Inactivation of the Snca gene in young mice by chronic injections of tamoxifen (TAM), a selective estrogen receptor modifier, has been shown to decrease the level of alpha-synuclein, a key peptide in the pathogenesis of Parkinson’s disease. In young mice, different time courses of the effect were observed in different brain areas, meaning associated disturbances in the intracerebral relations, namely in brain function after TAM-induced synucleinopathy. Methods: We analyzed electroencephalogram (EEG) coherence (“functional connectivity”) between the cortex (MC), putamen (Pt), and dopamine-producing brain regions (ventral tegmental area, VTA, and substantia nigra, SN) in two groups of two-month-old male mice. We compared EEG coherences in the conditional knockout Snca^flox/flox mice with those in their genetic background (C57Bl6J) one, two, and three months after chronic (for five days) intraperitoneal injections of TAM or the vehicle (corn oil). The EEG coherences in the TAM-treated group were compared with those in the alpha-synuclein knockout mice. Results: A significant suppression of EEG coherence in the TAM-treated mice versus the vehicle group was observed in all inter-structural relations, with the exception of MC-VTA at one and three months and VTA-SN at two months after the injections. Suppressive changes in EEG coherence were observed in the alpha-synuclein knockout mice as well; the changes were similar to those in TAM-treated mice three months after treatment. Conclusion: our data demonstrate a combined time-dependent suppressive effect induced by TAM on intracerebral EEG coherence.

Background: Alpha-synuclein is a key protein involved in the pathogenesis of Parkinson disease (PD). Its intermediate aggregated forms disturb the normal function of dopaminergic (DA) neurons. Furthermore, the loss of intraneuronal connections may precede nerve cell death in PD. Disturbance of presynaptic functions of alpha-synuclein and its family members, beta- and gamma-synuclein, can apparently be the first step in nigrostriatal system dysfunction. Based on their structure homology and subcellular localization, the three synuclein proteins could have overlapping functions. This also indicates necessitates to study each protein in isolation. Methods: We have established a unique mouse line with conditional knockout (KO) of alpha-synuclein inactivation on the background of gamma-synuclein KO. Results: During the early phase of alpha-synuclein loss of function, mice demonstrate reduced explorer activity, decreased gene expression of Mao-B in the midbrain, and transiently increased levels of beta-synuclein protein in the striatum after alpha-synuclein inactivation, as results, metabolism of dopamine stays unscathed. These changes can be caused by specific regulation of Mao-B by alpha-synuclein or can be a physiological reaction aimed at restoring alpha-synuclein levels. No significant changes in gene expression patterns of dopamine-related enzymes in the midbrain or protein levels in the striatum and midbrain were observed. Conclusions: Our data suggest that sudden alpha-synuclein depletion leads to an increase in beta-synuclein levels, likely as functional replacement. This result supports that beta-synuclein can compensate the loss of alpha-synuclein. In general, this process may characterize synapse reconstruction in early alpha-synuclein dysfunction with gamma-synuclein absence and form the basis for replacement therapeutic strategies in PD.

The aim of the work was behavioral and pathomorphological phenotyping of the mice knockout for the DYSF gene, which plays an important role in the development and progression of dysferlinopathy.Materials and methods. A B6.A-Dysf^prmd/GeneJ (Bla/J) mice subline was used in the work. During the study, a muscle activity was determined basing on the following tests: “Inverted grid”, “Grip strength”, “Wire Hanging”, “Weight-loaded swimming”, Vertical Pole”. Histological and immunofluorescent examinations of skeletal muscles (m. gastrocnemius, m. tibialis) were performed. The presence and distribution of the dysferlin protein was assessed, and general histological changes in the skeletal muscle characteristics of mice at the age of 12 and 24 weeks, were described. A morphometric analysis with the determination of the following parameters was performed: the proportion of necrotic muscle fibers; the proportion of fibers with centrally located nuclei; the mean muscle fiber diameter.Results. The “Grip strength” test and the “Weight-loaded swimming” test revealed a decrease in the strength of the forelimbs and endurance in the studied mice of the Bla/J subline compared to the control line. The safety of physical performance was checked using the “Wire Hanging” test and the “Vertical Pole” test, which showed a statistically significant difference between the studied mice and control. The coordination of movements and muscle strength of the limbs examined in the “Inverted Grid” test did not change in these age marks. Decreased grip strength of the forelimbs, decreased physical endurance with age, reflects the progression of the underlying muscular disease. Histological methods in the skeletal muscles revealed signs of a myopathic damage pattern: necrotic muscle fibers, moderate lympho-macrophage infiltration, an increase in the proportion of fibers with centrally located nuclei, and an increase in the average fiber diameter compared to the control. The dysferlin protein was not found out in the muscle tissues.Conclusion. Taking into account the results of the tests performed, it was shown that the absence of Dysf^-/^- gene expressionin Bla/J subline mice led to muscular dystrophy with the onset of the development of phenotypic disease manifestations at the age of 12 weeks and their peak at 24 weeks. Histopathological phenotypic manifestations of the disease are generally nonspecific and corresponded to the data of intravital pathoanatomical examination in diferlinopathy patients. The mice of the studied subline Bla/J are a representative model of dysferlinopathy and can be used to evaluate new therapeutic agents for the treatment of this disease.

Актуальность. Нарушение дофаминовой нейротрансмиссии характерно для многих нейродегенеративных заболеваний с моторной дисфункцией. Участие белков-синуклеинов в регуляции гомеостаза дофамина делает актуальным их изучение в контексте патологии. Целью исследования является характеристика изменений моторной функции мышей с различными комбинациями нокаутов по генам белков семейства синуклеинов с возрастом. Материалы и методы. Восемь групп животных проходили тестирование в автоматизированной системе CatWalk XT, а их мышечная сила оценивалась с помощью теста «Сила хвата» (Grip Strength Test Meter) в возрасте 6, 12, 18 и 24 месяца. Статистическая обработка данных проводилась в программе «GraphPad Prism Software 7.0». Результаты. Было выявлено достоверное снижение параметра «длина следа» с возрастом в группах животных без функционального альфа-синуклеина. Также у этих животных отмечены и другие нарушения походки. По данным нашего исследования, отсутствие бета- и гамма-синуклеина приводит к достоверному снижению массы тела и ухудшению силы хвата. Заключение. Показана важная роль белков семейства синуклеинов в ухудшении двигательной функции животных с возрастом и выявлены некоторые скрытые параметры, вносящие изменения в походку животных с различным комбинациями нокаутов по генам белков семейства синуклеинов. Actuality. Dopamine neurotransmission impairment is a common phenomenon in neurodegenerative diseases associated with motor dysfunction. The involvement of synuclein proteins in the regulation of dopamine homeostasis renders their study pertinent in the context of pathology. The aim of this study is to identify the impact of different combinations on motor function developed with ageing, in knockouts of synuclein family genes. Materials and methods. Eight groups of animals were tested in the CatWalk XT automated system, and their muscle strength was evaluated using the Grip Strength Test Meter at 6, 12, 18 and 24 months of age. Statistical analysis was carried out using GraphPad Prism Software 7.0. Results. A significant reduction in the ‘print length’ parameter with age was observed in the groups of animals lacking functional alpha-synuclein. Furthermore, other gait abnormalities were observed in these animals. Our study indicates that the absence of beta- and gamma-synuclein is associated with a significant reduction in body weight and impaired grip strength. Conclusion. The present study has demonstrated the pivotal role of synuclein family proteins in the impairment of motor function in animals with age. Furthermore, it has identified several hidden parameters that introduce changes in the gait of animals with different combinations of synuclein genes knockouts.

The aim of the work was to analyze the available therapeutic options for the conventional therapy of hereditary myopathies.Materials and methods. When searching for the material for writing a review article, such abstract databases as PubMed and Google Scholar were used. The search was carried out on the publications during the period from 1980 to September 2022. The following words and their combinations were selected as parameters for the literature selection: “myopathy”, “Duchenne”, “myodystrophy”, “metabolic”, “mitochondrial”, “congenital”, “symptoms”, “replacement”, “recombinant”, “corticosteroids”, “vitamins”, “tirasemtiv”, “therapy”, “treatment”, “evidence”, “clinical trials”, “patients”, “dichloracetate”.Results. Congenital myopathies are a heterogeneous group of pathologies that are caused by atrophy and degeneration of muscle fibers due to mutations in genes. Based on a number of clinical and pathogenetic features, hereditary myopathies are divided into: 1) congenital myopathies; 2) muscular dystrophy; 3) mitochondrial and 4) metabolic myopathies. At the same time, treatment approaches vary significantly depending on the type of myopathy and can be based on 1) substitution of the mutant protein; 2) an increase in its expression; 3) stimulation of the internal compensatory pathways expression; 4) restoration of the compounds balance associated with the mutant protein function (for enzymes); 5) impact on the mitochondrial function (with metabolic and mitochondrial myopathies); 6) reduction of inflammation and fibrosis (with muscular dystrophies); as well as 7) an increase in muscle mass and strength. The current review presents current data on each of the listed approaches, as well as specific pharmacological agents with a description of their action mechanisms.Conclusion. Currently, the following pharmacological groups are used or undergoing clinical trials for the treatment of various myopathies types: inotropic, anti-inflammatory and antifibrotic drugs, antimyostatin therapy and the drugs that promote translation through stop codons (applicable for nonsense mutations). In addition, metabolic drugs, metabolic enzyme cofactors, mitochondrial biogenesis stimulators, and antioxidants can be used to treat myopathies. Finally, the recombinant drugs alglucosidase and avalglucosidase have been clinically approved for the replacement therapy of metabolic myopathies (Pompe’s disease).

α-synuclein is one of the key molecular links in the pathogenesis of Parkinson’s disease. The accumulated data indicate that pathogenic mutations in theSncagene are associated with the development of neurodegenerative brain damage, indicating the relevance of studying the synuclein neurobiological role.The aimof the study was to create a genetically modified clone of mouse stem cells with a conditional knockout of humanized α-synuclein, which can be used for the reinjection into mouse blastocysts, as well as for basic and appliedin vitroresearch in the field of pathophysiology and neuropharmacology.Materials and methods.To create mouse stem cells with a conditional knockout of the humanizedSncagene, a previously obtained clone with the firstSncaexon flanked by LoxP sites, was used. The CRISPR/Cas9-mediated homologous recombination system with donor DNA oligonucleotides of the human sites of the corresponding gene sites was used to humanize the fourth and fifth exons. Cas9 nuclease, single guide RNA, and donor DNA were transfected into mouse cells.Results.An approach to obtaining clones of mouse genetically modified stem cells expressing pathological humanized α-synuclein, has been proposed and implemented. The resulting clones were plated on Petri dishes for propagation and a further genetic analysis. Clone 126-2F4 was found out carrying the necessary genetic modifications. The results obtained are fundamentally important not only for understanding the development of the pathological process in α-synucleinopathies, but which is more important, for the development of new therapeutic approaches that will stop the extension of the human α-synuclein aggregation pathology throughout the nervous system, and the validation of these approaches in preclinical trials.Conclusion.As a result of the study, a strategy for CRISPR/Cas9-assisted homologous recombination in the genome of mouse embryonic stem cells has been developed to create a fully humanizedSncagene encoding α-synuclein, and the clone genome of mouse embryonic stem cells has been edited using a CRISPR technology. The RNA and DNA oligonucleotides necessary for the creation of RNP complexes that carry out a directed homologous recombination in theSncalocus of the mouse genome have been synthesized. The developed cell clone can serve to create a line of genetically modified mice that serve as a test system for pathophysiological and neuropharmacological studies associated with synucleinopathies. Herewith, before the induction of the Cre-dependent recombination, this line is a representative model for studying a biological role of mutantSnca. At the same time, after a Cre-dependent knockout activation, it is possible to imitate the pharmacological inhibition of α-synuclein, which is of particular interest for applied research in neuropharmacology.