Emerging targets for addiction neuropharmacology: From mechanisms to therapeutics

Drug abuse represents a considerable burden of disease and has enormous economic impacts on societies. Over the years, few medications have been developed for clinical use. Their utilization is endowed with several limitations, including partial efficacy or significant side effects. On the other hand, the successful advancement of these compounds provides an important proof of concept for the feasibility of drug development programs in addiction. In recent years, a wealth of information has been generated on the psychological mechanisms, genetic or epigenetic predisposing factors, and neurobiological adaptations induced by drug consumption that interact with each other to contribute to disease progression. It is now clear that addiction develops through phases, from initial recreational use to excessive consumption and compulsive drug seeking, with a shift from positive to negative reinforcement driving motivated behaviors. A greater understanding of these mechanisms has opened new vistas in drug development programs. Researchers' attention has been shifted from investigation of classical targets associated with reward to biological substrates responsible for negative reinforcement, impulse loss of control, and maladaptive mechanisms resulting from protracted drug use. From this research, several new biological targets for the development of innovative therapies have started to emerge. This chapter offers an overview of targets currently under scrutiny for the development of new medications for addiction. This work is not exhaustive but rather it provides a few examples of how this research has advanced in recent years by virtue of studies carried out in our laboratory

Heterodimers of photoreceptor-specific nuclear receptor (PNR/NR2E3) and peroxisome proliferator-activated receptor (PPARγ) are disrupted by retinal disease-associated mutations

Photoreceptor-specific nuclear receptor (PNR/NR2E3) and Tailless homolog (TLX/NR2E1) are human orthologs of the NR2E group, a subgroup of phylogenetically related members of the Nuclear Receptor (NR) superfamily of transcription factors. We assessed the ability of these NRs to form heterodimers with other members of the human NRs representing all major subgroups. The TLX ligand binding domain (LBD) did not appear to form homodimers or interact directly with any other NR tested. The PNR LBD was able to form homodimers, but also exhibited robust interactions with the LBDs of PPARγ/NR1C3 and TRβ/NR1A2. The binding of PNR to PPARγ was specific for this paralog, as no interaction was observed with the LBDs of PPARαNR1C1 or PPARδNR1C2. In support of these findings, PPARγ and PNR were found to be co-expressed in human retinal tissue extracts and could be co-immunoprecipitated as a native complex. Selected sequence variants in the PNR LBD associated with human retinopathies, or a mutation in the dimerization region of PPARγ LBD associated with familial partial lipodystrophy type 3, were found to disrupt PNR/PPARγ complex formation. Wild type PNR, but not a PNR309G mutant, was able to repress PPARγ-mediated transcription in reporter assays. In summary our results reveal novel heterodimer interactions in the NR superfamily, suggesting previously unknown functional interactions of PNR with PPARγ and TRβ that have potential importance in retinal development and disease

Structural insights into regulation of nuclear receptors by ligands

Nuclear receptors are DNA-binding transcription factors, the transcriptional function of many of which depends on the binding of ligands, a feature that distinguishes nuclear receptors from other transcription factors. This review will summarize recent advances in our knowledge of the interaction between selected nuclear receptors and their cognate ligands

Pex11p Plays a Primary Role in Medium-Chain Fatty Acid Oxidation, a Process That Affects Peroxisome Number and Size in Saccharomyces cerevisiae

The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p has previously been implicated in peroxisome proliferation based on morphological observations of PEX11 mutant cells. Pex11p-deficient cells fail to increase peroxisome number in response to growth on fatty acids and instead accumulate a few giant peroxisomes. We report that mutants deficient in genes required for medium-chain fatty acid (MCFA) β-oxidation display the same phenotype as Pex11p-deficient cells. Upon closer inspection, we found that Pex11p is required for MCFA β-oxidation. Disruption of the PEX11 gene results in impaired formation of MCFA-CoA esters as measured in intact cells, whereas their formation is normal in cell lysates. The sole S. cerevisiae MCFA-CoA synthetase (Faa2p) remains properly localized to the inner leaflet of the peroxisomal membrane in PEX11 mutant cells. Therefore, the in vivo latency of MCFA activation observed in Pex11p-deficient cells suggests that Pex11p provides Faa2p with substrate. When PEX11 mutant cells are shifted from glucose to oleate-containing medium, we observed an immediate deficiency in β-oxidation of MCFAs whereas giant peroxisomes and a failure to increase peroxisome abundance only became apparent much later. Our observations suggest that the MCFA oxidation pathway regulates the level of a signaling molecule that modulates the number of peroxisomal structures in a cell

P465L-PPARγ mutation confers partial resistance to the hypolipidaemic action of fibrates

Familial partial lipodystrophic syndrome 3 (FPLD3) is associated with mutations in the transcription factor PPAR. One of these mutations, the P467L, confers a dominant negative effect. We and others have previously investigated the pathophysiology associated to this mutation using a humanised mouse model that recapitulated most of the clinical symptoms observed in human patients under different experimental conditions. One of the key clinical manifestations observed both in humans and mouse models is the ectopic accumulation of fat in the liver. Here, we dissect the molecular mechanisms that contribute to the excessive accumulation of lipids in the liver and characterise the negative effect of this PPAR mutation on the activity of PPAR in vivo when activated by fibrates. P465L mice have increased levels of insulin and free fatty acids (FFA), exhibit decreased levels of Very Low Density Lipoproteins (VLDL) when fed high fat diet (HFD) and partial impaired response to the hypolipidemic action of WY14643. This indicates that the deleterious effects of P465L-PPAR mutation may be augmented by their collateral negative effect on PPAR function.Wellcome Trust, MRC MDU (MC_UU_12012/2), FP7-MITIN (Integration of the System Models of Mitochondrial Function and Insulin Signaling and its Application in the Study of Complex Diseases) (Grant Agreement 223450) and H2020 EPoS (Elucidating Pathways of Steatohepatitis) (Grant Agreement 634413). Disease Model Core, Biochemistry Assay Lab and the Histology Core are funded by MRC_MC_UU_12012/5 and a Wellcome Trust Strategic Award [100574/Z/12/Z

Cordycepin Suppresses Expression of Diabetes Regulating Genes by Inhibition of Lipopolysaccharide-induced Inflammation in Macrophages

Background: It has been recently noticed that type 2 dia-betes (T2D), one of the most common metabolic diseases, causes a chronic low-grade inflammation and activation of the innate immune system that are closely involved in the pathogenesis of T2D. Cordyceps militaris, a traditional me-dicinal mushroom, produces a component compound, cordy-cepin (3’-deoxyadenosine). Cordycepin has been known to have many pharmacological activities including immuno-logical stimulating, anti-cancer, and anti-infection activities. The molecular mechanisms of cordycepin in T2D are not clear. In the present study, we tested the role of cordycepin on the anti-diabetic effect and anti-inflammatory cascades in LPS-stimulated RAW 264.7 cells. Methods: We confirmed the levels of diabetes regulating genes mRNA and protein of cytokines through RT-PCR and western blot analysis and fol

Effect of fenofibrate on microcirculation and wound healing in healthy and diabetic mice

<p>Abstract</p> <p>Objective</p> <p>Disturbances in wound healing in patients with hyperglycaemic blood sugar values are a common clinical problem. Recent studies identified PPARα-ligands as potential skin therapeutic agents. The aim of this study was to investigate the effects of oral fenofibrate treatment on dermal wound healing and micro-circulatory parameters in diabetic mice.</p> <p>Methods</p> <p>Dermal wounds were created in CD-1 mice. Mice were randomized into four treatment groups: diabetic mice treated (dbf) or not-treated with fenofibrate (dbnf). As controls served non-diabetic mice treated (ndf) or not-treated with fenofibrate (ndnf). At various points in time microcirculation was analyzed by intravital fluorescent microscopy to determine wound surface area, vessel diameter, plasma leakage, functional capillary density, and leukocyte/endothelium interaction.</p> <p>Results</p> <p>The dbf-mice showed a significantly increased diameter of the venules and the arterioles up to 3 days after wound creation compared to dbnf-mice. However, wound healing was not improved in dbf-compared to dbnf-mice. Surprisingly, all microcirculatory parameter (vessel diameter, plasma leakage and functional capillary density) were not deteriorated in dbnf-compared to ndnf-mice.</p> <p>Conclusion</p> <p>We confirm that high blood sugar values lead to a delayed wound healing, but this could not traced back to altered microcirculatory patterns. Furthermore, in dbf-mice an improved vasodilatatory function of small vessels could be detected, but with no substantial effect on wound healing. Further studies are needed to clarify, if topical application of fenofibrate might be beneficial.</p

A semi-quantitative RT-PCR method to measure the in vivo effect of dietary conjugated linoleic acid on porcine muscle PPAR gene expression

Conjugated linoleic acid (CLA) can activate (in vitro) the nuclear transcription factors known as the peroxisome proliferators activated receptors (PPAR). CLA was fed at 11 g CLA/kg of feed for 45d to castrated male pigs (barrows) to better understand long term effects of PPAR activation in vivo. The barrows fed CLA had lean muscle increased by 3.5% and overall fat reduced by 9.2% but intramuscular fat (IMF %) was increased by 14% (P < 0.05). To measure the effect of long term feeding of CLA on porcine muscle gene expression, a semi-quantitative RT-PCR method was developed using cDNA normalized against the housekeeping genes cyclophilin and β-actin. This method does not require radioactivity or expensive PCR instruments with real-time fluorescent detection. PPARγ and the PPAR responsive gene AFABP but not PPARα were significantly increased (P < 0.05) in the CLA fed pig’s muscle. PPARα and PPARγ were also quantitatively tested for large differences in gene expression by western blot analysis but no significant difference was detected at this level. Although large differences in gene expression of the PPAR transcriptional factors could not be confirmed by western blotting techniques. The increased expression of AFABP gene, which is responsive to PPAR transcriptional factors, confirmed that dietary CLA can induce a detectable increase in basal PPAR transcriptional activity in the live animal

Downregulation of peroxisome proliferator-activated receptors (PPARs) in nasal polyposis

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR) α, βδ and γ are nuclear receptors activated by fatty acid metabolites. An anti-inflammatory role for these receptors in airway inflammation has been suggested. METHODS: Nasal biopsies were obtained from 10 healthy volunteers and 10 patients with symptomatic allergic rhinitis. Nasal polyps were obtained from 22 patients, before and after 4 weeks of local steroid treatment (fluticasone). Real-time RT-PCR was used for mRNA quantification and immunohistochemistry for protein localization and quantification. RESULTS: mRNA expression of PPARα, PPARβδ, PPARγ was found in all specimens. No differences in the expression of PPARs were obtained in nasal biopsies from patients with allergic rhinitis and healthy volunteers. Nasal polyps exhibited lower levels of PPARα and PPARγ than normal nasal mucosa and these levels were, for PPARγ, further reduced following steroid treatment. PPARγ immunoreactivity was detected in the epithelium, but also found in smooth muscle of blood vessels, glandular acini and inflammatory cells. Quantitative evaluation of the epithelial immunostaining revealed no differences between nasal biopsies from patients with allergic rhinitis and healthy volunteers. In polyps, the PPARγ immunoreactivity was lower than in nasal mucosa and further decreased after steroid treatment. CONCLUSION: The down-regulation of PPARγ, in nasal polyposis but not in turbinates during symptomatic seasonal rhinitis, suggests that PPARγ might be of importance in long standing inflammations

Effects of Conjugated Linoleic Acid, Fish Oil and Soybean Oil on PPARs (α & γ) mRNA Expression in Broiler Chickens and Their Relation to Body Fat Deposits

An experiment was conducted on broiler chickens to study the effects of different dietary fats (Conjugated linoleic acid (CLA), fish oil, soybean oil, or their mixtures, as well as palm oil, as a more saturated fat), with a as fed dose of 7% for single fat and 3.5 + 3.5% for the mixtures, on Peroxisome Proliferator-Activated Receptors (PPARs) gene expression and its relation with body fat deposits. The CLA used in this experiment was CLA LUTA60 which contained 60% CLA, so 7% and 3.5% dietary inclusions of CLA LUTA60 were equal to 4.2% and 2.1% CLA, respectively. Higher abdominal fat pad was found in broiler chickens fed with a diet containing palm oil compared to chickens in the other experimental groups (P ≤ 0.05). The diets containing CLA resulted in an increased fat deposition in the liver of broiler chickens (P ≤ 0.05). The only exception was related to the birds fed with diets containing palm oil or fish oil + soybean oil, where contents of liver fat were compared to the CLA + fish oil treatment. PPARγ gene in adipose tissue of chickens fed with palm oil diet was up-regulated compared to other treatments (P ≤ 0.001), whereas no significant differences were found in adipose PPARγ gene expression between chickens fed with diets containing CLA, fish oil, soybean oil or the mixture of these fats. On the other hand, the PPARα gene expression in liver tissue was up-regulated in response to the dietary fish oil inclusion and the differences were also significant for both fish oil and CLA + fish oil diets compared to the diets with palm oil, soybean oil or CLA as the only oil source (P ≤ 0.001). In conclusion, the results of present study showed that there was a relationship between the adipose PPARγ gene up-regulation and abdominal fat pad deposition for birds fed with palm oil diet, while no deference was detected in n-3 and n-6 fatty acids, as well as CLA on PPARγ down regulation in comparison to a more saturated fat. When used on its own, fish oil was found to be a more effective fat in up-regulating hepatic PPARα gene expression and this effect was related to a less fat deposition in liver tissue. A negative correlation coefficient (−0.3) between PPARα relative gene expression and liver tissue fat content confirm the anti-lipogenic effect of PPARα, however, the change in these parameters was not completely parallel