Francesco Napolitano

Drug repositioning: a machine-learning approach through data integration

Existing computational methods for drug repositioning either rely only on the gene expression response of cell lines after treatment, or on drug-to-disease relationships, merging several information levels. However, the noisy nature of the gene expression and the scarcity of genomic data for many diseases are important limitations to such approaches. Here we focused on a drug-centered approach by predicting the therapeutic class of FDA-approved compounds, not considering data concerning the diseases. We propose a novel computational approach to predict drug repositioning based on state-of-the-art machine-learning algorithms. We have integrated multiple layers of information: i) on the distances of the drugs based on how similar are their chemical structures, ii) on how close are their targets within the protein-protein interaction network, and iii) on how correlated are the gene expression patterns after treatment. Our classifier reaches high accuracy levels (78%), allowing us to re-interpret the top misclassifications as re-classifications, after rigorous statistical evaluation. Efficient drug repurposing has the potential to significantly impact the whole field of drug development. The results presented here can significantly accelerate the translation into the clinics of known compounds for novel therapeutic uses.

d-Aspartate Prevents Corticostriatal Long-Term Depression and Attenuates Schizophrenia-Like Symptoms Induced by Amphetamine and MK-801

Since their discovery in the mammalian CNS, d-aspartate and d-serine have aroused a strong interest with regard to their role as putative neuromodulatory molecules. Whereas the functional role of d-serine as an endogenous coagonist of NMDA receptors (NMDARs) has been elucidated, the biological significance of d-aspartate in the brain is still mostly unclear. In the present study, we demonstrated that nonphysiological high levels of d-aspartate (1) increased in vivo NMDAR activity, (2) attenuated prepulse inhibition deficits induced by amphetamine and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate], (3) produced striatal adaptations of glutamate synapses resembling those observed after chronic haloperidol treatment, and (4) enhanced hippocampal NMDAR-dependent memory. This evidence was obtained using two different experimental strategies that produced an abnormal increase of endogenous d-aspartate levels in the mouse: a genetic approach based on the targeted deletion of the d-aspartate oxidase gene and a pharmacological approach based on oral administration of d-aspartate. This work provides in vivo evidence of a neuromodulatory role exerted by d-aspartate on NMDAR signaling and raises the intriguing hypothesis that also this d-amino acid, like d-serine, could be used as a therapeutic agent in the treatment of schizophrenia-related symptoms.

Rhes, a striatal-enriched small G protein, mediates mTOR signaling and L-DOPA–induced dyskinesia

L-DOPA–induced dyskinesia, the rate-limiting side effect in the therapy of Parkinsons disease, is mediated by activation of mammalian target of rapamycin (mTOR) signaling in the striatum. We found that Ras homolog enriched in striatum (Rhes), a striatal-specific protein, binds to and activates mTOR. Moreover, Rhes−/− mice showed reduced striatal mTOR signaling and diminished dyskinesia, but maintained motor improvement on L-DOPA treatment, suggesting a therapeutic benefit for Rhes-binding drugs.

DRD2/AKT1 interaction on D2 c-AMP independent signaling, attentional processing, and response to olanzapine treatment in schizophrenia

The D2/AKT1/GSK-3β signaling pathway has been involved in the downstream intracellular effects of dopamine, in the pathophysiology of cognitive deficits and related brain activity in schizophrenia, as well as in response to treatment with antipsychotics. Polymorphisms in the D2 (DRD2 rs1076560) and AKT1 (AKT1 rs1130233) genes have been associated with their respective protein expression and with higher-order cognition and brain function, including attention. Given the strong potential for their relationship, we investigated the interaction of these polymorphisms on multiple molecular and in vivo phenotypes associated with this signaling pathway. We measured AKT1 and GSK-3β proteins and phosphorylation in human peripheral blood mononuclear cells, functional MRI cingulate response during attentional control, behavioral accuracy during sustained attention, and response to 8 wk of treatment with olanzapine in a total of 190 healthy subjects and 66 patients with schizophrenia. In healthy subjects, we found that the interaction between the T allele of DRD2 rs1076560 and the A allele of AKT1 rs1130233 was associated with reduced AKT1 protein levels and reduced phosphorylation of GSK-3β, as well as with altered cingulate response and reduced behavioral accuracy during attentional processing. On the other hand, interaction of these two alleles was associated with greater improvement of Positive and Negative Syndrome Scale scores in patients with schizophrenia after treatment with olanzapine. The present results indicate that these functional polymorphisms are epistatically associated with multiple phenotypes of relevance to schizophrenia. Our results also lend support to further investigation of this downstream molecular pathway in the etiology and treatment of this disorder.

Antimicrobial and antioxidant properties of the essential oil of Salvia lanigera from Cyprus

The essential oil of aerial parts of Salvia lanigera Poir. (Lamiaceae) growing wild in Cyprus was obtained by hydrodistillation and was analysed by GC and GC-MS. A total of 67 compounds, representing 93.6% of the oil, were identified, and the major components were showed to be thymol (12.1%), hexadecanoic acid (6.0%), carvacrol and α-thujone (5.7%). The essential oil was assayed for its antioxidant and antimicrobial activities. Antimicrobial activity of the oil, evaluated using the broth dilution method, resulted higher against Gram-positive bacteria than the other referenced strains tested. Antioxidant activity of the oil was evaluated by using DPPH and FRAP methods together with three antioxidant standards, L-ascorbic acid, tert-butyl-4-hydroxy toluene (BHT) and gallic acid. The activity of the sample in both methods was higher than that of all of standards used at the same dose.

Dopamine D2 receptor dysfunction is rescued by adenosine A2A receptor antagonism in a model of DYT1 dystonia.

DYT1 dystonia is an inherited disease linked to mutation in the TOR1A gene encoding for the protein torsinA. Although the mechanism by which this genetic alteration leads to dystonia is unclear, multiple lines of clinical evidence suggest a link between dystonia and a reduced dopamine D2 receptor (D2R) availability. Based on this evidence, herein we carried out a comprehensive analysis of electrophysiological, behavioral and signaling correlates of D2R transmission in transgenic mice with the DYT1 dystonia mutation. Electrophysiological recordings from nigral dopaminergic neurons showed a normal responsiveness to D2-autoreceptor function. Conversely, postsynaptic D2R function in hMT mice was impaired, as suggested by the inability of a D2R agonist to re-establish normal corticostriatal synaptic plasticity and supported by the reduced sensitivity to haloperidol-induced catalepsy. Although an in situ hybridization analysis showed normal D1R and D2R mRNA expression levels in the striata of hMT mice, we found a significant decrease of D2R protein, coupled to a reduced ability of D2Rs to activate their cognate Go/i proteins. Of relevance, we found that pharmacological blockade of adenosine A2A receptors (A2ARs) fully restored the impairment of synaptic plasticity observed in hMT mice. Together, our findings demonstrate an important link between torsinA mutation and D2R dysfunction and suggest that A2AR antagonism is able to counteract the deficit in D2R-mediated transmission observed in mutant mice, opening new perspectives for the treatment of this movement disorder.

Interleukin-1β Causes Anxiety by Interacting with the Endocannabinoid System

Interleukin-1β (IL-1β) is involved in mood alterations associated with inflammatory illnesses and with stress. The synaptic basis of IL-1β-induced emotional disturbances is still unknown. To address the possible involvement of the endocannabinoid system in IL-1β-induced anxiety, we performed behavioral and neurophysiological studies in mice exposed to stress or to intracerebroventricular injections of this inflammatory cytokine or of its antagonist. We found that a single intracerebroventricular injection of IL-1β caused anxiety in mice, and abrogated the sensitivity of cannabinoid CB1 receptors (CB1Rs) controlling GABA synapses in the striatum. Identical behavioral and synaptic results were obtained following social defeat stress, and intracerebroventricular injection of IL-1 receptor antagonist reverted both effects. IL-1β-mediated inhibition of CB1R function was secondary to altered cholesterol composition within membrane lipid rafts, and required intact function of the transient receptor potential vanilloid 1 (TRPV1) channel, another element of the endocannabinoid system. Membrane lipid raft disruption and inhibition of cholesterol synthesis, in fact, abrogated IL-1β–CB1R coupling, and TRPV1−/− mice were indeed insensitive to the synaptic and behavioral effects of both IL-1β and stress. On the other hand, cholesterol enrichment of striatal slices mimicked the synaptic effects of IL-1β on CB1Rs only in control mice, while the same treatment was ineffective in slices prepared from TRPV1−/− mice. The present investigation identifies a previously unrecognized interaction between a major proinflammatory cytokine and the endocannabinoid system in the pathophysiology of anxiety.

New insights on the role of free d-aspartate in the mammalian brain

Free d-aspartate (d-Asp) occurs in substantial amounts in the brain at the embryonic phase and in the first few postnatal days, and strongly decreases in adulthood. Temporal reduction of d-Asp levels depends on the postnatal onset of d-aspartate oxidase (DDO) activity, the only enzyme able to selectively degrade this d-amino acid. Several results indicate that d-Asp binds and activates N-methyl-d-aspartate receptors (NMDARs). Accordingly, recent studies have demonstrated that deregulated, higher levels of d-Asp, in knockout mice for Ddo gene and in d-Asp-treated mice, modulate hippocampal NMDAR-dependent long-term potentiation (LTP) and spatial memory. Moreover, similarly to d-serine, administration of d-Asp to old mice is able to rescue the physiological age-related decay of hippocampal LTP. In agreement with a neuromodulatory action of d-Asp on NMDARs, increased levels of this d-amino acid completely suppress long-term depression at corticostriatal synapses and attenuate the prepulse inhibition deficits produced in mice by the psychotomimetic drugs, amphetamine and MK-801. Based on the evidence which points to the ability of d-Asp to act as an endogenous agonist on NMDARs and considering the abundance of d-Asp during prenatal and early life, future studies will be crucial to address the effect of this molecule in the developmental processes of the brain controlled by the activation of NMDARs.

Brain-Derived Neurotrophic Factor Controls Cannabinoid CB1 Receptor Function in the Striatum

The role of brain-derived neurotrophic factor (BDNF) in emotional processes suggests an interaction with the endocannabinoid system. Here, we addressed the functional interplay between BDNF and cannabinoid CB1 receptors (CB1Rs) in the striatum, a brain area in which both BDNF and CB1s play a role in the emotional consequences of stress and of rewarding experiences. BDNF potently inhibited CB1R function in the striatum, through a mechanism mediated by altered cholesterol metabolism and membrane lipid raft function. The effect of BDNF was restricted to CB1Rs controlling GABA-mediated IPSCs (CB1R(GABA)), whereas CB1Rs modulating glutamate transmission and GABAB receptors were not affected. The action of BDNF on CB1R(GABA) function was tyrosine kinase dependent and was complete even after receptor sensitization with cocaine or environmental manipulations activating the dopamine (DA)-dependent reward system. In mice lacking one copy of the BDNF gene (BDNF+/−), CB1R(GABA) responses were potentiated and were preserved from the action of haloperidol, a DA D2 receptor (D2R) antagonist able to fully abolish CB1R(GABA) function in rewarded animals. Haloperidol also enhanced BDNF levels in the striatum, suggesting that this neurotrophin may act as a downstream effector of D2Rs in the modulation of cannabinoid signaling. Accordingly, 5 d cocaine exposure both reduced striatal BDNF levels and increased CB1R(GABA) activity, through a mechanism dependent on D2Rs. The present study identifies a novel mechanism of CB1R regulation mediated by BDNF and cholesterol metabolism and provides some evidence that DA D2R-dependent modulation of striatal CB1R activity is mediated by this neurotrophin.

Decreased levels of d-aspartate and NMDA in the prefrontal cortex and striatum of patients with schizophrenia

The potential implication of a decrease in the function of N-methyl-d-aspartate receptors (NMDARs) in the pathophysiology of schizophrenia has long been hypothesised. Accordingly, compounds that inhibit the glycine-1 transporter or target the glycine-binding site of NMDARs, including the co-agonists D-serine and glycine, have shown promise in treating the symptoms of schizophrenia. Clinical interest for d-serine has also been supported by evidence for its abnormal metabolism in schizophrenic patients. Together with D-serine, another D-form amino acid, D-aspartate, exists in the brain of mammals. Synthesised by the enzyme aspartate racemase, D-aspartate is highly concentrated in the prenatal brain; after birth, its levels sharply decrease due to the catabolising activity of the enzyme D-aspartate oxidase. D-aspartate is able to stimulate NMDAR-dependent neurotransmission through direct action at the glutamate-binding site of NMDARs, thus functioning as an endogenous agonist for this subclass of glutamate receptors. In this study, we evaluated for the first time the content of D-aspartate and of its derivative, NMDA, in the post-mortem prefrontal cortex and striatum of schizophrenic patients. Moreover, in the same brain samples, we analysed the expression levels of the subunits that form NMDARs, which are the in vivo targets of D-aspartate and NMDA. Interestingly, we found that D-aspartate and NMDA are consistently decreased in schizophrenia brains compared to control brains. In the prefrontal cortex, this decrease is correlated with a marked downregulation of NMDAR subunits. Overall, these results agree with the innovative therapeutic research in schizophrenia that is aimed at targeting glutamatergic transmission via D-amino acids.