Fabio Pibiri

Nicotine decreases DNA methyltransferase 1 expression and glutamic acid decarboxylase 67 promoter methylation in GABAergic interneurons.

Tobacco smoking is frequently abused by schizophrenia patients (SZP). The major synaptically active component inhaled from cigarettes is nicotine, hence the smoking habit of SZP may represent an attempt to use nicotine self-medication to correct (i) a central nervous system nicotinic acetylcholine receptor (nAChR) dysfunction, (ii) DNA-methyltransferase 1 (DMT1) overexpression in GABAergic neurons, and (iii) the down-regulation of reelin and GAD67 expression caused by the increase of DNMT1-mediated hypermethylation of promoters in GABAergic interneurons of the telencephalon. Nicotine (4.5–22 μmol/kg s.c., 4 injections during the 12-h light cycle for 4 days) decreases DNMT1 mRNA and protein and increases GAD67 expression in the mouse frontal cortex (FC). This nicotine-induced decrease of DNMT1 mRNA expression is greater (80%) in laser microdissected FC layer I GABAergic neurons than in the whole FC (40%), suggesting selectivity differences for the specific nicotinic receptor populations expressed in GABAergic neurons of different cortical layers. The down-regulation of DNMT1 expression induced by nicotine in the FC is also observed in the hippocampus but not in striatal GABAergic neurons. Furthermore, these data show that in the FC, the same doses of nicotine that decrease DNMT1 expression also (i) diminished the level of cytosine-5-methylation in the GAD67 promoter and (ii) prevented the methionine-induced hypermethylation of the same promoter. Pretreatment with mecamylamine (6 μmol/kg s.c.), an nAChR blocker that penetrates the blood–brain barrier, prevents the nicotine-induced decrease of FC DNMT1 expression. Taken together, these results suggest that nicotine, by activating nAChRs located on cortical or hippocampal GABAergic interneurons, can up-regulate GAD67 expression via an epigenetic mechanism. Nicotine is not effective in striatal medium spiny GABAergic neurons that primarily express muscarinic receptors.

Decreased corticolimbic allopregnanolone expression during social isolation enhances contextual fear: A model relevant for posttraumatic stress disorder

Mice subjected to social isolation (3–4 weeks) exhibit enhanced contextual fear responses and impaired fear extinction. These responses are time-related to a decrease of 5α-reductase type I (5α-RI) mRNA expression and allopregnanolone (Allo) levels in selected neurons of the medial prefrontal cortex, hippocampus, and basolateral amygdala. Of note, the cued fear response was not different between group housed and socially isolated mice. In socially isolated mice, S-norfluoxetine, a selective brain steroidogenic stimulant (SBSS), in doses (0.45–1.8 μmol/kg) that increase brain Allo levels but fail to inhibit serotonin reuptake, greatly attenuates enhanced contextual fear response. SKF 105,111 (a potent 5α-RI inhibitor) decreases corticolimbic Allo levels and enhances the contextual fear response in group housed mice, which suggests that social isolation alters emotional responses by reducing the positive allosteric modulation of Allo at GABAA receptors in corticolimbic circuits. Thus, these procedures model emotional hyperreactivity, including enhanced contextual fear and impaired contextual fear extinction, which also is observed in posttraumatic stress disorder (PTSD) patients. A recent clinical study reported that cerebrospinal fluid Allo levels also are down-regulated in PTSD patients and correlate negatively with PTSD symptoms and negative mood. Thus, protracted social isolation of mice combined with tests of fear conditioning may be a suitable model to study emotional behavioral components associated with neurochemical alterations relating to PTSD. Importantly, drugs like SBSSs, which rapidly increase corticolimbic Allo levels, normalize the exaggerated contextual fear responses resulting from social isolation, suggesting that selective activation of neurosteroidogenesis may be useful in PTSD therapy.

Down-regulation of neurosteroid biosynthesis in corticolimbic circuits mediates social isolation-induced behavior in mice

Allopregnanolone (ALLO), synthesized by pyramidal neurons, is a potent positive allosteric modulator of the action of GABA at GABAA receptors expressing specific neurosteroid binding sites. In the brain, ALLO is synthesized from progesterone by the sequential action of two enzymes: 5α-reductase type I (5α-RI) and 3α-hydroxysteroid dehydrogenase (3α-HSD). In the cortex, hippocampus, and amygdala, these enzymes are colocalized in principal glutamatergic output neurons [Agís-Balboa RC, Pinna G, Zhubi A, Maloku E, Veldic M, Costa E, Guidotti A (2006) Proc Natl Acad Sci USA 103:14602–14607], but they are not detectable in GABAergic interneurons. Using RT-PCR and in situ hybridization, this study compares 5α-RI and 3α-HSD mRNA brain expression levels in group housed and in socially isolated male mice for 4 weeks. In these socially isolated mice, the mRNA expression of 5α-RI was dramatically decreased in hippocampal CA3 glutamatergic pyramidal neurons, dentate gyrus granule cells, glutamatergic neurons of the basolateral amygdala, and glutamatergic pyramidal neurons of layer V/VI frontal (prelimbic, infralimbic) cortex (FC). In contrast, 5α-RI mRNA expression failed to change in CA1 pyramidal neurons, central amygdala neurons, pyramidal neurons of layer II/III FC, ventromedial thalamic nucleus neurons, and striatal medium spiny and reticular thalamic nucleus neurons. Importantly, 3α-HSD mRNA expression was unchanged by protracted social isolation (Si). These data suggest that, in male mice, after 4 weeks of Si, the expression of 5α-RI mRNA, which is the rate-limiting-step enzyme of ALLO biosynthesis, is specifically down-regulated in glutamatergic pyramidal neurons that converge on the amygdala from cortical and hippocampal regions. In socially isolated mice, this down-regulation may account for the appearance of behavioral disorders such as anxiety, aggression, and cognitive dysfunction.

Neurosteroid Biosynthesis Regulates Sexually Dimorphic Fear and Aggressive Behavior in Mice

The neurosteroid allopregnanolone is a potent positive allosteric modulator of GABA action at GABAA receptors. Allopregnanolone is synthesized in the brain from progesterone by the sequential action of 5α-reductase type I (5α-RI) and 3α-hydroxysteroid dehydrogenase (3α-HSD). 5α-RI and 3α-HSD are co-expressed in cortical, hippocampal, and olfactory bulb glutamatergic neurons and in output neurons of the amygdala, thalamus, cerebellum, and striatum. Neither 5α-RI nor 3α-HSD mRNAs is expressed in glial cells or in cortical or hippocampal GABAergic interneurons. It is likely that allopregnanolone synthesized in principal output neurons locally modulates GABAA receptor function by reaching GABAA receptor intracellular sites through lateral membrane diffusion. This review will focus on the behavioral effects of allopregnanolone on mouse models that are related to a sexually dimorphic regulation of brain allopregnanolone biosynthesis. Animal models of psychiatric disorders, including socially isolated male mice or mice that receive a long-term treatment with anabolic androgenic steroids (AAS), show abnormal behaviors such as altered fear responses and aggression. In these animal models, the cortico-limbic mRNA expression of 5α-RI is regulated in a sexually dimorphic manner. Hence, in selected glutamatergic pyramidal neurons of the cortex, CA3, and basolateral amygdala and in granular cells of the dentate gyrus, mRNA expression of 5α-RI is decreased, which results in a downregulation of allopregnanolone content. In contrast, 5α-RI mRNA expression fails to change in the striatum medium spiny neurons and in the reticular thalamic nucleus neurons, which are GABAergic.By manipulating allopregnanolone levels in glutamatergic cortico-limbic neurons in opposite directions to improve [using the potent selective brain steroidogenic stimulant (SBSS) S-norfluoxetine] or induce (using the potent 5α-RI inhibitor SKF 105,111) behavioral deficits, respectively, we have established the fundamental role of cortico-limbic allopregnanolone levels in the sexually dimorphic regulation of aggression and fear. By selectively targeting allopregnanolone downregulation in glutamatergic cortico-limbic neurons, i.e., by improving the response of GABAA receptors to GABA, new therapeutics would offer appropriate and safe management of psychiatric conditions, including impulsive aggression, irritability, irrational fear, anxiety, posttraumatic stress disorders, and depression.

Two gene co-expression modules differentiate psychotics and controls

Schizophrenia (SCZ) and bipolar disorder (BD) are highly heritable psychiatric disorders. Associated genetic and gene expression changes have been identified, but many have not been replicated and have unknown functions. We identified groups of genes whose expressions varied together, that is co-expression modules, then tested them for association with SCZ. Using weighted gene co-expression network analysis, we show that two modules were differentially expressed in patients versus controls. One, upregulated in cerebral cortex, was enriched with neuron differentiation and neuron development genes, as well as disease genome-wide association study genetic signals; the second, altered in cerebral cortex and cerebellum, was enriched with genes involved in neuron protection functions. The findings were preserved in five expression data sets, including sets from three brain regions, from a different microarray platform, and from BD patients. From those observations, we propose neuron differentiation and development pathways may be involved in etiologies of both SCZ and BD, and neuron protection function participates in pathological process of the diseases.

Neurosteroids reduce social isolation-induced behavioral deficits: a proposed link with neurosteroid-mediated upregulation of BDNF expression.

The pharmacological action of selective serotonin reuptake inhibitor antidepressants may include a normalization of the decreased brain levels of the brain-derived neurotrophic factor (BDNF) and of neurosteroids such as the progesterone metabolite allopregnanolone, which are decreased in patients with depression and posttraumatic stress disorders (PTSD). The allopregnanolone and BDNF level decrease in PTSD and depressed patients is associated with behavioral symptom severity. Antidepressant treatment upregulates both allopregnanolone levels and the expression of BDNF in a manner that significantly correlates with improved symptomatology, which suggests that neurosteroid biosynthesis and BDNF expression may be interrelated. Preclinical studies using the socially isolated mouse as an animal model of behavioral deficits, which resemble some of the symptoms observed in PTSD patients, have shown that fluoxetine and derivatives improve anxiety-like behavior, fear responses and aggressive behavior by elevating the corticolimbic levels of allopregnanolone and BDNF mRNA expression. These actions appeared to be independent and more selective than the action of these drugs on serotonin reuptake inhibition. Hence, this review addresses the hypothesis that in PTSD or depressed patients, brain allopregnanolone levels, and BDNF expression upregulation may be mechanisms at least partially involved in the beneficial actions of antidepressants or other selective brain steroidogenic stimulant molecules.

Selective α4β2 nicotinic acetylcholine receptor agonists target epigenetic mechanisms in cortical GABAergic neurons.

Nicotine improves cognitive performance and attention in both experimental animals and in human subjects, including patients affected by neuropsychiatric disorders. However, the specific molecular mechanisms underlying nicotine-induced behavioral changes remain unclear. We have recently shown in mice that repeated injections of nicotine, which achieve plasma concentrations comparable to those reported in high cigarette smokers, result in an epigenetically induced increase of glutamic acid decarboxylase 67 (GAD67) expression. Here we explored the impact of synthetic α4β2 and α7 nAChR agonists on GABAergic epigenetic parameters. Varenicline (VAR), a high-affinity partial agonist at α4β2 and a lower affinity full agonist at α7 neuronal nAChR, injected in doses of 1–5 mg/kg/s.c. twice daily for 5 days, elicited a 30–40% decrease of cortical DNA methyltransferase (DNMT)1 mRNA and an increased expression of GAD67 mRNA and protein. This upregulation of GAD67 was abolished by the nAChR antagonist mecamylamine. Furthermore, the level of MeCP2 binding to GAD67 promoters was significantly reduced following VAR administration. This effect was abolished when VAR was administered with mecamylamine. Similar effects on cortical DNMT1 and GAD67 expression were obtained after administration of A–85380, an agonist that binds to α4β2 but has negligible affinity for α3β4 or α7 subtypes containing nAChR. In contrast, PNU–282987, an agonist of the homomeric α7 nAChR, failed to decrease cortical DNMT1 mRNA or to induce GAD67 expression. The present study suggests that the α4β2 nAChR agonists may be better suited to control the epigenetic alterations of GABAergic neurons in schizophrenia than the α7 nAChR agonists.

The combination of huperzine A and imidazenil is an effective strategy to prevent diisopropyl fluorophosphate toxicity in mice.

Diisopropyl fluorophosphate (DFP) causes neurotoxicity related to an irreversible inhibition of acetylcholinesterase (AChE). Management of this intoxication includes: (i) pretreatment with reversible blockers of AChE, (ii) blockade of muscarinic receptors with atropine, and (iii) facilitation of GABAA receptor signal transduction by benzodiazepines. The major disadvantage associated with this treatment combination is that it must to be repeated frequently and, in some cases, protractedly. Also, the use of diazepam (DZP) and congeners includes unwanted side effects, including sedation, amnesia, cardiorespiratory depression, and anticonvulsive tolerance. To avoid these treatment complications but safely protect against DFP-induced seizures and other CNS toxicity, we adopted the strategy of administering mice with (i) small doses of huperzine A (HUP), a reversible and long-lasting (half-life ≈5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for α5-containing GABAA receptors. Coadministration of HUP (50 μg/kg s.c., 15 min before DFP) with IMI (2 mg/kg s.c., 30 min before DFP) prevents DFP-induced convulsions and the associated neuronal damage and mortality, allowing complete recovery within 18–24 h. In HUP-pretreated mice, the ED50 of IMI to block DFP-induced mortality is ≈10 times lower than that of DZP and is devoid of sedation. Our data show that a combination of HUP with IMI is a prophylactic, potent, and safe therapeutic strategy to overcome DFP toxicity.

Enhanced fear responses in mice treated with anabolic androgenic steroids

Corticolimbic neurons express neurosteroid biosynthesis, which is altered during anabolic androgenic steroid (AAS) treatment. The brain circuits and neurons that underlie the behavioral deficits found after AAS treatment remain undefined. We studied the effects of testosterone propionate (testosterone) on fear conditioning responses and in primary output corticolimbic neurons on 5&agr;-reductase-type-I and 3&agr;-hydroxysteroid-dehydrogenase expression. Testosterone fails to change cued fear responses although it induces excessive contextual fear associated with corticolimbic 5&agr;-reductase-type-I mRNA expression downregulation in the prefrontal cortex, hippocampus, and basolateral amygdala glutamatergic neurons. Increased fear responses are abolished by normalizing corticolimbic allopregnanolone levels with allopregnanolone treatment (8 μmol/kg) or selective brain steroidogenic stimulants, including S-norfluoxetine (1.8 μmol/kg). Agents that increase corticolimbic allopregnanolone levels may be beneficial in treating AAS users.

Abstract 1279: Association between vitamin D levels and colorectal cancer in African Americans

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background. Colorectal cancer (CRC) is the fourth most commonly diagnosed cancer in the world and there is a sizeable disparity in CRC incidence and mortality between African Americans (AAs) and all other US racial groups that remains unexplained. Associations between vitamin D levels and CRC have been widely reported in many populations, but there are few studies in AAs. We hypothesized that vitamin D levels are lower in AAs with CRC. Methods. 401 subjects were prospectively ascertained in endoscopy clinics in the Illinois Medical District: 130 healthy controls, 136 individuals with adenomatous polyps, and 130 individuals with CRC; 270 of 401 subjects were AA. Clinical data included age, gender, and BMI (kg/m2); smoking and drinking status; anti-inflammatory medication use; education and income levels; physical exercise level. Levels of serum 25-(OH) vitamin D2 and D3 were measured by Quest Diagnostics using liquid chromatography and tandem mass spectroscopy. Thirty nine potentially functional SNPs from eight vitamin D-related genes (CYP2R1, CYP24A1, CYP27A1, CYP27B1, CYP3A4, DHCR7, GC, and VDR) were genotyped using the Sequenom MassARRAY platform. We tested associations between vitamin D and CRC using logistic regression models, adjusting for age, gender, and time of year of blood collection, and we tested each clinical covariate in the model. In addition, we tested associations between SNPs and vitamin D levels using logistic regression, adjusting for age, gender, and time of collection. Results. Levels of total vitamin D (D2 + D3) and of vitamin D3 were statistically significantly lower in AAs compared to Non-Hispanic Whites (NHW). Approximately 15% of AAs exhibited detectable levels of vitamin D2, which was evidence of supplementation; only 8% of NHWs supplemented. Supplementation was significantly associated with older ages, female gender, less alcohol use and less exercise. Vitamin D3 levels were 22% lower in AA CRC cases compared to AA controls (18.5 ng/ml vs 23.7 ng/ml; P <0.03); vitamin D3 levels were not significantly different in NHW CRC cases compared to controls (29.8 ng/ml vs. 28.2 ng/ml). Vitamin D3 levels in adenoma cases were not significantly different compared to controls in either group (23.5 ng/ml in AA adenoma cases and 27.3 ng/ml in NHW adenoma cases). The SNP rs1544410 in VDR and rs3829251 in DHCR7 had the smallest p values (0.008 and 0.013, respectively). The minor allele of rs16847024 in GC was associated with lower vitamin D3 levels; this same SNP was associated with left-sided CRC in AAs in a larger case-control study performed by our group. rs3829251 in DHCR7 has been previously associated with serum vitamin D levels in Han Chinese. Conclusion. These data demonstrated that vitamin D levels are associated with protection against CRC in AAs. SNPs in vitamin D-related genes may modulate vitamin D levels and risk of CRC. Citation Format: Sariya Siddiqi, Fabio Pibiri, Rosa Munoz Xicola, Rick A. Kittles, Xavier Llor, Sonia S. Kupfer, Nathan A. Ellis. Association between vitamin D levels and colorectal cancer in African Americans. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1279. doi:10.1158/1538-7445.AM2014-1279