Petr A. Slominsky

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.

A common 3‐bp deletion in the DYT1 gene in Russian families with early‐onset torsion dystonia

Hereditary torsion dystonia represent a clinically and genetically heterogeneous group of movement disorders. The most severe and frequent form of hereditary torsion dystonia is early‐onset generalized dystonia, DYT1. The DYT1 gene (Ozelius et al., 1997) encodes an ATP‐binding protein torsin A. A unique 3‐bp deletion (GAG) was found in the heterozygous state in almost all patients with early‐onset dystonia from different populations. We observed 39 patients with early‐onset generalized torsion dystonia belonging to 22 families from Russia. Seven families were of Ashkenazi Jewish (AJ) ethnic background, and other patients originated from the Slavonic population of Russia. The GAG deletion was identified in 24 affected persons from 15 families (68.2% of the families studied). In all the 7 families of AJ origin the disease was found to be caused by the deletion. In Slavs, the deletion was identified in 8 of 15 families (53%). In two deletion‐positive families we observed the co‐occurrence of typical early‐onset generalized dystonia and atypical phenotypes‐either isolated postural hand tremor or stutter. Hum Mutat 14:269, 1999.

Analysis of heavy neurofilament subunit gene polymorphism in Russian patients with sporadic motor neuron disease (MND)

Motor neuron disease (MND) results in the selective degeneration of motor neurons in the cerebral cortex, brain stem and spinal cord. The most common form of MND is amyotrophic lateral sclerosis (ALS). MND is complex and many genetic systems may be involved in the pathogenesis of this disease. Pathological and animal studies implicate neurofilament involvement in MND. The heavy subunit (NEFH) tail domain contains a repeated motif. In humans, there are two common variants: the 45 motif repeats long allele (L) and 44 motif repeats short allele (S). Previous studies have shown that the NEFH tail may be involved in the pathogenesis of MND. To investigate whether the L/S genotypes of the NEFH gene are associated with MND, we studied the frequency of L and S alleles in sporadic MND patients and a control population from Moscow. We observed a difference in SS genotype frequency between the control population and sporadic MND patients from Moscow. It was established that the SS genotype is sufficiently higher in sporadic MND patients. Moreover, we determined that patients with the SS genotype have the highest value of loss of the total clinical score. In summary, we conclude that the NEFH gene is involved in the pathogenesis of sporadic MND. The SS genotype represents a risk factor for the development and progression of sporadic MND in the Moscow population.

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.

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.

Cloning of Alu-containing cDNAs from human fibroblasts and identification of small Alu+ poly(A)+ RNAs in a variety of human normal and tumor cells

Two clones have been selected from a human fibroblast cDNA bank. By DNA sequencing the clones were shown to contain Alu elements located near the ends of the cDNA inserts. DNA of the clones was used for Northern blot hybridization analysis of a number of poly(A)‐containing RNAs from normal human tissues (brain, stomach, uterus, spleen, fibroblasts) and tumors (neurinoma, glioma, neuroblastoma, liposarcoma, adrenal cortex adenocarcinoma). All RNA samples reveal a heterodisperse distribution of Alu transcripts with discrete bands in the region of 7‐12 S RNA. The majority of these small poly(A)+ Alu+ RNAs contain Alu sequences only in one (canonical) orientation with functional signals including the split promoter for RNA polymerase III.

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.