when coexpressed with other GABA(A) subunits, the sole transfer of the alpha1TM2 segment to rho1 was not sufficient to form desensitizing receptors. All this suggests that the slow-desensitizing trait of rho1 receptors is determined by a combination of several interacting domains along the molecule.

Gamma-Hydroxybutyrate inhibits excitatory postsynaptic potentials in rat hippocampal slices.

Gamma-Hydroxybutyrate (GHB) has been shown to mimic different central actions of ethanol, to suppress alcohol withdrawal syndrome, and to reduce alcohol consumption both in rats and in humans. The aim of the present study was to determine if GHB shared with alcohol the ability to inhibit glutamate action at both NMDA and AMPA/kainate receptors. The NMDA or the AMPA/kainate receptors-mediated postsynaptic potentials were evoked in CA1 pyramidal neurons by stimulation of Schaffer-collateral commissural fibers in the presence of CGP 35348, bicuculline to block the GABA(B) and GABA(A) receptors, and 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX) or 30 microM DL-2-amino-5-phosphonovalerate (d-APV) to block AMPA/kainate or NMDA receptors, respectively. GHB (600 microM) produced a depression of both NMDA and AMPA/kainate receptors-mediated excitatory postsynaptic potentials with recovery on washout. The GHB receptors antagonist, NCS-382, at the concentration of 500 microM had no effect per se on these responses but prevented the depressant effect of GHB (600 microM) on the NMDA and AMPA/kainate-mediated responses. In the paired-pulse experiments, GHB (600 microM) depressed the amplitude of the first and the second evoked AMPA/kainate excitatory postsynaptic potentials, and significantly increased the paired-pulse facilitation (PPF). These results suggest that GHB inhibits excitatory synaptic transmission at Schaffer-collateral commissural-pyramidal neurons synapses by decreasing the probability of release of glutamate.

Antagonistic action of pitrazepin on human and rat GABA(A) receptors.

Pitrazepin, 3-(piperazinyl-1)-9H-dibenz(c,f) triazolo(4,5-a)azepin is a piperazine antagonist of GABA in a variety of electrophysiological and in vitro binding studies involving GABA and glycine receptors. In the present study we have investigated the effects of pitrazepin, and the GABA(A) antagonist bicuculline, on membrane currents elicited by GABA in Xenopus oocytes injected with rat cerebral cortex mRNA or cDNAs encoding alpha1beta2 or alpha1beta2gamma2s human GABA(A) receptor subunits. The three types of GABA(A) receptors expressed were reversibly antagonized by bicuculline and pitrazepin in a concentration-dependent manner. GABA dose-current response curves for the three types of receptors were shifted to the right, in a parallel manner, by increasing concentrations of pitrazepin. Schild analyses gave pA2 values of 6.42+/-0.62, n = 4, 6.41+/-1.2, n = 5 and 6.21+/-1.24, n = 6, in oocytes expressing rat cerebral cortex, alpha1beta2 or alpha1beta2gamma2s human GABA(A) receptors respectively (values are given as means +/- s.e. mean), and the Hill coefficients were all close to unity. All this is consistent with the notion that pitrazepin acts as a competitive antagonist of these GABA(A) receptors; and that their antagonism by pitrazepin is not strongly dependent on the subunit composition of the receptors here studied. Since pitrazepin has been reported to act also at the benzodiazepine binding site, we studied the effect of the benzodiazepine antagonist Ro 15-1788 (flumazenil) on the inhibition of alpha1beta2gamma2s receptors by pitrazepin. Co-application of Ro 15-1788 did not alter the inhibiting effect of pitrazepin. Moreover, pitrazepin did not antagonize the potentiation of GABA-currents by flunitrazepam. All this suggests that pitrazepin does not affect the GABA receptor-chloride channel by interacting with the benzodiazepine receptor site.

HPLC resolution of C5 chiral 4,5-dihydro-1,4-benzodiazepines: stereochemical characterization and enantioselective GABAA receptor binding.

HPLC resolution on chiral stationary phases has been successfully employed to obtain single enantiomers of C5 chiral 4,5-dihydro-1,4-benzodiazepines and to determine the enantiomeric composition of the collected stereoisomeric fractions. The absolute configuration of the prevailing enantiomer has been assigned on the basis of the circular dichroism spectra, as compared with that of the structural analogue (5R)- and (5S)-dihydrodiazepam. The single enantiomers, assayed for their binding to the central nervous system receptor, showed relatively low affinity but significant differences in displacing radioactively labelled flunitrazepam from specific benzodiazepine site. GABA shift experiments allowed the classification of these benzodiazepines as partial agonist or antagonist.

Allosteric modulation by single enantiomers of a C3-chiral 1,4-benzodiazepine of the gamma aminobutyric acid type A receptor channel expressed in Xenopus oocytes.

Xenopus laevis oocytes injected with Poly(A)(+)-RNA isolated from neuronal tissue express membrane proteins peculiar to the origin of mRNA. The translation of gamma aminobutyric acid type A (GABAA) receptors has been shown by dose/ response behavior of GABA and the reversible blockade of the GABA-induced current by picrotoxin. This current was analyzed quantitatively under two electrode voltage-clamp conditions. This methodology has been applied for the first time to study the functional properties of the receptor as a function of the stereochemistry of the ligands. The (+)-S and (-)-R enantiomers of a water-soluble benzodiazepine derivative, 7-chloro-1,3-dihydro-3-hemisuccinyloxy-5-phenyl-1,4-benzodiazep in-2-one (OXHEM), obtained by preparative high performance liquid chromatographic (HPLC) resolution on chiral stationary phase, act as agonists in the in vitro modulation of the chloride channel. The (+)-S-OXHEM enantiomer was the more active.

Naloxone blocks long-term depression of excitatory transmission in rat CA1 hippocampus in vitro.

In rat hippocampal slices, high intensity tetanic stimulation (two 1 s trains of 100 Hz separated by 20 s, 3-5X intensity of the test stimulus) of the Schaffer collateral-commissural (SCC) fibers induced a long-term depression (LTD) of the negative field excitatory postsynaptic potentials (fEPSP) in stratum radiatum of the CA1 region. The initial slope of the fEPSP, evoked by a single test shock applied to the SCC fibers, was depressed for a period longer than 40 min following such high intensity tetanic stimulation to this fiber system. However, the same tetanic stimulation delivered at low (test) intensity induced long-term potentiation (LTP) of the fEPSPs. Thus, similar patterns of stimulation can induce either LTP or LTD, depending on whether low- or high-intensity tetanic stimuli are delivered. The LTD induced by high strength tetanic stimulation was clearly blocked by the opioid antagonist naloxone (1 microM); however, the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoate (AP5; 50 microM) had no effect on the LTD. Our data suggest that the strong stimulation used for LTD induction may have activated other afferent fiber systems and/or local interneurons in addition to SCC fibers, such as the enkephalin-containing terminals of the perforant path (PP) projecting to the stratum lacunosum moleculare or opioid peptide-containing interneurons. Thus, the resulting release of endogenous opioid peptides could play a role in the cellular mechanisms involved in some forms of long-term synaptic depression.