Maria I. Shadrina

Effective quantitative real-time polymerase chain reaction analysis of the parkin gene ( PARK2 ) exon 1–12 dosage

BackgroundOne of the causes of Parkinsons disease is mutations in the PARK2 gene. Deletions and duplications of single exons or exon groups account for a large proportion of the gene mutations. Direct detection of these mutations can be used for the diagnosis of Parkinsons disease.MethodsTo detect these mutations, we developed an effective technique based on the real-time TaqMan PCR system, which allows us to evaluate the copynumbers of the PARK2 gene exons by comparing the intensity of the amplification signals from some exon of this gene with that of the β-globin gene (the internal control).ResultsWe analyzed rearrangements in exons 1–12 of the PARK2 gene in 64 patients from Russia with early-onset Parkinsons disease. The frequency of these mutations in our patients was 14%.ConclusionWe have developed a simple, accurate, and reproducible method applicable to the rapid detection of exon rearrangements in the PARK2 gene. It is suitable for the analysis of large patient groups, and it may become the basis for a diagnostic test.

miRNA expression is highly sensitive to a drug therapy in Parkinson's disease.

BACKGROUND miRNAs may play a role in the pathogenesis of Parkinsons disease. It is necessary to continue the search for new miRNAs that may affect the development of neurodegeneration in Parkinsons disease. METHODS 20 untreated patients with Parkinsons disease and 18 treated patients with Parkinsons disease (Hoehn and Yahr scores 1-2) were studied. An analysis of the levels of 11 miRNAs in the peripheral blood lymphocytes of patients was carried out using reverse transcription followed by real-time PCR. RESULTS The levels of miR-7, miR-9-3p, miR-9-5p, miR-129, and miR-132 were increased by more than three times in treated patients with Parkinsons disease compared with those of the controls. CONCLUSIONS It is probable that miRNAs are very sensitive to drug therapy and that the effects of therapy observed may be associated with changes in the levels of these miRNAs and their target genes in patients with Parkinsons disease.

ITPR1 gene p.Val1553Met mutation in Russian family with mild Spinocerebellar ataxia

BackgroundSpinocerebellar ataxias (SСAs) are a highly heterogeneous group of inherited neurological disorders. The symptoms of ataxia vary in individual patients and even within the same SCA subtype. A study of a four-generation family with autosomal dominant (AD) non-progressive SCA with mild symptoms was conducted. The genotyping of this family revealed no frequent pathogenic mutations. So the objective of this study was to identify the genetic causes of the disease in this family with the technology of whole-exome sequencing (WES).Methods and resultsWES, candidate variant analysis with further Sanger sequencing, mRNA secondary structure prediction, and RSCU analysis were performed; a heterozygous missense mutation in ITPR1 was identified.ConclusionOur study confirms the fact that ITPR1 gene plays a certain role in the pathogenesis of SCAs, and, therefore, we suggest that c.4657G>A p.Val1553Met) is a disease-causing mutation in the family studied.

Neurotrophin gene expression in rat brain under the action of Semax, an analogue of ACTH4-10

The heptapeptide Semax, an analogue of the N-terminal adrenocorticotropic hormone fragment (4-10) (ACTH(4-10)), has been shown to exert a number of neuroprotective effects. There are some investigations that connected these effects with the increase of neurotrophin gene expression under the peptide drug application in neuron cell cultures [M.I. Shadrina, O.V. Dolotov, I.A. Grivennikov, P.A. Slominsky, L.A. Andreeva, L.S. Inozemtseva, S.A. Limborska, N.F. Myasoedov, Rapid induction of neurotrophin mRNAs in rat glial cell cultures by Semax, an adrenocorticotropic hormone analogue, Neurosci. Lett. 308 (2001) (2) 115-118]. In this work, we examined the action of Semax on rapid changes of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) gene expression in vivo. Male Wistar rats were treated for 1h with Semax (50 microg/kg, single intranasal application) and neurotrophin gene expression in rat brain was analyzed by real-time polymerase chain reaction (PCR). It was revealed that an intranasal application of Semax increased the expression of both neurotrophin genes in rat hippocampus. Bdnf gene expression also increased in the brainstem and cerebellum. Ngf gene expression decreased in rat frontal cortex. Thus, Semax induces rapid, gene- and region-specific changes in neurotrophin gene expression in normal rat brain.

Association study of sporadic Parkinson's disease genetic risk factors in patients from Russia by APEX technology

Most patients with Parkinsons disease (PD) have sporadic form of the disease with a multifactorial etiology due to interactions between environmental conditions and the genetic constitution of the individuals. We have analyzed by APEX technology 50 single nucleotide polymorphisms (SNPs) in 19 genes related to cholecystokinin, serotonin, dopamine and opioid neurotransmission. Significant differences in the allele and genotype frequencies between the controls and PD patients were detected for four SNPs from three genes (serotonin 2A receptor (rs6311, P=0.043), Wolfram syndrome 1 (rs1801211, P=0.007), proopiomelanocortin (rs28930368, P=0.026 and rs2071345, P=0.027) genes). Two SNPs in proopiomelanocortin (POMC) gene were also associated with different clinical forms of PD. Our data suggest that at least three genes involved in neurotransmitter systems may have more specific role in genetic predisposition to PD.

Involvement of endocytosis and alternative splicing in the formation of the pathological process in the early stages of Parkinson's disease.

Parkinsons disease (PD) is the one of most widespread neurodegenerative pathologies. Because of the impossibility of studying the endogenous processes that occur in the brain of patients with PD in the presymptomatic stage, the mechanisms that trigger the disease remain unknown. Thus, the identification of the processes that play an important role in the early stages of the disease in these patients is extremely difficult. In this context, we performed a whole-transcriptome analysis of the peripheral blood of untreated patients with stage 1 PD (Hoehn-Yahr scale). We demonstrated a significant change in the levels of transcripts included in the large groups of processes associated with the functioning of the immune system and cellular transport. Moreover, a significant change in the splicing of genes involved in cellular-transport processes was shown in our study.

Expression of inflammation-related genes in mouse spleen under tuftsin analog Selank

Previous studies have shown that synthetic tuftsin analogue Selank causes a transcriptomic response in the rat hippocampus and in spleen cells and may participate in the regulation of inflammatory processes in the body. In this work we studied the effect of Selank and two of its fragments on the expression of genes involved in processes of inflammation. We analyzed the expression of 84 genes involved in processes of inflammation (e.g., chemokines, cytokines, and its receptors) in mouse spleen 6 and 24 h after Selank single intraperitoneal injection (100 μg/kg) using real-time PCR method. We found significant changes in the expression of 34 genes involved in inflammation processes. The detailed analysis of quantitative data showed that the Bcl6 gene, which plays a main role in the formation and development of the immune system, exhibited significant changes in its expression levels in response to injection of each of the peptides. Also, we observed expression changes for Bcl6 target and corepressor genes under the influence of Selank and its fragments. Our results showed that Selank and its fragments caused a number of alterations in the expression of genes involved in inflammation. The data obtained confirmed the participation of Selank in the processes of regulation of inflammation in the body. The complex biological effect of Selank may be partially determined by the systematic effect of this peptide on genomic expression.

Expression analysis of suppression of tumorigenicity 13 gene in patients with Parkinson's disease.

Parkinsons disease (PD) is the second most common progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons. Detecting changes in gene expression in untreated de novo patients with PD is important for understanding disease pathogenesis and for identifying biomarkers for preclinical stage of PD. In this study we investigate ST13 gene expression in the peripheral blood of different groups of patients with neurological diseases using reverse transcription reaction and real-time polymerase chain reaction (PCR). Our results suggest that the expression levels of ST13 cannot serve as a biomarker for early stages of PD.

Transcriptome Profile Changes in Mice with MPTP-Induced Early Stages of Parkinson's Disease.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. Despite progress in the study of the molecular, genetic, and pathogenic mechanisms of PD, it is unclear which processes trigger the development of the pathology associated with PD. Models of the presymptomatic and early symptomatic stages of PD induced by MPTP have been used to analyze changes in transcriptome profile in brain tissues, to identify specific patterns and mechanisms underlying neurodegeneration in PD. The whole-transcriptome analysis in the brain tissues of the mice with MPTP-induced PD showed that striatum is involved in the pathogenesis in the earliest stages and the processes associated with vesicular transport may be altered. The expression profiles of the genes studied in the substantia nigra and peripheral blood confirm that lymphocytes from peripheral blood may reflect processes occurring in the brain. These data suggest that messenger RNA (mRNA) levels in peripheral blood may provide potential biomarkers of the neurodegeneration occurring in PD. The changes in expression at the mRNA and protein levels suggest that Snca may be involved in neurodegeneration and Drd2 may participate in the development of the compensatory mechanisms in the early stages of PD pathogenesis. Our data suggest that the brain cortex may be involved in the pathological processes in the early stages of PD, including the presymptomatic stage.

The temporary dynamics of inflammation-related genes expression under tuftsin analog Selank action.

Previous studies have shown that synthetic tuftsin analogue Selank and its fragments cause a number of alterations in the expression of certain genes involved in inflammation in mouse spleen. In this work we studied the effect of Selank and its short fragment Gly-Pro on the temporary dynamics of C3, Casp1, Il2rg, and Xcr1 genes expression in mouse spleen after single intraperitoneal injection (100 μg/kg) of peptides using real-time PCR method. We found a significant 3-fold decrease in the C3 mRNA level just 30 min after Selank injection and similar alteration this gene mRNA level after Gly-Pro administration. A wave-like alteration in the Casp1 mRNA level was observed after Selank injection. We found a significant alteration in the mRNA level of the Il2rg gene at early time points after Selank and Gly-Pro administration and an almost equal reduction in the Xcr1 mRNA level 90 min after the administration of Selank and its fragment. Our results showed that, Selank and its short fragment Gly-Pro influence the expression of genes that mediate different types of immune responses, thereby maintaining the balance of the immune system. It should be noted that in most cases, there was a coincidence in the expression profiles of the studied genes after Selank and Gly-Pro administration. This might indicate an active contribution of the dipeptide to the final effect of Selank.