Hedgehog-interacting protein acts in the habenula to regulate nicotine intake.

Hedgehog-interacting protein (HHIP) sequesters Hedgehog ligands to repress Smoothened (SMO)-mediated recruitment of the GLI family of transcription factors. Allelic variation in HHIP confers risk of chronic obstructive pulmonary disease and other smoking-related lung diseases, but underlying mechanisms are unclear. Using single-cell and cell-type-specific translational profiling, we show that HHIP expression is highly enriched in medial habenula (MHb) neurons, particularly MHb cholinergic neurons that regulate aversive behavioral responses to nicotine. HHIP deficiency dysregulated the expression of genes involved in cholinergic signaling in the MHb and disrupted the function of nicotinic acetylcholine receptors (nAChRs) through a PTCH-1/cholesterol-dependent mechanism. Further, CRISPR/Cas9-mediated genomic cleavage of the Hhip gene in MHb neurons enhanced the motivational properties of nicotine in mice. These findings suggest that HHIP influences vulnerability to smoking-related lung diseases in part by regulating the actions of nicotine on habenular aversion circuits.

Gene editing reverses arrhythmia susceptibility in humanized PLN-R14del mice: modelling a European cardiomyopathy with global impact.

AIMS: A mutation in the phospholamban (PLN) gene, leading to deletion of Arg14 (R14del), has been associated with malignant arrhythmias and ventricular dilation. Identifying pre-symptomatic carriers with vulnerable myocardium is crucial because arrhythmia can result in sudden cardiac death, especially in young adults with PLN-R14del mutation. This study aimed at assessing the efficiency and efficacy of in vivo genome editing, using CRISPR/Cas9 and a cardiotropic adeno-associated virus-9 (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del. METHODS AND RESULTS: Humanized mice were generated expressing human wild-type (hPLN-WT) or mutant (hPLN-R14del) PLN in the heterozygous state, mimicking human carriers. Cardiac magnetic resonance imaging at 12 weeks of age showed bi-ventricular dilation and increased stroke volume in mutant vs. WT mice, with no deficit in ejection fraction or cardiac output. Challenge of ex vivo hearts with isoproterenol and rapid pacing unmasked higher propensity for sustained ventricular tachycardia (VT) in hPLN-R14del relative to hPLN-WT. Specifically, the VT threshold was significantly reduced (20.3 ± 1.2 Hz in hPLN-R14del vs. 25.7 ± 1.3 Hz in WT, P < 0.01) reflecting higher arrhythmia burden. To inactivate the R14del allele, mice were tail-vein-injected with AAV9.CRISPR/Cas9/gRNA or AAV9 empty capsid (controls). CRISPR-Cas9 efficiency was evaluated by droplet digital polymerase chain reaction and NGS-based amplicon sequencing. In vivo gene editing significantly reduced end-diastolic and stroke volumes in hPLN-R14del CRISPR-treated mice compared to controls. Susceptibility to VT was also reduced, as the VT threshold was significantly increased relative to controls (30.9 ± 2.3 Hz vs. 21.3 ± 1.5 Hz; P < 0.01). CONCLUSIONS: This study is the first to show that disruption of hPLN-R14del allele by AAV9-CRISPR/Cas9 improves cardiac function and reduces VT susceptibility in humanized PLN-R14del mice, offering preclinical evidence for translatable approaches to therapeutically suppress the arrhythmogenic phenotype in human patients with PLN-R14del disease.

Neurobiological Mechanisms of Nicotine Reward and Aversion.

Neuronal nicotinic acetylcholine receptors (nAChRs) regulate the rewarding actions of nicotine contained in tobacco that establish and maintain the smoking habit. nAChRs also regulate the aversive properties of nicotine, sensitivity to which decreases tobacco use and protects against tobacco use disorder. These opposing behavioral actions of nicotine reflect nAChR expression in brain reward and aversion circuits. nAChRs containing α4 and ß2 subunits are responsible for the high-affinity nicotine binding sites in the brain and are densely expressed by reward-relevant neurons, most notably dopaminergic, GABAergic, and glutamatergic neurons in the ventral tegmental area. High-affinity nAChRs can incorporate additional subunits, including ß3, α6, or α5 subunits, with the resulting nAChR subtypes playing discrete and dissociable roles in the stimulatory actions of nicotine on brain dopamine transmission. nAChRs in brain dopamine circuits also participate in aversive reactions to nicotine and the negative affective state experienced during nicotine withdrawal. nAChRs containing α3 and ß4 subunits are responsible for the low-affinity nicotine binding sites in the brain and are enriched in brain sites involved in aversion, including the medial habenula, interpeduncular nucleus, and nucleus of the solitary tract, brain sites in which α5 nAChR subunits are also expressed. These aversion-related brain sites regulate nicotine avoidance behaviors, and genetic variation that modifies the function of nAChRs in these sites increases vulnerability to tobacco dependence and smoking-related diseases. Here, we review the molecular, cellular, and circuit-level mechanisms through which nicotine elicits reward and aversion and the adaptations in these processes that drive the development of nicotine dependence. SIGNIFICANCE STATEMENT: Tobacco use disorder in the form of habitual cigarette smoking or regular use of other tobacco-related products is a major cause of death and disease worldwide. This article reviews the actions of nicotine in the brain that contribute to tobacco use disorder.

Addiction-related neuroadaptations following chronic nicotine exposure.

The addiction-relevant molecular, cellular, and behavioral actions of nicotine are derived from its stimulatory effects on neuronal nicotinic acetylcholine receptors (nAChRs) in the central nervous system. nAChRs expressed by dopamine-containing neurons in the ventral midbrain, most notably in the ventral tegmental area (VTA), contribute to the reward-enhancing properties of nicotine that motivate the use of tobacco products. nAChRs are also expressed by neurons in brain circuits that regulate aversion. In particular, nAChRs expressed by neurons in the medial habenula (mHb) and the interpeduncular nucleus (IPn) to which the mHb almost exclusively projects regulate the "set-point" for nicotine aversion and control nicotine intake. Different nAChR subtypes are expressed in brain reward and aversion circuits and nicotine intake is titrated to maximally engage reward-enhancing nAChRs while minimizing the recruitment of aversion-promoting nAChRs. With repeated exposure to nicotine, reward- and aversion-related nAChRs and the brain circuits in which they are expressed undergo adaptations that influence whether tobacco use will transition from occasional to habitual. Genetic variation that influences the sensitivity of addiction-relevant brain circuits to the actions of nicotine also influence the propensity to develop habitual tobacco use. Here, we review some of the key advances in our understanding of the mechanisms by which nicotine acts on brain reward and aversion circuits and the adaptations that occur in these circuits that may drive addiction to nicotine-containing tobacco products.

The use of high-throughput screening techniques to evaluate mitochondrial toxicity.

Toxicologists and chemical regulators depend on accurate and effective methods to evaluate and predict the toxicity of thousands of current and future compounds. Robust high-throughput screening (HTS) experiments have the potential to efficiently test large numbers of chemical compounds for effects on biological pathways. HTS assays can be utilized to examine chemical toxicity across multiple mechanisms of action, experimental models, concentrations, and lengths of exposure. Many agricultural, industrial, and pharmaceutical chemicals classified as harmful to human and environmental health exert their effects through the mechanism of mitochondrial toxicity. Mitochondrial toxicants are compounds that cause a decrease in the number of mitochondria within a cell, and/or decrease the ability of mitochondria to perform normal functions including producing adenosine triphosphate (ATP) and maintaining cellular homeostasis. Mitochondrial dysfunction can lead to apoptosis, necrosis, altered metabolism, muscle weakness, neurodegeneration, decreased organ function, and eventually disease or death of the whole organism. The development of HTS techniques to identify mitochondrial toxicants will provide extensive databases with essential connections between mechanistic mitochondrial toxicity and chemical structure. Computational and bioinformatics approaches can be used to evaluate compound databases for specific chemical structures associated with toxicity, with the goal of developing quantitative structure-activity relationship (QSAR) models and mitochondrial toxicophores. Ultimately these predictive models will facilitate the identification of mitochondrial liabilities in consumer products, industrial compounds, pharmaceuticals and environmental hazards.

Apremilast Induces Apoptosis of Human Colorectal Cancer Cells with Mutant KRAS.

BACKGROUND/AIM: We previously reported the crucial roles of oncogenic Kirsten rat sarcoma viral oncogene homologue (KRAS) in inhibiting apoptosis and disrupting cell polarity via the regulation of phosphodiesterase type 4B2 (PDE4B2) expression in human colorectal cancer (CRC) HCT116 cells in a three-dimensional culture (3DC). Here, we evaluated the effects of apremilast, a selective PDE4 inhibitor, on luminal apoptosis in 3DC and nude mice assay using HKe3 human CRC cells stably expressing wild-type (wt)PDE4B2 (HKe3-wtPDE4B2), mutant (mt)PDE4B2 (kinase dead) (HKe3-wtKRAS), wtKRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS). MATERIALS AND METHODS: Apoptosis was detected by immunofluorescence using confocal laser scanning microscopy or western blot in HKe3-wtPDE4B2, HKe3-mtPDE4B2, HKe3-wtKRAS and mtKRAS cells treated with or without apremilast in 3DC. Tumourigenicity was assessed in nude mice assay using these cells. RESULTS: Apremilast did not inhibit the proliferation of HKe3-wtPDE4B2 cells or HKe3-mtKRAS in two-dimensional cultures, whereas the number of apoptotic HKe3-wtPDE4B2 cells and HKe3-mtKRAS cells increased after apremilast treatment in 3DC, leading to formation of a luminal cavity. Tumour growth in nude mice was dramatically reduced by intraperitoneal injection of apremilast. Notably, a decreased level of caspase-1 expression was observed in HKe3-wtPDE4B2 and HKe3-mtKRAS cells. CONCLUSION: Apremilast induces tumour regression in nude mice, possibly by inducing caspase-1 expression.

Formoterol restores mitochondrial and renal function after ischemia-reperfusion injury.

Mitochondrial biogenesis may be an adaptive response necessary for meeting the increased metabolic and energy demands during organ recovery after acute injury, and renal mitochondrial dysfunction has been implicated in the pathogenesis of AKI. We proposed that stimulation of mitochondrial biogenesis 24 hours after ischemia/reperfusion (I/R)-induced AKI, when renal dysfunction is maximal, would accelerate recovery of mitochondrial and renal function in mice. We recently showed that formoterol, a potent, highly specific, and long-acting ß2-adrenergic agonist, induces renal mitochondrial biogenesis in naive mice. Animals were subjected to sham or I/R-induced AKI, followed by once-daily intraperitoneal injection with vehicle or formoterol beginning 24 hours after surgery and continuing through 144 hours after surgery. Treatment with formoterol restored renal function, rescued renal tubules from injury, and diminished necrosis after I/R-induced AKI. Concomitantly, formoterol stimulated mitochondrial biogenesis and restored the expression and function of mitochondrial proteins. Taken together, these results provide proof of principle that a novel drug therapy to treat AKI, and potentially other acute organ failures, works by restoring mitochondrial function and accelerating the recovery of renal function after injury has occurred.

cGMP-selective phosphodiesterase inhibitors stimulate mitochondrial biogenesis and promote recovery from acute kidney injury.

Recent studies demonstrate that mitochondrial dysfunction is a mediator of acute kidney injury (AKI). Consequently, restoration of mitochondrial function after AKI may be key to the recovery of renal function. Mitochondrial function can be restored through the generation of new, functional mitochondria in a process called mitochondrial biogenesis (MB). Despite its potential therapeutic significance, very few pharmacological agents have been identified to induce MB. To examine the efficacy of phosphodiesterase (PDE) inhibitors (PDE3: cAMP and cGMP activity; and PDE4: cAMP activity) in stimulating MB, primary cultures of renal proximal tubular cells (RPTCs) were treated with a panel of inhibitors for 24 hours. PDE3, but not PDE4, inhibitors increased the FCCP-uncoupled oxygen consumption rate (OCR), a marker of MB. Exposure of RPTCs to the PDE3 inhibitors, cilostamide and trequinsin, for 24 hours increased peroxisome proliferator-activated receptor γ coactivator-1α, and multiple mitochondrial electron transport chain genes. Cilostamide and trequinsin also increased mRNA expression of mitochondrial genes and mitochondrial DNA copy number in mice renal cortex. Consistent with these experiments, 8-Br-cGMP increased FCCP-uncoupled OCR and mitochondrial gene expression, whereas 8-Br-cAMP had no effect. The cGMP-specific PDE5 inhibitor sildenafil also induced MB in RPTCs and in vivo in mouse renal cortex. Treatment of mice with sildenafil after folic acid-induced AKI promoted restoration of MB and renal recovery. These data provide strong evidence that specific PDE inhibitors that increase cGMP are inducers of MB in vitro and in vivo, and suggest their potential efficacy in AKI and other diseases characterized by mitochondrial dysfunction and suppressed MB.

Telomeres and telomerase in renal health.

The role of telomeres and telomerase in human biology has been studied since the early 1990s because telomere attrition is implicated in various diseases including cardiovascular dysfunction, carcinogenesis, and the progression of acute kidney injury. Telomeric length is a reliable indicator of intrinsic biologic age and a surrogate for the mitotic clock. Because the prevalence of chronic kidney disease increases with age, telomere length and telomerase activity may play a role in its progression.

Comparative chronic liver toxicity of benzo[a]pyrene in two populations of the atlantic killifish (Fundulus heteroclitus) with different exposure histories.

BACKGROUND: The Atlantic Wood Industries Superfund site on the Elizabeth River (ER) in Portsmouth, Virginia, is contaminated with polycyclic aromatic hydrocarbons (PAHs) derived from creosote. Embryos and larvae of ER killifish (Fundulus heteroclitus) are refractory to the induction of enzymes regulated by the aryl hydrocarbon receptor including cytochrome P4501A (CYP1A) and are resistant to PAH-induced lethality and teratogenicity. However, adult ER killifish show a greater prevalence of hepatic and pancreatic tumors compared with those from reference sites. OBJECTIVES: We used controlled laboratory studies to determine if ER killifish are more or less sensitive to PAH-induced chronic hepatic toxicity than killifish from an uncontaminated site. METHODS: Larvae from the ER and a reference site on King's Creek (KC) were subjected to two 24-hr aqueous exposures of benzo[a]pyrene (BaP; 0-400 µg/L). At various time points, larvae were analyzed for CYP1A activity, BaP concentrations, nuclear and mitochondrial DNA damage, and liver pathology. RESULTS: CYP1A activity was induced by BaP in KC but not ER larvae, and KC larvae demonstrated a greater reduction in whole-body concentrations of BaP over time. Mitochondrial and nuclear DNA lesion frequency increased significantly in BaP-exposed KC larvae, but not in ER larvae. Nine months postexposure, KC juveniles exhibited significantly more hepatic foci of cellular alteration and only KC juveniles developed hepatocellular carcinomas. CONCLUSIONS: In addition to acquiring the heritable resistance to the acute teratogenic effects of PAHs, ER fish appear to have concomitantly developed resistance to chronic effects, including cancer.