Сell Physiology and Pathology Laboratory of Research and Development Center of Biomedical Photonics (10)

Planned project results:

• 2024:

1. cell cultures (healthy fibroblasts, mixed neuron-glial culture) with increased or decreased (after transfection with plasmids encoding RRK or RRX) polyphosphate content;
2. data of the relationship between the level of ΔΨM, autofluorescence of mitochondrial nicotinamide adenine dinucleotide (NADH), FAD++, as well as the rate of oxygen absorption and ATP synthesis in cell cultures with changes in the content of polyphosphate;
3. data of the levels of NADH and nicotinamide adenine dinucleotide phosphate (NADPH), reduced glutathione, NAD kinase activity in the studied cell models with different polyphosphate content;
4. data of the effect of changes in the level of polyphosphate in cellular models of neurodegenerative diseases with mutations leading to disruption of the processes of utilization of defective mitochondria (mutations in the PINK1 gene) on the functions of kinases and related parameters of mitochondrial functioning – function of the Na+/Ca++ exchanger, colocalization of mitochondria and lysosomes;
5. the method for evaluating the contribution of FAD++, which is part of various flavoproteins, to the total pool of FAD++ autofluorescence at the level of intact mitochondria, at the cellular level in healthy fibroblasts, mixed neuron-glial culture and on neurodegenerative diseases cell cultures.

• 2025:

1. data of changes in ATP levels in permeabilized cells under conditions of inhibition of enzymes possibly responsible for polyphosphate-dependent ATP synthesis, as well as changes in ATP levels in cells with different levels of glycolytic activity in response to the introduction of polyphosphate into the medium;
2. confocal images of cell cultures used in the work, stained with fluorescent probes specific to various organelles, as well as labeled antibodies to the identified enzymes;
3. results of western blotting of cell lysates using antibodies to the identified enzymes;
4. results of PCR analysis of the mRNA level of the identified enzymes;
5. data of the rate of polyphosphate-stimulated ATP synthesis under various conditions, parameters of enzymatic kinetics;
6. data of the rate of change in the level of polyphosphate and ATP in intact astrocytes and neurons in various variants of inhibition of ATP synthesis pathways;
7. the method for assessing the contribution of flavoprotein activity (FAD++, MAO) to the total pool of FAD++ autofluorescence at the tissue level in acute brain sections of healthy mice and mice of toxic neurodegenerative diseases models (in particular, Parkinson's disease).

• 2026:

1. data of the kinetics of fusion of synaptic vesicles with the presynaptic membrane, as well as changes in cytosolic calcium in response to external stimuli for Parkinson's diseasemodel cells with different levels of polyphosphate, as well as when treated with exogenous polyphosphate;
2. data of the nature of the development of calcium signaling in genetic and toxic models of taupathies when stimulated at different levels of polyphosphate;
3. information on the effect of the polyphosphate content on the threshold value of Ca++ concentration, leading to the discovery of mPTP;
4. the methodology for the differentiated assessment of the fluorescence intensity of FAD++, which is part of different flavoproteins, based on differences due to the peculiarities of the fluorescence lifetime of the coenzyme.

• 2027:

1. data of the rate and nature of oligomerization of α-synuclein, phosphorylated tau and β-amyloid in the extracellular medium with different polyphosphate content (using a polymer of different molecular weight);
2. in vitro data of the oligomerization (rate and nature of the structures formed) of α-synuclein in Parkinson's diseasecell models with different levels of polyphosphate;
3. data of the effect of changes in the content of intracellular polyphosphate on the viability of cellular models of neurodegenerative diseases, as well as the nature of developing cell death (apoptotic or necrotic) without and after external stimulation (due to dopamine, glutamate and other neurotransmitters).

• 2028:

1. comparative data of the level and rate of ATP formation and consumption in neuron-glial cultures during hypoxia modeling, as well as stimulation of the calcium signal during modulation of "classical" pathways, as well as polyphosphate-dependent ATP synthesis;
2. the results of determining the level of necrotic and apoptotic cell death when limiting oxidative phosphorylation by oxygen deprivation or activation of energy-consuming processes under conditions of modeling polyphosphate-associated ATP synthesis;
3. the level of metabolic activity, maturation, viability and morphology of the artificial tissues being developed on a polyphosphate-containing scaffold;
4. data of the effectiveness of healing various lesions of the skin of laboratory animals using a patch/bandages with polyphosphate based on studies using the assessment of tissue perfusion with blood, tissue saturation and the normalized amplitude of NADH fluorescence;
5. volumetric structures with actively proliferating model cells obtained by bioprinting, with the determination of the rate of proliferation, as well as the metabolic status of cells.

Ensuring the functioning of the healthcare system is one of the priorities of the state policy of the Russian Federation (according to forecasts, the volume of federal budget expenditures on healthcare in 2024 will amount to 1.62 trillion rubles (4.6% of the total planned expenditure). Within the framework of the national Healthcare project, the costs will amount to 289.9 billion rubles, and within the framework of the state program "Development of healthcare" in 2024-2026 – at least 3.99 trillion rubles). It should be noted that neurodegenerative diseasesforms a serious burden on other sectors of the social sphere related to the support of people affected by these pathologies. In this regard, the research planned within the framework of the project, which will not only provide new fundamental knowledge about the mechanisms of neurodegeneration, but also develop an understanding of the natural mechanisms of neuroprotection, as well as identify promising pharmacological targets, can form the basis for the development and introduction of domestic drugs based on physiologically active substances to correct the condition or complete restoration of brain functions in patients with neurodegenerative diseases, most of whom are incurable today.

The study planned for implementation corresponds to the priority areas of projects of technological sovereignty and projects of structural adaptation of the economy of the Russian Federation, approved by Decree of the Government of the Russian Federation No. 603 dated 04/15/2023, namely the direction "medical industry" in terms of the production of materials, dressings and similar products, including those coated with medicines. Using the unique potential of polyphosphate can ensure the creation of materials with high tissue regeneration efficiency, reducing both recovery time and patient suffering, as well as avoiding negative side effects.