4-Hydroxynonenal impairs miRNA maturation in heart failure via Dicer post-translational modification.

BACKGROUND AND AIMS: Developing novel therapies to battle the global public health burden of heart failure remains challenging. This study investigates the underlying mechanisms and potential treatment for 4-hydroxynonenal (4-HNE) deleterious effects in heart failure. METHODS: Biochemical, functional, and histochemical measurements were applied to identify 4-HNE adducts in rat and human failing hearts. In vitro studies were performed to validate 4-HNE targets. RESULTS: 4-HNE, a reactive aldehyde by-product of mitochondrial dysfunction in heart failure, covalently inhibits Dicer, an RNase III endonuclease essential for microRNA (miRNA) biogenesis. 4-HNE inhibition of Dicer impairs miRNA processing. Mechanistically, 4-HNE binds to recombinant human Dicer through an intermolecular interaction that disrupts both activity and stability of Dicer in a concentration- and time-dependent manner. Dithiothreitol neutralization of 4-HNE or replacing 4-HNE-targeted residues in Dicer prevents 4-HNE inhibition of Dicer in vitro. Interestingly, end-stage human failing hearts from three different heart failure aetiologies display defective 4-HNE clearance, decreased Dicer activity, and miRNA biogenesis impairment. Notably, boosting 4-HNE clearance through pharmacological re-activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) using Alda-1 or its improved orally bioavailable derivative AD-9308 restores Dicer activity. ALDH2 is a major enzyme responsible for 4-HNE removal. Importantly, this response is accompanied by improved miRNA maturation and cardiac function/remodelling in a pre-clinical model of heart failure. CONCLUSIONS: 4-HNE inhibition of Dicer directly impairs miRNA biogenesis in heart failure. Strikingly, decreasing cardiac 4-HNE levels through pharmacological ALDH2 activation is sufficient to re-establish Dicer activity and miRNA biogenesis; thereby representing potential treatment for patients with heart failure.

Influence of the dose of ketamine used on schizophrenia-like symptoms in mice: A correlation study with TH, GAD67, and PPAR-γ.

Schizophrenia is a chronic, debilitating mental illness that has not yet been completely understood. In this study, we aimed to investigate the effects of different doses of ketamine, a non-competitive NMDA receptor antagonist, on the positive- and negative-like symptoms of schizophrenia. We also explored whether these effects are related to changes in the immunoreactivity of GAD67, TH, and PPAR-γ in brain structures. To conduct the study, male mice received ketamine (20-40 mg/kg) or its vehicle (0.9 % NaCl) intraperitoneally for 14 consecutive days. We quantified stereotyped behavior, the time of immobility in the forced swimming test (FST), and locomotor activity after 7 or 14 days. In addition, we performed ex vivo analysis of the immunoreactivity of GAD, TH, and PPAR-γ, in brain tissues after 14 days. The results showed that ketamine administration for 14 days increased the grooming time in the nose region at all tested doses. It also increased immobility in the FST at 30 mg/kg doses and decreased the number of rearing cycles during stereotyped behavior at 40 mg/kg. These behavioral effects were not associated with changes in locomotor activity. We did not observe any significant alterations regarding the immunoreactivity of brain proteins. However, we found that GAD and TH were positively correlated with the number of rearing during the stereotyped behavior at doses of 20 and 30 mg/kg ketamine, respectively. GAD was positively correlated with the number of rearing in the open field test at a dose of 20 mg/kg. TH was inversely correlated with immobility time in the FST at a dose of 30 mg/kg. PPAR-γ was inversely correlated with the number of bouts of stereotyped behavior at a dose of 40 mg/kg of ketamine. In conclusion, the behavioral alterations induced by ketamine in positive-like symptoms were reproduced with all doses tested and appear to depend on the modulatory effects of TH, GAD, and PPAR-γ. Conversely, negative-like symptoms were associated with a specific dose of ketamine.

Exercise preserves physical fitness during aging through AMPK and mitochondrial dynamics.

Exercise is a nonpharmacological intervention that improves health during aging and a valuable tool in the diagnostics of aging-related diseases. In muscle, exercise transiently alters mitochondrial functionality and metabolism. Mitochondrial fission and fusion are critical effectors of mitochondrial plasticity, which allows a fine-tuned regulation of organelle connectiveness, size, and function. Here we have investigated the role of mitochondrial dynamics during exercise in the model organism Caenorhabditis elegans. We show that in body-wall muscle, a single exercise session induces a cycle of mitochondrial fragmentation followed by fusion after a recovery period, and that daily exercise sessions delay the mitochondrial fragmentation and physical fitness decline that occur with aging. Maintenance of proper mitochondrial dynamics is essential for physical fitness, its enhancement by exercise training, and exercise-induced remodeling of the proteome. Surprisingly, among the long-lived genotypes we analyzed (isp-1,nuo-6, daf-2, eat-2, and CA-AAK-2), constitutive activation of AMP-activated protein kinase (AMPK) uniquely preserves physical fitness during aging, a benefit that is abolished by impairment of mitochondrial fission or fusion. AMPK is also required for physical fitness to be enhanced by exercise, with our findings together suggesting that exercise may enhance muscle function through AMPK regulation of mitochondrial dynamics. Our results indicate that mitochondrial connectivity and the mitochondrial dynamics cycle are essential for maintaining physical fitness and exercise responsiveness during aging and suggest that AMPK activation may recapitulate some exercise benefits. Targeting mechanisms to optimize mitochondrial fission and fusion, as well as AMPK activation, may represent promising strategies for promoting muscle function during aging.

Ex vivo and in vitro inhibitory potential of Kava extract on monoamine oxidase B activity in mice.

Background and aim: This study investigated the effect of Kava extract (Piper methysticum), a medicinal plant that has been worldly used by its anxiolytic effects, on monoamine oxidase (MAO) activity of mice brain after 21 days of treatment as well as anxiolytic and locomotor behavior. Furthermore, the in vitro inhibitory profile of Kava extract on MAO-B activity of mouse brain was evaluated. Experimental procedure: Mice were treated with Kava extract (10, 40, 100 and 400 mg/kg) for 21 days by gavage. After behavioral analysis (plus maze test and open field), MAO activity in different mouse brain structures (cortex, hippocampus, region containing the substantia nigra and striatum) were performed. MAO-B inhibitory profile was characterized in vitro. Results: The treatment with Kava extract (40 mg/kg) increased the percentage of entries of mice into the open arms. Ex vivo analysis showed an inhibition on MAO-B activity caused by Kava extract in cortex (10 mg/kg) and in the region containing the substantia nigra (10 and 100 mg/kg). In vitro, Kava extract also reversibly inhibited MAO-B activity with IC50 = 14.62 µg/mL and, increased Km values at the concentrations of 10 and 30 µg/mL and decreased Vmax value at 100 µg/mL. Conclusion: Kava extract showed different effects on MAO-B isoform depending on the brain structure evaluated. Therefore, the use of Kava extract could be promissory in pathologies where MAO-B is the pharmacological target.

Kava decreases the stereotyped behavior induced by amphetamine in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Kava extract (Piper methysticum) is a phytotherapic mainly used for the treatment of anxiety. Although the reported effects of Kava drinking improving psychotic symptoms of patients when it was introduced to relieve anxiety in aboriginal communities, its effects on models of psychosis-like symptoms are not investigated. AIM OF THE STUDY: To investigate the effects of Kava extract on behavioral changes induced by amphetamine (AMPH) and its possible relation with alterations in monoamine oxidase (MAO) activity. MATERIALS AND METHODS: Mice received vehicle or Kava extract by gavage and, 2 h after vehicle or AMPH intraperitoneally. Twenty-five minutes after AMPH administration, behavioral (elevated plus maze, open field, stereotyped behavior, social interaction and Y maze) and biochemical tests (MAO-A and MAO-B activity in cortex, hippocampus and striatum) were sequentially evaluated. RESULTS: Kava extract exhibited anxiolytic effects in plus maze test, increased the locomotor activity of mice in open field test and decreased MAO-A (in cortex) and MAO-B (in hippocampus) activity of mice. Kava extract prevented the effects of AMPH on stereotyped behavior and, the association between Kava/AMPH increased the number of entries into arms in Y maze test as well as MAO-B activity in striatum. However, Kava extract did not prevent hyperlocomotion induced by AMPH in open field test. The social interaction was not modified by Kava extract and/or AMPH. CONCLUSION: The results showed that Kava extract decreased the stereotyped behavior induced by AMPH at the same dose that promotes anxiolytic effects, which could be useful to minimize the psychotic symptoms in patients.

Haloperidol Interactions with the dop-3 Receptor in Caenorhabditis elegans.

Haloperidol is a typical antipsychotic drug commonly used to treat a broad range of psychiatric disorders related to dysregulations in the neurotransmitter dopamine (DA). DA modulates important physiologic functions and perturbations in Caenorhabditis elegans (C. elegans) and, its signaling have been associated with alterations in behavioral, molecular, and morphologic properties in C. elegans. Here, we evaluated the possible involvement of dopaminergic receptors in the onset of these alterations followed by haloperidol exposure. Haloperidol increased lifespan and decreased locomotor behavior (basal slowing response, BSR, and locomotion speed via forward speed) of the worms. Moreover, locomotion speed recovered to basal conditions upon haloperidol withdrawal. Haloperidol also decreased DA levels, but it did not alter neither dop-1, dop-2, and dop-3 gene expression, nor CEP dopaminergic neurons' morphology. These effects are likely due to haloperidol's antagonism of the D2-type DA receptor, dop-3. Furthermore, this antagonism appears to affect mechanistic pathways involved in the modulation and signaling of neurotransmitters such as octopamine, acetylcholine, and GABA, which may underlie at least in part haloperidol's effects. These pathways are conserved in vertebrates and have been implicated in a range of disorders. Our novel findings demonstrate that the dop-3 receptor plays an important role in the effects of haloperidol.

Effects of CATECHIN on reserpine-induced vacuous chewing movements: behavioral and biochemical analysis.

This study evaluated the effect of (+)-catechin, a polyphenolic compound, on orofacial dyskinesia (OD) induced by reserpine in mice. The potential modulation of monoaminoxidase (MAO) activity, tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD67) immunoreactivity by catechin were used as biochemical endpoints. The interaction of catechin with MAO-A and MAO-B was determined in vitro and in silico. The effects of catechin on OD induced by reserpine (1 mg/kg for 4 days, subcutaneously) in male Swiss mice were examined. After, catechin (10, 50 or 100 mg/kg, intraperitoneally) or its vehicle were given for another 20 days. On the 6th, 8th, 15th and 26th day, vacuous chewing movements (VCMs) and locomotor activity were quantified. Biochemical markers (MAO activity, TH and GAD67 immunoreactivity) were evaluated in brain structures. In vitro, catechin inhibited both MAO isoforms at concentrations of 0.34 and 1.03 mM being completely reversible for MAO-A and partially reversible for MAO-B. Molecular docking indicated that the catechin bound in the active site of MAO-A, while in the MAO-B it interacted with the surface of the enzyme in an allosteric site. In vivo, reserpine increased the VCMs and decreased the locomotor activity. Catechin (10 mg/kg), decreased the number of VCMs in the 8th day in mice pre-treated with reserpine without altering other behavioral response. Ex vivo, the MAO activity and TH and GAD67 immunoreactivity were not altered by the treatments. Catechin demonstrated a modest and transitory protective effect in a model of OD in mice.

Manganese in the Diet: Bioaccessibility, Adequate Intake, and Neurotoxicological Effects.

Manganese (Mn) is an essential element that participates in several biological processes. Mn serves as a cofactor for several enzymes, such as glutamine synthetase and oxidoreductases, that have an important role in the defense of the organisms against oxidative stress. The diet is the main source of Mn intake for humans, and adequate daily intake levels for this metal change with age. Moreover, in higher amounts, Mn may be toxic, mainly to the brain. Here, we provide an overview of Mn occurrence in food, addressing its bioaccessibility and discussing the dietary standard and recommended intake of Mn consumption. In addition, we review some mechanisms underlying Mn-induced neurotoxicity.

The effects of manganese overexposure on brain health.

Manganese (Mn) is the twelfth most abundant element on the earth and an essential metal to human health. Mn is present at low concentrations in a variety of dietary sources, which provides adequate Mn content to sustain support various physiological processes in the human body. However, with the rise of Mn utility in a variety of industries, there is an increased risk of overexposure to this transition metal, which can have neurotoxic consequences. This risk includes occupational exposure of Mn to workers as well as overall increased Mn pollution affecting the general public. Here, we review exposure due to air pollution and inhalation in industrial settings; we also delve into the toxic effects of manganese on the brain such as oxidative stress, inflammatory response and transporter dysregulation. Additionally, we summarize current understandings underlying the mechanisms of Mn toxicity.

Thimerosal inhibits Drosophila melanogaster tyrosine hydroxylase (DmTyrH) leading to changes in dopamine levels and impaired motor behavior: implications for neurotoxicity.

Thimerosal (THIM) is a well-established antifungal and antiseptic agent widely used as a preservative in vaccines. Recent studies identified the neurotoxic effects of THIM, including malfunction of the monoaminergic system. However, the underlying cytotoxic mechanisms are not well understood. Here we used the fruit fly Drosophila melanogaster to investigate the mechanisms of THIM-induced neurotoxicity. We focused on the dopaminergic system, and the rate-limiting enzyme tyrosine hydroxylase (DmTyrH), to test the hypothesis that THIM can impair dopamine (DA) homeostasis and subsequently cause dysfunction. We studied the effect of THIM by feeding 1-2 day old flies (both sexes) food supplemented with 25 µM THIM for 4 days and determined THIM-induced effects on survival, oxidative stress, and metabolic activity based on MTT assay and acetylcholinesterase (AChE) activity. Our results demonstrate that D. melanogaster exposed to THIM present changes in DmTyrH expression and activity, together with altered DA levels that led to impaired motor behavior. These phenotypes were accompanied by an increase in oxidative stress, with a decrease in MTT reduction, in AChE activity, and also in survival rate. These findings suggest an initiating and primary role for THIM-mediated DmTyrH dysfunction that leads to impaired DA function and behavioral abnormalities, ultimately causing oxidative stress-related neurotoxicity.

In vitro and in vivo evaluation of enzymatic and antioxidant activity, cytotoxicity and genotoxicity of curcumin-loaded solid dispersions.

Curcumin, the main bioactive polyphenolic compound in Curcuma longa L. rhizomes has a wide range of bioactive properties. Curcumin presents low solubility in water and thus limited bioavailability, which decreases its applicability. In this study, cytotoxic effects of curcumin solid dispersions (CurSD) were evaluated against tumor (breast adenocarcinoma and lung, cervical and hepatocellular carcinoma) and non-tumor (PLP2) cells, while cytotoxic and genotoxic effects were evaluated in Allium cepa. The effect of the CurSD on the acetylcholinesterase (AChE), butyrylcholinesterase (BChE), glutathione S-transferase (GST), and monoamine oxidase (MAO A-B) enzymes was determined, as well as its capacity to inhibit the oxidative hemolysis (OxHLIA) and the formation of thiobarbituric acid reactive substances (TBARS). CurSD are constituted by nanoparticles that are readily dispersible in water, and inhibited 24% and 64% of the AChE and BChE activity at 100 µM, respectively. GST activity was inhibited at 30 µM while MAO-A and B activity were inhibited at 100 µM. CurSD showed cytotoxicity against all the tested tumor cell lines without toxic effects for non-tumor cells. No cytotoxic and genotoxic potential was detected with the Allium cepa test. CurSD maintained the characteristics of free curcumin on the in vitro modulation of important enzymes without appreciable toxicity.

Protective effect of (-)-α-bisabolol on rotenone-induced toxicity in Drosophila melanogaster.

(-)-α-Bisabolol (BISA) is a sesquiterpene alcohol, which has several recognized biological activities, including anti-inflammatory, anti-irritant, and antibacterial properties. In the present study, we investigated the influence of BISA (5, 25, and 250 µmol/L) on rotenone (500 µmol/L)-induced toxicity in Drosophila melanogaster for 7 days. BISA supplementation significantly decreased rotenone-induced mortality and locomotor deficits. The loss of motor function induced by rotenone correlated with a significant change in stress response factors; it decreased thiol levels, inhibited mitochondria complex I, and increased the mRNA expression of antioxidant marker proteins such as superoxide dismutase (SOD), catalase (CAT), and the keap1 gene product. Taken together, our findings indicate that the toxicity of rotenone is likely due to the direct inhibition of complex I activity, resulting in a high level of oxidative stress. Dietary supplementation with BISA affected the expression of SOD mRNA only at a concentration of 250 µmol/L, and did not affect any other parameter measured. Our results showed a protective effect of BISA on rotenone-induced mortality and locomotor deficits in Drosophila; this effect did not correlate with mitochondrial complex I activity, but may be related to the antioxidant protection afforded by eliminating superoxide generated as a result of rotenone-induced mitochondrial dysfunction.

Harpagophytum Procumbens Ethyl Acetate Fraction Reduces Fluphenazine-Induced Vacuous Chewing Movements and Oxidative Stress in Rat Brain.

Long-term treatment with fluphenazine is associated with manifestation of extrapyramidal side effects, such as tardive dyskinesia. The molecular mechanisms related to the pathophysiology of TD remain unclear, and several hypotheses, including a role for oxidative stress, have been proposed. Harpagophytum procumbens is an herbal medicine used mainly due to anti-inflammatory effects, but it also exhibits antioxidant effects. We investigated the effect of ethyl acetate fraction of H. procumbens (EAF HP) in fluphenazine-induced orofacial dyskinesia by evaluating behavioral parameters at different times (vacuous chewing movements (VCM's) and locomotor and exploratory activity), biochemical serological analyses, and biochemical markers of oxidative stress of the liver, kidney, cortex, and striatum. Chronic administration of fluphenazine (25 mg/kg, intramuscular (i.m) significantly increased the VCMs at all analyzed times (2, 7, 14, and 21 days), and this was inhibited by EAF HP (especially at a dose of 30 mg/kg). Fluphenazine decreased locomotion and exploratory activity, and EAF HP did not improve this decrease. Fluphenazine induced oxidative damage, as identified by changes in catalase activity and ROS levels in the cortex and striatum, which was reduced by EAF HP, especially in the striatum. In the cortex, EAF HP was protective against fluphenazine-induced changes in catalase activity but not against the increase in ROS level. Furthermore, EAF HP was shown to be safe, since affected serum biochemical parameters or parameters of oxidative stress in the liver and kidney. These findings suggest that the H. procumbens is a promising therapeutic agent for the treatment of involuntary oral movements.

Behavioral and neurochemical effects induced by reserpine in mice.

RATIONALE: Reserpine, a monoamine-depleting agent, which irreversibly and non-selectively blocks the vesicular monoamine transporter, has been used as an animal model to study several neurological disorders, including tardive dyskinesia and Parkinson's disease. OBJECTIVE: The purpose of this study was to examine if motor deficits induced by reserpine in mice could be related to alterations in the expression of dopaminergic system proteins such as tyrosine hydroxylase (TH) and dopamine transporter (DAT) and in the activity of monoamine oxidase (MAO). METHODS: Mice received either vehicle or reserpine (0.1, 0.5, or 1 mg/kg, s.c.) for four consecutive days. Two, 20, or 60 days after reserpine withdrawal, behavioral, and neurochemical changes were evaluated. RESULTS: Reserpine at a dose of 0.5 and 1 mg/kg increased vacuous chewing movements (VCMs) and reduced locomotion. Behavioral changes were accompanied by reduction in TH immunoreactivity in the striatum evaluated on days 2 and 20 after the last injection of 1 mg/kg reserpine. Furthermore, negative correlations were found between VCM and MAO-A or MAO-B on day 2 and TH striatal immunoreactivity on day 20 after the last injection of 1 mg/kg reserpine. A positive correlation was observed between VCMs and DAT immunoreactivity in the substantia nigra on day 2 after the last injection of 0.5 mg/kg reserpine. CONCLUSIONS: These findings suggest that the pharmacological blockage of vesicular monoamine transporter (VMAT) by reserpine caused neurochemical and behavioral alterations in mice.

Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis.

G(M1) gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal beta-d-galactosidase (EC 3.2.1.23) and consequent storage of undegraded G(M1) ganglioside. To characterize the genetic mutation responsible for feline G(M1) gangliosidosis, the normal sequence of feline beta-galactosidase cDNA first was defined. The feline beta-galactosidase open reading frame is 2010 base pairs, producing a protein of 669 amino acids. The putative signal sequence consists of amino acids 1-24 of the beta-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human beta-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal beta-galactosidase was sequenced, the mutation responsible for feline G(M1) gangliosidosis was defined as a G to C substitution at position 1448 of the open reading frame, resulting in an amino acid substitution at arginine 483, known to cause G(M1) gangliosidosis in humans. Feline beta-galactosidase messenger RNA levels were normal in cerebral cortex, as determined by quantitative RT-PCR assays. Although enzymatic activity is severely reduced by the mutation, a full-length feline beta-galactosidase cDNA restored activity in transfected G(M1) fibroblasts to 18-times normal. beta-Galactosidase protein levels in G(M1) tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant beta-galactosidase protein did not reach the lysosome. Additionally, G(M1) cat fibroblasts demonstrated increased expression of glucose-related protein 78/BiP and protein disulfide isomerase, suggesting that the unfolded protein response plays a role in pathogenesis of feline G(M1) gangliosidosis.

An inversion of 25 base pairs causes feline GM2 gangliosidosis variant.

In G(M2) gangliosidosis variant 0, a defect in the beta-subunit of lysosomal beta-N-acetylhexosaminidase (EC 3.2.1.52) causes abnormal accumulation of G(M2) ganglioside and severe neurodegeneration. Distinct feline models of G(M2) gangliosidosis variant 0 have been described in both domestic shorthair and Korat cats. In this study, we determined that the causative mutation of G(M2) gangliosidosis in the domestic shorthair cat is a 25-base-pair inversion at the extreme 3' end of the beta-subunit (HEXB) coding sequence, which introduces three amino acid substitutions at the carboxyl terminus of the protein and a translational stop that is eight amino acids premature. Cats homozygous for the 25-base-pair inversion express levels of beta-subunit mRNA approximately 190% of normal and protein levels only 10-20% of normal. Because the 25-base-pair inversion is similar to mutations in the terminal exon of human HEXB, the domestic shorthair cat should serve as an appropriate model to study the molecular pathogenesis of human G(M2) gangliosidosis variant 0 (Sandhoff disease).