Meclofenoxate Inhibits Aggregation of Alpha-synuclein in vitro.

BACKGROUND: α-Synuclein, a natively disordered protein, is a key component of Lewy bodies, the ubiquitinated protein aggregates which are the pathological hallmark of Parkinson's disease (PD). Meclofenoxate (centrophenoxine) is a nootropic drug which has shown beneficial therapeutic effects in various neuronal diseases. Administration of meclofenoxate enhanced levels of dopamine and improved motor function in animal models of Parkinson's disease (PD). Evidence suggested that dopamine interacts with and modulates α-synuclein aggregation. OBJECTIVE: The aim of this work was to investigate whether the observed positive effect of addition of meclofenoxate, a nootropic agent, on dopamine level, could be correlated with its effect on aggregation of α-synuclein. METHODS: Purification of recombinant human α-synuclein was performed by anion exchange chromatography. The purified protein was incubated in the absence and presence of meclofenoxate and was analyzed for aggregation by Thioflavin T fluorescence spectroscopy. Conformational changes in α-synuclein were monitored by fluorescence spectroscopy and fluorescence quenching studies using a neutral quencher. Secondary structure analysis of α-synuclein was monitored by circular dichroism spectroscopy. RESULTS: Recombinant human α-synuclein was expressed and purified by anion-exchange chromatography. Incubation of α-synuclein with meclofenoxate led to lowering aggregation in a concentration-dependent manner. Reduction in formation of oligomers was seen which suggested the formation of an off-pathway species which did not give rise to an aggregation-competent entity. Fluorescence quenching studies revealed that the additive distorted the native conformation of α- synuclein, leading to the formation of lower amounts of aggregation-prone species. CONCLUSION: In the presence of higher concentrations of meclofenoxate, α-synuclein undergoes a change in its conformation. This change is not dependent on the concentration of the additive. This non-native conformer promotes the formation of a species which does not undergo further aggregation. Our study provides a mechanistic explanation of the earlier observation that meclofenoxate has a beneficial effect on progression of PD in animal models.

The Effect of Meclofenoxate on the Transcriptome of Aging Brain of Nothobranchius guentheri Annual Killifish.

Annual fish of the genus Nothobranchius are promising models for aging research. Nothobranchius reproduces typical aspects of vertebrate aging, including hallmarks of brain aging. Meclofenoxate (MF) is a well-known compound that can enhance cognitive performance. The drug is prescribed for asthenic conditions, trauma, and vascular diseases of the brain. It is believed that MF is able to delay age-dependent changes in the human brain. However, until now, there has been no study of the MF effect on the brain transcriptome. In the present work, we performed an RNA-Seq study of brain tissues from aged Nothobranchius guentheri, which were almost lifetime administered with MF, as well as young and aged control fish. As expected, in response to MF, we revealed significant overexpression of neuron-specific genes including genes involved in synaptic activity and plasticity, neurotransmitter secretion, and neuron projection. The effect was more pronounced in female fish. In this aspect, MF alleviated age-dependent decreased expression of genes involved in neuronal activity. In both treated and untreated animals, we observed strong aging-associated overexpression of immune and inflammatory response genes. MF treatment did not prevent this effect, and moreover, some of these genes tended to be slightly upregulated under MF treatment. Additionally, we noticed upregulation of some genes associated with aging and cellular senescence, including isoforms of putative vascular cell adhesion molecule 1 (VCAM1), protein O-GlcNAcase (OGA), protein kinase C alpha type (KPCA), prolow-density lipoprotein receptor-related protein 1 (LRP1). Noteworthy, MF treatment was also associated with the elevated transcription of transposons, which are highly abundant in the N. guentheri genome. In conclusion, MF compensates for the age-dependent downregulation of neuronal activity genes, but its effect on aging brain transcriptome still cannot be considered unambiguously positive.

Chemical Compensation of Mitochondrial Phospholipid Depletion in Yeast and Animal Models of Parkinson's Disease.

We have been investigating the role that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) content plays in modulating the solubility of the Parkinson's disease protein alpha-synuclein (α-syn) using Saccharomyces cerevisiae and Caenorhabditis elegans. One enzyme that synthesizes PE is the conserved enzyme phosphatidylserine decarboxylase (Psd1/yeast; PSD-1/worms), which is lodged in the inner mitochondrial membrane. We previously found that decreasing the level of PE due to knockdown of Psd1/psd-1 affects the homeostasis of α-syn in vivo. In S. cerevisiae, the co-occurrence of low PE and α-syn in psd1Δ cells triggers mitochondrial defects, stress in the endoplasmic reticulum, misprocessing of glycosylphosphatidylinositol-anchored proteins, and a 3-fold increase in the level of α-syn. The goal of this study was to identify drugs that rescue this phenotype. We screened the Prestwick library of 1121 Food and Drug Administration-approved drugs using psd1Δ + α-syn cells and identified cyclosporin A, meclofenoxate hydrochloride, and sulfaphenazole as putative protective compounds. The protective activity of these drugs was corroborated using C. elegans in which α-syn is expressed specifically in the dopaminergic neurons, with psd-1 depleted by RNAi. Worm populations were examined for dopaminergic neuron survival following psd-1 knockdown. Exposure to cyclosporine, meclofenoxate, and sulfaphenazole significantly enhanced survival at day 7 in α-syn-expressing worm populations whereby 50-55% of the populations displayed normal neurons, compared to only 10-15% of untreated animals. We also found that all three drugs rescued worms expressing α-syn in dopaminergic neurons that were deficient in the phospholipid cardiolipin following cardiolipin synthase (crls-1) depletion by RNAi. We discuss how these drugs might block α-syn pathology in dopaminergic neurons.

Aging of cell membranes: facts and theories.

This chapter is intended to outline the main results of a research trend realized by the author during the last 45 years, focused on the main role played by the cell membrane in the aging process. It is a very wide field; therefore, the reader cannot expect in this limited space a detailed description, but will be given a wide, interdisciplinary insight into the main facts and theories regarding cellular aging. The central idea described here is the concept called the membrane hypothesis of aging (MHA). The history, the chemical roots, physicochemical facts, biophysical processes, as well as the obligatory biochemical consequences are all touched in by indicating the most important sources of detailed knowledge for those who are more interested in the basic biology of the aging process. This chapter covers also the available anti-aging interventions on the cell membrane by means of the centrophenoxine treatment based on the MHA. It also briefly interprets the possibilities of a just developing anti-aging method by using the recombinant human growth hormone, essential basis of which is the species specificity, and the general presence of receptors of this hormone in the plasma membrane of all types of cells.

Biolistic transformation of wheat with centrophenoxine as a synthetic auxin.

Cereal crops, including bread wheat (Triticum aestivum L.), are an important staple food worldwide. With a growing global population, it is evident that current crop production will not meet the rising demands being placed on modern agriculture. Efforts to improve crop yield and stress-tolerance by traditional breeding are labor intensive, time consuming, and highly dependent upon the ability to capture existing and novel genetic variation from a restricted genetic pool. Genetic engineering of crop species is one of several alternatives to traditional breeding for the introduction of novel genetic variation. This recently established technology has proved useful for the introduction of novel traits like pest resistance and herbicide tolerance. As a universal tool for genetic transformation, the Biolistic Gene Gun allows for the genomic integration of novel gene sequences from various sources into a whole host of living organisms.In this chapter, we present a novel and detailed protocol for the Biolistic Transformation of bread wheat that uses the pharmaceutical compound, Centrophenoxine (CPX). The application of CPX as the main auxin-like plant growth regulator in cereal genetic transformation replaces the potent but more toxic herbicide 2,4-D.

Effect of centrophenoxine against rotenone-induced oxidative stress in an animal model of Parkinson's disease.

Oxidative stress has been implicated in the etiology of Parkinson's disease (PD). The important biochemical features of PD, being profound deficit in dopamine (DA) content, reduced glutathione (GSH), and enhanced lipid peroxidation (LPO) in dopaminergic (DA-ergic) neurons resulting in oxidative stress, mitochondrial dysfunction and apoptosis. Rotenone-induced neurotoxicity is a well acknowledged preclinical model for studying PD in rodents as it produces selective DA-ergic neuronal degeneration. In our previous study, we have shown that chronic administration of rotenone to rats is able to produce motor dysfunction, which increases progressively with rotenone treatment and centrophenoxine (CPH) co-treatment is able to attenuate these motor defects. The present study was carried out to evaluate the antioxidant potential of CPH against rotenone-induced oxidative stress. Chronic administration of rotenone to SD rats resulted in marked oxidative damage in the midbrain region compared to other regions of the brain and CPH co-treatment successfully attenuated most of these changes. CPH significantly attenuated rotenone-induced depletion in DA, GSH and increase in LPO levels. In addition, the drug prevented the increase in nitric oxide (NO) and citrulline levels and also enhanced the activity of catalase and superoxide dismutase (SOD). Histological analysis carried out using hematoxylin and eosin staining has indicated severe damage to mid brain in comparison to cortex and cerebellum and this damage is attenuated by CPH co-treatment. Our results strongly indicate the possible therapeutic potential of centrophenoxine as an antioxidant in Parkinson's disease and other movement disorders where oxidative stress is a key player in the disease process.

[Effects of nicotinic cholinoreceptor ligands and nootropic drugs on the spontaneous exploratory activity in a labyrinth in mice].

We have studied the effects of nicotine (0.125, 0.25, and 0.5 mg/kg) and mecamylamine (0.5, 1.5, and 3 mg/kg) in comparison to reference cognition-enhancing drugs piracetam (100 and 300 mg/kg) and meclofenoxate (20, 50, and 100 mg/kg) administered to male C57BL mice intraperitoneally 30 min prior to behavioral test. The behavioral drug effect was evaluated as influencing the activity in visiting arms of a closed plus-maze. Piracetam (300 mg/kg) and meclofenoxate (100 mg/kg) improved the exploratory activity. Mecamylamine (0.5 mg/kg) also improved the exploratory activity, while nicotine (0.5 mg/kg) deteriorated it.

Behavioral alterations in rotenone model of Parkinson's disease: attenuation by co-treatment of centrophenoxine.

Rotenone, a potent specific inhibitor of mitochondrial complex-1, appears to reproduce the behavioral features of Parkinson's disease in rats. It destroys dopaminergic neurons selectively, causing deficiency of dopamine in striatum which leads to impaired motor functions. Oxidative stress generated as a result of mitochondrial dysfunction and metabolism of dopamine has been implicated as an important factor in the etiology of Parkinson's disease. Present study explores the potential of centrophenoxine (a well known anti-aging and antioxidant drug) against rotenone induced motor dysfunction. Sprague Dawley male rats were administered with rotenone on a daily basis by subcutaneous injection of dose: 2 mg/kg body weight over a period of 35 days. Data showed impaired motor function, significant increase in catalepsy, decrease in locomotor activity and decrease in muscle activity. Dopamine content of rotenone treated animals was found to decrease significantly and lipid peroxidation was found to increase significantly in rotenone treated animals when compared with co-treated group. Co-treatment with centrophenoxine (100 mg/kg i.p. for 35 days) significantly attenuated the extent of motor dysfunction and changes in the level of dopamine and lipid peroxidation induced by rotenone toxicity. Thus, the present study provides evidence that centrophenoxine co-treatment attenuates rotenone induced motor dysfunction by virtue of its antioxidant action.

[Effects of piracetam and meclofenoxate on the brain NMDA and nicotinic receptors in mice with different exploratory efficacy in the cross maze test].

A population of outbred mice of the ICR strain was divided into two subpopulations according to their high (EH mice) or low (EL mice) exploratory efficacy in the closed cross maze test. In addition, the EH and EL mice differed in the number of binding sites of (i) [G-3H]-MK-801 with NMDA receptors from hippocampus and (ii) [G-3H]-nicotine with nicotine cholinoreceptors (nACh) from neocortex. A subchronic administration of the cognition enhancer piracetam (200 mg/kg, once per day for 5 days) increased by 70% the number of binding sites of NMDA receptors in the EL mice. At the same time, this treatment decreased the density of neocortical nACh receptors in both EL and EH mice (by 55% and 40%, respectively). A subchronic administration of the cognition enhancer and anti-oxidant meclofenoxate (100 mg/kg, once per day for 5 days) also decreased the density of neocortical nACh receptors in both EL and EH mice (by 48% and 20%, respectively). However, meclofenoxate also increased by 41% the number of binding sites of NMDA receptors in the EH mice.

Further evidence of centrophenoxine mediated protection in aluminium exposed rats by biochemical and light microscopy analysis.

The environmental agent aluminium has been intensively investigated in the initiation and progression of various neurological disorders and the role of oxidative stress in these disorders is a widely discussed phenomenon. In this light, the present study is focused on the role of aluminium in mediating oxidative stress, which may help in better understanding its role in neuronal degeneration. Further, we have exploited a known anti-aging drug centrophenoxine to explore its potential in the conditions of metal induced oxidative damage. Aluminium was administered orally at a dose level of 100 mg/kg b.wt./day for a period of 6 weeks followed by a post treatment of centrophenoxine at a dose level of 100 mg/kg b.wt./day for another 6 weeks. Following aluminium exposure, a significant increase in lipid peroxidation levels (estimated by MDA) were observed which was accompanied by a decrease in reduced glutathione content in both cerebrum and cerebellum of rat brain. Post treatment of centrophenoxine significantly reduced the lipid peroxidation levels and also increased the reduced glutathione content in both the regions. Histologically observed marked deteriorations in the organization of various cellular layers in both cerebrum and cerebellum were observed after aluminium administration. Centrophenoxine treated animals showed an appreciable improvement in the histoarchitecture of the cellular layers. Our results indicate that centrophenoxine has an antioxidant potential and should be examined further in aluminium toxic conditions.

Evidence for centrophenoxine as a protective drug in aluminium induced behavioral and biochemical alteration in rat brain.

Potential use of various nootropic drugs have been a burning area of research on account of various physical and chemical insult in brain under different toxicological conditions. One of the nootropic drug centrophenoxine, also known as an anti-aging drug has been exploited in the present experiment under aluminium toxic conditions. Aluminium was administered by oral gavage at a dose level of 100 mg/Kg x b x wt/day for a period of six weeks. To elucidate the region specific response, study was carried out in two different regions of brain namely cerebrum and cerebellum. Following aluminium exposure, a significant decrease in the activities of enzymes namely Hexokinase, Lactate dehydrogenase, Succinate dehydrogenase, Mg(2+) dependent ATPase was observed in both the regions. Moreover, the activity of acetylcholinesterase was also reported to be significantly decreased. Post-treatment with centrophenoxine was able to restore the altered enzyme activities and the effect was observed in both the regions of brain although the activity of lactate dehydrogenase and acetylcholinesterase did not register significant increase in the cerebellum region. Further, centrophenoxine was able to improve the altered short-term memory and cognitive performance resulted from aluminium exposure. From the present study, it can be concluded that centrophenoxine has a potential and can be exploited in other toxicological conditions also.

Modulatory effects of centrophenoxine on different regions of ageing rat brain.

The debilitating consequences of age-related brain deterioration are widespread and extremely costly in terms of quality of life and longevity. Free radical induced damage is thought to be responsible, at least in part, for the degenerative effects of aging. This may be largely due to high-energy requirements, high oxygen consumption, high tissue concentration of iron and low of antioxidant enzymes in brain. Therefore, supplementing an external source of free radical scavenger would greatly benefit in ameliorating the free radical damage which may thus be beneficial in aging. In the present study, an important nootropic agent Centrophenoxine, which has an easy access to brain, has been administered to aged animals (16 months old). Rats aged 6 months (young group) and 16 months old (old group) were chosen for the study. Both groups were administered Centrophenoxine (dissolved in physiological saline) intraperitoneally once a day for 6 weeks. Our study indicates an increased activity of Catalase, Superoxide Dismutase, Glutathione reductase, as well as an increase in the reduced, oxidized, and total glutathione content thus resulting in an altered redox state. A substantial increase in the malondialdehyde content was also reported as a result of aging. Whereas, following Centrophenoxine administration (100 mg/kg body weight/day, injected i.p) alterations in the activities of Superoxide dismutase, Glutathione reductase as well as in the reduced and oxidized glutathione content was reported in aged rat brain. Lipid peroxidation was also reported to be significantly decreased in aged animals after Centrophenoxine supplementation for 6 weeks. The use of an extraneous antioxidant substance may prove beneficial in combating the conditions of oxidative stress in ageing brain.

Centrophenoxine improves chronic cerebral ischemia induced cognitive deficit and neuronal degeneration in rats.

AIM: To study the effects of centrophenoxine (CPH, meclofenoxate) on chronic cerebral hypoperfusion induced deficits in rats. METHODS: Chronic hypoperfusion in rats was performed by permanent bilateral ligation of the common carotid arteries. Morris water maze was used to measure spatial memory performance. Spectrophotometrical techniques were used to assay SOD, GPx activities, MDA content, TXB2, and 6-keto-PGF1alpha levels. Morphological change was examined by HE staining. The expression of Bax and p53 protein were assayed by immunohistochemistry analysis. RESULTS: Chronic hypoperfusion in rats resulted in spatial memory impairments shown by longer escape latency and shorter time spent in the target quadrant. These behavioral dysfunction were accompanied by increase in SOD and GPx activities, the content of MDA, the levels of pro-inflammatory mediators (TXB2, 6-keto-PGF1alpha), overexpression of Bax and P53 protein, and delayed degeneration of neurons in cortex and hippocampus. Oral administration of CPH (100 mg/kg, once per day for 37 d) markedly improved the memory impairment, reduced the increase in antioxidant enzyme activities, MDA content and the levels of pro-inflammatory mediators to their normal levels, and attenuated neuronal damage. CONCLUSION: The abilities of CPH to attenuate memory deficits and neuronal damage after ischemia may be beneficial in cerebrovascular type dementia.

Pharmacological interventions against aging through the cell plasma membrane: a review of the experimental results obtained in animals and humans.

As was shown in a recent review by this author (Ann. N.Y. Acad. Sci., 928: 187-199, 2001), oxyradicals cannot be considered only as harmful by-products of the oxidative metabolism, but living cells and organisms implicitly require their production. This idea is supported by numerous facts and arguments, the most important of which is that the complete inhibition of the oxyradical production by KCN (or by any block of respiration) kills the living organisms long before the energy reserves would be exhausted. This new theoretical approach not only helps our understanding of the normal functions of the living organisms, such as the basic memory mechanisms in the brain cells, but also helps in identifying the site-specific, radical-induced damaging mechanisms that represent the undesirable side effects of oxygen free radicals. First of all, these effects make the cell plasma membrane vulnerable and cause a series of intracellular functional disorders, as described by the membrane hypothesis of aging (MHA). The logical way for any antiaging intervention therefore should be to increase the available number of loosely bound electrons inside the plasma membrane that are easily accessible for OH(*) free radical scavenging. The present review summarizes the available knowledge regarding the theory of the use of membrane-related antiaging pharmaca, like centrophenoxine (CPH), tested in both animal experiments and human clinical trials. A modified, developed version of CPH coded as BCE-001 is also reported.

The effect of meclofenoxate with ginkgo biloba extract or zinc on lipid peroxide, some free radical scavengers and the cardiovascular system of aged rats.

Aged rats are highly prone to many physiological changes such as blood pressure and heart rate. These changes could be due to modification in membrane phospholipid composition of their blood vessels. Lipid peroxide in vivo has been identified as a basic deteriorative reaction in cellular mechanisms of aging in human. The effect of a nootropic drug, meclofenoxate (MF) or its combination with extract of ginkgo biloba (EGb-761) or zinc (Zn) on malondialdehyde (MDA) product as an index of endogenous lipid peroxidation; phospholipid; glutathione (GSH) and protein thiols (PrSHs) contents as well as superoxide dismutase (SOD) activity in blood, brain, heart and liver of 24-month-old male rats was investigated. Aged rats were treated with MF once daily at oral doses of 100 mg kg-1 body wt. alone or with either EGb at a dose of 150 mg kg-1 body wt. or Zn at 10.5 mg kg-1 body wt. for 4 weeks. This study showed that aging caused a higher increment in MDA level of brain and heart than liver and plasma accompanied with reduction in brain and heart phospholipid contents as well as alteration of the antioxidant systems as compared to 4-month-old rats. Treatment of aged rats with MF alone or combined with either EGb or Zn caused improvement in the measured free radical scavengers especially in brain and heart tissues. Our results also showed that both EGb and Zn induced a significant potential effect of MF action on blood pressure and heart rate. The results were explained in the light of the antioxidant properties of EGb and Zn. Thus it is concluded that EGb and Zn have a beneficial role with MF in diminishing cumulative oxidative changes in aging.

[Biomedical aspects of health preservation strategy: role of catecholaminergic neuronal populations]. / Biomeditsinskie aspekty strategii sokhraneniia zdorov'ia: rol' katekholaminergicheskikh neironal'nykh populiatsii.

Lifestyle, environmental factors, genetics, and medical care are the main factors that determine the health status of man. Of particular attention are biological mechanisms ensuring the body's adaptation to constantly changing environmental conditions. The noradrenergic neuronal populations, the sympathetic nervous system in particular, modulate metabolic processes and supports a variety of activities, making them relevant to changing living conditions. There is a clear correlation between the life span and the number of sympathetic nerve cells functioning during postnatal ontogenesis. The exposures that reduce the activity of peripheral and central noradrenergic neurons and slow down aging processes in them loosen the relationships between the inner and outer world to prevent hyperactivity and to prolong life.

Centrophenoxine activates acetylcholinesterase activity in hippocampus of aged rats.

Age-related changes in the acetylcholinesterase activity were measured in the hippocampus, brain stem and cerebellum of rats (aged 4, 8, 16 and 24 months). The age-dependent decrease in the enzyme activity first appeared in the hippocampus; the brain stem was affected later while the cerebellum remained unaffected. Centrophenoxine, usually considered as an ageing reversal drug and also regarded as a neuroenergeticum in human therapy, increased the acetylcholinesterase activity in the hippocampus of aged rats, the activity was also elevated in the brain stem but no in the cerebellum. The acetylcholinesterase-stimulating influence of the drug is likely to be implicated in the pharmacological reversal of the age related decline of the cholinergic system. This effect of the drug may also mediate its effects on cognitive and neuronal synaptic functions.

[Comparative quantitative pharmacological-EEG analysis of the effects of psychostimulants]. / Sravnitel'nyi kolichestvennyi farmako-EEG-analiz deistviia psikhostimuliruiushchikh sredstv.

Amphetamine, caffeine, sydnocarb, meclofenoxate, adapromine, midantan, and nomifensine were studied for their effects on bioelectrical activity and Fourier EEG power spectra of the sensomotor cortex, dorsal hippocamp and lateral hypothalamus of freely behaving awake rats. The drop in the absolute power of all frequency ranges with the enhanced power of fast beta 1,2-ranges was common to the action of psychostimulants. In addition to the common properties, specific features of their action were revealed. Amphetamine, meclofenoxate, and nomifensine were found to increase the amplitude of the dominant peak in the theta-range and amphetamine shifts the frequency of the dominant peak to the region of faster ranges. The agents-induced electrophysiological changes correspond to the varying degrees of activation of the central nervous system, causing the optimization of behavioral functions, abolition of fatigue and drowsiness and enhancing physical and mental working capacity.

In vitro and in vivo effect of the nootropic agent adafenoxate on the 5-HT1 sites in different rat brain structures.

1. The effect of the nootropic agent adafenoxate (a structural analogue of meclofenoxate) on the binding parameters of 5-HT1 receptors in vitro and in vivo in rat cerebral cortex, striatum, hippocampus and hypothalamus was studied. 2. The chronic (100 mg/kg per os for 7 days) adafenoxate treatment produced a significant (24.6%) decrease in the density of 5-HT1 sites in the hippocampus. 3. In vitro adafenoxate inhibited specific [3H]5-HT binding with equal potency in all the regions studied with IC50s in the microM range. 4. It is suggested that the decrease in the density of the 5-HT1 sites in rat hippocampus might contribute to the nootropic action of adafenoxate.