Evidence for effective structure-based neuromodulatory effects of new analogues of neurosteroid allopregnanolone.

The neurosteroid allopregnanolone (AP) modulates neuroendocrine/neurobiological processes, including hypothalamic-pituitary-adrenocortical activities, pain, anxiety, neurogenesis and neuroprotection. These observations raised the hope of developing AP-based therapies against neuroendocrine and/or neurodegenerative disorders. However, the pleiotropic actions of AP, particularly its cell-proliferation-promoting effects, hamper the development of selective/targeted therapies. For example, although AP-induced neurogenesis may serve to compensate neuronal loss in degenerative brains, AP-evoked cell-proliferation is contraindicated for steroid-sensitive cancer patients. To foster progress, we synthesised 4 novel AP analogues of neurosteroids (ANS) designated BR053 (12-oxo-epi-AP), BR297 (O-allyl-epi-AP), BR351 (O-allyl-AP) and BR338 (12-oxo-AP). First, because AP is well-known as allosteric modulator of GABAA receptors (GABAA-R), we used the electrophysiological patch-clamp technique to determine the structure-activity relationship of our ANS on GABAA-activated current in NCB20 cells expressing functional GABAA-R. We found that the addition of 12-oxo-group did not significantly change the respective positive or negative allosteric effects of 3α-AP or 3ß-(epi)-AP analogues. Importantly, substitution of the 3α-hydroxyl-group by 3α-O-allyl highly modified the ANS activities. Unlike AP, BR351 induced a long-lasting desensitisation/inhibition of GABAA-R. Interestingly, replacement of the 3ß-hydroxyl by 3ß-O-allyl (BR297) completely reversed the activity from negative to positive allosteric action. In a second step, we compared the actions of AP and ANS on SH-SY5Y neuronal cell viability/proliferation using MTT-reduction assays. Different dose-response curves were demonstrated for AP and the ANS. By contrast to AP, BR297 was totally devoid of cell-proliferative effect. Finally, we compared AP and ANS abilities to protect against oxidative stress-induced neuronal death pivotally involved in neurodegenerative diseases. Both BR351 and BR297 had notable advantages over AP in protecting SH-SY5Y cells against oxidative stress-induced death. Thus, BR297 appears to be a potent neuroprotective compound devoid of cell-proliferative activity. Altogether, our results suggest promising perspectives for the development of neurosteroid-based selective and effective strategies against neuroendocrine and/or neurodegenerative disorders.

Behavioral and electromyographic assessment of oxaliplatin-induced motor dysfunctions: Evidence for a therapeutic effect of allopregnanolone.

The antineoplastic oxaliplatin (OXAL) is pivotal for metastatic cancer treatments. However, OXAL evokes sensory and motor side-effects including pain, muscle weakness, motor nerve fiber dysfunctions/neuropathies that significantly impact patients' lives. Therefore, preclinical investigations are struggling to characterize effective analgesics against OXAL-induced painful/sensory symptoms but surprisingly, OXAL-evoked motor dysfunctions received little attention although these neurological symptoms are also disabling for patients. Here, we validated a rat model of OXAL-induced motor neuropathy by using (i) behavioral methods as the wire suspension and balance beam tests to assess muscle weakness and (ii) electrophysiological techniques to record the gastrocnemius electromyography (EMG). The conductance velocity of motor fibers was reduced and compound muscle action potential (CMAP) duration increased in OXAL-treated rats, leading to CMAP dispersion with no modification of the area under the curve, reflecting a heterogeneous demyelination of motor fibers. Functional motor unit analysis revealed a 50 % decrease of their estimated number which was compensated by a motor unit size increase. OXAL-induced motor weakness appeared as a combined consequence of motor fiber demyelination and motor axonopathy. Because we previously observed that allopregnanolone (AP) counteracted OXAL-evoked painful/sensory symptoms, we evaluated its action against OXAL-induced motor neurological dysfunctions. AP treatment successfully corrected motor behaviors, conductance velocity, CMAP duration, motor unit number (MUN) and motor unit size altered by OXAL-chemotherapy. These results, which are the first to show that AP efficiently rescues OXAL-induced motor neuropathy, consolidate the idea that AP-based therapy may be relevant for the treatment of both sensory and motor peripheral neuropathies.

Chronic inflammatory demyelinating polyradiculoneuropathy: A new animal model for new therapeutic targets.

Animal models are fundamental to advance knowledge of disease pathogenesis and to test/develop new therapeutic strategies. Most of the current knowledge about the pathogenic mechanisms underpinning autoimmune demyelination processes implicating autoantigens has been obtained using the Experimental Autoimmune Neuritis (EAN) animal model. The most widely used EAN model is obtained by active immunization of Lewis rats using a peptide, P0 (180-199), issuing from the major peripheral nervous system myelin protein. But this model mimics only the classical monophasic acute form of demyelinating polyradiculoneuropathy, i.e. Guillain-Barré syndrome (GBS). We developed a new model by immunizing Lewis rats using the same immunodominant neuritogenic peptide P0 (180-199) but this time with its S-palmitoyl derivative, S-palm P0 (180-199). All of the animals immunized with the S-palm P0 (180-199) peptide developed a chronic relapsing-remitting form of the disease corresponding to the electrophysiological criteria of demyelination (slow sensory nerve conduction velocity, prolonged motor nerve latency, partial motor nerve conduction blocks) with axon degeneration. These findings were confirmed by immunohistopathology study and thus, appear to mimic human chronic inflammatory demyelinating polyradiculopathy (CIDP). This new model opens up new avenues of research for testing new anti-inflammatory and neuroprotective therapeutic strategies.

[Put sound, but the sense, the multilinguisms].

Exchanges, travel, intercountry adoption which has become an increasingly common practice, uprooting, etc. have disrupted the compositions of increasingly multilingual socie­ties and families. The development of research in cognitive sciences and the birth of bilingualism as a field of study since the 1970s, together with recent advances in linguistics, psycho­logy, neuroimaging and speech therapy have made it possible to better describe the neuropsychological function and develop­ment of the bilingual person, particularly in the development of the normo and hearing-impaired child, in both language and behavior. It also involves other pathological situations such as stuttering and autism seen in Alzheimer's disease. Bilingualism is now a recognized cultural, social, educational and professio­nal substantial asset. Children who are able to express them­selves in more than one language see their metalinguistic skills increase and show a stronger attachment to their parent's culture of origin.

Potential role of allopregnanolone for a safe and effective therapy of neuropathic pain.

Because the treatment and management of neuropathic pain are extremely complicated, the characterization of novel analgesics and neuroprotectors with safe toxicological profiles is a crucial need to develop efficient therapies. Several investigations revealed that the natural neurosteroid allopregnanolone (AP) exerts analgesic, neuroprotective, antidepressant and anxiolytic effects. These effects result from AP ability to modulate GABA(A), glycine, L- and T-type calcium channels. It has been shown that AP treatment induced beneficial actions in humans and animal models with no toxic side effects. In particular, a multi-parametric analysis revealed that AP efficiently counteracted chemotherapy-evoked neuropathic pain in rats. It has also been demonstrated that the modulation of AP-producing enzyme, 3α-hydroxysteroid oxido-reductase (3α-HSOR), in the spinal cord regulates thermal and mechanical pain thresholds of peripheral nerve injured neuropathic rats. The painful symptoms were exacerbated by intrathecal injections of provera (pharmacological inhibitor of 3α-HSOR) which decreased AP production in the spinal cord. By contrast, the enhancement of AP concentration in the intrathecal space induced analgesia and suppression of neuropathic symptoms. Moreover, in vivo siRNA-knockdown of 3α-HSOR expression in healthy rat dorsal root ganglia increased thermal and mechanical pain perceptions while AP evoked a potent antinociceptive action. In humans, blood levels of AP were inversely associated with low back and chest pain. Furthermore, oral administration of AP analogs induced antinociception. Altogether, these data indicate that AP, which possesses a high therapeutic potential and a good toxicological profile, may be used to develop effective and safe strategies against chronic neuropathic pain.

Calcium and cAMP signaling induced by gamma-hydroxybutyrate receptor(s) stimulation in NCB-20 neurons.

The NCB-20 neurohybridoma cells differentiated with dibutyryl-cyclic-AMP represent an interesting model to study several components of the gamma-hydroxybutyrate (GHB) system in brain. In particular, an active Na(+)-dependent uptake and a depolarization-evoked release of GHB is expressed by these cells, together with high affinity specific binding sites for this substance. However, only little is known about cellular mechanisms following GHB receptor(s) stimulation in these neurons. Electrophysiological data indicate that GHB can differently affect Ca(2+) currents. L-type calcium channels were typically inhibited by GHB when NCB-20 cells were depolarized. In contrast, when NCB-20 cells were at resting potential, GHB induced a specific Ca(2+) entry through T-type calcium channels. In this study, we investigated the effect induced on cytosolic free Ca(2+) level and cAMP production by GHB receptor(s) stimulated with micromolar concentrations of GHB or structural analogues of GHB. Ca(2+) movements studied by cellular imaging were dose-dependently increased but disappeared for GHB concentrations >25 microM. In addition, nanomolar doses of GHB inhibited forskolin-stimulated adenylate cyclase. This effect was also rapidly desensitized at higher GHB concentrations. Acting as an antagonist, NCS-382 decreased GHB receptor(s) mediated cAMP and calcium signals. The agonist NCS-356 mimicked GHB effects which were not affected by the GABA(B) receptor antagonist CGP-55-845. Our results reveal the occurrence of Ca(2+)-dependent adenylate cyclase inhibition in NCB-20 neurons after GHB receptor(s) stimulation by GHB concentrations <50 microM. Above this dose, GHB effects were inactivated. In addition, at GHB concentrations exceeding 50 microM, GTP-gammaS binding was also reduced, confirming the desensitization of GHB receptor(s). Taken together, these results support the existence in NCB-20 neurons of GHB receptors belonging to GPCR family that may recruit various G protein subtypes.

Gamma-hydroxybutyrate receptor function determined by stimulation of rubidium and calcium movements from NCB-20 neurons.

Gamma-Hydroxybutyrate is derived from GABA in brain and plays specific functional roles in the CNS. It is thought to exert a tonic inhibitory control on dopamine and GABA release in certain brain areas, through specific gamma-hydroxybutyrate receptors. Apart from modifying certain calcium currents, the specific transduction mechanism induced by stimulation of gamma-hydroxybutyrate receptors remains largely unknown. We investigated the possible contribution of K(+) channels to the hyperpolarization phenomena generally induced by gamma-hydroxybutyrate in brain, by monitoring (86)Rb(+) movements in a neuronal cell line (NCB-20 cells), which expresses gamma-hydroxybutyrate receptors. Physiological concentrations of gamma-hydroxybutyrate (5-25 microM) induce a slow efflux of (86)Rb(+), which peaks at 5-15 min and returns to baseline levels 20 min later after constant stimulation. This effect can be reproduced by the gamma-hydroxybutyrate receptor agonist NCS-356 and blocked by the gamma-hydroxybutyrate receptor antagonist 6,7,8,9-tetrahydro-5-[H]-benzocycloheptene-5-ol-4-ylidene. The GABA(B) receptor antagonist CGP 55845 has no effect on gamma-hydroxybutyrate-induced (86)Rb(+) efflux. The pharmacology of this gamma-hydroxybutyrate-dependent efflux of (86)Rb(+) is in favor of the involvement of tetraethylammonium and charybdotoxin insensitive, apamin sensitive Ca(2+) activated K(+) channels, identifying them as small conductance calcium activated channels. We demonstrated a gamma-hydroxybutyrate dose-dependent entry of calcium ions into NCB-20 neuroblastoma cells at resting potential. Electrophysiological data showed that this Ca(2+) entry corresponded mainly to a left-hand shift of the current/voltage relation of the T-type calcium channel. This process must at least partially trigger small conductance calcium activated channel activation leading to gamma-hydroxybutyrate-induced hyperpolarization.

Homocystinuria in the Arab population of Israel: identification of two novel mutations using DGGE analysis.

This study describes, for the first time, a thorough genetic investigation in Israeli Arab homocystinuric patients. By using a DGGE methodology and sequencing we were able to identify the disease causing mutation in all. Of the mutations that were detected, two are novel: a 785C>G transversion in exon 7 (T262R) and a 5-bp deletion in the 5' of IVS17 including the T in the +2 position that is crucial for correct splicing (g18327-18331del5). In spite of the highly consanguineous nature of this population several different mutations were found. This may suggest that the mutations arose only recently in the population. The results of our study would enable early prenatal diagnosis, genetic counseling and screening for the mutations in population at risk. Hum Mutat 16:372, 2000.

Mutation at codon 210 (V210I) of the prion protein gene in a North African patient with Creutzfeldt-Jakob disease.

A point mutation at codon 210 of the prion protein gene (PRNP), resulting in the substitution of isoleucine for valine (V210I) has been found in a 54-year-old Moroccan patient affected with Creutzfeldt-Jakob disease (CJD). This patient is the first carrier of the PRNP V210I mutation reported from North Africa. The clinical presentation of the patient was rather similar to that seen in classical CJD, except that unusual early sensory symptoms were observed. The mother of the proband, aged 72, is a further example of an asymptomatic elderly carrier of the PRNP V210I mutation, suggesting an incomplete penetrance of the disease.

Recurrent hyperbilirubinaemia, a feature of familial Mediterranean fever: report of a child and review of the literature.

Recurrent hyperbilirubinaemia was described as a feature of familial Mediterranean fever in the 1950s and early 1960s. However, over the last 33 years only one case has been published. We present a 12-year-old Arab boy who developed recurrent hyperbilirubinaemia in the course of familial Mediterranean fever. His response to colchicine was excellent. Review of the literature reveals that hyperbilirubinaemia of familial Mediterranean fever has a distinct clinical picture characterized by concurrent peritonitis, minimal jaundice and short duration. Factors contributing to the paucity of reports in recent literature are discussed.

Neurochemical and electrophysiological evidence for the existence of a functional gamma-hydroxybutyrate system in NCB-20 neurons.

Clonal neurohybridoma NCB-20 cells express a valproate-insensitive succinic semialdehyde reductase activity that transforms succinic semialdehyde into gamma-hydroxybutyrate. This activity (1.14+/-0.16 nmol/min/mg protein) was similar to the lowest activity existing in adult rat brain. [3H]gamma-Hydroxybutyrate labels a homogeneous population of sites on NCB-20 cell membranes (Kd=250+/-44.4nM, Bmax=180+/-16.2fmol/mg protein) that apparently represents specific gamma-hydroxybutyrate binding sites characterized previously on brain cell membranes. Finally, an Na+-dependent uptake of [3H]gamma-hydroxybutyrate was expressed in NCB-20 cells with a Km of 35+21.1 microM and a Vmax of 80+/-14.2 pmol/min/mg protein. A three-day treatment with 1 mM dibutyryl-cyclic-AMP induced a three-fold increase in the cellular succinic semialdehyde reductase activity. In parallel, a K+-evoked release of [3H]gamma-hydroxybutyrate occurred. This release was Ca2+ dependent and was not present in undifferentiated cells. Cyclic-AMP treatment induced a decrease of [3H]gamma-hydroxybutyrate binding sites, which could be due to spontaneous gamma-hydroxybutyrate release. Patch-clamp experiments carried out on differentiated NCB-20 cells revealed the presence of Ca2+ conductances which were partially inhibited by 50 microM gamma-hydroxybutyrate. This gamma-hydroxybutyrate-induced effect was blocked by the gamma-hydroxybutyrate receptor antagonist NCS-382, but not by the GABA(B) antagonist CGP-55845. These results demonstrate the presence of an active gamma-hydroxybutyratergic system in NCB-20 cells which possesses the ability to release gamma-hydroxybutyrate. These cells express specific gamma-hydroxybutyrate receptors which modulate Ca2+ currents independently of GABA(B) receptors.

Permeation and gating of alpha1 glycine-gated channels expressed at low and high density in Xenopus oocyte.

When a high density of alpha1-subunit glycine receptor (GlyR) is expressed in Xenopus oocytes, two populations of channels can be distinguished according to their apparent affinity for glycine which differs 5- to 6-fold. To compare the open pore diameter of these channels, the relative permeability of formate with respect to chloride (P(formate)/P(Cl)) was determined in bionic conditions. For the low-affinity GlyR P(formate)/P(Cl) was comparable to that reported for glycine-gated channels in cultured spinal cord and hippocampal neurons. In contrast, the high-affinity GlyR had a 56% larger P(formate)/P(Cl). In addition, the open probability of the channels was differentially sensitive to voltage. These results show that the high expression of alpha1 GlyR resulted in two populations of GlyR which differed not only in the affinity to agonists but also in permeation and gating mechanisms.

Expression of the human glycine receptor alpha 1 subunit in Xenopus oocytes: apparent affinities of agonists increase at high receptor density.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel, which mediates post-synaptic inhibition in spinal cord and other brain regions. Heterologous expression of the ligand binding alpha subunits of the GlyR generates functional agonist-gated chloride channels that mimic most of the pharmacological properties of the receptor in vivo. Here, nuclear injection into Xenopus oocytes of a human alpha 1 subunit cDNA, engineered for efficient expression, was used to create GlyR channels over a wide density range, resulting in whole-cell glycine currents of 10 nA to 25 microA. Notably, the pharmacology of these channels changed at high expression levels, with the appearance of a novel receptor subpopulation of 5- to 6-fold higher apparent agonist affinity at current values > 4 microA. The low-affinity receptors were readily blocked by nM concentrations of the competitive antagonist strychnine, whereas the high-affinity receptors were more resistant to antagonism by this alkaloid. Picrotoxinin, a chloride channel blocker, inhibited both GlyR populations with equal potency. Our data suggest that receptor interactions, occurring at high receptor density, modify the agonist response of the GlyR. This phenomenon may contribute to neurotransmitter efficacy at fast synapses.

Cloning and expression of the 58 kd beta subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) mediates post-synaptic inhibition in spinal cord and other regions of the CNS. Purified mammalian GlyR contains two membrane-spanning subunits 48 kd (alpha) and 58 kd (beta) plus a 93 kd receptor-associated cytoplasmic protein. Here, the primary structure of the beta subunit was deduced from cDNAs isolated from rat spinal cord and brain cDNA libraries. The predicted amino acid sequence exhibits 47% identity to the previously characterized rat alpha 1 polypeptide. Northern blot analysis revealed high levels of beta subunit transcripts in postnatal spinal cord, cerebellum, and cortex. Nuclear injection into Xenopus oocytes of a beta subunit cDNA engineered for efficient expression generated weak glycine-activated chloride currents that were insensitive to the classic GlyR antagonist, strychnine. Our data indicate a differential expression of GlyR alpha and beta subunits in the rat nervous system and support a structural role of the beta polypeptide in the native receptor complex.

Structure and functional expression of cloned rat serotonin 5HT-2 receptor.

A complementary DNA (cDNA) encoding a serotonin receptor with 51% sequence identity to the 5HT-1C subtype was isolated from a rat brain cDNA library by homology screening. Transient expression of the cloned cDNA in mammalian cells was used to establish the pharmacological profile of the encoded receptor polypeptide. Membranes from transfected cells showed high-affinity binding of the serotonin antagonists spiperone, ketanserin and mianserin, low affinity for haloperidol (a dopamine D2 receptor antagonist), 8-OH-DPAT as well as MDL-72222 and no detectable binding of [3H]serotonin. This profile is consonant with the 5HT-2 subtype of serotonin receptors. In agreement with this assignment, serotonin increased the intracellular Ca2+ concentration and activated phosphoinositide hydrolysis in transfected mammalian cells. The agonist also elicited a current flow, blocked by spiperone, in Xenopus oocytes injected with in vitro synthesized RNA containing the cloned nucleotide sequences.

Small-conductance chloride channels activated by calcium on cultured endocrine cells from mammalian pars intermedia.

Porcine intermediate lobe (IL) endocrine cells maintained in primary culture have been studied using patch-clamp derived configurations to record unitary activity on outside-out vesicles. Solutions were devised so as to record Cl current in isolation and to fix cytoplasmic Ca concentration [Ca]i between 0.1 microM and 3 microM. Between [Ca]i 0.5 and 1 microM, the chloride permeability was restricted to single events with a small amplitude, that varied linearly with the membrane potential. Mean slope conductance of this chloride channel was 2.5 pS. Single channel analysis yielded two mean open time values of 10 and 55 ms at -80 mV. Relaxations of chloride currents on outside-out patches was examined at different [Ca]i. Relaxation was negligible at 0.15 microM [Ca]i, whereas at higher [Ca]i, the current exhibited relaxation in response to voltage jumps the kinetic of which could be fitted by two exponentials. At 0.5 microM [Ca]i, the fast relaxation time constant was shown to be voltage insensitive with a value of about 10 ms. The slow relaxation time constant had a mean value of 75 ms at -60 mV and increased with membrane depolarization with a twofold change over 120 mV. Another voltage effect was to favour the slow opening mode at the more depolarized potentials: the ratio of fast to slow relaxations being 5:1 at -60 mV as compared to 1:1 at +80 mV). Finally the estimated probability of opening (po) linearly increased with voltage. po displayed a bell-shaped dependence on [Ca]i, so that full activation of the channels was not achieved.

Spontaneous and GABA-evoked chloride channels on pituitary intermediate lobe cells and their internal Ca requirements.

On porcine intermediate lobe (IL) endocrine cells, spontaneously opening chloride channels have been studied and compared to GABA-A activated chloride channels. Elementary currents were recorded mainly from outside-out patches excised from IL cells maintained in culture for 1-4 weeks. Spontaneous inward currents were observed in Cs-loaded cells after replacing Na in the extracellular medium by the impermeant ion choline. This activity, at an internal calcium concentration of 10(-8) M corresponded to a channel for chloride ions with a main conductance level of 26 pS, and substates around 11 pS. The sequence of permeabilities to halides was I greater than Br greater than Cl. These conductance characteristics were common to the GABA-operated channels which also showed a main conductance substate of 23-31 pS. The open time of the 26 pS level mostly encountered in spontaneous activity, was distributed along two modes: one, the most frequent, around 1 ms, and the other around 4 ms. This latter mode was the predominant one observed during GABA and isoguvacine applications but in addition a bursting activity of 19 ms duration was also seen. Specific GABA-A receptor antagonists (bicuculline and SR42641, 1 microM) blocked activity evoked by GABA (1-10 microM), but did not affect spontaneous events. These spontaneous Cl events were only observed in a restricted range of internal Ca concentrations, i.e. between 1 nM and 0.1 microM, and were practically abolished at Cai 1 microM. The GABA-induced activity of Cl channels was also Ca-sensitive, being reduced when Cai reached 1 microM.

Intracellular effectors and modulators of GABA-A and GABA-B receptors: a commentary.

The inhibitory neurotransmitter GABA activates two receptor subtypes that can be distinguished by their pharmacology. The GABA-A site is competitively antagonized by bicuculline and exclusively coupled to a chloride channel. The GABA-B receptor, for which baclofen is the only specific agonist, is resistant to bicuculline inhibition and, depending upon its localization, will activate K currents and/or inhibit Ca currents. Both electrophysiological and biochemical approaches have been applied to the study of each receptor. The membrane and intracellular components that to date have been implicated in GABA-B activation are discussed: G proteins, adenylate cyclase and intracellular calcium levels. This latter factor is also discussed with respect to GABA-A receptor action.