Mechanism and therapeutic targets of the involvement of a novel lysosomal proton channel TMEM175 in Parkinson's disease.

Parkinson's disease (PD), recognized as the second most prevalent neurodegenerative disease in the aging population, presents a significant challenge due to the current lack of effective treatment methods to mitigate its progression. Many pathogenesis of PD are related to lysosomal dysfunction. Moreover, extensive genetic studies have shown a significant correlation between the lysosomal membrane protein TMEM175 and the risk of developing PD. Building on this discovery, TMEM175 has been identified as a novel potassium ion channel. Intriguingly, further investigations have found that potassium ion channels gradually close and transform into hydrion "excretion" channels in the microenvironment of lysosomes. This finding was further substantiated by studies on TMEM175 knockout mice, which exhibited pronounced motor dysfunction in pole climbing and suspension tests, alongside a notable reduction in dopamine neurons within the substantia nigra compacta. Despite these advancements, the current research landscape is not without its controversies. In light of this, the present review endeavors to methodically examine and consolidate a vast array of recent literature on TMEM175. This comprehensive analysis spans from the foundational research on the structure and function of TMEM175 to expansive population genetics studies and mechanism research utilizing cellular and animal models.A thorough understanding of the structure and function of TMEM175, coupled with insights into the intricate mechanisms underpinning lysosomal dysfunction in PD dopaminergic neurons, is imperative. Such knowledge is crucial for pinpointing precise intervention targets, thereby paving the way for novel therapeutic strategies that could potentially alter the neurodegenerative trajectory of PD.

Causal relationship between circulating insulin-like growth factor-1 and Parkinson's disease: a two-sample Mendelian randomization study.

Background: Linear associations between circulating insulin-like growth factor-1 (IGF-1) levels and Parkinson's disease (PD) have been evidenced in observational studies. Yet, the causal relationship between IGF-1 levels and PD remains obscure. We conducted Mendelian randomization to examine the correlation between genetically predicted IGF-1 levels and PD. Methods: By reviewing genome-wide association studies (GWAS) that are publicly accessible, we uncovered SNPs linked to both serum concentrations of IGF-1 and PD. A two-sample Mendelian randomization (MR) analysis was carried out to evaluate the individual effect of IGF-1 on PD. Results: In a primary causal effects model in MR analysis, employing the inverse-variance weighted (IVW) method, IGF-1 levels exhibited a notable association with the risk of PD (OR, 1.020, 95% CI, 1.003-1.038, p = 0.0215). Multiple evaluations revealed that horizontal pleiotropy was improbable to distort the main results (MR-Egger: P PD intercept =0.719), and no bias was detected by leave-one-out analysis. Conclusion: This study unearthed evidence indicating that heightened IGF-1 levels might be causally correlated with an increased risk of PD.

Flexible Large-Area Graphene Films of 50-600 nm Thickness with High Carrier Mobility.

Bulk graphene nanofilms feature fast electronic and phonon transport in combination with strong light-matter interaction and thus have great potential for versatile applications, spanning from photonic, electronic, and optoelectronic devices to charge-stripping and electromagnetic shielding, etc. However, large-area flexible close-stacked graphene nanofilms with a wide thickness range have yet to be reported. Here, we report a polyacrylonitrile-assisted 'substrate replacement' strategy to fabricate large-area free-standing graphene oxide/polyacrylonitrile nanofilms (lateral size ~ 20 cm). Linear polyacrylonitrile chains-derived nanochannels promote the escape of gases and enable macro-assembled graphene nanofilms (nMAGs) of 50-600 nm thickness following heat treatment at 3,000 °C. The uniform nMAGs exhibit 802-1,540 cm2 V-1 s-1 carrier mobility, 4.3-4.7 ps carrier lifetime, and > 1,581 W m-1 K-1 thermal conductivity (nMAG-assembled 10 µm-thick films, mMAGs). nMAGs are highly flexible and show no structure damage even after 1.0 × 105 cycles of folding-unfolding. Furthermore, nMAGs broaden the detection region of graphene/silicon heterojunction from near-infrared to mid-infrared and demonstrate higher absolute electromagnetic interference (EMI) shielding effectiveness than state-of-the-art EMI materials of the same thickness. These results are expected to lead to the broad applications of such bulk nanofilms, especially as micro/nanoelectronic and optoelectronic platforms.

Discovery of Potent and Selective MTH1 Inhibitors for Oncology: Enabling Rapid Target (In)Validation.

We describe the discovery of three structurally differentiated potent and selective MTH1 inhibitors and their subsequent use to investigate MTH1 as an oncology target, culminating in target (in)validation. Tetrahydronaphthyridine 5 was rapidly identified as a highly potent MTH1 inhibitor (IC50 = 0.043 nM). Cocrystallization of 5 with MTH1 revealed the ligand in a Φ-cis-N-(pyridin-2-yl)acetamide conformation enabling a key intramolecular hydrogen bond and polar interactions with residues Gly34 and Asp120. Modification of literature compound TH287 with O- and N-linked aryl and alkyl aryl substituents led to the discovery of potent pyrimidine-2,4,6-triamine 25 (IC50 = 0.49 nM). Triazolopyridine 32 emerged as a highly selective lead compound with a suitable in vitro profile and desirable pharmacokinetic properties in rat. Elucidation of the DNA damage response, cell viability, and intracellular concentrations of oxo-NTPs (oxidized nucleoside triphosphates) as a function of MTH1 knockdown and/or small molecule inhibition was studied. Based on our findings, we were unable to provide evidence to further pursue MTH1 as an oncology target.

An amorphous carbon nitride/NiO/CoN-based composite: a highly efficient nonprecious electrode for supercapacitors and the oxygen evolution reaction.

Due to their features of low cost, good corrosion resistance and environmental friendliness, transition metal oxides/nitrides are among the most promising materials for energy storage and conversion. Meanwhile, graphitic carbon nitride is a non-metallic polymer that has been widely used in the environmental and energy conversion fields due to its abundant precursor species and simple process of synthesis. In this study, an amorphous carbon nitride/NiO/CoN-based composite (Ni-Co-CN) is in situ fabricated via simple one-step pyrolysis; it displays high capacitive performance and efficient electrocatalytic capability for the oxygen evolution reaction (OER). Specifically, the optimized Ni-Co-CN electrode shows an ultra-high areal specific capacitance of 18.8 F cm-2 at 2 mA cm-2 in 3 M KOH electrolyte, and it retains 91.4% of its areal specific capacitance even after 10 000 cycles of CV scans. Upon being used as an electrocatalyst in the OER process, the overpotential of Ni-Co-CN can reach 195 mV versus a reference hydrogen electrode (RHE) at 10 mA cm-2, which is far lower than those of most reported Ni/Co-based catalysts. Additionally, the potential loss of Ni-Co-CN electrode is less than 1% after a long-term durability test over 60 h. The experimental results integrated with density functional theoretical calculations reveal that the excellent performance of the Ni-Co-CN self-supported electrode can be ascribed to the fast redox reduction of multi-valent transition metal ions, abundant surface defects and plentiful nano-scaled porous structures. This work provides a promising strategy for exploring methods to combine economic Ni/Co-based compounds with carbon-based materials to obtain low-cost yet efficient electrode materials for electrochemical energy storage and conversion.

Excellent Adsorptive Behaviors of ZnO Nanoparticle-Deposited Activated Carbon Covered with Nano-Graphene Oxide.

In the paper, ZnO-deposited activated carbon composite (ZnO-AC) was firstly prepared in a simple two-step preparation process, and then covered with nano-graphene oxide to give the NGO-ZnOAC composite. The successful deposition of ZnO and NGO on the AC surface was demonstrated by various experiments, and the ZnO nanoparticles showed a mean diameter size mainly within about 10 nm. The specific surface area of the NGO-AC and NGO-ZnO-AC decreased from 67.74 m²/g of the parent AC to 32.54 and 11.43 m²/g, respectively. The fabricated NGO-ZnO-AC showed excellent adsorptive behaviors towards CrO2-4 and Cu2+ ions, outperforming both ZnO-AC and AC.

EDTA-bonded multi-connected carbon-dots and their Eu3+ complex: preparation and optical properties.

EDTA-bonded multi-connected carbon-dots (EDTA-C-dots) were prepared from carbon dot precursors and complexed with Eu3+ to give Eu3+-coordinated EDTA-bonded multi-connected carbon dots (Eu-EDTA-C-dots). Whereas EDTA-C-dots were readily soluble in DMSO, Eu-EDTA-C-dots could not be easily dissolved in DMSO, water, or other common organic solvents. The newly prepared materials were thoroughly characterized. The X-ray diffraction results showed that no crystalline phase of Eu oxides (europium oxide or europium hydroxide) could be observed in Eu-EDTA-C-dots. The infrared and UV-Vis spectra showed that coordination with Eu3+ ions did not damage the structure of the EDTA-C-dots. It was found that EDTA could be easily grafted on the surface of carbon dots and EDTA had minimal influence on the photoluminescence of the carbon dot matrix. In contrast, the existence of Eu3+ ions strongly quenched the photoluminescence of Eu-EDTA-C-dots. The measured and fitted decay lifetime indicated that Eu-EDTA-C-dots possessed two photoluminescence decay processes, i.e., radiative recombination and non-radiative recombination.

Facile and green fabrication of cellulosed based aerogels for lampblack filtration from waste newspaper.

In this study, the lightweight, hydrophobic and porous cellulose-based aerogels (CAGs) were synthesized through a freeze-drying process using waste newspaper as the only raw material. After crosslinking with glutaraldehyde and treatment with trimethylchlorosilane (TMCS) using a simple thermal chemical vapor deposition process, the resulting CAGs became hydrophobic and oleophilic. Furthermore, the as-prepared CAGs exhibited a low density (17.4-28.7mgcm-3) and mesoporous inner-structure. All these properties attributed the novel aerogel not only with a good adsorption capability of oils and organic solvents, including kerosene, nitrobenzene, and chloroform, but also an excellent filtration capacity of lampblack.

Targeting LOXL2 for cardiac interstitial fibrosis and heart failure treatment.

Interstitial fibrosis plays a key role in the development and progression of heart failure. Here, we show that an enzyme that crosslinks collagen-Lysyl oxidase-like 2 (Loxl2)-is essential for interstitial fibrosis and mechanical dysfunction of pathologically stressed hearts. In mice, cardiac stress activates fibroblasts to express and secrete Loxl2 into the interstitium, triggering fibrosis, systolic and diastolic dysfunction of stressed hearts. Antibody-mediated inhibition or genetic disruption of Loxl2 greatly reduces stress-induced cardiac fibrosis and chamber dilatation, improving systolic and diastolic functions. Loxl2 stimulates cardiac fibroblasts through PI3K/AKT to produce TGF-ß2, promoting fibroblast-to-myofibroblast transformation; Loxl2 also acts downstream of TGF-ß2 to stimulate myofibroblast migration. In diseased human hearts, LOXL2 is upregulated in cardiac interstitium; its levels correlate with collagen crosslinking and cardiac dysfunction. LOXL2 is also elevated in the serum of heart failure (HF) patients, correlating with other HF biomarkers, suggesting a conserved LOXL2-mediated mechanism of human HF.

Inhibitions of late INa and CaMKII act synergistically to prevent ATX-II-induced atrial fibrillation in isolated rat right atria.

AIMS: Increases in late Na(+) current (late INa) and activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) are associated with atrial arrhythmias. CaMKII also phosphorylates Nav1.5, further increasing late INa. The combination of a CaMKII inhibitor with a late INa inhibitor may be superior to each compound alone to suppress atrial arrhythmias. Therefore, we investigated the effect of a CaMKII inhibitor in combination with a late INa inhibitor on anemone toxin II (ATX-II, a late INa enhancer)-induced atrial arrhythmias. METHODS AND RESULTS: Rat right atrial tissue was isolated and preincubated with either the CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP), the late INa inhibitor GS458967, or both, and then exposed to ATX-II. ATX-II increased diastolic tension and caused fibrillation of isolated right atrial tissue. AIP (0.3µmol/L) and 0.1µmol/L GS458967 alone inhibited ATX-II-induced arrhythmias by 20±3% (mean±SEM, n=14) and 34±5% (n=13), respectively, whereas the two compounds in combination inhibited arrhythmias by 81±4% (n=10, p<0.05, vs either AIP or GS458967 alone or the calculated sum of individual effects of both compounds). AIP and GS458967 also attenuated the ATX-induced increase of diastolic tension. Consistent with the mechanical and electrical data, 0.3µmol/L AIP and 0.1µmol/L GS458967 each inhibited ATX-II-induced CaMKII phosphorylation by 23±3% and 32±4%, whereas the combination of both compounds inhibited CaMKII phosphorylation completely. CONCLUSION: The effects of an enhanced late INa to induce arrhythmic activity and activation of CaMKII in atria are attenuated synergistically by inhibitors of late INa and CaMKII.

Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII.

AIMS: Enhanced cardiac late Na current (late INa) and increased sarcoplasmic reticulum (SR)-Ca(2+)-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late INa and SR-Ca(2+)-leak in atrial cardiomyocytes (CMs). METHODS AND RESULTS: In murine atrial CMs, SR-Ca(2+)-leak was increased by the late INa enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca(2+)/calmodulin-dependent protein kinase II (Autocamide-2-related inhibitory peptide), protein kinase A (H89), or late INa (Ranolazine or Tetrodotoxin) all prevented ATX-II-dependent SR-Ca(2+)-leak. The SR-Ca(2+)-leak induction by ATX-II was not detected when either the Na(+)/Ca(2+) exchanger was inhibited (KBR) or in CaMKIIδc-knockout mice. FRET measurements revealed increased cAMP levels upon ATX-II stimulation, which could be prevented by inhibition of adenylyl cyclases (ACs) 5 and 6 (NKY 80) but not by inhibition of phosphodiesterases (IBMX), suggesting PKA activation via an AC-dependent increase of cAMP levels. Western blots showed late INa-dependent hyperphosphorylation of CaMKII as well as PKA target sites at ryanodine receptor type-2 (-S2814 and -S2808) and phospholamban (-Thr17, -S16). Enhancement of late INa did not alter Ca(2+)-transient amplitude or SR-Ca(2+)-load. However, upon late INa activation and simultaneous CaMKII inhibition, Ca(2+)-transient amplitude and SR-Ca(2+)-load were increased, whereas PKA inhibition reduced Ca(2+)-transient amplitude and load and additionally slowed Ca(2+) elimination. In atrial CMs from patients with atrial fibrillation, inhibition of late INa, CaMKII, or PKA reduced the SR-Ca(2+)-leak. CONCLUSION: Late INa exerts distinct effects on Ca(2+) homeostasis in atrial myocardium through activation of CaMKII and PKA. Inhibition of late INa represents a potential approach to attenuate CaMKII activation and decreases SR-Ca(2+)-leak in atrial rhythm disorders. The interconnection with the cAMP/PKA system further increases the antiarrhythmic potential of late INa inhibition.

Thioredoxin-2 inhibits mitochondrial reactive oxygen species generation and apoptosis stress kinase-1 activity to maintain cardiac function.

BACKGROUND: Thioredoxin 2 (Trx2) is a key mitochondrial protein that regulates cellular redox and survival by suppressing mitochondrial reactive oxygen species generation and by inhibiting apoptosis stress kinase-1 (ASK1)-dependent apoptotic signaling. To date, the role of the mitochondrial Trx2 system in heart failure pathogenesis has not been investigated. METHODS AND RESULTS: Western blot and histological analysis revealed that Trx2 protein expression levels were reduced in hearts from patients with dilated cardiomyopathy, with a concomitant increase in ASK1 phosphorylation/activity. Cardiac-specific Trx2 knockout mice develop spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, reduced ventricular wall thickness, and a progressive decline in left ventricular contractile function, resulting in mortality due to heart failure by ≈4 months of age. The progressive decline in cardiac function observed in cardiac-specific Trx2 knockout mice was accompanied by the disruption of mitochondrial ultrastructure, mitochondrial membrane depolarization, increased mitochondrial reactive oxygen species generation, and reduced ATP production, correlating with increased ASK1 signaling and increased cardiomyocyte apoptosis. Chronic administration of a highly selective ASK1 inhibitor improved cardiac phenotype and reduced maladaptive left ventricular remodeling with significant reductions in oxidative stress, apoptosis, fibrosis, and cardiac failure. Cellular data from Trx2-deficient cardiomyocytes demonstrated that ASK1 inhibition reduced apoptosis and reduced mitochondrial reactive oxygen species generation. CONCLUSIONS: Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial reactive oxygen species production and ASK1-dependent apoptosis. Inhibition of ASK1 represents a promising therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure.

Intracellular Na+ overload causes oxidation of CaMKII and leads to Ca2+ mishandling in isolated ventricular myocytes.

An increase of late Na(+) current (INaL) in cardiac myocytes can raise the cytosolic Na(+) concentration and is associated with activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and alterations of mitochondrial metabolism and Ca(2+) handling by sarcoplasmic reticulum (SR). We tested the hypothesis that augmentation of INaL can increase mitochondrial reactive oxygen species (ROS) production and oxidation of CaMKII, resulting in spontaneous SR Ca(2+) release and increased diastolic Ca(2+) in myocytes. Increases of INaL and/or of the cytosolic Na(+) concentration led to mitochondrial ROS production and oxidation of CaMKII to cause dysregulation of Ca(2+) handling in rabbit cardiac myocytes.

Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM), the most common mendelian heart disorder, remains an orphan of disease-specific pharmacological treatment because of the limited understanding of cellular mechanisms underlying arrhythmogenicity and diastolic dysfunction. METHODS AND RESULTS: We assessed the electromechanical profile of cardiomyocytes from 26 HCM patients undergoing myectomy compared with those from nonfailing nonhypertrophic surgical patients by performing patch-clamp and intracellular Ca(2+) (Ca(2+)(i)) studies. Compared with controls, HCM cardiomyocytes showed prolonged action potential related to increased late Na(+) (I(NaL)) and Ca(2+) (I(CaL)) currents and decreased repolarizing K(+) currents, increased occurrence of cellular arrhythmias, prolonged Ca(2+)(i) transients, and higher diastolic Ca(2+)(i). Such changes were related to enhanced Ca(2+)/calmodulin kinase II (CaMKII) activity and increased phosphorylation of its targets. Ranolazine at therapeutic concentrations partially reversed the HCM-related cellular abnormalities via I(NaL) inhibition, with negligible effects in controls. By shortening the action potential duration in HCM cardiomyocytes, ranolazine reduced the occurrence of early and delayed afterdepolarizations. Finally, as a result of the faster kinetics of Ca(2+)(i) transients and the lower diastolic Ca(2+)(i), ranolazine accelerated the contraction-relaxation cycle of HCM trabeculae, ameliorating diastolic function. CONCLUSIONS: We highlighted a specific set of functional changes in human HCM myocardium that stem from a complex remodeling process involving alterations of CaMKII-dependent signaling, rather than being a direct consequence of the causal sarcomeric mutations. Among the several ion channel and Ca(2+)(i) handling proteins changes identified, an enhanced I(NaL) seems to be a major contributor to the electrophysiological and Ca(2+)(i) dynamic abnormalities of ventricular myocytes and trabeculae from patients with HCM, suggesting potential therapeutic implications of I(NaL) inhibition.

Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice.

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.

Inhibition of aldehyde dehydrogenase-2 suppresses cocaine seeking by generating THP, a cocaine use-dependent inhibitor of dopamine synthesis.

There is no effective treatment for cocaine addiction despite extensive knowledge of the neurobiology of drug addiction. Here we show that a selective aldehyde dehydrogenase-2 (ALDH-2) inhibitor, ALDH2i, suppresses cocaine self-administration in rats and prevents cocaine- or cue-induced reinstatement in a rat model of cocaine relapse-like behavior. We also identify a molecular mechanism by which ALDH-2 inhibition reduces cocaine-seeking behavior: increases in tetrahydropapaveroline (THP) formation due to inhibition of ALDH-2 decrease cocaine-stimulated dopamine production and release in vitro and in vivo. Cocaine increases extracellular dopamine concentration, which activates dopamine D2 autoreceptors to stimulate cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) in primary ventral tegmental area (VTA) neurons. PKA and PKC phosphorylate and activate tyrosine hydroxylase, further increasing dopamine synthesis in a positive-feedback loop. Monoamine oxidase converts dopamine to 3,4-dihydroxyphenylacetaldehyde (DOPAL), a substrate for ALDH-2. Inhibition of ALDH-2 enables DOPAL to condense with dopamine to form THP in VTA neurons. THP selectively inhibits phosphorylated (activated) tyrosine hydroxylase to reduce dopamine production via negative-feedback signaling. Reducing cocaine- and craving-associated increases in dopamine release seems to account for the effectiveness of ALDH2i in suppressing cocaine-seeking behavior. Selective inhibition of ALDH-2 may have therapeutic potential for treating human cocaine addiction and preventing relapse.

Suppression of heavy drinking and alcohol seeking by a selective ALDH-2 inhibitor.

BACKGROUND: Inherited human aldehyde dehydrogenase 2 (ALDH-2) deficiency reduces the risk for alcoholism. Kudzu plants and extracts have been used for 1,000 years in traditional Chinese medicine to treat alcoholism. Kudzu contains daidzin, which inhibits ALDH-2 and suppresses heavy drinking in rodents. Decreased drinking due to ALDH-2 inhibition is attributed to aversive properties of acetaldehyde accumulated during alcohol consumption. However, daidzin can reduce drinking in some rodents without necessarily increasing acetaldehyde. Therefore, a selective ALDH-2 inhibitor might affect other metabolic factors involved in regulating drinking. METHODS: Aldehyde dehydrogenase 2 inhibitors were synthesized based on the co-crystal structure of ALDH-2 and daidzin. We tested the efficacy of a highly selective reversible ALDH-2 inhibitor, CVT-10216, in models of moderate and high alcohol drinking rats. We studied 2-bottle choice and deprivation-induced drinking paradigms in Fawn Hooded (FH) rats, operant self-administration in Long Evans (LE), FH, and inbred P (iP) rats and in cue-induced reinstatement in iP rats. We also assayed blood acetaldehyde levels as well as dopamine (DA) release in the nucleus accumbens (NAc) and tested possible rewarding/aversive effects of the inhibitor in a conditioned place preference (CPP) paradigm. RESULTS: CVT-10216 increases acetaldehyde after alcohol gavage and inhibits 2-bottle choice alcohol intake in heavy drinking rodents, including deprivation-induced drinking. Moreover, CVT-10216 also prevents operant self-administration and eliminates cue-induced reinstatement of alcohol seeking even when alcohol is not available (i.e., no acetaldehyde). Alcohol stimulates DA release in the NAc, which is thought to contribute to increased drinking and relapse in alcoholism. CVT-10216 prevents alcohol-induced increases in NAc DA without changing basal levels. CVT-10216 does not show rewarding or aversive properties in the CPP paradigm at therapeutic doses. CONCLUSION: Our findings suggest that selective reversible ALDH-2 inhibitors may have therapeutic potential to reduce excessive drinking and to suppress relapse in abstinent alcoholics.

Up-regulation of AGS3 during morphine withdrawal promotes cAMP superactivation via adenylyl cyclase 5 and 7 in rat nucleus accumbens/striatal neurons.

Effective medical treatment of opiate addiction is limited by a high relapse rate in abstinent addicts. Opiate withdrawal causes cAMP superactivation, but the underlying molecular mechanisms are not clear. Recent evidence implicates an activator of G-protein signaling 3 (AGS3) in opiate addiction. We found previously that during a 10-min activation of opioid receptors, AGS3 binds G alpha(i)-GDP to promote free G betagamma stimulation of adenylyl cyclase (AC) 2 and 4, and/or inactivate G alpha(i) inhibitory function, thereby transiently enhancing cAMP-dependent protein kinase A (PKA) activity. In contrast, we report here that in nucleus accumbens/striatal neurons, morphine withdrawal induces cAMP superactivation, which requires up-regulation of AGS3. cAMP increases as a function of withdrawal time, by approximately 20% at 10 min and 75% at 5 h. However, cAMP superactivation does not require G betagamma. Instead, adenosine A2A receptor activation of G alpha(s/olf) seems to initiate cAMP superactivation and promote AGS3 up-regulation. Elevated AGS3 binds to G alpha(i) to prevent its inhibition on AC activation. Moreover, withdrawal-induced increases in cAMP/PKA activate phospholipase C and epsilon protein kinase C to further stimulate AC5 and AC7, causing cAMP superactivation. Our findings identify a critical role for AC 5 and 7 and A2A receptors for up-regulation of AGS3 in morphine withdrawal-induced cAMP superactivation.

Dopamine and ethanol cause translocation of epsilonPKC associated with epsilonRACK: cross-talk between cAMP-dependent protein kinase A and protein kinase C signaling pathways.

We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) involve both epsilon protein kinase C (epsilonPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of epsilonPKC and the relationship to PKA activation. In the present study, we used a new epsilonPKC antibody, 14E6, that selectively recognized active epsilonPKC when not bound to its anchoring protein epsilonRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated epsilonPKC and induced translocation of both epsilonPKC and its anchoring protein, epsilonRACK to a new cytosolic site. The selective epsilonPKC agonist, pseudo-epsilonRACK, activated epsilonPKC but did not cause translocation of the epsilonPKC/epsilonRACK complex to the cytosol. These data suggest a step-wise activation and translocation of epsilonPKC after NPA or ethanol treatment, where epsilonPKC first translocates and binds to its RACK and subsequently the epsilonPKC/epsilonRACK complex translocates to a new subcellular site. Direct activation of PKA by adenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), prostaglandin E1, or the adenosine A2A receptor is sufficient to cause epsilonPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between epsilonPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.