Effect of neurosteroids on a model lipid bilayer including cholesterol: An Atomic Force Microscopy study.

Amphiphilic molecules which have a biological effect on specific membrane proteins, could also affect lipid bilayer properties possibly resulting in a modulation of the overall membrane behavior. In light of this consideration, it is important to study the possible effects of amphiphilic molecule of pharmacological interest on model systems which recapitulate some of the main properties of the biological plasma membranes. In this work we studied the effect of a neurosteroid, Allopregnanolone (3α,5α-tetrahydroprogesterone or Allo), on a model bilayer composed by the ternary lipid mixture DOPC/bSM/chol. We chose ternary mixtures which present, at room temperature, a phase coexistence of liquid ordered (Lo) and liquid disordered (Ld) domains and which reside near to a critical point. We found that Allo, which is able to strongly partition in the lipid bilayer, induces a marked increase in the bilayer area and modifies the relative proportion of the two phases favoring the Ld phase. We also found that the neurosteroid shifts the miscibility temperature to higher values in a way similarly to what happens when the cholesterol concentration is decreased. Interestingly, an isoform of Allo, isoAllopregnanolone (3ß,5α-tetrahydroprogesterone or isoAllo), known to inhibit the effects of Allo on GABAA receptors, has an opposite effect on the bilayer properties.

Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study.

Amphiphilic molecules supposed to affect membrane protein activity could strongly interact also with the lipid component of the membrane itself. Neurosteroids are amphiphilic molecules that bind to plasma membrane receptors of cells in the central nervous system but their effect on membrane is still under debate. For this reason it is interesting to investigate their effects on pure lipid bilayers as model systems. Using the micropipette aspiration technique (MAT), here we studied the effects of a neurosteroid, allopregnanolone (3α,5α-tetrahydroprogesterone or Allo) and of one of its isoforms, isoallopregnanolone (3ß,5α-tetrahydroprogesterone or isoAllo), on the physical properties of pure lipid bilayers composed by DOPC/bSM/chol. Allo is a well-known positive allosteric modulator of GABAA receptor activity while isoAllo acts as a non-competitive functional antagonist of Allo modulation. We found that Allo, when applied at nanomolar concentrations (50-200 nM) to a lipid bilayer model system including cholesterol, induces an increase of the lipid bilayer area and a decrease of the mechanical parameters. Conversely, isoAllo, decreases the lipid bilayer area and, when applied, at the same nanomolar concentrations, it does not affect significantly its mechanical parameters. We characterized the kinetics of Allo uptake by the lipid bilayer and we also discussed its aspects in relation to the slow kinetics of Allo gating effects on GABAA receptors. The overall results presented here show that a correlation exists between the modulation of Allo and isoAllo of GABAA receptor activity and their effects on a lipid bilayer model system containing cholesterol.

Design, stereoselective synthesis, configurational stability and biological activity of 7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide.

Chiral 5-arylbenzothiadiazine derivatives have recently attracted particular attention because they exhibit an interesting pharmacological activity as AMPA receptor (AMPAr) positive modulators. However, investigations on their configurational stability suggest a rapid enantiomerization in physiological conditions. In order to enhance configurational stability, preserving AMPAr activity, we have designed the novel compound (R,S)-7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide bearing a pyrrolo moiety coupled with the 5-(furan-3-yl) substituent on benzothiadiazine core. A stereoselective synthesis was projected to obtain single enantiomer of the latter compound. Absolute configuration was assigned by X-ray crystal structure. Patch clamp experiments evaluating the activity of single enantiomers as AMPAr positive allosteric modulator showed that R stereoisomer is the active component. Molecular modeling studies were performed to explain biological results. An on-column stopped-flow bidimensional recycling HPLC procedure was applied to obtain on a large scale the active enantiomer with enantiomeric enrichment starting from the racemic mixture of the compound.

Molecular modeling studies, synthesis, configurational stability and biological activity of 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide.

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPArs) has led to a search for new AMPAr positive modulators. Among them, 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide (1) has attracted particular attention, because it is one of the most active benzothiadiazine-derived positive modulators of the AMPA receptor. It possesses two stereogenic centers, C3 and C6, thus it can exist as four stereoisomers. In this work, preliminary in silico studies suggested that 1 interacts stereoselectively with AMPArs. Single stereoisomers of 1 were prepared in order to evaluate their biological activity. However, studies regarding the configurational stability of the investigated compounds suggested a rapid epimerization at C3 in aqueous solvents, and we can expect the same reaction in vivo. Thus, electrophysiological experiments were performed on the two epimeric mixtures, (3∗,6R)- and (3∗,6S)- 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide, in order to evaluate their activities as positive allosteric modulators of AMPArs. The obtained data suggest that the (3∗,6S) epimeric mixture is the most active in positively modulating AMPArs, confirming in silico results.

Thyroid hormones reduce nicotinic receptor mediated currents in SH-SY5Y neuroblastoma cells.

BACKGROUND: Thyroid hormones (THs) are crucial for maturation and functioning of mammalian CNS. THs "classical" signaling involves nuclear receptors binding but also their non genomic actions, as rapid modulators of cell activity, are widely recognized. Since THs imbalance affects cognition and the cholinergic system is deeply involved in learning and memory processes we have studied THs effects at the level of the nicotinic acetylcholine receptors (nAchR). METHODS: We used the patch-clamp technique to analyze T3 and T4 modulation of nicotine (NIC)-mediated current in SH-SY5Y neuroblastoma cells. RESULTS: Both hormones decreased NIC-evoked current in a dose dependent fashion. The antagonism was reversible, not competitive and not blocked by Tetrac, an integrin αVß3 receptor antagonist. A similar effect was detected with the endogenous agonist Acetylcholine. THs potencies were higher at 100 µM NIC (IC50 = 4.6 ± 2 µM for T3 and 4.8 ± 2 µM for T4) compared to those measured at 10 µM NIC (IC50 = 10 ± 4 µM for T3 and 8 ± 4 µM for T4). Furthermore, the efficacy of THs reached almost 90% at 100 µM NIC while was about 30 % at 10 µM NIC. THs inhibited nAchR-mediated currents by enhancing receptor desensitization and this effect was more pronounced at high agonist concentrations. CONCLUSIONS: Our results make light on a new non genomic activity of THs at the level of nAchR. This mechanism of action  of THs  can provide  a new explanation for the cognitive deficits associated with tyroid dysfunction.

A novel class of allosteric modulators of AMPA/Kainate receptors.

The rapid hydrolysis in vivo of IDRA21 to 2-amino-5-chlorobenzensulfonamide has been demonstrated by microdialysis experiments. The IDRA21 metabolite possess in vitro a biological activity similar to that of IDRA21 itself. Taking 2-amino-5-chlorobenzensulfonamide as lead compound, a novel class of AMPAR positive allosteric modulators has been prepared.

Functional in vitro characterization of CR 3394: a novel voltage dependent N-methyl-D-aspartate (NMDA) receptor antagonist.

Using the patch-clamp technique, we studied the effect of two novel adamantane derivatives, N-[2-(3,5-dimethyl-1-adamantyl)ethyl] guanidine (CR 3391) and N-[2-(3,5-dimethyl-1-adamantyl) ethyl]acetamidine (CR 3394), on NMDA receptors expressed in cortical neuron cultures. Our data show that CR 3391 and CR 3394 reduce NMDA-evoked currents (IC50 = 1.7 +/- 0.6 microM and 6.7 +/- 1.5 microM, respectively). This antagonism is non-competitive and is completely reversible. The effect of CR 3394, like that of memantine, was strongly voltage dependent. HEK293 cells expressing NR1a/NR2B recombinant NMDA receptors and immature neurons (DIV 8-9) were more sensitive to CR 3394 antagonism than NR1a/NR2A expressing cells and DIV 15 neurons. CR 3394 also reduced the duration and amplitude of miniature excitatory post-synaptic currents mediated exclusively by NMDA receptors (NMDA-mEPSCs). Both memantine and CR 3394 inhibited NMDA-evoked [3H]norepinephrine release from rat hippocampal slices in a concentration-dependent manner with similar potency. CR 3394, but not memantine, increased cathecholamine resting release at low micromolar concentrations. Moreover, in an in vitro model of neurotoxicity, CR 3394 strongly reduced glutamate- and NMDA-induced neuronal death. Taken together, our data highlight pharmacological features of CR 3394 in vitro that prompt us to further evaluate it as a candidate for the treatment of neurodegenerative disorders.

Positive allosteric modulators of α7 nicotinic acetylcholine receptors affect neither the function of other ligand- and voltage-gated ion channels and acetylcholinesterase, nor ß-amyloid content.

The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and ß-amyloid (Aß) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50=26±6µM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2-4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aß42 in an Alzheimer's disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets.

PKCε and allopregnanolone: functional cross-talk at the GABAA receptor level.

Changes in GABAergic inhibition occur during physiological processes, during response to drugs and in various pathologies. These changes can be achieved through direct allosteric modifications at the γ-amino butyric acid (GABA) type A (GABAA) receptor protein level, or by altering the synthesis, trafficking and stability of the receptor. Neurosteroids (NSs) and protein kinase C (PKC) are potent modulators of GABAA receptors and their effects are presumably intermingled, even though evidence for this hypothesis is only partially explored. However, several PKC isoforms are able to phosphorylate the GABAA receptor, producing different functional effects. We focused on the ε isoform, that has been correlated to the sensitivity of the GABAA receptor to allosteric modulators and whose expression may be regulated in peripheral sensory neurons by NSs. The cross-talk between PKC-ε and NSs, leading to changes in GABAA receptor functionality, is considered and discussed in this perspective.

IDRA-21, a positive AMPA receptor modulator, inhibits synaptic and extrasynaptic NMDA receptor mediated events in cultured cerebellar granule cells.

IDRA-21 (7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide) reduces alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptors desensitisation in vitro and restores learning and cognitive impairment in vivo. In this study, we show that in cerebellar granule cells (CGCs) in culture IDRA-21 reduces N-methyl-d-aspartate receptor (NMDAR) whole-cell currents. The effect is neither competitive nor voltage-dependent. The reduction of NMDA currents is stronger at low glycine concentrations suggesting an interaction with this site. IDRA-21 shortens miniature excitatory postsynaptic currents mediated by NMDARs (NMDA-mEPSCs) in CGCs grown in low potassium with no effect on peak amplitudes. By using fast glutamate application onto CGCs nucleated patches, we found that IDRA-21 decreases both decay time constant and amplitude of the current. Experiments performed on recombinant NMDAR expressed in HEK 293 cells showed that IDRA-21 was more effective on NR1a-NR2B than NR1a-NR2A receptors highlighting a subunit selectivity of the drug. Our findings make light on a novel target for IDRA-21: NMDA receptors function is negatively modulated and the different action at the level of extrasynaptic and synaptic receptors could be ascribed to a partial selectivity for NR2B subunits.

Synthesis of 3,4-dihydro-2H-1,2,4-benzo-thiadiazine 1,1-dioxide derivatives as potential allosteric modulators of AMPA/kainate receptors.

A series of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide derivatives were synthesized and evaluated for their activity as allosteric modulators of kainate-activated currents in primary cultures of cerebellar granule neurons. Substitution of different groups at the 3-position of the benzothiadiazine ring distinguished between positive and negative allosteric modulatory properties.

Apigenin modulates GABAergic and glutamatergic transmission in cultured cortical neurons.

Using the patch-clamp technique, we studied the modulation of ionotropic gamma-aminobutyric acid (GABA) and glutamate neurotransmission by apigenin, a flavonoid with sedative and antidepressant activity. Apigenin reversibly reduced GABA-evoked currents mediated by alpha1beta2gamma2 receptors expressed in HEK293 cells. Amplitude and frequency of spontaneous postsynaptic inhibitory currents (sIPSCs) mediated by GABA(A) receptors were also decreased by apigenin in cultured cortical neurons. The flavonoid was almost inactive on alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) mediated currents while it reduced N-methyl-D-aspartate (NMDA) receptor mediated responses with a half maximal inhibiting concentration (IC50) of 10 microM. The flavonoid inhibited also peak amplitude and frequency of spontaneous postsynaptic excitatory currents (sEPSCs). Finally, apigenin is neuroprotective against glutamate-induced neurotoxicity in cerebellar and cortical neurons in culture. Our data reveal the antagonistic effect of apigenin on GABA and NMDA channels. While the inhibition on GABA receptor cannot explain the effects of the drug in vivo our data on NMDA channels reveal a new target of apigenin. A reduction of the network excitability could thus account for the sedative effects. Furthermore, our data suggest a potential neuroprotective activity of apigenin.

Simultaneous determination of pregnenolone sulphate, dehydroepiandrosterone and allopregnanolone in rat brain areas by liquid chromatography-electrospray tandem mass spectrometry.

Neurosteroids (NSs) are well known modulators of neuronal activity and by binding to different neuronal receptors are responsible for a broad spectrum of biological and pathophysiological conditions. Here, a sensitive liquid chromatographic-electrospray ionization-tandem mass spectrometric method (LC-ESI-MS/MS) has been developed and validated for the simultaneous determination in rat brain areas of three NSs, i.e. pregnenolone sulphate (PS), dehydroepiandrosterone (DHEA) and allopregnanolone (AP). NSs were extracted with methanol-formic acid, purified by Hybrid-SPE cartridges and subjected to LC-ESI-MS/MS without any preliminary derivatization or deconjugation procedure. Quantitation was performed by multiple reaction monitoring mode with the internal standard method, using deuterium-labelled analogues of the analyzed NSs. The proposed method provided for the first time a direct quantitative determination of PS without hydrolysis; in particular, PS concentrations were found significantly (p<0.01) higher in hippocampus, the brain area associated primarily with memory, than in cortical tissue of control rats, suggesting the important role of this NS in the process of memory formation. The developed method could be successfully applied to quantify simultaneously PS, DHEA and AP levels in brain tissue in order to study their changes during various neurodegenerative diseases and to investigate the role of PS in the brain.

Novel modulatory effects of neurosteroids and benzodiazepines on excitatory and inhibitory neurons excitability: a multi-electrode array recording study.

The balance between glutamate- and GABA-mediated neurotransmission in the brain is fundamental in the nervous system, but it is regulated by the "tonic" release of a variety of endogenous factors. One such important group of molecules are the neurosteroids (NSs) which, similarly to benzodiazepines (BDZs), enhance GABAergic neurotransmission. The purpose of our work was to investigate, at in vivo physiologically relevant concentrations, the effects of NSs and BDZs as GABA modulators on dissociated neocortical neuron networks grown in long-term culture. We used a multi-electrode array (MEA) recording technique and a novel analysis that was able to both identify the action potentials of engaged excitatory and inhibitory neurons and to detect drug-induced network up-states (burst). We found that the NSs tetrahydrodeoxycorticosterone (THDOC) and allopregnanolone (ALLO) applied at low nanomolar concentrations, produced different modulatory effects on the two neuronal clusters. Conversely, at high concentrations (1 µM), both NSs, decreased excitatory and inhibitory neuron cluster excitability; however, even several hours after wash-out, the excitability of inhibitory neurons continued to be depressed, leading to a network long-term depression (LTD). The BDZs clonazepam (CLZ) and midazolam (MDZ) also decreased the network excitability, but only MDZ caused LTD of inhibitory neuron cluster. To investigate the origin of the LTD after MDZ application, we tested finasteride (FIN), an inhibitor of endogenous NSs synthesis. FIN did not prevent the LTD induced by MDZ, but surprisingly induced it after application of CLZ. The significance and possible mechanisms underlying these LTD effects of NSs and BDZs are discussed. Taken together, our results not only demonstrate that ex vivo networks show a sensitivity to NSs and BDZs comparable to that expressed in vivo, but also provide a new global in vitro description that can help in understanding their activity in more complex systems.

5-Arylbenzothiadiazine Type Compounds as Positive Allosteric Modulators of AMPA/Kainate Receptors.

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPAr) has led to the search for new AMPAr positive modulators. On the basis of crystallographic data of the benzothiadiazines binding mode in the S1S2 GluA2 dimer interface, a set of 5-aryl-2,3-dihydrobenzothiadiazine type compounds has been synthesized and tested. Electrophysiological results suggested that 5-heteroaryl substituents on the benzothiadiazine core like 3-furanyl and 3-thiophenyl dramatically enhance the activity as positive modulators of AMPAr with respect to IDRA21 and cyclothiazide. Mouse brain microdialysis studies have suggested that 7-chloro-5-(3-furyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide crosses the blood-brain barrier after intraperitoneal injection. Biological results have been rationalized by a computational docking simulation that it has currently employed to design new AMPAr-positive modulator candidates.

GABA(A) receptor neurotransmission dysfunction in a mouse model of social isolation-induced stress: possible insights into a non-serotonergic mechanism of action of SSRIs in mood and anxiety disorders.

Protracted social isolation in laboratory animals causes stress, which induces a variety of behavioral abnormalities including increased aggressiveness, anxiety-related behaviors, cognitive deficits and hyper locomotion. Many of these disorders are similar to the symptoms found in psychiatric disorders, such as depression, anxiety, premenstrual dysphoria and posttraumatic stress disorders (PTSD). Recent studies have demonstrated that male mice that have been socially isolated for more than 4 weeks show: (a) reduced responsiveness of GABA(A) receptors (GABA(A)-R) to the administrations of GABA mimetic drugs at GABA(A)-R; (b) downregulated biosynthesis of 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP) (allopregnanolone: ALLO), a neurosteroid with a potent positive allosteric modulatory effect on the action of GABA on GABA(A)-R; and (c) alterations in the expression of GABA(A)-R subunits (i.e. a decrease of alpha1/alpha2 and gamma2 subunits and an increase of alpha4 and alpha5 subunits). The selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLX) and its congener norfluoxetine (Nor-FLX), when administered systemically at nmol/kg doses, normalize the reduced content of brain ALLO and the reduced responsiveness of GABA(A)-R to GABA mimetic drugs (i.e. pentobarbital) and also attenuate aggressive behavior in socially isolated mice in a stereospecific manner. Although these compounds inhibit ex vivo serotonin reuptake into brain tissue, their SSRI activities require high micromol/kg dose ranges and are not stereospecific. These studies suggest that in socially isolated mice, abnormalities of GABA(A)-R signal transduction are attributable to the downregulation of ALLO production and to a switch in heteropentameric GABA(A)-R subunit assembly composition. Hence, the normalization of ALLO biosynthesis may be a new target for the development of drugs effective for psychiatric disorders related to neurosteroid biosynthesis downregulation.

Design of 1-substituted 2-arylmethyl-4,5-methylenedioxybenzene derivatives as antiseizure agents.

A series of 1-substituted 2-[(4-aryl)-methyl]-4,5-methylenedioxybenzene derivatives (13-25), structurally related to model compound 5 (2-[(4-aminophenyl)-(4-methylsemicarbazono)-methyl]-4,5-methylenedioxyphenylacetic acid methyl ester), were synthesized and tested as anticonvulsant agents in DBA/2 mice against sound-induced seizures. The new compounds possess anticonvulsant properties lower than those of prototype 5 but, in some instances, comparable to that of GYKI 52466, a well-known noncompetitive AMPA receptor antagonist.