Sulev Koks

Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting 1% of the population over 60 years of age. PD pathology is characterised by the loss of dopaminergic neurons from the substantia nigra and the formation of neuronal inclusions called Lewy bodies. The development of PD involves a complex interaction of genetic and environmental factors. Many of the genetic factors affecting disease progression have been identified but there is much still to be determined. This is particularly true for those factors not causative of the disease, but progression and severity of the disease once diagnosed. We aim to address both genetic and transcriptomic changes which we can bioinformatically identify in the Parkinson’s Progression Markers Initiative (PPMI) cohort which was established to identify PD progression markers to help understand disease aetiology and ultimately aid in the development of novel therapeutics. Methods Genetic data, longitudinal clinical data and transcriptomic data has been obtained from the Parkinson’s Progression Markers Initiative (PPMI) cohort (www.ppmi-info.org). The DESeq2 package in R will be used to detect statistically significant differences in the gene expression profiles between the different genotypes which we are currently delineating to correlate with specific clinical progression markers. This will allow us to align genetic variation with blood transcriptomic changes with disease progression. Approach for statistical analysis Once we have identified targets using DESeq2, their association with PD progression will be analysed in the longitudinal analysis using a linear mixed-effects model combined with FDR correction, measuring the changes in phenotypic scores during follow-up visits that will later be analysed.

Chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis that leads to a mixture of euploid and aneuploid cells within the same human embryo during in vitro fertilization (IVF). However, the rate of CIN in naturally conceived embryos is largely unknown, because it is impossible to study human embryos in vivo. Here, we developed and applied a novel haplarithmisis-based method to characterize allelic architecture of DNA samples derived from the placenta and cord blood of the same pregnancy. Specifically, we scrutinized genome-wide single nucleotide polymorphism profiles in DNA from the father, mother, placenta and neonate umbilical cord blood of 55 families (quartets), of which 26 and 29 quartets were from natural and IVF pregnancies, respectively. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo genomic alterations occur at similar rates in IVF and naturally conceived neonates. The findings confirm that IVF treatment has no detrimental effect on the chromosomal constitution of fetal or placental lineages.

Tuuma siirdamise teel kloonimise efektiivsus on väga madal, sageli on elussündide osakaal alla 1%(Watanabe, 2013). Embrüonaalses arengus on väga oluline etapp doonorraku tuuma ümberprogrammeerimine, mis ei pruugi kloonimise puhul toimuda täies ulatuses. Puuduliku ümberprogrammeerimise tulemusena peetub kloonembrüote kasv erinevates arenguetappides ning lõpeb sageli loote surmaga (Chitwood jt, 2013; Graf jt, 2014). Suurem osa probleemidest avaldub varastes arenguetappides. Kuid ka hilisemas arengus võib avalduda kõrvalekaldeid normaalsest arengust–sageli esineb kloontiinuste puhul platsentoomide suurenemist ja nende arvu vähenemist, suurenenud läbimõõduga nabanööri ja ka vasikad ise on keskmisest suuremad. Sellist nähtust nimetatakse suure järglase sündroomiks (Chitwood jt, 2013; Graf jt, 2014).

Recurrent miscarriage (RM) is defined as ≥3 consecutive pregnancy losses and occurs in 1–3% of fertile couples. No specific early biomarkers with high predictive value of threatening miscarriage have been identified. Using GeneChips (Affymetrix), we profiled whole-genome differential gene expression in RM placental tissue compared to uncomplicated pregnancies matched for gestational age. In cases of RM, significantly increased placental mRNA expression of TNF-related apoptosis-inducing ligand (TRAIL; P<0.005) and S100A8 (P<0.001) encoding for inflammatory marker calprotectin (S100A8/A9) was confirmed by Taqman RT-qPCR. Using ELISA, the concentrations of soluble TRAIL and calprotectin in maternal serum in normal first trimester (n=35) and failed pregnancies (early, n=18 and late miscarriage, n=4; tubal pregnancy, n=11) were determined. When compared to normal first trimester pregnancy, significantly higher maternal serum concentration of sTRAIL was detected at the RM event (P<0.001), in women with tubal pregnancy (P<0.05), and in pregnant women, who developed an unpredicted miscarriage 2–50 days after prospective serum sampling (P<0.05). Maternal serum levels of calprotectin were neither diagnostic nor prognostic to early pregnancy failures (P>0.05). sTRAIL may have a potential as a predictive biomarker in maternal serum for early pregnancy complications.

Brain and skin are both initially part of the ectoderm during prenatal development, but they are separated during neurogenesis. There is growing evidence that brain and skin are functionally related and skin pigmentation and several behavioral traits, such as anxiety, are coinherited. Here we describe a novel protein, Myg1 (Melanocyte proliferating gene 1, also known as Gamm1). Myg1 is known as putative phosphoesterase that is under-expressed in mouse malignant melanoma compared to autonomously growing mouse melanocytes. In our lab we have found that Myg1 is most significantly overexpressed gene in the amygdaloid area of rats in response to anxiety reaction induced by cat odor. Using real time Q-PCR we detected Myg1 transcript in all 10 areas of the brain we examined. WebQTL analysis revealed that the expression level of Myg1 (located in chr 12) is strongly regulated by a certain locus in chr 19 that includes several QTL markers: Dark skin 1, Dopamine transporter density and Open field activity and conditioned avoidance. Thus Myg1 could be involved both in regulating skin pigmentation and behavioral responses. We also explored Myg1 expression in the skin samples of vitiligo patients. Vitiligo is a skin disease in which pigment cells (melanocytes) are destroyed or stop producing melanin, resulting in white patches on the skin. We found that Myg1 expression is significantly (p<0.01) increased in both damaged and normal skin of vitiligo patients (n=28) compared to the skin samples of the control group (n=21). We propose that Myg1 is involved in neuroendocrine stress response system that has both central (HPA-axis) and peripheral (skin response to stress) versions. Using YFP-tag we have seen that Myg1 is predominantly expressed in the cell nucleus, but is in a low rate also present in cytoplasm. The precise role of Myg1 in the neural system and in the pigmentation pathway remains to be elucidated.

Previous studies suggest that cholecystokinin (CCK) is implicated in the modulation of pain sensitivity and memory functions. We used CCK2 receptor deficient homozygous (-/-) and heterozygous (+/-) mice and their wild type (+/+) littermates of 129sv/C57BL6 background and assessed their pain sensitivity and learning abilities in two different learning paradigms: passive avoidance test and Morris water maze. It has been found that mice without CCK2 receptors display hyposensitivity in Von Frey filament test and in plantar analgesia test and impaired memory functions in Y-maze paradigm. We found that in passive avoidance test, CCK2 receptor deficient -/- and +/- mice showed normal learning abilities compared to +/+ mice, if electric shocks of higher magnitude (0.4 mA or 0.6 mA for 2 s) were used, but displayed almost twice shorter entrance latencies (indicative of impaired memory) on Day 2 in case an electric shock of 0.2 mA for 2 s was used on Day 1. On Day 9 (long-term memory test), +/+ mice, subjected to stronger (0.4 mA or 0.6 mA for 2 s) electric shocks on Day 1, displayed somewhat decreased latencies, but the memory of -/- and +/- mice had even improved compared to Day 2. In Morris water maze, we established no statistically significant differences between -/-, +/- and +/+ CCK2 receptor deficient mice in neither the speed of learning, short-term memory (1 day and 5 days), nor long-term memory (14 days). Thus, the partly impaired performance of -/- and +/- mice in passive avoidance test (0.2 mA) can be explained by lower pain sensitivity, not impaired memory functions, because in three other conditions (passive avoidance with 0.4 mA and 0.6 mA, Morris water maze) no differences were established between the genotypes. The results are in good concordance with earlier experiments showing that CCK2 receptor deficient mice have lower pain sensitivity, but do not support previous studies that have established impaired memory functions in CCK2 receptor deficient mice.

Neuropeptide cholecystokinin (CCK) has been shown to suppress ethanol intake, but it is not clear if CCK-2 receptors play any role in this effect. In the present study, locomotor and sedative effects of acute ethanol administration, but also voluntary ethanol intake and preference were studied in male and female mice with genetic invalidation of CCK-2 receptors (-/-). Ethanol 0.5g/kg induced significant reduction of locomotor activity in male mice lacking CCK-2 receptors not habituated to motility boxes, as compared to their wild-type littermates (+/+). This effect was significantly modified by previous habituation to testing conditions. No such effect was observed in female mice. However, in female wild-type mice habituated to motility boxes ethanol 1.0g/kg induced significant locomotor suppression compared to female mice lacking CCK-2 receptors. Duration of loss of righting reflex (ethanol dose 4.0 g/kg) was not altered in mice lacking CCK-2 receptors. However, blood ethanol concentration at regain of righting reflex was significantly lower in female mice lacking CCK-2 receptors compared to their wild-type littermates. Ethanol intake, but not preference, was increased in mice with invalidation of CCK-2 receptor gene independent of gender. Current results indicate altered behavioural effects of ethanol and modified ethanol intake in mice lacking CCK-2 receptors. Gender-dependent neurochemical alterations possibly underlying these effects are discussed.

The effect of cat odour exposure was studied on the behaviour and neuropeptide gene expression in rodents. Exposure to the cat odour suppressed the exploratory activity of male Wistar rats in the elevated zero-maze. Treatment with morphine (1 mg/kg) increased the exploratory behaviour of rats in an unfamiliar environment. Previous exposure of animals to the cat odour completely abolished this stimulating action of morphine. Cat odour exposure induced a significant increase in the expression of Pomc1, Oprm1 and Cck genes in the brain structures related to anxiety and motivation. The similar study was also performed in female 129Sv/C57/Bl6 mice. The exposure of wild-type and homozygous CCK2 receptor deficient mice to the cat odour did not reveal substantial differences between the two genotypes. However, the following exposure of mice to the elevated plus-maze established differences as homozygous mice displayed increased exploratory activity in the plus-maze. The cat odour exposure significantly reduced exploratory activity only in homozygous mice. Together with the increased exploratory activity in homozygous mice the expression of Oprm1 gene was elevated in the frontal cortex and mesencephalon. Thus, the reduced anxiety of homozygous mice seems to be related to an increased tone of opioid system in the brain. The exposure of mice to cat odour did not change the gene expression in wild-type mice, whereas in homozygous animals a significantly increased expression of the Mc3r gene in the frontal cortex and temporal lobe, and the Pomc1 gene in the temporal lobe, mesencephalon and mesolimbic area was established. Therefore, it is likely that the increased function of melanocortin system is responsible for the anxiogenic effect of cat odour exposure in homozygous mice. In conclusion, the experiments conducted on rodents demonstrate that the cat odour exposure induced anxiety is accompanied by the compensatory changes in the activity of opioid and melanocortin systems.

Endocannabinoid system consists of two receptors (CB1, CB2), their endogeneous ligands and the enzymes responsible for their synthesis and degradation. In our experiment we have exposed rats to cat odour to induce the ethologically relevant anxiety reaction in rats. The elevated zero-maze was also performed to test alterations of the emotional behaviour. The animals were killed and RNA was isolated from different brain regions. cDNA expression of genes involved in synthesis or degradation of endocananbinoids (NAPE-PLD, FAAH, PLC and DAGLa, MGLL and receptor CB1) were analyzed in hippocampus, in amygdala and in mesolimbic structure using quantitative real-time PCR. In control group the highest gene exspression level was in CB1 receptors. The FAAH ekspression was 2, 6 times higher in amygdala compared to mesolimbic structure. The MGLL expression in contrary was 6 times higher in mesolimbic structure compared to amygdala. DAGLa expression was 2 times higher than NAPE-PLD expression and there has been also shown that the level of 2-AG in brain is higher than the level of anandamide. Unexpectedly, there were no significant gene expression changes in amygdala and in hipocampus in these animals exposed to cat-odour compared to the control group rats. Also, no differences of the gene expression were in rats preformed zero-maze compared to the control group. But, we found some gene expression changes in mesolimbic structure. There was higher (1, 4 times) CB1 gene expression level rats exposed to cat odour compared to the control group. Also, the expression of NAPE-PLD was 1, 6 times higher than the control group and the level of FAAH expression was 1, 8 times higher. Therefore, there is possible, that the endocannabionid system is more important in mesolimbic structure and anandamide, but not 2-AG synthesis mediates anxiety-related behaviour.