Background/Objectives: MicroRNAs are key post-transcriptional regulators involved in various diseases. Despite its status as the gold standard, real-time RT-PCR faces challenges arising from high sequence homology among closely related microRNAs and the substantial biomaterial required to enrich small RNA fractions. This study aimed to develop an optimized protocol for simultaneous analysis of microRNA and mRNA expression from a single total RNA sample using mouse (Mus musculus) brain tissue, avoiding dependence on pre-designed commercial assay panels. Methods: We optimized a real-time RT-PCR workflow enabling simultaneous analysis of mature microRNAs and mRNAs from a single total RNA sample. Modifications include a redesigned universal reverse primer, LNA-modified TaqMan probes, and omission of the 65 °C denaturation step during reverse transcription. The method was validated for five microRNAs in mouse brain tissue. Results: The assay showed high specificity, discriminating closely related miR-125a-5p and miR-125b-5p with a ΔCt difference of 6.7 ± 1.2 cycles. Co-analysis with Oligo(dT)18 and Random hexamer primers did not interfere with microRNA detection. Conclusions: The developed approach enables reliable detection of closely related microRNAs and parallel analysis of different RNA types, which is particularly important for studying regulatory networks when working with limited amounts of biomaterial. This protocol provides a complementary, accessible option for targeted studies in resource-limited settings or for non-cataloged miRNA targets.

(1) Background: Although the rhesus macaque (Macaca mulatta) is widely maintained in captive populations for translational research, reliable molecular tools for monitoring their physiological status remain limited-a gap that directly affects population management and health monitoring. In particular, no validated panel of housekeeping genes (HKGs) is currently available for expression analysis at the mRNA level in peripheral blood of the species. (2) Methods: Peripheral blood samples from healthy male rhesus macaques aged 4-7 years were used. The expression stability of candidate HKGs was evaluated using RefFinder based on cycle threshold (Ct) values obtained from real-time PCR. (3) Results: A panel of 29 candidate HKGs was compiled. After multistage bioinformatic and experimental selection, 22 genes were excluded. Expression stability analysis of the remaining seven genes (AHSA1, B4GALT3, CYB5R1, HPCAL1, PSMD6, SSR2, and VPS72) identified B4GALT3 and PSMD6 as the most stable. (4) Conclusions: This study presents the experimentally validated set of HKGs for expression studies in Macaca mulatta. Based on the results of expression stability analysis, the B4GALT3 and PSMD6 genes can be recommended as reference genes for studying changes in gene expression in peripheral blood.

The deficiency of glucocerebrosidase (GCase) encoded by the GBA1 gene, leads to the autosomal recessive Gaucher disease and highly increased risk of developing Parkinson’s disease (PD). In order to study the effect of GCase dysfunction on neurodegeneration, we evaluated the GCase activity, lysosphingolipid content, extent of dopaminergic neuron degeneration in the substantia nigra (SN), and levels of dopamine (DA) and total and oligomeric α-synuclein (α-Syn) in the brain of mice with the presymptomatic stage of parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in combination with a single injection of the GCase selective inhibitor conduritol-β-epoxide (CBE) (100 mg/kg body weight). A single injection of CBE led to a 50

ANXA2 is a multifunctional member of the annexin protein family, implicated in vesicular transport, antioxidant defense, and actin remodeling. Its role in oncogenesis is actively investigated, notably in glioblastoma, astrocytoma, and breast cancer. However, a growing body of literature explores ANXA2’s involvement in neurodegenerative processes. The evidence suggests a potential contribution of ANXA2 to the pathogenesis of primary and secondary tauopathies, as well as Parkinson’s disease. It is crucial to note that the majority of these findings are correlative and necessitate further experimental validation. This review therefore presents a comprehensive analysis of data pertaining to ANXA2’s involvement in various cellular processes, the disruption of which contributes to neurological pathologies.

Myelin is a highly specialized form of plasma membrane. Gradual loss of myelin is one of the characteristic features of both age-related and pathological changes in the brain. This review seeks to characterize the spectrum of genes whose products take part in the formation and functioning of myelin in the CNS and to assess their possible contributions to the pathogenesis of Parkinson’s disease (PD). Analysis of published data obtained on model objects and patients with PD identified several myelin protein genes which may be associated with the development of PD. The genes for PLP1, MOBP, FA2H, and HSPA8 are the most promising for studies of their contributions to the pathogenesis of PD.

Myelin is a highly specialized form of the plasma membrane. The gradual loss of myelin is typical for both age-related and pathological changes in the brain. In this review, we attempted to characterize the spectrum of genes which products are involved in the formation and functioning of CNS myelin, as well as to assess their possible contribution to the pathogenesis of PD. Analysis of currently published data obtained from model objects and patients with PD has identified several myelin protein genes that may be associated with the development of PD. The PLP1, MOBP, FA2H, and HSPA8 genes are the most promising for studying their contribution to the pathogenesis of PD.

Active study of the molecular mechanisms of the occurrence and development of various cardiac pathologies using the assessment of gene expression by real-time PCR is necessary to solve the problem of finding and selecting adequate reference genes. A search is conducted for the most stably expressed potential reference genes in the intravital myocardial samples of patients with severe heart failure due to hypertrophic cardiomyopathy or coronary heart disease. Gene-expression analysis is performed using reverse transcription and real-time PCR. Gene-expression stability is assessed using the RefFinder resource. It is shown that the genes expressed most stably in human myocardium in the case of heart failure are PSMD6, PSMD7, SARS1, and AARS1. In this regard, we recommend including them in the gene panel when searching for and validating potential reference genes to assess the expression of candidate genes in myocardial tissues.

The use of various model organisms has made a huge contribution to understanding the causes and mechanisms of disease development and the study of pathological processes occurring during the development of diseases of the human cardiovascular system, and, in particular, hypertrophic cardiomyopathy (HCM). The optimal solution in the study of primary molecular disturbances is the use of cellular models such as induced pluripotent stem cells (iPSCs), primary rodent cardiomyocytes (CMs), and immortalized lines. In this review, we have focused on the most commonly used cell models, including freshly isolated adult and neonatal rodent CMs, and on the commercially available immortalized cell lines (HL-1, AC16, and H9c2). In order to assess the adequacy of these lines as CM models for studying human myocardial pathologies, a comparative analysis of phenotypic characteristics (morphology, metabolism, calcium homeostasis, etc.) and the nuances of practical use (availability, response to hypertrophic inducers, transfection, etc.) was carried out. The latest published data on the use of these models to assess the pathogenicity of HCM-associated mutations, as well as to screen the effectiveness of developed therapeutic drugs, are also summarized.

The zebrafish (Danio rerio) is one of the promising objects for modeling pathological conditions. A large number of such works are carried out at the transcriptomic level. Transcriptomic studies require the use of stable housekeeping genes (HKGs), and so we selected candidate HKGs and analyzed their stability in D. rerio under normal conditions. The work was carried out using D. rerio of the AB line whose embryos were sampled at three time points: the hatching stage (2 days postfertilization, dpf), and early larval stages (5 and 9 dpf). Analysis of HKG expression levels was performed using real-time polymerase chain reaction. Further analysis of the obtained Ct results was carried out in the RefFinder program. Different HKGs are suitable for assessing changes in gene expression at different stages of D. rerio development. When assessing changes in gene expression at the hatching stage (2 dpf), the optimal pair of HKGs is aars1 and polr2f; for the early larval stage (5 dpf), it is aars1 and psmd6; and for 9 dpf, it is bcat2 and actb1. During transcriptomic studies conducted at several developmental stages simultaneously, it is necessary to use a single selected pair of HKGs for all the studied stages of zebrafish development: aars1 and lsm12b. According to the data obtained, aars1 and lsm12b are the most stable HKGs for all the early developmental stages of D. rerio studied. At the same time, we first showed that aars1 is the most stable HKG for the early stages of the development. The second most stable HKG is lsm12b. Our data on the stability of the lsm12b gene are consistent with previously published data.

Mutations in the MYBPC3 gene are currently believed to lead to the development of hypertrophic cardiomyopathy (HCM) in the majority of genetically determined cases. However, despite many years of research, both in worldwide and in Russia in particular, the genetic landscape of HCM is still insufficiently studied. Moreover, the insufficient study of genetically determined cases of HCM in the Russian population does not allow us to study the possible relation of the phenotypic characteristics of HCM patients with certain pathogenic variants of the genome of these patients. In this regard, the purpose of our work was to study the prevalence of rare pathogenic variants rs730880711 and rs397515974 of the MYBPC3 gene in HCM patients from Russia and to assess the effect of these mutations on the severity of this disease. The sample included 180 patients with moderate HCM and 137 patients with severe HCM. Analysis of the genotypes of rs730880711 (NC_000011.10:g.47342928_47342929insG; V453X) and rs397515974 (NC_000011.10:g.47337452G>C; Y847X) variants in the MYBPC3 gene was carried out in genomic DNA samples isolated from peripheral blood by real-time PCR. The performed analysis of the prevalence of rare pathogenic variants rs730880711 and rs397515974 in the MYBPC3 gene in patients with moderate and severe forms of HCM from Russia showed that the frequency of each mutation was 0.003. Both pathogenic variants were identified in individuals with the moderate disease. Thus, the indicated mutations are extremely rare in HCM patients from Russia and do not make a significant contribution to the development of this disease in the Russian population.

Parkinson’s disease (PD) is one of the most common human neurodegenerative diseases. Belated diagnoses of PD and late treatment are caused by its elongated prodromal phase. Thus, searching for new candidate genes participating in the development of the pathological process in the early stages of the disease in patients who have not yet received therapy is relevant. Changes in mRNA and protein levels have been described both in the peripheral blood and in the brain of patients with PD. Thus, analysis of changes in the mRNA expression in peripheral blood is of great importance in studying the early stages of PD. This work aimed to analyze the changes in MEF2C, SLC22A4, P2RY12, and LRRN3 gene expression in the peripheral blood of patients in the early stages of PD. We found a statistically relevant and PD-specific change in the expression of the LRRN3 gene, indicating a disruption in the processes of neuronal regeneration and the functioning of synapses. The data obtained during the study indicate that this gene can be considered a potential biomarker of the early stages of PD.

Parkinson’s disease (PD) is one of the most common progressive neurodegenerative diseases. An important feature of the disease is its long latent period, which necessitates search for prognostic biomarkers. One method of identifying biomarkers of PD is to study changes in gene expression in peripheral blood of the patients in early stages of the disease and have not been treated. In this study, we analyzed relative mRNA levels of the genes GRIPAP1, DLG4, KIF1B, NGFRAP1, and NRF1, which are associated with neurotransmitter transport, apoptosis, and mitochondrial dysfunction, in the peripheral blood of PD patients using reverse transcription and real-time PCR with TaqMan probes. The results of this study suggest that the GRIPAP1 and DLG4 genes could be considered as potential biomarkers for the early clinical stages of Parkinson’s disease. The data obtained may indicate that NGFRAP1 is involved in pathogenesis of both PD and other neurodegenerative diseases. Furthermore, in the early clinical stages of the disease we studied, the KIF1B and NRF1 genes were found not to be involved in PD pathogenesis at the expression level.

Parkinson’s disease (PD) is characterized by a long prodromal period, during which patients often have sleep disturbances. The histaminergic system and circadian rhythms play an important role in the regulation of the sleep–wake cycle. Changes in the functioning of these systems may be involved in the pathogenesis of early stages of PD and may be age-dependent. Here, we have analyzed changes in the expression of genes associated with the regulation of the sleep–wake cycle (Hnmt, Hrh1, Hrh3, Per1, Per2, and Chrm3) in the substantia nigra (SN) and striatum of normal male mice of different ages, as well as in young and adult male mice with an MPTP-induced model of the early symptomatic stage (ESS) of PD. Age-dependent expression analysis in normal mouse brain tissue revealed changes in Hrh3, Per1, Per2, and Chrm3 genes in adult mice relative to young mice. When gene expression was examined in mice with the MPTP-induced model of the ESS of PD, changes in the expression of all studied genes were found only in the SN of adult mice with the ESS model of PD. These data suggest that age is a significant factor influencing changes in the expression of genes associated with sleep–wake cycle regulation in the development of PD.

Hypertrophic cardiomyopathy (HCM) is considered the most common inherited heart disease. HCM is a highly heterogeneous disease from the genetic point of view. However, the cause of the disease remains unclear, despite the large number of pathogenic variants detected in more than a quarter of patients. Therefore, studying the prevalence of pathogenic variants associated with HCM especially in the Russian population is still relevant. In this context, the objective of the research survey was to assess the contribution of pathogenic variants rs200411226 and rs397515905 in the MYBPC3 gene, leading to substitutions R495Q, R495W, and R495G, to the development of HCM in the Russian population. The sample included 224 patients with HCM of varying severity. The genotypes of variants rs200411226 (NM_000256.3:c.1484G>A) and rs397515905 (NM_000256.3:c.1483C>T/G)) in the MYBPC3 gene (R495Q, R495W, and R495G) were analyzed for all the patients with real-time PCR. The survey analysis of the prevalence of these pathogenic variants has shown that the R495Q and R495W mutations in the survey sample occur with a frequency of 0.4% for each mutation, which is generally comparable with the frequencies of these pathogenic variants in the other populations. In addition, R495Q and R495W mutations do not appear to cause a severe form of the disease. Survey testing failed to identify the R495G pathogenic variant in the survey sample of HCM patients. Therefore, these mutations themselves as a cause for HCM progression do not have any significant prevalence among the Russian population.

Mutations in the GBA1 gene represent the major genetic risk factor for Parkinson's disease (PD). The lysosomal enzyme beta-glucocerebrosidase (GCase) encoded by the GBA1 gene participates in both the endolysosomal pathway and the immune response. Disruption of these mechanisms is involved in PD pathogenesis. However, molecular mechanisms of PD associated with GBA1 mutations (GBA-PD) are unknown today in particular due to the partial penetrance of GBA1 variants in PD. The modifiers of GBA1 penetrance have not been elucidated. We characterized the transcriptomic profiles of cells from the substantia nigra (SN) of mice with co-injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and selective inhibitor of GCase activity (conduritol-β-epoxide, (CBE)) to mimic PD bearing GCase dysfunction (MPTP+CBE), mice treated with MPTP, mice treated with CBE and control mice treated with injection of sodium chloride (NaCl) (vehicle). Differential expression analysis, pathway enrichment analysis, and outlier detection were performed. Functional clustering of differentially represented transcripts revealed more processes associated with the functioning of neurogenesis, inflammation, apoptosis and autophagy in MPTP+CBE and MPTP mice than in vehicle mice, with a more pronounced alteration of autophagy processes in MPTP+CBE mice than in MPTP mice. The PI3K-Akt-mTOR signaling pathway may be considered a potential target for therapy in PD with GCase dysfunction.

Aim To determine specific clinical characteristics caused by a combination of the rs397516037 pathogenic variant in the myosin-binding protein C (MTBPC3) and the rs749628307 polymorphic variant in the vinculin (VCL) gene in a Russian family of carriers and to evaluate the contribution of the rs749628307 polymorphic variant in the VCL gene to the development of hypertrophic cardiomyopathy (HCMP).Material and methods The family under study included one healthy person and 3 patients with HCMP. A targeted analysis of proband's exome was performed. A structural alignment for both forms of the VCL protein, the canonical form and the form with p.Arg230His substitution, was performed.Results The pathogenic rs397516037 variant and the potentially pathogenic rs749628307 variant were detected in the proband and several family members. A possibly damaging variant rs749628307 was detected in the proband and several family members evaluated in this study. The structural alignment confirmed that the rs749628307 variant did not alter the protein structure significantly and could not cause an impairment or loss of the protein function.Conclusion This study demonstrated that apparently the rs749628307 variant in the VCL gene does not affect the protein structure in a pathogenetically significant way, neither does it affect the severity and form of the clinical manifestations of HCMP; therefore, it cannot be considered as pathogenic.

Mutations in the glucocerebrosidase gene (GBA1), which encodes the lysosomal enzyme glucocerebrosidase (GCase), can cause Gaucher disease, an autosomal recessive disease, and increase the risk of Parkinson’s disease (PD). The risk of developing PD for carriers of homozygous and heterozygous GBA1 mutations increases by 8–10 times, but not all carriers develop PD during their lifetime. Additionally, GBA-associated PD (GBA-PD) represents 10 to 30% of all forms of parkinsonism. The development mechanism of GBA-PD remains unknown. A decrease in GCase activity and accumulation of lysosphingolipids in patients with GBA-PD was shown by us and other researchers [1, 2]. GCase dysfunction is thought to result in impaired autophagy and accumulation of the alpha-synuclein protein, which is a crucial process in neurodegeneration in PD. Several techniques based on modeling parkinsonism in mice with GCase dysfunction were used to study the impact of GCase dysfunction on DA neuron neurodegeneration [3, 4]. In this study, we evaluated GCase activity, lysosphingolipids level, and the degree of neurodegeneration in DA-neurons of the substantia nigra’s compact (SNc) and reticular part (SNr), as well as the levels of dopamine and alpha-synuclein (total and oligomeric) in the brains of mice with a double “soft” neurotoxic model induced by the introduction of the neurotoxin 1-methyl-4-phenyl-1. This is the first time such an evaluation has been made. The presymptomatic stage of parkinsonism induced by 2,3,6-tetrahydropyridine (MPTP) involved the double administration of 12 μg/kg at a 2-hour interval, in combination with a single injection of the selective GCase inhibitor conduritol-B-epoxide (CBE) at a dose of 100 mg/kg. Additionally, we compared the transcriptomes of primary macrophage cultures from GBA-PD patients [5] with the transcriptome of SN brain tissue in model mice. We demonstrated that a singular injection of CBE resulted in a 50% decrease in GCase activity in the mouse brain and an elevation in lysosphingolipid levels. Additionally, the introduction of both MPTP and CBE led to an increase in the level of oligomeric forms of alpha-synuclein in the striatum. Simultaneously, degeneration of DA neurons in SNc, assessed by tyrosine hybroxylase (TH) immunohistochemistry 14 days after injection, was comparable to MPTP and CBE (decreasing to 50 and 60%, respectively). The neurotoxic model, when combined, demonstrates a significantly greater reduction in dopamine concentration, accumulation of total alpha-synuclein in the striatum, and more severe neurodegeneration of DA neurons in SNr (70% compared to 45% with MPTP administration). A comparison of differential gene expression in primary macrophage cultures from patients with GBA-PD and controls revealed a reduction in the expression of genes associated with neurogenesis, such as JUNB, NR4A2, and EGR1. In both the GBA-PD patient group (TRIM13, BCL6) and the MPTP-induced parkinsonism mouse group with GCase dysfunction (MPTP+CBE), genes related to the PI3K-Akt-mTOR signaling pathway, which regulates autophagy, were found to be activated. These genes include Pdk4, Sgk, and Ppp2r3d. The data obtained indicates that dysfunctional GCase leads to the accumulation of toxic forms of alpha-synuclein and degeneration of DA neurons, similar to the effects of small doses of MPTP. Combining neurotoxins (MPTP+CBE) causes a greater accumulation of alpha-synuclein and a higher degree of neuron degeneration. Transcriptomic analysis conducted on GBA-PD patients’ cells and a combined neurotoxic mouse model (MPTP+CBE) brain revealed modifications in gene expression of autophagy regulation. Approaches focused on enhancing GCase activity and autophagy exhibit potential in developing neuroprotective agents.