Mildronate and its Neuroregulatory Mechanisms : Targeting the Mitochondria, Neuroinflammation, and Protein Expression

This review for the first time summarizes the data obtained in the neuropharmacological studies of mildronate, a drug previously known as a cardioprotective agent. In different animal models of neurotoxicity and neurodegenerative diseases, we demonstrated its neuroprotecting activity. By the use of immunohistochemical methods and Western blot analysis, as well as some selected behavioral tests, the new mechanisms of mildronate have been demonstrated: a regulatory effect on mitochondrial processes and on the expression of nerve cell proteins, which are involved in cell survival, functioning, and inflammation processes. Particular attention is paid to the capability of mildronate to stimulate learning and memory and to the expression of neuronal proteins involved in synaptic plasticity and adult neurogenesis. These properties can be useful in neurological practice to protect and treat neurological disorders, particularly those associated with neurodegeneration and a decline in cognitive functions.publishersversionPeer reviewe

Mildronate's protective effects in the peripheral nervous system : Stavudine-induced neuropathy and formalin-induced inflammation

Funding Information: This work was supported by the Latvian Council of Science Grant Nr. 05-1418; ESF Grant ESS2004/3; Contract No. 453, between the Latvian Institute of Organic Synthesis and the Joint Stock Company “Grindex”; Contract No. 2377, between the University of Latvia and the Joint Stock Company “Grindex”; and a L’ORÉAL Latvian “For Women In Science” fellowship with the support of the Latvian National Commission for UNESCO and the Latvian Academy of Sciences.Mildronate, previously known as a cardioprotective drug, recently was found to normalise mitochondrial processes by preventing the dysfunction of complex I in rat liver mitochondria. Previously we have shown also the ability of mildronate to prevent pathologies in the central nervous system by normalizing the expression of different signalling molecules in brain tissue. This allowed us to suggest that mildronate may possess a beneficial role also in peripheral nervous system pathologies. The present study was designed to assess the peripheral tissue damage caused by anti-HIV drug stavudine, as well as pain and inflammation caused by formalin. For this demonstration, we investigated the influence of mildronate: (1) on decreased myelin expression and increased neuron degeneration in rat sciatic nerve tissue caused by stavudine; and (2) on formalin-induced inflammation in mice. We found that mildronate protected the stavudine-induced degeneration of neurons in rat peripheral sciatic nerve without a significant influence on demyelination. In a formalin test, mildronate showed anti-inflammatory action comparable to that of indomethacin, a reference drug. The present results show that mildronate is capable of regulating peripheral nerve damage and peripheral inflammatory responses. We suggest that the multifunctional effects of mildronate can be attributed to its ability to regulate mitochondrial processes. The obtained data indicate protective effects of mildronate in different peripheral neurological pathologies.publishersversionPeer reviewe

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

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 Alzheimer's 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 (0.01-0.05mg/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

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

Intranasal Administration of Extracellular Vesicles Derived from Human Teeth Stem Cells Improves Motor Symptoms and Normalizes Tyrosine Hydroxylase Expression in the Substantia Nigra and Striatum of the 6-Hydroxydopamine-Treated Rats

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting millions of people worldwide. At present, there is no effective cure for PD; treatments are symptomatic and do not halt progression of neurodegeneration. Extracellular vesicles (EVs) can cross the blood–brain barrier and represent promising alternative to the classical treatment strategies. In the present study, we examined therapeutic effects of intranasal administration of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on unilateral 6‐hydroxydopamine (6‐OHDA) medial forebrain bundle (MFB) rat model of PD. CatWalk gait tests revealed that EVs effectively suppressed 6‐OHDA‐induced gait impairments. All tested gait parameters (stand, stride length, step cycle, and duty cycle) were significantly improved in EV‐treated animals when compared with 6‐OHDA‐lesion group rats. Furthermore, EVs slowed down numbers of 6‐OHDA‐induced contralateral rotations in apomorphine test. Improvements in motor function correlated with normalization of tyrosine hydroxylase expression in the striatum and substantia nigra. In conclusion, we demonstrated, for the first time, the therapeutic efficacy of intranasal administration of EVs derived from SHEDs in a rat model of PD induced by 6‐OHDA intra‐MFB lesion. Our findings could be potentially exploited for the development of new treatment strategies against PD

Neuro-, cardio- and hepatoprotective effects of mitochondria-targeted compounds in anti-HIV drug toxicity models

ANOTĀCIJA Promocijas darbā pētītas dažādu savienojumu vielas – mildronāts (azabutirobetaīna klase) un 1,4-dihidropiridīnu klases vielas – cerebrokrasts (ar tipisku biciklisku struktūru), glutapirons un tauropirons (abi aminoskābju saturoši) – kas spēj regulēt šūnu mitohondriālos procesus. Kā toksiskās mitohondriju disfunkciju izraisošas modeļvielas izmantoti anti-HIV preparāti – azidotimidīns, stavudīns, lamivudīns, indinavīrs un efavirēns. Eksperimenti veikti izolētos žurku aknu mitohondrijos, kā arī peļu neiro-, kardio- un hepatotoksicitātes modeļos. Audos ex vivo noteikta morfoloģiskā aina un dažādu marķieru ekspresija, kas liecina par iekaisumu (kodola faktors kappa Bp65 jeb NF-κBp65, inducējamā slāpekļa oksīda sintāze jeb iNOS, apoptozi (kaspāze-3, šūnas apoptozes jutības proteīns jeb CAS), mitohondriju (citohroma c oksidāze) un glijas astrocītu (glijas fibrilārais skabais proteīns jeb GFAP) funkcijām. Pirmo reizi esam parādījuši mildronāta spēju in vitro protektēt mitohondriālos procesus kompleksa I līmenī. Iegūtie dati pārliecinoši parāda, ka vielas, īpaši mildronāts un cerebrokrasts uzrāda izteiktu citoprotektīvu darbību, novēršot iekaisuma un deģeneratīvos procesus peļu sirds, smadzeņu un aknu audos. Aminoskābes saturošie 1,4-dihidropiridīna atvasinājumi glutapirons un tauropirons izraisīja darbības duālismu – gan iekaisumu un apoptozes veidošanos, gan protektīvu darbību, kas liek domāt, ka mitohondriji nav glutapirona un tauropirona galvenie mērķi šūnā. Mildronāta un cerebrokrasta protektīvie efekti dažādos šūnas funkciju un signālmolekulu līmeņos ļauj spriest, ka to vielu struktūru elementiem (farmakoforiem) ir būtiska loma mitohondriju aizsargāšanā. Mūsu rezultāti apstiprina darba koncepciju, ka mitohondriju protekcija ir jauna farmakoterapeitiska stratēģija dažādu patoloģiju (t.sk.neirodeģenetīvu slimību) profilaksē un ārstēšanā.ANNOTATION The present study investigates four different compounds – mildronate, a representative of aza-butyrobetaine class, and atypical 1,4-dihydropyridine (DHP) derivatives: cerebrocrast (with bicyclic structure), glutapyrone and tauropyrone (both amino acid-containing DHPs), which are capable to regulate mitochondrial processes. Anti-HIV drugs – azidothymidine, stavudine, lamivudine, indinavir and efavirenz are used as mitochondria-compromizing substances. Experiments are carried out in isolated rat liver mitochondria, as well as in mice tissues of neuro-, cardio- and hepatotoxicity models by evaluating tissue morphology and the expression of markers indicating inflammation (nuclear factor kappa Bp65 or NF-κBp65, inducible nitric oxide synthase or iNOS), apoptosis (caspase-3, cellular apoptosis susceptibility protein or CAS), mitochondrial (cytochrome c oxidase) and glial astrocyte (glial fibrillary acidic protein or GFAP) functions. For the first time we have demonstrated that mildronate is capable to protect isolated rat liver mitochondria from azidothymidine-induced toxicity. The obtained data convincingly show cytoprotective action of the tested compounds (particularly mildronate and cerebrocrast) by protecting inflammatory and degenerative processes in mice cardiac, brain and liver tissues. Amino acid-containing DHPs glutapyrone and tauropyrone showed a dualism of action – the inflammation- and apoptosis-promoting and the protective action, suggesting that their action is directed to other than mitochondria target. The protective effects of mildronate and cerebrocrast manifested at different levels of cell functioning and molecules allow us to suggest that these compounds comprise the structure elements (pharmacophores) that play a significant role in mitochondrial protection. These data confirm the concept that mitochondria-targeting can be considered as novel pharmacotherapeutic strategy for the prophylaxis and treatment of different pathologies, particularly these of neurodegenerative diseases

Search for stroke-protecting agents in endothelin-1-induced ischemic stroke model in rats

Background and Objective. Ischemic stroke may initiate a reperfusion injury leadingto brain damage cascades where inflammatory mechanisms play a major role. Therefore, thenecessity for the novel stroke-protecting agents whose the mechanism of action is focused on their anti-inflammatory potency is still on the agenda for drug designers. Our previous studies demonstrated that cerebrocrast (a 1,4-dihydropyridine derivative) and mildronate (a representative of the aza-butyrobetaine class) possessed considerable anti-inflammatory and neuroprotective properties in different in vitro and in vivo model systems.The present study investigated their stroke-protecting ability in an endothelin-1 (ET-1)-induced ischemic stroke model in rats. Material and Methods. Male Wistar rats were pretreated (for 7 days, per os) with cerebrocrast (0.1 mg/kg), mildronate (100 mg/kg), or their combination, followed by the intracerebral injection of ET-1. Functional and behavioral tests were carried out up to 14 days after the ET-1 injection. Ex vivo, the number of degenerated neurons and the infarction size in the cerebral cortical tissue were assessed histologically. Results. Cerebrocrast and mildronate effectively normalized ET-1-induced disturbances in neurological status, improved the muscle tone, and decreased the number of degenerated cortical cells. Both drugs also reduced the infarction size, and cerebrocrast showed at least a 2-fold higher activity than mildronate. The combination of both drugs did not cause a more pronounced effect in comparison with the action of drugsadministered separately. Conclusions. The 1,4-dihydropyridine and aza-butyrobetaine structures may serve for the design of novel stroke-protecting agents to prevent severe neurological poststroke consequences.publishersversionPeer reviewe

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. Methods The effects of taurine and tauropyrone, as well as of the 1,4-dihydropyridine moiety were compared in in-vitro experiments to determine the binding to GABA receptors and influence on mitochondrial processes (isolated rat liver mitochondria), and in in-vivo tests to assess the influence on behavioural effects caused by the GABA-A receptor ligands, bicuculline, diazepam and ethanol. Key findings Unlike taurine, tauropyrone did not display binding activity for the GABA-A receptor, and only taurine (but not tauropyrone) at low doses (0.1, 1.0 and 10 mg/kg) antagonised the bicuculline-induced convulsion effect. Taurine and tauropyrone had no effect on diazepam myorelaxing action, and they both exerted a comparable 'anti-ethanol' effect (shortening of the ethanol-sleeping time). Taurine and tauropyrone did not influence processes of mitochondrial bioenergetics. Conclusions The action of tauropyrone at the level of the GABA-A receptor differs qualitatively from that of taurine, probably because of its 1,4-dihydropyridine moiety, which may hinder access to the GABA-A receptor GABA site. Tauropyrone does not show improved pharmacological efficacy in in-vitro and in-vivo studies in comparison with taurine.publishersversionPeer reviewe