The delayed effect of rotenone on the relative content of brain isatin-binding proteins of rats with experimental parkinsonism.

Isatin (indoldione-2,3) is an endogenous biological regulator found in the brain, peripheral tissues, and biological fluids of humans and animals. Its biological activity is realized via isatin-binding proteins, many of which were identified during proteomic profiling of the brain of mice and rats. A number of these proteins are related to the development of neurodegenerative diseases. Previously, using a model of experimental Parkinsonism induced by a seven-day course of rotenone injections, we have observed behavioral disturbances, as well as changes in the profile and relative content of brain isatin-binding proteins. In this study, we have investigated behavioral responses and the relative content of brain isatin-binding proteins in rats with rotenone-induced Parkinsonism 5 days after the last administration of this neurotoxin. Despite the elimination of rotenone, animals exhibited motor and coordination impairments. Proteomic profiling of isatin-binding proteins revealed changes in the relative content of 120 proteins (the relative content of 83 proteins increased and that of 37 proteins decreased). Comparison of isatin-binding proteins characterized by the changes in the relative content observed in the brain right after the last injection of rotenone (n=16) and 5 days later (n=11) revealed only two common proteins (glyceraldehyde-3-phosphate dehydrogenase and subunit B of V-type proton ATPase). However, most of these proteins are associated with neurodegeneration, including Parkinson's and Alzheimer's diseases.

Neuroprotective effects of isatin and afobazole in rats with rotenone-induced Parkinsonism are accompanied by increased brain levels of Triton X-100 soluble alpha-synuclein.

Effects of the endogenous neuroprotector isatin and the pharmacological drug afobazole (exhibiting neuroprotective properties) on behavioral reactions and quantitative changes in the brain proteomic profile have been investigated in rats with experimental rotenone Parkinsonism. A single dose of isatin (100 mg/kg subcutaneously on the last day of a 7-day course of rotenone administration) improved the motor activity of rats with rotenone-induced Parkinsonism in the open field test (horizontal movements) and the rotating rod test. Afobazole (10 mg/kg intraperitoneally, daily during the 7-day course of rotenone administration) reduced the manifestations of rigidity and postural instability. Proteomic analysis, performed using brain samples obtained the day after the last administration of rotenone and neuroprotectors, revealed similar quantitative changes in the brain of rats with rotenone Parkinsonism. An increase in the relative content of 65 proteins and a decrease in the relative content of 21 proteins were detected. The most pronounced changes - an almost ninety-fold increase in the alpha-synuclein content - were found in the brains of rats treated with isatin. In animals of the experimental groups treated with "Rotenone + Isatin", as well as "Rotenone + Afobazole", the increase in the relative content of this protein in the brain was almost 60 and 50 times higher than the control values. Taking into consideration the known data on the physiological role of alpha-synuclein, an increase in the content of this protein in the brain upon administration of neuroprotectors to animals with rotenone Parkinsonism may represent a compensatory reaction, at least in the early stages of this disease and the beginning of its treatment.

Quantitative changes of brain isatin-binding proteins of rats with the rotenone-induced experimental parkinsonism.

Isatin (indoldione-2,3) is an endogenous regulator found in humans and animals. It exhibits a broad range of biological activity mediated by numerous isatin-binding proteins. Isatin produces neuroprotective effects in several experimental models of diseases, including Parkinsonism induced by the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).Rotenone (a neurotoxin used to modeling Parkinson's disease in rodents) causes significant changes in the profile of isatin-binding proteins of rat brain. Comparative proteomic identification of brain proteins of control rats and the rats with the rotenone-induced Parkinsonian syndrome (PS) revealed significant quantitative changes of 86 proteins under the influence of rotenone. This neurotoxin mainly caused the increase of the quantity of proteins involved in signal transduction and regulation of enzyme activity (24), proteins involved in cytoskeleton formation and exocytosis (23), and enzymes involved in energy generation and carbohydrate metabolism (19). However, only 11 of these proteins referred to isatin-binding proteins; the content of eight of them increased while the content of three proteins decreased. This suggests that the dramatic change of the profile of isatin-binding proteins, found in the development of the rotenone-induced PS, comes from changes in the state of the pre-existing molecules of proteins, rather than altered expression of corresponding genes.

Experimental Study of Antidepressant Properties of Afobazole.

We studied the effects of oral administration of Afobazole in a dose of 10 mg/kg for 5 days on depressive-like behavior of male C57BL/6 mice in the tail suspension test in comparison with amitriptyline (10 mg/kg) or fluoxetine (20 mg/kg) treatment. Afobazole produced an antidepressant effect similar to amitriptyline, but inferior to fluoxetine. The σ1 receptor antagonist BD-1047 in a dose of 5 mg/kg blocked the antidepressant effect of Afobazole, which indicates the involvement of σ1 receptors in the antidepressant effect of the drug.

[Characteristics of behavioral reactions and the profile of brain isatin-binding proteins of rats with the rotenone-induced experimental parkinsonism]. / Osobennosti povedencheskikh reaktsii i profilia izatin-sviazyvaiushchikh belkov mozga u krys s indutsirovannym rotenonom éksperimental'nym parkinsonizmom.

The neurotoxins rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (МPTP) are used for modeling Parkinson's disease in animals (PD). They induce the mitochondrial respiratory chain dysfunction, which leads to the dopaminergic (DA) neuron degeneration. The advantage of the rotenone model consists in ability of rotenone to cause neurodegeneration showing symptoms and molecular biological characteristics similar to those of PD. Isatin (indoldione-2,3) is an endogenous regulator found in tissues and biological fluids of humans and animals. It exhibits a broad range of biological activity mediated by numerous isatin-binding proteins. In this work we have investigated behavioral reactions and profiles of brain isatin-binding proteins of rats with Parkinson's syndrome (PS) in comparison with the corresponding parameters of MPTP-induced Parkinsonism in mice. Systemic injection of rotenone caused severe PS comparable with the effect of MPTP injection. It was accompanied by significant body weight loss, death, oligokinesia, muscular rigidity, and postural instability of animals. In spite of the same pathogenic basis of PS caused by rotenone and MPTP, the molecular mechanisms of their action differ. In the case of rotenone-induced PS, the pool of isatin-binding proteins common of the control rats and the rats with PS (146) significantly exceeded the pool of the common proteins of control mice and mice with PS induced by MPTP, whether right after neurotoxin injection (27), or (all the more) in a week after the MPTP injection (14). The comparison of isatin-binding proteins specific of the animals with MPTP-induced PS and with the rotenone-induced PS (as compared with the control animals) revealed total absence of proteins common of these two models of PD. It is to be noted that both neurotoxins particularly affected the proteins participating in the signal transmission and enzyme activity regulation. The changes of the profile of isatin-binding proteins in response to the injection of rotenone suggest that the neuroprotector isatin could also influence positively in the case of the rotenone model of PD.

[Changes in the mitochondrial subproteome of mouse brain Rpn13-binding proteins induced by the neurotoxin MPTP and the neuroprotector isatin]. / Izmenenie mitokhondrial'nogo subproteoma Rpn13-sviazyvaiushchikh belkov mozga myshi pod deistviem neirotoksina MFTP i neiroprotektora izatina.

Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson's disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle play an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al. (2020) Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 14, 297-305). In this study we have investigated the effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroprotector isatin on the mitochondrial subproteome of Rpn13-binding proteins of the mouse brain. Administration of MPTP (30 mg/kg) to animals caused movement disorders typical of PD, while pretreatment with isatin (100 mg/kg, 30 min before MPTP) reduced their severity. At the same time, the injection of MPTP, isatin, or their combination (isatin + MPTP) had a significant impact on the total number and the composition of Rpn13-binding proteins. The injection of MPTP decreased the total number of Rpn13-binding proteins in comparison with control, and the injection of isatin prior to MPTP or without MPTP caused an essential increase in the number of Rpn13-binding proteins, mainly of the functional group of proteins participating in the protein metabolism regulation, gene expression, and differentiation. Selected biosensor validation confirmed the interaction of Rpn13 subunit of proteasome with some proteins (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, histones H2A and H2B) revealed while proteomic profiling. The results obtained testify that under the conditions of experimental MPTP-induced parkinsonism the neuroprotective effect of isatin may be aimed at the interaction of mitochondria with the components of UPS.

[The effect of a neuroprotective dose of isatin or deprenyl to mice on the profile of brain isatin-binding proteins]. / Izmenenie profilia izatin-sviazyvaiushchikh belkov mozga myshei pri odnokratnom vvedenii neiroprotektornoi dozy izatina ili deprenila.

Isatin (indol-2,3-dione), an endogenous biofactor found in the brain, peripheral tissues and biological body fluids of humans and animals, exhibits a wide range of biological and pharmacological activities. They are realized via interaction with numerous isatin-binding proteins. Some of these proteins identified during proteomic profiling of the brain are involved in the development of neurodegenerative pathology. In the context of the neuroprotective effect, the effect of isatin is comparable to the effects of deprenyl (selegiline), a pharmacological agent used for treatment of Parkinson's disease. In this study, we have investigated the effect of a single dose administration of isatin (100 mg/kg) and deprenyl (10 mg/kg) to mice on the profile of the brain isatin-binding proteins. Comparative proteomic analysis of brain isatin-binding proteins of mice treated with isatin or deprenyl resulted in identification of a representative group of proteins (n=200) sensitive to the administration of these substances. The change in the profile of isatin-binding proteins may be obviously attributed to accumulation of isatin and deprenyl in the brain and their interaction with target proteins; this prevents protein binding to the affinity sorbent. Thus identified brain isatin-binding proteins of the control animals obviously represent specific targets that interact directly with isatin (and also with deprenyl) in vivo. Isatin or deprenyl administered to animals interact with these proteins and thus inhibit their binding to the affinity sorbent (immobilized isatin analogue).

Afobazole Alleviates Cognitive Rigidity in Experimental Model of Autism Spectrum Disorders.

Afobazole (10 mg/kg) alleviated cognitive rigidity in BALB/c mice, a phenotypic model of autism spectrum disorders. It improved spatial memory and retraining in T-maze with drinking reinforcement and restored the retrieval of acquired skill during reversal learning in Morris water maze.

[Effect of afobazole and levodopa on the activity of proline-specific proteinases and adenosine deaminase in blood serum and brain structures of rats with experimental Parkinson's syndrome induced by systemic administration of rotenone]. / Vliianie afobazola i levodopy na aktivnost' prolinspetsificheskikh proteinaz i adenozindezaminazy v syvorotke krovi i strukturakh golovnogo mozga krys s éksperimental'nym parkinsonicheskim sindromom, vyzvannym sistemnym vvedeniem rotenona.

Rats with experimental Parkinson's syndrome induced by seven-day intraperitoneal administration of rotenone at a dose of 2.75 mg/kg have an increased activity of prolylendopeptidase (EC 3.4.21.26, PREP) in blood serum and a decreased activity of adenosine deaminase (EC 3.5.4.4, ADA) in serum and in the prefrontal cortex. PREP and ADA activity in other brain structures (in the striatum, hypothalamus and hippocampus) did not change; dipeptidyl peptidase IV activity (EC 3.4.14.5, DPP-4, CD26) also remained constant in serum and in all the brain structures investigated. Afobazole and levodopa, which exhibit antiparkinsonian activity in this model of Parkinson's syndrome, decrease elevated PREP activity in serum and increase reduced ADA activity in the prefrontal cortex of rats with the experimental pathology. Meanwhile, treatment with the study drugs was associated with a decrease of ADA activity in the other brain structures.

[The effect of neurotoxin MPTP administration to mice on the proteomic profile of brain isatin-binding proteins]. / Vliianie neirotoksina MFTP na proteomnyi profil' izatin-sviazyvaiushchikh belkov mozga myshei.

Isatin (indole-2,3-dione) is an endogenous indole found in the mammalian brain, peripheral organs and body fluids. It acts as a neuroprotector, which decreases manifestation of locomotor impairments in animal models of Parkinson's disease. A wide range of biological activity of isatin is associated with interaction of this regulator with numerous isatin-binding proteins. The aim of this study was to investigate the profile of brain isatin-binding proteins in mice with MPTP-induced Parkinsonism (90 min and seven days after administration of this neurotoxin). A single dose administration of MPTP (30 mg/kg, ip.) was accompanied by locomotor impairments in the open field test 90 min after administration; seven days after MPTP administration locomotor activity of mice significantly improved but did not reach the control level. Five independent experiments on proteomic profiling of isatin-binding proteins resulted in confident identification of 96±12 proteins. Development of MPTP-induced locomotor impairments was accompanied by a significant decrease in the number of isatin-binding proteins (63±6; n=5; p<0.01). Seven days after MPTP administration the total number of identified proteins increased and reached the control level (132±34; n=4). The profiles of isatin-binding proteins were rather specific for each group of mice: in the control group these proteins (which were not found in both groups of MPTP-treated mice) represented more than 70% of total proteins. In the case of MPTP treated mice this parameter was 60% (90 min after MPTP administration) and >82% (seven days after MPTP administration). The major changes were found in the groups of isatin-binding proteins involved into cytoskeleton formation and exocytosis, regulation of gene expression, cell division and differentiation and also proteins involved in signal transduction.

Activities of Proline-Specific Proteinases in the Serum and Cerebrospinal Fluid of Rats with the Fetal Valproate Syndrome.

In 60-day-old Wistar rats with fetal valproate syndrome, the brain to body weight ratio was higher by 9.4% and activity of dipeptidyl peptidase IV in the serum and cerebrospinal fluid was higher by 18.4 and 40.6%, respectively, than in healthy controls. Activity of prolylendopeptidase in the serum and cerebrospinal fluid in rats with the fetal valproate syndrome did not differ from the control.

[A comparative study of the effects of mexidolum and mildronatum on the physical performance of experimental animals]. / Sravnitel'noe issledovanie vliianiia meksidola i mildronata na fizicheskuiu rabotosposobnost' v éksperimente.

AIM: To evaluate the effects of mexidolum on physical performance using acute and subchronic administration in experimental animals. MATERIAL AND METHODS: The investigation was carried out using 123 male white outbred mice. The forced swim test was used to assess the effects of the drugs on the physical performance of mice. RESULTS AND CONCLUSION: A single intraperitoneal administration of 50 and 100 mg/kg mexidolum and subchronic intraperitoneal administration of 100 mg/kg mexidolum significantly enhances the physical performance of animals in the forced swim test. Subchronic intraperitoneal administration of 100 mg/kg of the comparison drug mildronatum enhances the physical performance of animals, while intraperitoneal administration at a lower dose (50 mg/kg) has no effect. The effect of mexidolum at a dose of 50 and 100 mg/kg is comparable with the effect of mildronatum in a dose of 100 mg/kg.

Brain Mitochondrial Subproteome of Rpn10-Binding Proteins and Its Changes Induced by the Neurotoxin MPTP and the Neuroprotector Isatin.

Mitochondria play an important role in molecular mechanisms of neuroplasticity, adaptive changes of the brain that occur in the structure and function of its cells in response to altered physiological conditions or development of pathological disorders. Mitochondria are a crucial target for actions of neurotoxins, causing symptoms of Parkinson's disease in various experimental animal models, and also neuroprotectors. Good evidence exists in the literature that mitochondrial dysfunction induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) influences functioning of the ubiquitin-proteasomal system (UPS) responsible for selective proteolytic degradation of proteins from various intracellular compartments (including mitochondria), and neuroprotective effects of certain antiparkinsonian agents (monoamine oxidase inhibitors) may be associated with their effects on UPS. The 19S proteasomal Rpn10 subunit is considered as a ubiquitin receptor responsible for delivery of ubiquitinated proteins to the proteasome proteolytic machinery. In this study, we investigated proteomic profiles of mouse brain mitochondrial Rpn10-binding proteins, brain monoamine oxidase B (MAO B) activity, and their changes induced by a single-dose administration of the neurotoxin MPTP and the neuroprotector isatin. Administration of isatin to mice prevented MPTP-induced inactivation of MAO B and influenced the profile of brain mitochondrial Rpn10-binding proteins, in which two pools of proteins were clearly recognized. The constitutive pool was insensitive to neurotoxic/neuroprotective treatments, while the variable pool was specifically influenced by MPTP and the neuroprotector isatin. Taking into consideration that the neuroprotective dose of isatin used in this study can result in brain isatin concentrations that are proapoptotic for cells in vitro, the altered repertoire of mitochondrial Rpn10-binding proteins may thus represent a part of a switch mechanism from targeted elimination of individual (damaged) proteins to more efficient ("global") elimination of damaged organelles and whole damaged cells.

Anti-Parkinsonian Activity of Hemantane on a Model of Hemiparkinsonian Syndrome in Rats.

Hemantane demonstrated a pronounced antiparkinsonian activity in the model of hemiparkinsonian syndrome provoked in rats by unilateral intracerebral injection of 6-hydroxydopamine, which was comparable to efficacy of levodopa in decreasing the duration of cataleptogenic state and the degree of akinesia of the contralesional forelimb assessed in the cylinder test. In the stepping test, hemantane exerted a long-term effect in contrast to levodopa, which diminished its beneficial action to the treatment day 21. In the swing test, the behavior normalized only by hemantane.

Experimental investigation of pharmacodynamics and pharmacokinetics of carbamazepine nanoemulsion.

Anticonvulsant activity and pharmacokinetics of nanoemulsion and unmodified substance of carbamazepine were compared in experiments on mice. Carbamazepine nanoemulsion demonstrated significant anticonvulsant activity and was superior to unmodified substance of carbamazepine against seizures induced by maximum electric shock and picrotoxin. Relative bioavailability of carbamazepine after administration of nanoemulsion was 160% compared to unmodified substance. Carbamazepine nanoemulsion more effectively penetrated through BBB by 1.5 times in comparison with unmodified substance.

Noopept normalizes parameters of the incretin system in rats with experimental diabetes.

Experiments on adult Wistar rats with streptozotocin-induced diabetes showed that antihyperglycemic activity of an original nootropic and neuroprotective drug Noopept (N-phenylacetyl-L-prolylglycine ethyl ester) is more pronounced under conditions of oral application than after intraperitoneal injection. These data provided a basis for studying the effect of Noopept on major indexes of the incretin system. Streptozotocin was shown to decrease the concentrations of incretin GLP-1 and insulin in the blood. Noopept had a normalizing effect on these parameters. This influence of Noopept was not related to the inhibition of a major enzyme metabolizing incretins (dipeptidyl peptidase IV). A reference drug sitagliptin also increased the contents of incretins and insulin, which was associated with the inhibition of dipeptidyl peptidase IV. It is known that GLP-1 increases NGF expression in the insular system. Our results suggest that the increase in incretin activity contributes to the antiapoptotic effect of Noopept on pancreatic ß cells. The mechanism for an increase in blood GLP-1 level after oral application of Noopept requires further investigations.

[Influence of hemantane in injectable dosage form on levodopa-induced dyskinesia in rats with model parkinsonian syndrome].

Levodopa-induced heavy dyskinesia was modeled in rats with severe hemiparkinsonian syndrome induced by injection of 6-hydroxydopamine in the left medial forebrain bundle. It is established that the antidyskinetic effect of the injectable dosage form of a new antiparkinsonian drug hemantane (5 mg/kg) after a single intravenous administration is weaker than that of the most effective in clinical practice antidyskinetic drug amantadine (20 mg/kg). However, after five days of treatment, the effect of hemantane injections exceeded that of amantadine.

Comparative activity of proline-containing dipeptide noopept and inhibitor of dipeptidyl peptidase-4 sitagliptin in a rat model of developing diabetes.

Developing diabetes was modeled on adult male Wistar rats by repeated intraperitoneal injections of streptozotocin in a subdiabetogenic dose of 30 mg/kg for 3 days. Proline-containing dipeptide drug Noopept or a standard diabetic drug dipeptidyl peptidase-4 inhibitor sitagliptin was administered per os in a dose of 5 mg/kg before each injection of the toxin and then for 16 days after streptozotocin course. In active control group, spontaneously increase glucose level and reduced tolerance to glucose load (1000 mg/kg intraperitoneally) were observed on the next day after the third administration of toxin. Basal glucose level decreased by day 16, but glucose tolerance remained impaired. Noopept normalized the basal blood glucose level and tolerance to glucose load on the next day after administration of streptozotocin. The effect of Noopept persisted to the end of the experiment. At early terms of the experiment, sitagliptin was somewhat superior to Noopept by the effect on baseline glucose level, but was inferior by the influence on glucose tolerance.. By the end of the experiment, Noopept significantly (by 2 times) surpassed sitagliptin by its effect on glucose tolerance.

[Effect of hemantane on bioelectric activity of brain in mice with MPTP-induced Parkinson syndrome].

A single intraperitoneal injection of MPTP neurotoxin (30 mg/kg) in C57BL/6 mice causes desynchronization of EEG with a decrease of theta-1 activity and a growth of beta activity in the interval of 15-30 Hz. Subchronic administration of the new antiparkinsonian drug hemantane (injection form) in a dose of 10 mg/kg makes the power of MPTP-induced beta oscillations less pronounced and leads to its reliable decrease within 24 h. This effect of hemantane administration was manifested during the entire period of observations.

Efficiency of noopept in streptozotocin-induced diabetes in rats.

We studied the effects of new nootropic and neuroprotective drug Noopept (N-phenylacetyl-L-prolylglycine ethyl ester) in various dosage regimens on the dynamics of glycemia, body weight, and pain sensitivity in rats receiving diabetogenic toxin streptozotocin. In experimental diabetic rats, Noopept alleviated glycemia and weight loss and normalized enhanced pain sensitivity. The normalizing effect of Noopept was most pronounced when it was administered as a preventive agent prior to injection of the toxin. Both preventive and therapeutic administration of Noopept (delayed injections included) significantly weakened the examined metabolic effects of diabetogenic toxin. Possible mechanisms of the antidiabetic action of Noopept are analyzed.