Flexible and Convergent Enantioselective Total Synthesis of (R)-Juglanaloids A and B: Two Phthalide Spiro Alkaloids with Potential Alzheimer's Disease Inhibitory Activity.

Juglanaloids A and B are recently isolated natural products characterized by an unprecedented spiro bicyclic isobenzofuranone-tetrahydrobenzazepinone framework and a promising antiamyloid activity. Here reported is a straightforward convergent total synthesis of these natural products, which were obtained in high enantiomeric purity (94% and >99% ee for juglanaloids A and B, respectively) through an eight-step longest linear sequence, based on an efficient and reliable enantioselective phase-transfer-catalyzed alkylation step. Considering the interesting biological activity of juglanaloids, this convenient, highly enantioselective, flexible, and predictable synthetic strategy promises to be a powerful tool for accessing potentially bioactive spiro bicyclic phthalide-tetrahydrobenzazepinone derivatives.

Inter- and Intra-Subunit Butanol/Isoflurane Sites of Action in the Human Glycine Receptor.

Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl2 (10 µM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model for alcohol/anesthetic interaction with human GLRA1.

Chronic Intermittent Ethanol Regulates Hippocampal GABA(A) Receptor Delta Subunit Gene Expression.

Chronic ethanol consumption causes structural and functional reorganization in the hippocampus and induces alterations in the gene expression of gamma-aminobutyric acid type A receptors (GABAARs). Distinct forced intermittent exposure models have been used previously to investigate changes in GABAAR expression, with contrasting results. Here, we used repeated cycles of a Chronic Intermittent Ethanol paradigm to examine the relationship between voluntary, dependence-associated ethanol consumption, and GABAAR gene expression in mouse hippocampus. Adult male C57BL/6J mice were exposed to four 16-h ethanol vapor (or air) cycles in inhalation chambers alternated with limited-access two-bottle choice between ethanol (15%) and water consumption. The mice exposed to ethanol vapor showed significant increases in ethanol consumption compared to their air-matched controls. GABAAR alpha4 and delta subunit gene expression were measured by qRT-PCR at different stages. There were significant changes in GABAAR delta subunit transcript levels at different time points in ethanol-vapor exposed mice, while the alpha4 subunit levels remained unchanged. Correlated concurrent blood ethanol concentrations suggested that GABAAR delta subunit mRNA levels fluctuate depending on ethanol intoxication, dependence, and withdrawal state. Using a vapor-based Chronic Intermittent Ethanol procedure with combined two-bottle choice consumption, we corroborated previous evidences showing that discontinuous ethanol exposure affects GABAAR delta subunit expression but we did not observe changes in alpha4 subunit. These findings indicate that hippocampal GABAAR delta subunit expression changes transiently over the course of a Chronic Intermittent Ethanol paradigm associated with voluntary intake, in response to ethanol-mediated disturbance of GABAergic neurotransmission.

Proteomic approaches and identification of novel therapeutic targets for alcoholism.

Recent studies have shown that gene regulation is far more complex than previously believed and does not completely explain changes at the protein level. Therefore, the direct study of the proteome, considerably different in both complexity and dynamicity to the genome/transcriptome, has provided unique insights to an increasing number of researchers. During the past decade, extraordinary advances in proteomic techniques have changed the way we can analyze the composition, regulation, and function of protein complexes and pathways underlying altered neurobiological conditions. When combined with complementary approaches, these advances provide the contextual information for decoding large data sets into meaningful biologically adaptive processes. Neuroproteomics offers potential breakthroughs in the field of alcohol research by leading to a deeper understanding of how alcohol globally affects protein structure, function, interactions, and networks. The wealth of information gained from these advances can help pinpoint relevant biomarkers for early diagnosis and improved prognosis of alcoholism and identify future pharmacological targets for the treatment of this addiction.

Integration of miRNA and protein profiling reveals coordinated neuroadaptations in the alcohol-dependent mouse brain.

The molecular mechanisms underlying alcohol dependence involve different neurochemical systems and are brain region-dependent. Chronic Intermittent Ethanol (CIE) procedure, combined with a Two-Bottle Choice voluntary drinking paradigm, represents one of the best available animal models for alcohol dependence and relapse drinking. MicroRNAs, master regulators of the cellular transcriptome and proteome, can regulate their targets in a cooperative, combinatorial fashion, ensuring fine tuning and control over a large number of cellular functions. We analyzed cortex and midbrain microRNA expression levels using an integrative approach to combine and relate data to previous protein profiling from the same CIE-subjected samples, and examined the significance of the data in terms of relative contribution to alcohol consumption and dependence. MicroRNA levels were significantly altered in CIE-exposed dependent mice compared with their non-dependent controls. More importantly, our integrative analysis identified modules of coexpressed microRNAs that were highly correlated with CIE effects and predicted target genes encoding differentially expressed proteins. Coexpressed CIE-relevant proteins, in turn, were often negatively correlated with specific microRNA modules. Our results provide evidence that microRNA-orchestrated translational imbalances are driving the behavioral transition from alcohol consumption to dependence. This study represents the first attempt to combine ex vivo microRNA and protein expression on a global scale from the same mammalian brain samples. The integrative systems approach used here will improve our understanding of brain adaptive changes in response to drug abuse and suggests the potential therapeutic use of microRNAs as tools to prevent or compensate multiple neuroadaptations underlying addictive behavior.

Neurobiological signatures of alcohol dependence revealed by protein profiling.

Alcohol abuse causes dramatic neuroadaptations in the brain, which contribute to tolerance, dependence, and behavioral modifications. Previous proteomic studies in human alcoholics and animal models have identified candidate alcoholism-related proteins. However, recent evidences suggest that alcohol dependence is caused by changes in co-regulation that are invisible to single protein-based analysis. Here, we analyze global proteomics data to integrate differential expression, co-expression networks, and gene annotations to unveil key neurobiological rearrangements associated with the transition to alcohol dependence modeled by a Chronic Intermittent Ethanol (CIE), two-bottle choice (2BC) paradigm. We analyzed cerebral cortices (CTX) and midbrains (MB) from male C57BL/6J mice subjected to a CIE, 2BC paradigm, which induces heavy drinking and represents one of the best available animal models for alcohol dependence and relapse drinking. CIE induced significant changes in protein levels in dependent mice compared with their non-dependent controls. Multiple protein isoforms showed region-specific differential regulation as a result of post-translational modifications. Our integrative analysis identified modules of co-expressed proteins that were highly correlated with CIE treatment. We found that modules most related to the effects of CIE treatment coordinate molecular imbalances in endocytic- and energy-related pathways, with specific proteins involved, such as dynamin-1. The qRT-PCR experiments validated both differential and co-expression analyses, and the correspondence among our data and previous genomic and proteomic studies in humans and rodents substantiates our findings. The changes identified above may play a key role in the escalation of ethanol consumption associated with dependence. Our approach to alcohol addiction will advance knowledge of brain remodeling mechanisms and adaptive changes in response to drug abuse, contribute to understanding of organizational principles of CTX and MB proteomes, and define potential new molecular targets for treating alcohol addiction. The integrative analysis employed here highlight the advantages of systems approaches in studying the neurobiology of alcohol addiction.

Positively correlated miRNA-mRNA regulatory networks in mouse frontal cortex during early stages of alcohol dependence.

BACKGROUND: Although the study of gene regulation via the action of specific microRNAs (miRNAs) has experienced a boom in recent years, the analysis of genome-wide interaction networks among miRNAs and respective targeted mRNAs has lagged behind. MicroRNAs simultaneously target many transcripts and fine-tune the expression of genes through cooperative/combinatorial targeting. Therefore, they have a large regulatory potential that could widely impact development and progression of diseases, as well as contribute unpredicted collateral effects due to their natural, pathophysiological, or treatment-induced modulation. We support the viewpoint that whole mirnome-transcriptome interaction analysis is required to better understand the mechanisms and potential consequences of miRNA regulation and/or deregulation in relevant biological models. In this study, we tested the hypotheses that ethanol consumption induces changes in miRNA-mRNA interaction networks in the mouse frontal cortex and that some of the changes observed in the mouse are equivalent to changes in similar brain regions from human alcoholics. RESULTS: miRNA-mRNA interaction networks responding to ethanol insult were identified by differential expression analysis and weighted gene coexpression network analysis (WGCNA). Important pathways (coexpressed modular networks detected by WGCNA) and hub genes central to the neuronal response to ethanol are highlighted, as well as key miRNAs that regulate these processes and therefore represent potential therapeutic targets for treating alcohol addiction. Importantly, we discovered a conserved signature of changing miRNAs between ethanol-treated mice and human alcoholics, which provides a valuable tool for future biomarker/diagnostic studies in humans. We report positively correlated miRNA-mRNA expression networks that suggest an adaptive, targeted miRNA response due to binge ethanol drinking. CONCLUSIONS: This study provides new evidence for the role of miRNA regulation in brain homeostasis and sheds new light on current understanding of the development of alcohol dependence. To our knowledge this is the first report that activated expression of miRNAs correlates with activated expression of mRNAs rather than with mRNA downregulation in an in vivo model. We speculate that early activation of miRNAs designed to limit the effects of alcohol-induced genes may be an essential adaptive response during disease progression.

Molecular targets of alcohol action: Translational research for pharmacotherapy development and screening.

Alcohol abuse and dependence are multifaceted disorders with neurobiological, psychological, and environmental components. Research on other complex neuropsychiatric diseases suggests that genetically influenced intermediate characteristics affect the risk for heavy alcohol consumption and its consequences. Diverse therapeutic interventions can be developed through identification of reliable biomarkers for this disorder and new pharmacological targets for its treatment. Advances in the fields of genomics and proteomics offer a number of possible targets for the development of new therapeutic approaches. This brain-focused review highlights studies identifying neurobiological systems associated with these targets and possible pharmacotherapies, summarizing evidence from clinically relevant animal and human studies, as well as sketching improvements and challenges facing the fields of proteomics and genomics. Concluding thoughts on using results from these profiling technologies for medication development are also presented.

Dynamin-1 co-associates with native mouse brain BKCa channels: proteomics analysis of synaptic protein complexes.

In every synapse, a large number of proteins interact with other proteins in order to carry out signaling and transmission in the central nervous system. In this study, we used interaction proteomics to identify novel synaptic protein interactions in mouse cortical membranes under native conditions. Using immunoprecipitation, immunoblotting, and mass spectrometry, we identified a number of novel synaptic protein interactions involving soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), calcium-activated potassium channel (BKCa) alpha subunits, and dynamin-1. These novel interactions offer valuable insight into the protein-protein interaction network in intact synapses that could advance understanding of vesicle trafficking, release, and recycling.

Chronic vagus nerve stimulation induces neuronal plasticity in the rat hippocampus.

Vagus nerve stimulation (VNS) is used to treat pharmacotherapy-resistant epilepsy and depression. However, the mechanisms underlying the therapeutic efficacy of VNS remain unclear. We examined the effects of VNS on hippocampal neuronal plasticity and behaviour in rats. Cell proliferation in the hippocampus of rats subjected to acute (3 h) or chronic (1 month) VNS was examined by injection of bromodeoxyuridine (BrdU) and immunohistochemistry. Expression of doublecortin (DCX) and brain-derived neurotrophic factor (BDNF) was evaluated by immunofluorescence staining. The dendritic morphology of DCX+ neurons was measured by Sholl analysis. Our results show that acute VNS induced an increase in the number of BrdU+ cells in the dentate gyrus that was apparent 24 h and 3 wk after treatment. It also induced long-lasting increases in the amount of DCX immunoreactivity and in the number of DCX+ neurons. Neither the number of BrdU+ cells nor the amount of DCX immunoreactivity was increased 3 wk after the cessation of chronic VNS. Chronic VNS induced long-lasting increases in the amount of BDNF immunoreactivity and the number of BDNF+ cells as well as in the dendritic complexity of DCX+ neurons in the hippocampus. In contrast to chronic imipramine treatment, chronic VNS had no effect on the behaviour of rats in the forced swim or elevated plus-maze tests. Both chronic and acute VNS induced persistent changes in hippocampal neurons that may play a key role in the therapeutic efficacy of VNS. However, these changes were not associated with evident behavioural alterations characteristic of an antidepressant or anxiolytic action.

Vagus nerve stimulation increases norepinephrine concentration and the gene expression of BDNF and bFGF in the rat brain.

Vagus nerve stimulation therapy, effective for treatment-resistant epilepsy, has recently been approved also for treatment-resistant depression; nevertheless, the molecular mechanism(s) underlying its therapeutic action remains unclear. Given that neurotrophic factors and monoamines could play a crucial role in the pathophysiology of depression, we tested whether vagus nerve stimulation increases the expression of brain-derived neurotrophic factor, fibroblast growth factor, and nerve growth factor as well as the concentration of norepinephrine in the rat brain. Rats were implanted with a vagus nerve stimulator device and the effects of acute stimulation were evaluated on the growth factors mRNA levels and norepinephrine concentration by ribonuclease protection assay and microdialysis, respectively. We found that acute vagus nerve stimulation increased the expression of brain-derived neurotrophic factor and fibroblast growth factor in the hippocampus and cerebral cortex, decreased the abundance of nerve growth factor mRNA in the hippocampus, and, similar to the antidepressant drug venlafaxine, increased the norepinephrine concentration in the prefrontal cortex. This study demonstrates that acute vagus nerve stimulation triggers neurochemical and molecular changes in the rat brain involving neurotransmitters and growth factors known to play a crucial role in neuronal trophism. These new findings contribute to the elucidation of the molecular mechanisms underlying the therapeutic actions of vagus nerve stimulation in both treatment-resistant depression and epilepsy.

Flumazenil selectively prevents the increase in alpha(4)-subunit gene expression and an associated change in GABA(A) receptor function induced by ethanol withdrawal.

The actions of ethanol on gamma-aminobutyric acid type A (GABA(A)) receptors are still highly controversial issues but it appears that some of its pharmacological effects may depend on receptor subunit composition. Prolonged ethanol exposure produces tolerance and dependence and its withdrawal alters GABA(A) receptor subunit gene expression and function. Whereas benzodiazepines are clinically effective in ameliorating ethanol withdrawal symptoms, work in our laboratory showed that benzodiazepines also prevent, in vitro, some of the ethanol withdrawal-induced molecular and functional changes of the GABA(A) receptors. In the present work, we investigated the effects, on such changes, of the benzodiazepine receptor antagonist flumazenil that can positively modulate alpha(4)-containing receptors. We here report that flumazenil prevented both the ethanol withdrawal-induced up-regulation of the alpha(4)-subunit and the increase in its own modulatory action. In contrast, flumazenil did not inhibit ethanol withdrawal-induced decrease in alpha(1)- and delta-subunit expression as well as the corresponding decrease in the modulatory action on GABA(A) receptor function of both the alpha(1)-selective ligand zaleplon and the delta-containing receptor preferentially acting steroid allopregnanolone. These observations are the first molecular and functional evidence that show a selective inhibition by flumazenil of the up-regulation of alpha(4)-subunit expression elicited by ethanol withdrawal.

Plastic neuronal changes in GABA(A) receptor gene expression induced by progesterone metabolites: in vitro molecular and functional studies.

Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons is affected by endogenous modulators of receptor function such as neuroactive steroids. Neuroactive steroids such as the progesterone metabolite allopregnanolone might thus exert differential effects on GABA(A) receptor plasticity in neurons, likely accounting for some of the physiological actions of these compounds. Here we summarise experimental data obtained in vitro that show how fluctuations in the concentration of progesterone regulate both the expression and function of GABA(A) receptors. The data described in this manuscript are in agreement with the notion that fluctuations in the concentrations of progesterone and its metabolite allopregnanolone play a major role in the temporal pattern of expression of various subunits of the GABA(A) receptor. Thus, rapid and long-lasting increases or decreases in the concentrations of these steroid derivatives observed in physiological and patho-physiological conditions, or induced by pharmacological treatments, might elicit selective changes in GABA(A) receptor gene expression and function in specific neuronal populations. Given both the importance of GABA(A) receptors in the regulation of neuronal excitability and the large fluctuations in the plasma and brain concentrations of neuroactive steroids associated with physiological conditions and the response to environmental stimuli, these compounds are likely among the most relevant endogenous modulators that could affect emotional and affective behaviors.

Distinct patterns of expression and regulation of GABA receptors containing the delta subunit in cerebellar granule and hippocampal neurons.

Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABA(A)R) for gamma-aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABA(A)R plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the delta subunit of GABA(A)Rs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of delta subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down-regulated delta subunit expression in cerebellar granule neurons and gradually normalized that in hippocampal neurons. These effects of ethanol exposure and withdrawal were accompanied by corresponding functional changes in GABA(A)Rs. GABA(A)Rs containing the delta subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABA(A)Rs that contain the delta subunit in different neuronal types.

Modulation of GABA(A) receptor gene expression by allopregnanolone and ethanol.

Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons is affected by endogenous modulators of receptor function such as neuroactive steroids. This effect of steroids appears to be mediated through modulation of GABA(A) receptor signalling mechanisms that control the expression of specific receptor subunit genes. Furthermore, the specific outcomes of such signalling appear to differ among neurons in different regions of the brain. Neuroactive steroids such as the progesterone metabolite allopregnanolone might thus exert differential effects on GABA(A) receptor plasticity in distinct neuronal cell populations, likely accounting for some of the physiological actions of these compounds. Here we summarise experimental data obtained both in vivo and in vitro that show how fluctuations in the concentration of allopregnanolone regulate both the expression and function of GABA(A) receptors and consequently affect behaviour. Such regulation is operative both during physiological conditions such as pregnancy and lactation as well as in pharmacologically induced states such as pseudopregnancy and long-term treatment with steroid derivatives or anxiolytic-hypnotic drugs. Accordingly, long-lasting exposure of GABA(A) receptors to ethanol, as well as its withdrawal, induces marked effects on receptor structure and function. These results suggest the possible synergic action between endogenous steroids and ethanol in modulating the functional activity of specific neuronal populations.

Ethanol withdrawal-induced up-regulation of the alpha2 subunit of the GABAA receptor and its prevention by diazepam or gamma-hydroxybutyric acid.

The gamma-aminobutyric acid type A (GABA(A)) receptor is an important pharmacological target of ethanol. The effect of ethanol withdrawal on the expression of the alpha(2) subunit of this receptor was examined with rat cerebellar granule cells in primary culture. Long-term exposure of these cells to ethanol (100 mM, 5 days) did not affect the abundance of the mRNA for the alpha(2) subunit, as revealed by an RNase protection assay. In contrast, subsequent ethanol withdrawal for 3 h induced a marked increase in the amount of this mRNA (2.6-fold) as well as in that of the encoded polypeptide (2.2-fold), the latter revealed by immunoblot analysis. Exposure of the cells to gamma-hydroxybutyric acid (100 mM) during ethanol withdrawal prevented the increase in the amounts of both the alpha(2) mRNA and polypeptide, whereas similar treatment with diazepam (10 microM) blocked the increase in the abundance of the alpha(2) polypeptide but not that in the amount of the alpha(2) mRNA. The effect of gamma-hydroxybutyric acid was not blocked by the competitive GABA(B) receptor antagonist SCH 50911(10 microM). Given that the alpha(2) subunit of the GABA(A) receptor mediates the anxiolytic action of benzodiazepines, its up-regulation during discontinuation of long-term ethanol exposure might be relevant to the therapeutic efficacy of these drugs in the treatment of anxiety associated with ethanol withdrawal.

Changes in GABA(A) receptor gene expression induced by withdrawal of, but not by long-term exposure to, ganaxolone in cultured rat cerebellar granule cells.

The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanolone, on the function and expression of GABA(A) receptors were determined. Electrophysiological recordings demonstrated that ganaxolone potentiated with a potency and efficacy similar to those of allopregnanolone the Cl- currents evoked by GABA at recombinant human GABA(A) receptors (comprising alpha1beta2gamma2L or alpha2beta2gamma2L subunit assemblies) expressed in Xenopus oocytes. Exposure of cultured rat cerebellar granule cells to 1 microM ganaxolone for 5 days had no effect on the abundance of mRNAs encoding the alpha1, alpha2, alpha3, alpha4, alpha5, gamma2L, or gamma2S subunits of the GABA(A) receptor. Withdrawal of ganaxolone after such long-term treatment, however, induced an increase in the abundance of alpha2, alpha4, and alpha5 subunit mRNAs and a decrease in the amounts of alpha1, gamma2L, and gamma2S subunit mRNAs. These changes were maximal 3 to 6 h after drug withdrawal and were reversible, being no longer apparent after 24 h. These results suggest that long-term exposure of cerebellar granule cells to ganaxolone does not affect the sensitivity of the GABA(A) receptor to several positive modulators. Nevertheless, the reduction in the amounts of the alpha1 and gamma2 subunit mRNAs together with the increase in the abundance of the alpha4 subunit mRNA induced by abrupt discontinuation of long-term treatment with ganaxolone suggest that withdrawal of this drug might result in a reduced response to classic benzodiazepines.