Jolanta Pupure

Mitochondria as the target for mildronate's protective effects in azidothymidine (AZT)-induced toxicity of isolated rat liver mitochondria.

Previously mildronate, an aza‐butyrobetaine derivative, was shown to be a cytoprotective drug, through its mechanism of action of inhibition of carnitine palmitoyltransferase‐1, thus protecting mitochondria from long‐chain fatty acid accumulation and subsequent damage. Recently in an azidothymidine (AZT)‐induced cardiotoxicity model in vivo (in mice), we have found mildronates ability of protecting heart tissue from nuclear factor κB abnormal expression. Preliminary data also demonstrate cerebro‐ and hepatoprotecting properties of mildronate in AZT‐toxicity models. We suggest that mildronate may target its action predominantly to mitochondria. The present study in isolated rat liver mitochondria was designed to clarify mitochondrial targets for mildronate by using AZT as a model compound. The aim of this study was to investigate: (1) whether mildronate may protect mitochondria from AZT‐induced toxicity; and (2) which is the most critical target in mitochondrial processes that is responsible for mildronates regulatory action. The results showed that mildronate protected mitochondria from AZT‐induced damage predominantly at the level of complex I, mainly by reducing hydrogen peroxide generation. Significant protection of AZT‐caused inhibition of uncoupled respiration, ADP to oxygen ratio, and transmembrane potential were also observed. Mildronate per se had no effect on the bioenergetics, oxidative stress, or permeability transition of rat liver mitochondria. Since mitochondrial complex I is the first enzyme of the respiratory electron transport chain and its damage is considered to be responsible for different mitochondrial diseases, we may account for mildronates effectiveness in the prevention of pathologies associated with mitochondrial dysfunctions. Copyright

Neuroprotective properties of mildronate, a mitochondria-targeted small molecule.

Mildronate, a representative of the aza-butyrobetaine class of drugs with proven cardioprotective efficacy, was recently found to prevent dysfunction of complex I in rat liver mitochondria. The present study demonstrates that mildronate also acts as a neuroprotective agent. In a mouse model of azidothymidine (anti-HIV drug) neurotoxicity, mildronate reduced the azidothymidine-induced alterations in mouse brain tissue: it normalized the increase in caspase-3, cellular apoptosis susceptibility protein (CAS) and iNOS expression assessed by quantitative and semi-quantitative analysis. Mildronate also normalized the changes in cytochrome c oxidase (COX) expression, reduced the expression of glial fibrillary acidic protein (GFAP) and cellular infiltration. The present results show that the neuroprotective action of mildronate results at least partially from anti-neurodegenerative (anti-apoptotic) and anti-inflammatory mechanisms. It might be suggested that the molecular conformation of mildronate can facilitate its easy binding to mitochondria, and regulate the expression of different signal molecules, hence maintaining cellular signaling and survival.

Neuroprotective Properties of Mildronate, a Small Molecule, in a Rat Model of Parkinson’s Disease

Previously, we have found that mildronate [3-(2,2,2-trimethylhydrazinium) propionate dihydrate], a small molecule with charged nitrogen and oxygen atoms, protects mitochondrial metabolism that is altered by inhibitors of complex I and has neuroprotective effects in an azidothymidine-neurotoxicity mouse model. In the present study, we investigated the effects of mildronate in a rat model of Parkinson’s disease (PD) that was generated via a unilateral intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). We assessed the expression of cell biomarkers that are involved in signaling cascades and provide neural and glial integration: the neuronal marker TH (tyrosine hydroxylase); ubiquitin (a regulatory peptide involved in the ubiquitin-proteasome degradation system); Notch-3 (a marker of progenitor cells); IBA-1 (a marker of microglial cells); glial fibrillary acidic protein, GFAP (a marker of astrocytes); and inducible nitric oxide synthase, iNOS (a marker of inflammation). The data show that in the 6-OHDA-lesioned striatum, mildronate completely prevented the loss of TH, stimulated Notch-3 expression and decreased the expression of ubiquitin, GFAP and iNOS. These results provide evidence for the ability of mildronate to control the expression of an array of cellular proteins and, thus, impart multi-faceted homeostatic mechanisms in neurons and glial cells in a rat model of PD. We suggest that the use of mildronate provides a protective effect during the early stages of PD that can delay or halt the progression of this neurodegenerative disease.

Comparative study of taurine and tauropyrone: GABA receptor binding, mitochondrial processes and behaviour

Objectives  Taurine, a sulfur‐containing amino acid, has high hydrophilicity and is poorly absorbed. Tauropyrone, a taurine‐containing 1,4‐dihydropyridine derivative, is suggested to have greater activity than taurine owing to improved physicochemical properties that facilitate delivery of the compound to target cells. The aim of this study was to determine whether the 1,4‐dihydropyridine moiety in tauropyrone improves the pharmacological efficacy of taurine in vitro and in vivo.

Distinct Influence of Atypical 1,4‐Dihydropyridine Compounds in Azidothymidine‐Induced Neuro‐ and Cardiotoxicity in Mice Ex Vivo

This study demonstrates the effective protection by compounds of atypical 1,4-dihydropyridine (DHP) series cerebrocrast, glutapyrone and tauropyrone against neuro- and cardiotoxicity caused by the model compound azidothymidine, a well-known mitochondria-compromising anti-HIV drug. In previous in vitro experiments, we have demonstrated distinct effects of these DHP compounds to influence mitochondrial functioning. In the present in vivo experiments, DHP compounds were administered intraperitoneally in mice daily for 2 weeks, per se and in combinations with azidothymidine at doses: azidothymidine 50 mg/kg; cerebrocrast 0.1 mg/kg; glutapyrone 1 mg/kg; and tauropyrone 1 mg/kg. At the end of the experiment, mice were killed, heart and brain tissues were removed and examined ex vivo histopathologically and immunohistochemically. NF-kappaBp65 and caspase-3 were used as the markers indicating inflammatory and apoptotic events, respectively. Cerebrocrast (dicyclic structure) was the most potent DHP, which effectively reduced azidothymidine-induced overexpression of NF-kappaBp65 and caspase-3 in mouse myocardium and brain cortex. Glutapyrone per se increased the number of caspase-3-positive cells in the brain, whereas it reduced NF-kappaBp65 and caspase-3 expression in cardiac tissue caused by azidothymidine. Tauropyrone showed dual action: per se it increased caspase-3 in the brain and NF-kappaBp65 expression in the heart, but it considerably reduced these activations in azidothymidine-treated mice. This study provides the first demonstration of a distinct pharmacological action for atypical DHP compounds in cardiac and brain tissues. The dicyclic structure of cerebrocrast is considered beneficial for neuro- and cardioprotection at least in part via mitochondrial targeting and consequent regulation of inflammatory and apoptotic processes.

γ1- and γ2-melanocyte stimulating hormones induce central anxiogenic effects and potentiate ethanol withdrawal responses in the elevated plus-maze test in mice

Little is known about the endogenous functions of gamma1- and gamma2-melanocyte stimulating hormones (gamma1- and gamma2-MSH). Although gamma-MSHs bind to melanocortin receptor subtypes 3 and 4, we have previously shown that these peptides also influence non-melanocortinergic processes, such as dopaminergic and GABAergic. The aim of this study was to determine the effects of gamma1- and gamma2-MSH (at doses 0.3, 1 and 2 nmol/mouse/5 microl) on the anxiety levels in mice in elevated plus maze. Three experimental paradigms were performed to assess the effects of peptides on: a) ethanol withdrawal; b) acute ethanol-induced anxiolytic action; c) peptides per se. We used ethanol as the model substance, since its action involves either dopaminergic/GABAergic or melanocortinergic processes. gamma-MSHs were administered intracisternally in mice and behavioural responses were assessed in the elevated plus maze test. This study provides the first demonstration of an anxiogenic effect of gamma1- and gamma2-MSH, their synergistic/additive effect on ethanol withdrawal-induced anxiety behaviour, and an antagonism of peptides involved in the anxiolytic action of ethanol. Furthermore, results suggest that gamma-MSHs belong to an anxiogenic peptide family that may play an important role in anxiety disorders as well as in the development of alcohol dependence and/or alcohol withdrawal-induced behaviours.

Very low doses of muscimol and baclofen ameliorate cognitive deficits and regulate protein expression in the brain of a rat model of streptozocin-induced Alzheimer’s disease

Abstract Recent studies devoted to neuroprotection have focused on the role of the gamma‐aminobutyric acid (GABA) system in regulating neuroinflammatory processes which play a key role in the neurodegenerative processes observed in Alzheimers disease (AD) by inducing glial cell overactivation and impairing neurotransmission. Data on the efficacy of classical GABA‐A and GABA‐B receptor agonists (muscimol and baclofen, respectively) in animal models of AD are not available. Moreover, no published studies have examined the ability of optimal doses of these compounds to prevent neuroinflammation, the alterations in neurotransmission and cognitive deficits. In the present study, we used a non‐transgenic rat model of AD obtained by intracerebroventricular streptozocin (STZ) injection and assessed the effects of muscimol and baclofen at very low doses (0.01–0.05 mg/kg) on spatial memory and the expression of cortical and hippocampal proteins related to neuroinflammation, namely proteins involved in astroglial functions (glial fibrillary acidic protein, GFAP), GABA synthesis (GABA synthesizing enzyme, glutamic acid decarboxylase 67, GAD67) and acetylcholine degradation (acetylcholine esterase). The presented study demonstrated that in a rat model of STZ‐induced AD both muscimol and baclofen at the tested doses exerted memory‐enhancing and anti‐inflammatory effects, as well as normalization of acetylcholine esterase and GABA expression. We suggested that the function of very low doses of GABA receptor agonists differs from typical GABA‐related inhibition and may be mediated by the allosteric sites of GABA receptors or other non‐specific cell regulatory pathways.

1,4-Dihydropyridines as Tools for Mitochondrial Medicine Against Oxidative Stress and Associated Metabolic Disorders

Various 1, 4-dihydropyridines (DHPs) could affect basic and cell-type specific mitochondrial functions in different way and at various extent either as protectors or as harmful compounds, depending on their lipophylicity and chemical modifications of the DHP nucleus. Hence, several DHPs may affect: 1) mitochondrial bioenergetics as well as enzymatic activities including electron transport chain reactions and organic acids consumption rate ; 2) mitochondrial lipid peroxidation, production of reactive oxygen species and reactive nitrogen species ; 3) antioxidant enzymes: glutathione-S-transferase, superoxide dismutase, catalase ; 4) mitochondrial and cellular membranotropic and/or physico-chemical properties: incorporation into mitochondrial membrane, alteration of membrane lipid organization, thermotropic phase transition profile and membrane lateral heterogeneity ; 5) chemo-osmotic processes (mitochondrial permeability transition, swelling/contraction/aggregation) ; 6) mitochondrial viability, protecting mitochondria against toxic effects of doxorubicin, MPP+, mixture of rotenone/oligomycin, etc.) ; 7) implication of some DHPs in the regulation of mitochondria-mediated heme biosynthetic pathways was checked also in respect to potential differences between their effects on normal and malignant cells. The effects of DHPs on mitochondria of different cellular origin (hepatic, cardiac, neuronal, brain, muscle) were observed.

GABAA agonist muscimol ameliorates learning/memory deficits in streptozocin-induced Alzheimer’s disease non-transgenic rat model

Background: GABAergic inhibitory action regulates learning/memory processes and contributes to neurotransmission (Gong et al., 2009). Existing evidence suggests GABAergic system is involved in pathophysiology of Alzheimer’s disease (AD) via inhibitory interneuron deficits (Verret et al., 2012) and decrease in functional GABAA receptors (Limon et al., 2012). In vitro, GABA and muscimol (GABAA receptor agonist) blocked neuronal death induced by Aβ in rat hippocampal and cortical neurons (Paula-Lima et al., 2005). Our concept: low doses of muscimol may prevent learning/memory deficits in intracerebroventricular (icv) streptozocin (STZ)-induced AD non-transgenic rat model. Methods. Wistar male rats (280±20 g) were pre-treated with saline (control) or muscimol (0.01 and 0.05 mg/kg) for 3 days. On day 4, rats received icv STZ (100 μg/ml) or aCSF. From day 18, rats received muscimol or saline for 4 days; rat spatial learning and memory were assessed in water maze test (4 trials/day) by recording the time to reach the hidden platform (escape latency). A probe trial without platform was carried out 24 h after the training trials, and the number of platform zone crossings has been recorded. Results. STZ statistically increased the escape latency vs. control group (p<0.0001). Muscimol at both doses significantly decreased the escape latency in STZ rats vs. STZ, reversing STZ effect by about 90% on days 3 and 4 (p<0.0001). In probe trial, the number of platform crossings in muscimol+STZ rats’ was significantly increased vs. STZ rats. Muscimol at both doses per se showed values comparable to control. Conclusions. Obtained data suggest that icv STZ significantly decreased rat spatial learning and memory and learning ability. Muscimol at both low doses significantly improved rats’ learning and memory abilities in both normal and AD-type rats. One may suggest that intensification of GABAergic processes may be a useful pharmacotherapeutic strategy to halt early memory decline in AD.

PROTECTIVE EFFECTS OF MILDRONATE IN INDINAVIR- AND EFAVIRENZ-INDUCED TOXICITY IN MICE

Protective effects of mildronate in indinavir- and efavirenz-induced toxicity in mice Previously we showed that mildronate effectively protected mice heart tissue against the toxic influence of anti-HIV drugs azidothymidine, stavudine and lamivudine, which belong to nucleosideanalogue reverse transcriptase inhibitor (NRTI) class. Recently we also demonstrated that mildronate protected isolated rat liver mitochondria against mitochondrial damage caused by azidothymidine. The present study was devoted to examine the possible protective effectiveness of mildronate in cardio-, hepato- and neurotoxicity models caused by anti-HIV drugs of other classes: indinavir, a representative of protease inhibitor (PI) class, and efavirenz, a non-nucleoside-analogue reverse transcriptase inhibitor (NNRTI). Drugs were administered intraperitoneally for two weeks, at the dose of 50 mg/kg of anti-HIV drugs and 100 mg/kg for mildronate. Afterwards, mice heart, liver and brain tissue were examined morphologically and immunohistochemically. The results showed that indinavir in heart tissue caused inflammatory and degenerative changes, manifested as increased expression of nuclear factor kappaBp65 (NF-kBp65), as well as cardiomyocyte necrosis and cellular infiltration. Efavirenz did not cause pathological changes in mice heart tissue, whereas it induced marked expression of caspase-3 and glial fibrillary acidic protein (GFAP) in mice brain tissue and small degenerative alterations in mice liver tissue. The data obtained show mildronates protective properties in indinavir-induced cardiotoxicity and efavirenz-induced neurotoxicity. Mildronāta efekti indinavīra un efavirēna izraisītajos toksicitātes modeļos pelēm Pētījuma uzdevums bija noskaidrot mildronāta spēju pasargāt peļu sirds, smadzeņu un aknu audus no anti-HIV vielu indinavīra (proteāzes inhibitora) un efavirēna (ne-nukleozīdu analogu reversās transkriptāzes inhibitora) izraisītajām morfologiskajām un imunohistoķīmiskajām pārmaiņām. Indinavīrs (50 mg/kg, divu nedēļu intraperitoneāla ievadīšana) peļu sirds audos izraisīja iekaisuma un degeneratīvas pārmaiņas, kas izpaudās kā palielināta kodola faktora kappaBp65 (NF-κBp65) ekspresija, kardiomiocītu nekroze un šūnu infiltrācija. Efavirēns (50 mg/kg, divu nedēļu intraperitoneāla ievadīšana) neizraisīja patologiskas izmaiņas peļu sirds audos, taču izraisīja izteiktu apoptozi un astrogliozi smadzeņu audos un nelielas degeneratīvas pārmaiņas peļu aknu audos. Mildronāts (100 mg/kg, ievadīts intraperitoneāli vienlaicīgi ar anti-HIV vielām) uzrādīja protektējošas īpašības, būtiski samazinot indinavīra un efavirēna efektus tajos audos, kuros šīs vielas uzrādīja toksicitāti. Iegūtie dati, no vienas puses, demonstrē pētīto anti-HIV vielu atšķirīgo ietekmi uz dažādu audu funkcijām, un, no otras puses, atšķirīgo mildronāta protektējošo efektu, kam var būt terapeitiska nozīme, izmantojot mildronātu kā indinavīra kardiotoksicitātes un efivarēna neirotoksicitātes protektētāju.