Technology of genomic balancing of chromatin of autologous hematopoietic stem cells for gene therapy of fatal immune-mediated diseases of civilization, extended life expectancy and sudden human death prevention.

A biotechnology for personalized ex vivo gene therapy based on molecular genomic balancing of hematopoietic stem cell (HSC) chromatin with nucleosome monomers of human genomic DNA (hDNAnmr) has been developed and implemented in the clinic to change (to "correct") mutant chromosome loci genomes of dominant HSC clones that form mono- and oligoclonal hematopoiesis during aging and major (oncological, cardiovascular, neurodegenerative and autoimmune) fatal immune-mediated diseases of civilization. A fundamentally new biotechnological approach has been applied to the delivery of genetic material into eukaryotic stem and progenitor cells by establishing an artificial "recombinogenic situation" in them to induce homologous recombination (equivalent replacement) of mutant DNA regions with healthy hDNAnmr. In experimental preclinical trials, the effectiveness of genomic balancing technology has been proven to reduce the risk of sudden death in old animals and to increase the lifespan of outbred mice by 30% and Wistar rats by 57%. The improvement in their quality of life, compared with the control, is explained by an increase in the telomeric regions of the HSCs and HPCs chromosomes by 1.5-2 times. The potential of the technology to slow down the hereditary neurodegenerative diseases on the model of amyotrophic lateral sclerosis is shown. The effectiveness of this technology in clinical practice is presented on the example of a terminal patient with stage 4 neuroendocrine cancer. This technology used in the treatment of a number of oncological, neurodegenerative, autoimmune and hereditary diseases with clonal hematopoiesis is able to arrest the progression of the disease, prevent its recurrence, prolong the active life of a person, increase the average life expectancy and prevent sudden death.

[Changes in the Activities and Contents of Individual Forms of Proteasomes in Samples of the Cerebral Cortex during Pathology Development in 5xFAD Mice].

The ubiquitin-proteasome system (UPS) provides hydrolysis of most intracellular proteins in proteasomes. There are various forms of proteasomes that differ, among other things, in the set of proteolytic subunits and the presence of activators. Alzheimer's disease (AD) is characterized by disturbances in the functional state of the UPS. At the same time, an increase in the expression of certain forms of proteasomes, in particular, proteasomes containing immune subunits (nonconstitutive proteasomes), has been shown. Here, we studied dynamic changes in the expression of catalytic proteasome subunit genes and corresponding proteins in the cerebral cortex of animals using a mouse model of AD (5xFAD transgenic mice). Increases by 4 and 6 folds in transcripts of the PSMB9 and PSMB8 genes encoding immune proteasome subunits were detected, as well as a significant increase in the content of immune ß-subunits (by 2.8 folds, ß1i; 2.2 folds, ß2i) in samples from 5xFAD mice at the age of 380 days, compared with samples from mice at 60 days of age. Moreover, the activation of both 20S and 26S proteasomes containing immune subunits were revealed in samples from 380 days old 5xFAD mice by electrophoresis in native conditions. This indicates activated synthesis of the immune subunits and assembly of nonconstitutive proteasomes at the terminal stage of pathology development. The obtained data, in combination with the available literature, indicate that the activation of nonconstitutive proteasomes is a universal phenomenon characteristic of various animal models of AD, which may reflect both the development of neuroinflammation and adaptive processes in tissues induced by the accumulation of toxic protein aggegates.

[Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging].

Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the ß5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the ß5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit ß5; increased expression of genes encoding immune proteasome subunits ß5i and ß1i. In tissue homogenates of aged mice, an increase in the content of immune subunits ß1i and ß2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the ß5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the ß5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

[The role of sphingolipids in pathogenesis of amyotrophic lateral sclerosis]. / Rol' sfingolipidov v patogeneze bokovogo amiotroficheskogo skleroza.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by selective degeneration of motor neurons of the spinal cord and motor cortex and brain stem. The key features of the course of this disease are excitotoxicity, oxidative stress, mitochondrial dysfunction, neuro-inflammatory and immune reactions. Recently, the mechanisms of programmed cell death (apoptosis), which may be responsible for the degeneration of motor neurons in this disease, have been intensively studied. In this regard, sphingolipids, which are the most important sources of secondary messengers that transmit cell proliferation, differentiation and apoptosis signals, and are involved in the development of neuroinflammatory and immune responses, are of particular interest in the context of ALS pathogenesis. The review provides information from domestic and foreign authors on the involvement of various sphingolipids (sphingomyelins, ceramides, sphingosine, sphinganin, sphingosine-1-phosphate, galactosylceramides, glucosylceramides, gangliosides) in the development of pro-inflammatory reactions and apoptosis of motor neurons in ALS. The authors discuss the prospects of using new drugs that control the metabolism of sphingolipids for the treatment of ALS.

[Structures of the Mouse Central Nervous System Contain Different Quantities of Proteasome Gene Transcripts].

Proteasomes are multisubunit complexes that degrade most intracellular proteins. Three of the 14 subunits of the 20S proteasome, specifically ß1, ß2, and ß5, demonstrate catalytic activity and hydrolyze peptide bonds after acidic, basic, and hydrophobic amino acids, respectively. Within proteasome, the constitutive catalytic subunits ß1, ß2, and ß5 can be substituted by the immune ßli, ß2i, and ß5i subunits, respectively. However, proteasomes do not always contain all the immune subunits at once; some proteasomes contain both immune and constitutive catalytic subunits simultaneously. Incorporation of immune subunits modifies the pattern of peptides produced by proteasomes. This is essential for antigen presentation and cellular response to stress as well as for a number of intracellular signaling pathways. We have developed a quantitative PCR-based system for the determination of the absolute levels of murine constitutive and immune proteasome subunits gene expression. Using the obtained system, we have estimated the expression levels of genes encoding proteasome subunits in the mouse central nervous system (CNS) tissues. We have shown that the quantity of transcripts of proteasome catalytic subunits in different CNS structures differed significantly. These data allow us to assume that the studied brain regions can be divided into two groups, with relatively "high" (cerebral cortex and spinal cord) and "low" (hippocampus and cerebellum) levels of proteasome subunit genes expression. Moreover, it was possible to distinguish structures with similar and significantly different gene expression profiles of proteasome catalytic subunits. Thus, the gene expression profiles in the cortex, spinal cord, and cerebellum were similar, but different from the expression profile in the hippocampus. Based on the obtained data, we suggest that there are differences in the proteasome pool, as well as in the functional load on the ubiquitin-proteasome system in different parts of the CNS.

[The role of lipids in the pathogenesis of lateral amyotrophic sclerosis]. / Rol' lipidov v patogeneze bokovogo amiotroficheskogo skleroza.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by selective degeneration of motor neurons of the motor cortex, brain stem and brain stem. Mutations in genes coding for SOD1, C9ORF72, TDP-43, FUS and others are associated with ALS and result in abnormal processing and transport of RNA as well as changes in the dynamics of cytoskeleton. In addition, a sharp change in the metabolism of various lipid classes, including phospholipids, fatty acids, sphingolipids, etc., was detected. This review describes changes in lipid content and activity of enzymes involved in their metabolism in ALS animal models as well as in patients. Changes in the metabolism of fatty acids, phospholipids, cholesterol and its derivatives are reviewed in detail. The prospects of searching for new drugs among modulators of lipid metabolism enzymes are discussed.

Promising Molecular Targets for Pharmacological Therapy of Neurodegenerative Pathologies.

Drug development for the treatment of neurodegenerative diseases has to confront numerous problems occurring, in particular, because of attempts to address only one of the causes of the pathogenesis of neurological disorders. Recent advances in multitarget therapy research are gaining momentum by utilizing pharmacophores that simultaneously affect different pathological pathways in the neurodegeneration process. The application of such a therapeutic strategy not only involves the treatment of symptoms, but also mainly addresses prevention of the fundamental pathological processes of neurodegenerative diseases and the reduction of cognitive abilities. Neuroinflammation and oxidative stress, mitochondrial dysfunction, dysregulation of the expression of histone deacetylases, and aggregation of pathogenic forms of proteins are among the most common and significant pathological features of neurodegenerative diseases. In this review, we focus on the molecular mechanisms and highlight the main aspects, including reactive oxygen species, the cell endogenous antioxidant system, neuroinflammation triggers, metalloproteinases, α-synuclein, tau proteins, neuromelanin, histone deacetylases, presenilins, etc. The processes and molecular targets discussed in this review could serve as a starting point for screening leader compounds that could help prevent or slow down the development of neurodegenerative diseases.

Changes in the Metabolism of Sphingoid Bases in the Brain and Spinal Cord of Transgenic FUS(1-359) Mice, a Model of Amyotrophic Lateral Sclerosis.

The aim of this study was to evaluate changes in the content of sphingoid bases - sphingosine (SPH), sphinganine, and sphingosine-1-phosphate (SPH-1-P) - and in expression of genes encoding enzymes involved in their metabolism in the brain structures (hippocampus, cortex, and cerebellum) and spinal cord of transgenic FUS(1-359) mice. FUS(1-359) mice are characterized by motor impairments and can be used as a model of amyotrophic lateral sclerosis (ALS). Lipids from the mouse brain structures and spinal cord after 2, 3, and 4 months of disease development were analyzed by chromatography/mass spectrometry, while changes in the expression of the SPHK1, SPHK2, SGPP2, SGPL1, ASAH1, and ASAH2 genes were assayed using RNA sequencing. The levels of SPH and sphinganine (i.e., sphingoid bases with pronounced pro-apoptotic properties) were dramatically increased in the spinal cord at the terminal stage of the disease. The ratio of the anti-apoptotic SPH-1-P to SPH and sphinganine sharply reduced, indicating massive apoptosis of spinal cord cells. Significant changes in the content of SPH and SPH-1-P and in the expression of genes related to their metabolism were found at the terminal ALS stage in the spinal cord. Expression of the SGPL gene (SPH-1-P lyase) was strongly activated, while expression of the SGPP2 (SPH-1-P phosphatase) gene was reduced. Elucidation of mechanisms for the regulation of sphingolipid metabolism in ALS will help to identify molecular targets for the new-generation drugs.

[The association of gamma-synuclein autoantibodies with the polymorphism in exon 4 of the coding gene]. / Sviaz' nalichiia autoantitel k gamma-sinukleinu s polimorfizmom v 4-m ékzone kodiruiushchego gena.

AIM: To analyze the polymorphism in exon 4 of the gamma-synuclein gene (SNCG) in patients with autoantibodies against the gamma-synuclein protein. MATERIAL AND METHODS: To identify autoantibodies against gamma-synuclein, the serum from patients with chronic cerebral ischemia and cervical osteochondrosis was used. All patients were women of the Slavic ethnic group, mean age 61±5 years. The isolated genomic DNA was used to determine the point mutation in exon 4 by the restriction endonuclease HphI and subsequent sequencing of the resulting fragments to confirm the results. RESULTS AND CONCLUSION: Antibodies against gamma-synuclein were identified in 2 patients with chronic cerebral ischemia and 3 with cervical osteochondrosis. All five patients had a T to A substitution at position 371, which was detected by the restriction endonuclease HphI resulting in a hydrolysis of the amplicon and the formation of two fragments. The subsequent sequencing of exon 4 of the SNCG revealed no other mutations and confirmed the T to A substitution. This single nucleotide polymorphism results in the amino acid substitution of glutamic acid to valine at position 110 (out of 127), changing its physicochemical properties and the ability to form aggregates as well as post-translational modifications. The obtained results provide grounds for further association studies of SNCG polymorphism in patients with various diseases of the nervous system.

Gamma-Carbolines Derivatives As Promising Agents for the Development of Pathogenic Therapy for Proteinopathy.

Uncontrolled protein aggregation, accompanied by the formation of specific inclusions, is a major component of the pathogenesis of many common neurodegenerative diseases known as proteinopathies. The intermediate products of this aggregation are toxic to neurons and may be lethal. The development strategy of pathogenic therapy for proteinopathy is based on the design of drugs capable of both inhibiting proteinopathy progression and increasing the survival of affected neurons. The results of a decade-long research effort at leading Russian and international laboratories have demonstrated that Dimebon (Latrepirdine), as well as a number of its derivatives from a gamma-carboline group, show a strong neuroprotective effect and can modulate the course of a neurodegenerative process in both in vitro and in vivo model systems. The accumulated data indicate that gamma-carbolines are promising compounds for the development of pathogenic therapy for proteinopathies.

[Dimebon delays the onset of symptoms of FUS-proteinopathy in transgenic mice]. / Otsrochennyi debiut simptomaticheskoi stadii FUS-proteinopatii u transgennykh myshei pod deistviem dimebona.

AIM: To evaluate an effect of dimebon on the onset of symptomatic stage in FUS.1-513 transgenic mice - a new genetic model of neurodegeneration, and to study the dynamics of disease progression in the terminal stage. MATERIAL AND METHODS: The study was carried out on males of line FUS1-513 with the contribution of genes from CD1 strains. Mice of the experimental group (n=28) received dimebon with water in the concentration of 70 mcg/ml starting from the 35th day of life. The control group (n=25) did not receive the drug. Age, body mass of animals at the start of symptomatic stage and duration of symptomatic stage were assessed. RESULTS: Application of dimebon can delay the onset of the manifestation of clinical symptoms of the neurodegenerative process in the experimental group (127.6±4.6 days) compared to the control group (110.6±4.2 days). The body mass was similar in both groups. CONCLUSION: Dimebon leads to an increase in the duration of presymptomatic stage and delays the manifestation of clinical symptoms. The changes in the dynamics of the pathological process in the symptomatic stage are not detected.

Detection of autoantibodies to potentially amyloidogenic protein, gamma-synuclein, in the serum of patients with amyotrophic lateral sclerosis and cerebral circulatory disorders.

In this study, we analyzed serum for the presence of antibodies to gamma-synuclein in patients with amyotrophic lateral sclerosis (ALS) compared to the control group of patients with other neurological diseases and healthy control donors. As a result, antibodies against gamma-synuclein are not an ALS-specific feature and have been identified in patients with ALS as well as in the control group patients. Patients with the impaired cerebral circulation showed increased incidence of autoantibodies to gamma-synuclein, yet the difference lacks statistical representativeness due to limited sample size.

Glutamate release and uptake processes are altered in a new mouse model of amyotrophic lateral sclerosis.

In this paper, we showed that in the cortex of mice expressing an abberant form of FUS protein that model amyotrophic lateral sclerosis (ALS), the processes of KCl-induced and basal [(3)H]glutamate release and uptake are altered at the presymptomatic stage as compared to the non-transgenic littermates. The change in these three parameters in transgenic animals causes excitotoxicity, which, in turn, may lead to massive loss of motor neurons and the onset of ALS symptoms.

The role of alpha-synuclein in the development of the dopaminergic neurons in the substantia nigra and ventral tegmental area.

Alpha-synuclein is a presynaptic protein of vertebrates that belongs to the family of synucleins. Normal functions of synucleins remain unknown. Alpha-synuclein is one of the causative factors of the familial and idiopathic forms of Parkinson's disease (PD). The progressive loss of dopaminergic (DA) neurons is characteristic of PD and the most severe damage occurs in the substantia nigra (SN). This leads to an erraticism of the synthesis and synaptic secretion of the neurotransmitters, subsequently resulting in the loss of the connections between brain areas. This work shows that alpha-synuclein is directly involved in the formation of the mature DA neurons of the midbrain at different stages of the ontogenesis and these findings are consistent with data obtained in other studies. Thus, alpha-synuclein may have a varying modulating effect on the growth dynamics and the fate of populations of DA neurons.

The new line of genetically modified mice with constitutive knockout of the gene alpha synuclein to study pathogenetic aspects of differential loss of dopaminergic neurons .

The purpose: This study investigated the role of alpha-synuclein in the development of dopaminergic neurons. Methods: In this study a new SNCA knockout mouse line has been used to model the deficiency of alpha-synuclein function. In the knockout and control mice the dynamics of the formation of two distinct populations of dopaminergic neurons differently affected in patients with PD was studied by the comparative morphometric analysis. Results: Here, we revealed a prominent modulating effect of alpha-synuclein on the developing DA neurons in substantia nigra (SN) which is the most affected region in PD patients. Yet, alpha-synuclein had no effect on the formation of DA neurons in ventral tegmental area which is much less susceptible to degeneration in PD patients. Conclusion: The new line of knockout mice is a convenient model for studying pathophysiologic aspects of selective impairment of DA neurons.

The Y-Box Binding Protein 1 Suppresses Alzheimer's Disease Progression in Two Animal Models.

The Y-box binding protein 1 (YB-1) is a member of the family of DNA- and RNA binding proteins. It is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation. Previously, YB-1 was detected in neurons of the neocortex and hippocampus, but its precise role in the brain remains undefined. Here we show that subchronic intranasal injections of recombinant YB-1, as well as its fragment YB-11-219, suppress impairment of spatial memory in olfactory bulbectomized (OBX) mice with Alzheimer's type degeneration and improve learning in transgenic 5XFAD mice used as a model of cerebral amyloidosis. YB-1-treated OBX and 5XFAD mice showed a decreased level of brain ß-amyloid. In OBX animals, an improved morphological state of neurons was revealed in the neocortex and hippocampus; in 5XFAD mice, a delay in amyloid plaque progression was observed. Intranasally administered YB-1 penetrated into the brain and could enter neurons. In vitro co-incubation of YB-1 with monomeric ß-amyloid (1-42) inhibited formation of ß-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with ß-amyloid prevents formation of filaments that are responsible for neurotoxicity and neuronal death. Our data are the first evidence for a potential therapeutic benefit of YB-1 for treatment of Alzheimer's disease.

Dimebon reduces the levels of aggregated amyloidogenic protein forms in detergent-insoluble fractions in vivo.

Aggregation of proteins liable to assembling into fibrils with subsequent formation of amyloid incorporations is an important component in the pathogenesis of many neurodegenerative diseases. Dimebon, a Russian drug, reduces the content of detergent-insoluble fibrillar forms of synuclein, the main protein component of pathological incorporations in neurons of transgenic mouse strain used in the study.