Beata Kaźmierczak

The influence of heroin abuse on glutathione-dependent enzymes in human brain.

Heroin is an illicit narcotic abused by millions of people worldwide. In our earlier studies we have shown that heroin intoxication changes the antioxidant status in human brain. In the present work we continued our studies by estimating the effect of heroin abuse on reduced glutathione (GSH) and enzymes related to this cofactor, such as glutathione S-transferase detoxifying electrophilics (GST) and organic peroxides (as Se-independent glutathione peroxidase-GSHPx), and Se-dependent glutathione peroxidase (Se-GSHPx) specific mainly for hydrogen peroxide. Studies were conducted on human brains obtained from autopsy of 9 heroin abusers and 8 controls. The level of GSH and the activity of glutathione-related enzymes were determined spectrophotometrically. The expression of GST pi on mRNA and protein level was studied by RT-PCR and Western blotting, respectively. The results indicated significant increase of GST and GSHPx activities, unchanged Se-GSHPx activity, and decreased level of GSH in frontal, temporal, parietal and occipital cortex, brain stem, hippocampus, and white matter of heroin abusers. GST pi expression was increased on both mRNA and protein levels, however the increase was lower in brain stem than in other regions. Heroin affects all regions of human brain, and especially brain stem. Its intoxication leads to an increase of organic rather then inorganic peroxides in various brain regions. Glutathione S-transferase plays an important role during heroin intoxication, however its protective effect is lower in brain stem than in brain cortex or hippocampus.

SMN1 gene duplications are more frequent in patients with progressive muscular atrophy.

Abstract Survival Motor Neuron 1 (SMN1) is a causative gene for autosomal recessive infantile and juvenile proximal spinal muscular atrophy. SMN1 duplications have recently been found to increase susceptibility to amyotrophic lateral sclerosis. The role of centromeric SMN copy (SMN2) has been postulated in progressive muscular atrophy (PMA). The aim of this study was to analyse the SMN1 and SMN2 copy number variations in patients with PMA. SMN1 and SMN2 genotype was studied in 87 patients with PMA, diagnosed at the Department of Neurology, Medical University of Warsaw, between 1992 and 2012 and in 600 healthy controls. Results demonstrated that three copies of SMN1 were found in 8.1% of PMA patients and in 24% of PMA patients with disease duration above 48 months compared to 4.6% of the general population. Patients with three SMN1 copies had a limb onset, lower median age of onset and longer disease duration compared to patients with two SMN1 copies. There were no significant differences in the SMN2 copy numbers. In conclusion, the increased copy number of SMN1 may be a susceptibility factor to PMA and influence the clinical phenotype.

Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

Dynactin is a complex motor protein involved in the retrograde axonal transport disturbances of which may lead to amyotrophic lateral sclerosis (ALS). Mice with hSOD1G93A mutation develop ALS-like symptoms and are used as a model for the disease studies. Similar symptoms demonstrate Cra1 mice, with Dync1h1 mutation. Dynactin heavy (DCTN1) and light (DCTN3) subunits were studied in the CNS of humans with sporadic ALS (SALS), mice with hSOD1G93A (SOD1/+), Dync1h1 (Cra1/+), and double (Cra1/SOD1) mutation at presymptomatic and symptomatic stages. In SALS subjects, in contrast to control cases, expression of DCTN1-mRNA but not DCTN3-mRNA in the motor cortex was higher than in the sensory cortex. However, the mean levels of DCTN1-mRNA and protein were lower in both SALS cortexes and in the spinal cord than in control structures. DCTN3 was unchanged in brain cortexes but decreased in the spinal cord on both mRNA and protein levels. In all SALS tissues immunohistochemical analyses revealed degeneration and loss of neuronal cells, and poor expression of dynactin subunits. In SOD1/+ mice both subunits expression was significantly lower in the frontal cortex, spinal cord and hippocampus than in wild-type controls, especially at presymptomatic stage. Fewer changes occurred in Cra1/SOD1 and Cra1/+ mice.It can be concluded that in sporadic and SOD1-related ALS the impairment of axonal retrograde transport may be due to dynactin subunits deficiency and subsequent disturbances of the whole dynein/dynactin complex structure and function. The Dync1h1 mutation itself has slight negative effect on dynactin expression and it alleviates the changes caused by SOD1G93A mutation.

Tau Isoforms Expression in Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Tau is a protein involved in regulation of microtubule stability, axonal differentiation and transport. Alteration of retrograde transport may lead to motor neuron degeneration. Thus alternative mRNA splicing and expression of tau isoforms were studied in a transgenic mouse model harboring the human SOD1 G93A mutation. The studies were performed on cortex, hippocampus and spinal cord of 64- and 120-day-old animals (presymptomatic and symptomatic stage) and wild type controls. Exon 10 was found in all studied tissues. The 2N isoform containing exons 2 and 3 (+2+3) and the 1N (+2−3) predominated over the 0N (−2−3) in brain regions of the studied mice. The 2N expression was significantly lower in cortex and hippocampus of symptomatic animals compared to analogue control tissues. The decrease in 2N expression resulted in lower levels of total tau mRNA and tau protein. No changes in tau expression were observed in spinal cord of studied animals.

GSTP1 Polymorphisms and their Association with Glutathione Transferase and Peroxidase Activities in Patients with Motor Neuron Disease

Glutathione S-transferase pi (GSTP1) is a crucial enzyme in detoxification of electrophilic compounds and organic peroxides. Together with Se-dependent glutathione peroxidase (Se-GSHPx) it protects cells against oxidative stress which may be a primary factor implicated in motor neuron disease (MND) pathogenesis. We investigated GSTP1 polymorphisms and their relationship with GST and Se-GSTPx activities in a cohort of Polish patients with MND. Results were correlated with clinical phenotypes. The frequency of genetic variants for GSTP1 exon 5 (I105V) and exon 6 (A114V) was studied in 104 patients and 100 healthy controls using real-time polymerase chain reaction. GST transferase activity was determined in serum with 1-chloro-2,4-dinitrobenzene, its peroxidase activity with cumene hydroperoxide, and Se-GSHPx activity with hydrogen peroxide. There were no differences in the prevalence of GSTP1 polymorphism I105V and A114V between MND and controls, however the occurrence of CT variant in codon 114 was associated with a higher risk for MND. GSTP1 polymorphisms were less frequent in classic ALS than in progressive bulbar palsy. In classic ALS C* (heterozygous I /V and A /V) all studied activities were significantly lower than in classic ALS A* (homozygous I /I and A/A). GST peroxidase activity and Se-GSHPx activity were lower in classic ALS C* than in control C*, but in classic ALS A* Se-GSHPx activity was significantly higher than in control A*. It can be concluded that the presence of GSTP1 A114V but not I105V variant increases the risk of MND, and combined GSTP1 polymorphisms in codon 105 and 114 may result in lower protection of MND patients against the toxicity of electrophilic compounds, organic and inorganic hydroperoxides.

Recurrent K3E mutation in Cu/Zn superoxide dismutase gene associated with amyotrophic lateral sclerosis

Abstract Cu/Zn superoxide dismutase (SOD1) gene mutations are the most frequently reported genetic causes of amyotrophic lateral sclerosis (ALS). The objective of the study was to describe a clinical phenotype and haplotype background of Polish and Japanese ALS patients harbouring the K3E SOD1 mutation. The K3E mutation was identified by direct sequencing, high resolution melting analysis or high-throughput microarray-based resequencing system. Microsatellite polymorphic markers flanking SOD1 were genotyped in members of six kindreds and two SALS patients. Results demonstrated that the K3E mutation was responsible for classic ALS. The median age of onset was 54 years. The clinical phenotype did not substantially differ between SALS and FALS cases of either ethnic origin, with some intrafamiliar variabilities. There was a limb onset in 92% of patients. In patients with bulbar syndrome, dysphagia predominated over dysarthria. Respiratory insufficiency was found in 61.1% of patients (19–84 months after the first symptoms onset). Median survival was 101 months with age of death ranging from 45 to 77 years. K3E was the most frequent SOD1 mutation among Polish FALS patients. It originated independently, on different haplotype background in the Polish and Japanese populations. In conclusion, recurrent K3E mutation results in a relatively slowly progressing limb onset ALS with classic phenotype.

Validation of qPCR reference genes in lymphocytes from patients with amyotrophic lateral sclerosis

Quantitative polymerase chain reaction (qPCR) is the most specific and reliable method for determination of mRNA gene expression. Crucial point for its accurate normalization is the choice of appropriate internal control genes (ICGs). In the present work we determined and compare the expression of eight commonly used ICGs in lymphocytes from 26 patients with amyotrophic lateral sclerosis (ALS) and 30 control subjects. Peripheral blood mononuclear cells (PBMCs) before and after immortalization by EBV transfection (lymphoblast cell lines—LCLs) were used for qPCR analysis. LCLs were studied before and after liquid nitrogen cryopreservation and culturing (groups LCL1 and LCL2, respectively). qPCR data of 8 ICGs expression was analyzed by BestKeeper, NormFinder and geNorm methods. All studied genes (18SRNA, ACTB, B2M, GUSB,GAPDH, HPRT1, MT-ATP6 and RPS17) were expressed in PBMCs, whereas only first four in LCLs. LCLs cryopreservation had no effect on ICGs expression. Comprehensive ranking indicated RPS17 with MT-ATP6 as the best ICGs for qPCR in PBMCs of control and ALS subjects, and RPS17 with 18RNA or MT-ATP6 in LCLs from ALS. In PBMCs 18RNA shouldn’t be used as ICG.

Aquaporin-4: A novel diagnostic biomarker for seronegative neuromyelitis optica

Neuromyelitis optica (NMO) and its spectrum disorders (NMOSDs) are a heterogenous group of demyelinating diseases, distinguished from others by the presence of NMO-IgG (immunoglobulin G). The results of recent studies revealed however, that some NMO/NMOSD patients are seronegative and cannot be distinguished from multiple sclerosis (MS) by NMO-IgG assessment.1 This differentiation is essential, as inadequately treated NMO or NMOSD has a poor prognosis. Also immunomodulation, now commonly used in treatment of early stages of MS, has either no influence on the course of NMO or may even worsen it.2 Other immunosuppressive agents used in more severe cases of MS, such as natalizumab, might also worsen the course of NMO.3 These data suggest that there is an increasing need for other biomarkers that might help to distinguish NMO/NMOSD from other demyelinating disorders. The aim of our study was to compare aquaporin-4 (AQP4) concentrations in the serum of NMO/ NMOSD and MS patients, as well as in controls, in order to establish whether it can be used as diagnostic biomarker for NMO/NMOSD and MS.

Alteration of Motor Protein Expression Involved in Bidirectional Transport in Peripheral Blood Mononuclear Cells of Patients with Amyotrophic Lateral Sclerosis

Background: Sporadic amyotrophic lateral sclerosis (SALS) is a fatal motor neuron degenerative disease of unclear pathogenesis. Disturbances of intracellular transport are possible causes of the disease. Objective: We evaluated the expression of motor proteins involved in the anterograde (kinesins KIF1B, KIF5C) and retrograde (KIFC3, dynactin subunits DCTN1 and DCTN3) intracellular transport in peripheral blood mononuclear cells (PBMCs). Materials and Methods: PBMCs were obtained from 74 SALS patients with different clinical phenotypes, 65 blood donors (healthy control I), and 29 cases with other neurological diseases (disease control II) divided into subgroups IIA (atypical parkinsonism) and IIB (ALS-mimicking disorders). mRNA expression was studied by real-time qPCR, and protein level by Western blotting. Results: In SALS, KIF5C and KIFC3 expression was significantly lower and DCTN1 higher than in control I, and dependent of age. KIF1B expression was significantly higher in SALS than in subgroup IIB, whereas DCTN1 and DCTN3 were higher in SALS than in subgroup IIA. All changes in the studied proteins were statistically significant in classic ALS but not in progressive muscular atrophy. Conclusion: In SALS, and especially in classic ALS, the changes in motor protein expression may alter bidirectional intracellular transport in PBMCs. More studies are needed to find out whether the levels of KIF5C and DCTN1 may be useful in ALS diagnosis, and whether KIF1B expression may discriminate ALS from ALS-mimicking disorders.