The Research Institute of Molecular Medicine and Pathobiochemistry was established at the Krasnoyarsk State Medical University named after Prof. V. F. Voino-Yasenetsky on December 8, 2006 on the basis of the Interdepartmental biochemical research laboratory. Since 2012, the Institute has been functioning as a Center for Collective Use (CCU) of the Russian Federation researching "Living systems" for the implementation of interdisciplinary research and educational projects. The organizer and head of the Research Institute is Doctor of Medical Science, Professor A.B. Salmina, E.A. Pozhilenkova is the Executive Director of the CCU - Ph.D., Associate Professor. Currently, the Research Institute is a base for the implementation of modern research technologies, training of postgraduates and masters (KrasSMU, SFU, other medical universities of the Russian Federation), and interdisciplinary, including international, scientific projects.

Currently, the average age of the employees of the Research Institute is 36 years. The team of the Research Institute includes 8 doctors of science and 13 candidates of science. Among members of the Research Institute there are three persons after long-term internships in Japan (including two persons who graduated and received Ph.D. in Japan), one foreign Professor (Japan), two visit-Professors of University of Kanazawa (Japan), one Professor Emeritus, University of Niigata (Japan), more than ten persons that had the target internships abroad (Japan, Italy, Germany, Finland, Norway, UK, USA). The Research Institute includes a collaborating center of the Scientific Center of Neurology of the Russian Academy of Sciences (Moscow), two international laboratories (Russian-Italian Laboratory of Medical Genetics, Russian-Japanese laboratory of the social brain). For 10 years of the Research Institute's existence, there were more than 30 theses defenses on its basis, including 8 PhD; more than 330 scientific articles and monographs were published, including more than 70 papers, abstracted in the Web of Science database; more than 30 scientific events, including International Congress on Neuroscience (2014), scientific-practical seminars and conferences on translational medicine, integrative neuroscience (2006-2012), modern methods of research in molecular medicine and neuroscience (2015-2019) were organized; we have done the research on more than 30 grants: grants of the President of RF supporting the leading scientific schools (3), young candidates of Sciences (2), young doctors of Sciences (2), grants of Russian Scientific Fund (2), grants of RFFR, Krasnoyarsk Regional Fund for the Support of Scientific and Scientific-Technical Activities, Fund of Innovation, etc., which provide the Research Institute with the modern research equipment.

Key research competencies of the Research Institute of Molecular Medicine and Pathobiochemistry of the KrasSMU named after Prof. V. F. Voino-Yasenetsky

Main scientific partners: Research Center of Neurology of the Russian Academy of Sciences (Moscow), Lomonosov Moscow State University (Moscow), National Research Saratov State University named after N.G.Chernyshevsky (Saratov), Siberian Federal University (Krasnoyarsk), Universities of Kanazawa and Kyushu (Japan), Charite University (Germany), University of Bristol (UK), Institute of Medical Genetics MAGI (Italy), University of Turin (Italy).

In 2011, the team of researchers of the Institute and members of the scientific school of A.B. Salmina (supervisor), A.I. Inzhutova, N.A. Malynovskaya, O.S. Okuneva, A.V. Morgun were awarded with the RF Government Prize in science and technology for young scientists for 2010 for the development of new technologies for the management of molecular mechanisms of intercellular communication in translational medicine. Also, there is a team of scientific school "New management technologies for molecular mechanisms of intercellular communication in translational medicine", which was supported three times by the Council on Grants of the President of the Russian Federation as the leading scientific school of Russia in the field of ''Medicine" in 2014-2015, 2016-2017, 2018-2019, (head of the school - A.B. Salmina).

Main current projects

We established new features of integrative brain functions realization affected by external stimuli within so called ''enriched (multistimuli) environment''. It was shown that enriched environment does not affect cognitive functions of a damaged brain (animals with experimental Alzheimer's disease) or aging brain (physiological aging), however, positively affects the preservation of social memory and social interest in cases of physiological aging and experimental Alzheimer's disease. For the first time new influencing mechanisms of enriched environment on neurogenesis and sinaptogenesis in physiological aging and experimental Alzheimer's disease were found. Our study shows that enriched environment effectively launches apoptosis and early stages of cell proliferation, but cannot affect later stages, associated with cells neuronal phenotype acquisition in Alzheimer's disease experimental model. For the first time we demonstrated features of Arc3.1 and Cx43 expression in cortical, hippocampus and amygdala cells in experimental Alzheimer's disease and physiological aging, including those in enriched environment condition. We also demonstrated the influence of enriched environment factors on parental and exploratory behavior in animals, and also registered features of mature and aging rats behavior in enriched environment. Also, we established features of enriched environment influence on neurogenic niche cells proliferation in vitro in animals affected by physiological aging and experimental Alzheimer's disease, and also demonstrated the possibilities of usage of enriched cybernetic environment paradigm for neurorehabilitation. Currently, we explore mechanisms of local insulin resistance development in brain tissue in Alzheimer's disease experimental model, associated with aberrant expression of IRS, Glut.

Another important field of our study is exploration of aberrant neurogenesis mechanisms in neuroinflammation. These studies were conducted with the usage of modern models of Alzheimer's disease (5xFAD etc.) and, also, on animals which were not able to express RAGE, NLRP3, CD38. For the first time we detected that training promotes the increase in inflammasome expression in stem cells and neuroblasts in neurogenic niches in healthy brain, however neurodegeneration of Alzheimer's type leads to dysfunction of this mechanism. We, also, found out the features of neuroplastic violations (neurogenesis, behavioural characteristics) in animals without expression of NLRP3 inflammasome, RAGE receptors. We acquired new data regarding H2S production via CSE in brain neurogenic niches. We also acquired new data regarding the role of NAD+-converting enzymes CD38, CD157 in regulation of neurogenesis. We presented an idea regarding the compound of local microenvironment in neurogenic niches, that became the basis for development of in vitro model of neurogenic niche, the research is continuing.

Part of our study is dedicated to neurogenesis in immature, mature and aging brain with analysis of events, happening in brain neurogenic niches in the way of progenitors' migration and their involvement in cells ensembles including those affected by external modulating factors (effects of neurotoxic substances, stress, hypoxia and ischemia). We studied mechanisms of neuroinflammation development via CD38- and P2X7 in experimental Parkinson's disease [O.L. Lopatina et al., 2019; Ya.V. Gorina et al., 2018; Yu.K. Komleva et al., 2018; V.V. Salmin et al., 2017; V.V. Salmin et al., 2017; V.V. Kazakova et al., 2017; Ya.V. Gorina et al., 2017; E.A. Pozhilenkova, 2017 A.F. Bezdenezhnykh et al., 2016; M.G. Sadovsky et al., 2016; E.A. Pozhilenkova et al., 2016; Yu.K. Komleva et al., 2016; Yu.K. Komleva et al., 2015; A.B. Salmina et al., 2015; N.V. Kuvacheva, 2015; N.A. Malinovskaya et al., 2015; A.B. Salmina et al., 2014; Yu.K. Komleva et al., 2014; N.A. Malinovskaya et al., 2014; Yu.K. Komleva et al., 2013; N.V. Kuvacheva et al., 2013; Yu.K. Komleva et al., 2013; Yu.K. Komleva, 2013; S.M. Cherepanov et al., 2012; Yu.K. Komleva et al., 2012].

Another area of our research focuses on the role of neuropeptides (in particular, oxytocin, OT) in regulating of social behavior. A significant amount of research was performed earlier with Japanese colleagues in studying the CD38 role in the regulation of oxytocin secretion in the hypothalamic-pituitary system and the control of social behavior in mammals. For the first time, together we showed that CD38 in hypothalamus and pituitary cells is responsible for the oxytocin secretion, and violation of this mechanism is one of the mechanisms of autism development (the results are published in the scientific journal ''Nature'', 2007). In our collaborative work with Japanese colleagues, it has been proved that CD38-knockout animals can be an adequate experimental model of social behavior disorders [Jin et al., 2007; Higashida et al., 2010; Lopatina et al., 2010; Lopatina et al., 2013; Salmina et al., 2013]. In collaboration with Japanese partners, it was shown that the NAD+- converting enzyme - NAD+ - glycohydrolase/CD38 - is a key regulator of oxytocin secretion in the hypothalamic-pituitary system, and violation of this molecule's expression is responsible for the formation of the autistic phenotype in experimental animals [Jin et al., 2007; Higashida et al., 2010; Salmina et al., 2010]. It was found that OT-mediated ADP-ribosyl cyclase activity and an increase in [Ca2+] i were sensitive to the modulation of protein kinase C (PKC) activity and the production of cyclic ADP-ribose (cADPR) in the hypothalamus and the posterior pituitary lobe. The results, obtained in experiments with male mice, confirm the independence of PKC- and cADPR-dependent mechanisms of autoregulation from the sex, and their specificity not only for female individuals, but also for males, influencing the implementation of social behavior [Lopatina et al, 2008]. Oxytocin levels in blood plasma and tissues were analyzed in female mice of both genotypes (CD38+/+, CD38-/-) with different parental experience. The accumulation of parental experience led to a three-fold increase in plasma oxytocin levels in female wild-type mice and a two-fold increase in female CD38-deficient mice. This observation in CD38+/+ female mice may be the result of increased oxytocin release from the neurohypophysis and increased synthesis of oxytocin in the hypothalamus, which is confirmed by low levels of oxytocin in the neurohypophysis and high activity of ADP-ribosyl cyclase in hypothalamus cells [Lopatina et al., 2010]. It was previously shown that the OT anxiolytic effect in social stress is prominent in the group of "subordinate" mice, compared with the group of "dominant" mice, which was associated with different levels of OT secretion in the hypothalamic-pituitary system (greater secretion in "subordinates" in the social hierarchy of mice) [O. Lopatina, 2017]. Recent results show that the key regulators of oxytocin transport to brain tissue are protein glycation end-product receptors (RAGE) expressed in endothelial cells of cerebral microvessels [Y. Yamamoto et al., 2019]. A similar, RAGE-mediated mechanism of transepithelial transport can be performed in intestinal cells in the early neonatal period [H. Higashida et al., 2017]. In addition, in our work [O.L. Lopatina et al., 2019], we showed the key role of oxytocin in regulating the balance of excitation and inhibition in the context of microenvironment formation in cerebral neurogenic niches.

Currently, a project for assessing the role of oxytocin in the talent development in adolescents and in the formation of compliance in patients has been launched. Another project of developing a device and method for registering skin autofluorescence to assess the degree of accumulation of RAGE and soluble forms of RAGE (sRAGE) in patients with diabetes mellitus is, also, under research.

An important area of research carried out at the Research Institute of Molecular Medicine and Pathobiochemistry is the development of new models of the blood-brain barrier (BBB) and neurogenic niche in vitro. The main results of our studies and development in this field are the creation of an original three-cell model of the neurovascular brain unit and BBB in vitro using stem cells as a source for obtaining perivascular glia and neuronal cells (in static and dynamic/microfluidic configurations), applicable for testing permeability, studying the mechanisms of barrier-genesis, developing methods for controlled management of BBB permeability; development of bioscaffolds with a given topology obtained by laser photopolymerization methods and a concentration gradient of pro-angiogenic factors, as well as original microfluidic chambers supporting cerebral angiogenesis and barrier-genesis in vitro. In particular, we have developed a new protocol for cell obtaining, as well as the models of the neurovascular unit/blood-brain barrier in vitro, based on the use of progenitor brain cells, experimentally proved the participation of CD38, Cx43, Pgp, and SLC family proteins in damage and dysregulation of neuron-astroglial metabolic coupling in perinatal brain damage, and obtained new data on markers of cell damage of the neurovascular brain unit (neurons, astrocytes, cerebral endotheliocytes) in correlation to the clinical symptoms of perinatal hypoxic-ischemic brain damage [E.D. Khilazheva, 2015; A.V. Morgun, 2015; V.V. Salmin, 2016; A.V. Morgun et al., 2016; N.A. Malinovskaya et al., 2016; N.V. Kuvacheva et al., 2016; V.A. Ruzaeva et al., 2017; V.V. Salmin, 2017; Yu.K. Komleva et al., 2018, N.A. Malinovskaya et al., 2018; A.B. Salmina et al., 2019].

We have studied some new mechanisms of intercellular interactions in the neurovascular brain unit and the BBB with the participation of proteins-components of close contacts, slit contacts, pro- and anti-angiogenic factors of astroglial origin (lactate, growth factors, amyloid), according to the stage of ontogenesis (developing and mature brain), as well as in brain disorders (perinatal hypoxia-ischemia, Alzheimer's type neurodegeneration, early life stress), the use of protocols of optogenetics for directed modulation of astrocyte-endothelial interactions in the BBB in vitro. In particular, we demonstrated in vitro and in vivo the completeness of barrier-genesis in the early postnatal period of development in experimental animals, features of damage processes of barrier-genesis in hypoxia and under early life stress associated with changes in protein expression of close contacts and caveolin, proteins, which participate in HIF-1-related cellular signaling systems, the change of production of factors with pro-and anti-angiogenic cell activity of astroglial nature (VEGF, TS, MMP2, MMP9) and their receptors on the cerebral endothelial cells, changes in the expression of transport-regulating proteins, lactate reception in cells of the neurovascular brain unit (MCT1, MCT4, CD147, GPR81); identified key differences in the expression of these molecules in hypoxic brain damage and after early life stress; established mechanisms of influence of perivascular astroglia cells on endothelial cells of cerebral microvessels, mediated by the production and secretion of lactate, which has a pro-angiogenic effect with the identification of the gene expression profile activated astroglia.

For the first time it was shown that lactate stimulates the processes of proliferation and subsequent specialization of the BBB cells in vitro by increasing expression of VEGFR2, GPR81, RAGE in endothelial cells and perivascular astroglia, defining the reception of pro-angiogenic molecules. We, also, studied the role of lactate receptors GPR81 in metabolic conjugation of astroglia and cerebral endothelial cells, and applied protocols for targeted optogenetic modulation of the astrocyte-endothelial interface in the neurovascular brain unit; the role of beta-amyloid and astrocyte gamma secretase activity in the regulation of cerebral angiogenesis and barrier-genesis was demonstrated. It was found that maintaining of the integrity of the endothelial layer in the developing BBB can be achieved with preserved glycolysis in astrocytes and the implementation of the biological GPR81 activity of lactate receptors. Some mechanisms of neuroinflammation development with the participation of cells-components of the neurovascular brain unit and BBB in neurodegenerative, infectious diseases, as well as in brain development disorders were observed [N.V. Kuvacheva, 2013; A.B. Salmina, 2014; A.V. Morgun, 2014; A.V. Morgun, 2015; A.B. Salmina, 2015; N.A. Malinovskaya, 2016; V.A. Ruzaeva, 2016; A.V. Morgun, 2016; A.N. Shuvaev, 2016; A. Tohidpour, 2017; A.V. Morgun, 2017; E.B. Boitsova, 2017; N.A. Malinovskaya et al., 2017; A.V. Morgun et al., 2019].

Finally, the Research Institute of Molecular Medicine and Pathobiochemistry is developing new optical methods for monitoring the state of cells and tissues in vivo and in vitro using autofluorescence spectroscopy [V.V. Salmin, 2012; V.V. Salmin, 2014; P.V. Lavrentyev, 2015; K.A. Shapovalov, 2017; et al.], new methods for using cold plasma to correct the functional state of endothelial and epithelial cells, studying the role of astroglia in the pathogenesis of spinocerebellar ataxia type 1 [Takahashi H. et al., 2017; A.N. Shuvaev et al., 2016; Takahashi H. et al., 2015]; researching a neuroprotective effect of hypoxia and hypercapnia [P.P Tregub et al., 2019; P.P Tregub et al., 2016]; studying the mechanisms and markers of angiogenesis disorders and endothelial dysfunction formation in cardiovascular, reproductive, and respiratory systems disorders [N.G. Sazonova et al., 2017; Yu.S. Vinnik et al., 2017; M.Yu. Yurieva et al., 2017; S.V. Chubarova et al., 2017; M.G. Mamaeva et al., 2016; O.V. Zimnitskaya et al., 2015; Yu.S. Vinnik et al., 2014; M.G. Mamaeva et al., 2014; E.A. Sobko et al., 2014; I.A. Solovyova et al., 2013; S.V. Chubarova et al., 2013; E.A. Sobko et al., 2013; Yu.S. Vinnik et al., 2012].

Cell models of the blood-brain barrier and neurogenic niche in vitro for biomedical applications

The only cell models of the blood-brain barrier in vitro (BBB in vitro) in the Russian Federation using primary cultures of rodent brain cells (endothelial cells, neurons and astrocytes) based on culture tablets with a semi-permeable membrane have been developed at KrasSMU named after Prof. V.F. Voino-Yasenetsky. It is possible to simultaneously conduct several experiments on a single tablet.

The main purpose of the BBB model in vitro is to conduct research to assess the permeability of candidate drugs from the blood to the central nervous system, to study the features of damage and recovery of brain cells in normal and pathological modeling (hypoxia, inflammation, chronic neurodegenerative diseases, toxic damage, cancer), to reproduce personalized models for choosing treatment tactics for diseases associated with BBB damage, as well as to develop new methods for controlling the barrier permeability - the need to open it (to deliver drugs to the brain tissue) or close it (in the case of pathologically enhanced permeability).

For the first time, based on the BBB model in vitro, an original model of the brain neurogenic niche (microphysiological system) was created, which allows to conduct research on cellular and molecular mechanisms of regulation of neurogenesis in norm and neurodegeneration, brain development disorders, as well as to develop new approaches for correction of the neurological deficits associated with neurogenesis disorders, and to solve the problems of regenerative medicine.

Models of BBB and neurogenic niche in vitro are presented in two versions – static and dynamic (microfluidic). The models were tested in research carried out by scientists of the Saratov State University named after N. G. Chernyshevsky, Kazan State Medical University, Lomonosov Moscow State University and other institutions of the Russian Federation.

Basic market segment of the product: research and development of new methods and technologies for diagnostics and therapy for the needs of clinical and theoretical medicine; comparative research on preclinical and clinical evaluation of drugs. Possible consumers of cell models are research centers and institutes, pharmaceutical companies, and research departments of the Universities.

The main advantages of the cell models of the BBB and neurogenic niche in vitro: 1) a standardized approach to assessing the barrier permeability, high reproducibility of the obtained results; 2) determining the metabolic and functional changes occurring in the BBB and neurogenic niche, with the assessment of intercellular interactions in normal and pathological conditions; 3) simulating the transport properties of the blood-brain barrier, critical for evaluating the effectiveness of new methods of managing BBB permeability; 4) ease of use, reducing time during pharmacological studies.