Aleksandra Szybinska

Proliferation and apoptosis of human CD8(+)CD28(+) and CD8(+)CD28(-) lymphocytes during aging.

It is commonly believed that the age-related decrease in the ratio CD28(+)/CD28(-) among CD8(+) T cells reflects replicative senescence of the lymphocytes. To verify this claim we measured the proliferation of CD8(+)CD28(+) and CD8(+)CD28(-) subsets by flow cytometry after PHA treatment of mononuclear lymphocytes from donors of different age, including centenarians. The fraction of CD28(+) cells decreases from ca. 80 to 40% (young to centenarians, respectively) with increasing age of the donors. Stimulation by PHA results in an increase in the ratio of CD28(+) relative to CD28(-) in all age groups. We found that not only CD8(+)CD28(+) but also CD8(+)CD28(-) cells were capable of proliferation. Moreover, the fraction of proliferation-competent CD28(-) cells was higher in the older donors compared with the younger ones. While PHA treatment led to apoptosis (as measured by DNA content and caspase-3 activation) of more than 20% of all lymphocytes, in the CD8(+) subset only ca. 10% died, irrespective of their CD28 status. Altogether, we showed over-representation of proliferating CD8(+)CD28(-) cells in aged people, which might not be particularly prone to undergo apoptosis.

Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

Mutations in presenilin 1 (PS1), which are the major cause of familial Alzheimers disease (FAD), are involved in perturbations of cellular Ca2+ homeostasis. Attenuation of capacitative Ca2+ entry (CCE) is the most often observed alteration of Ca2+ homeostasis in cells bearing FAD PS1 mutations. However, molecular mechanisms underlying this CCE impairment remains elusive. We demonstrate that cellular levels of STIM1 and STIM2 proteins, which are key players in CCE, depend on presenilins. We found increased level of STIM1 and decreased level of STIM2 proteins in mouse embryonic fibroblasts lacking presenilins. Fura-2 ratiometric assays revealed that CCE is enhanced in these cells after Ca2+ stores depletion by thapsigargin treatment. In turn, overexpression of PS1 with FAD mutations in HEK293 cells led to an attenuation of CCE. Although, no changes in STIM protein levels were observed in these HEK293 cells, FAD mutations in endogenous PS1 in human B lymphocytes resulted in a decreased expression of STIM2 in parallel to an attenuation of CCE. Our experiments showing that knock-out of presenilins in MEF cells and FAD mutations in endogenous PS1 in lymphocytes affect both CCE and the cellular level of STIM proteins open new perspectives for studies on CCE in FAD.

Conformationally altered p53 : a novel Alzheimer's disease marker?

The identification of biological markers of Alzheimers disease (AD) can be extremely useful to improve diagnostic accuracy and/or to monitor the efficacy of putative therapies. In this regard, peripheral cells may be of great importance, because of their easy accessibility. After subjects were grouped according to diagnosis, the expression of conformationally mutant p53 in blood cells was compared by immunoprecipitation or by a cytofluorimetric assay. In total, 104 patients with AD, 92 age-matched controls, 15 patients with Parkinsons disease and 9 with other types of dementia were analyzed. Two independent methods to evaluate the differential expression of a conformational mutant p53 were developed. Mononuclear cells were analyzed by immunoprecipitation or by flow-cytometric analysis, following incubation with a conformation-specific p53 antibody, which discriminates unfolded p53 tertiary structure. Mononuclear cells from AD patients express a higher amount of mutant-like p53 compared to non-AD subjects, thus supporting the study of conformational mutant p53 as a new putative marker to discriminate AD from non-AD patients. We also observed a strong positive correlation between the expression of p53 and the age of patients. The expression of p53 was independent from the length of illness and from the Mini Mental State Examination value.

Conformational altered p53 as an early marker of oxidative stress in Alzheimer's disease.

In order to study oxidative stress in peripheral cells of Alzheimers disease (AD) patients, immortalized lymphocytes derived from two peculiar cohorts of patients, referring to early onset AD (EOSAD) and subjects harboured AD related mutation (ADmut), were used. Oxidative stress was evaluated measuring i) the typical oxidative markers, such as HNE Michel adducts, 3 Nitro-Tyrosine residues and protein carbonyl on protein extracts, ii) and the antioxidant capacity, following the enzymatic kinetic of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRD). We found that the signs of oxidative stress, measured as oxidative marker levels, were evident only in ADmut but not in EOSAD patients. However, oxidative imbalance in EOSAD as well as ADmut lymphocytes was underlined by a reduced SOD activity and GRD activity in both pathological groups in comparison with cells derived from healthy subjects. Furthermore, a redox modulated p53 protein was found conformational altered in both EOSAD and ADmut B lymphocytes in comparison with control cells. This conformational altered p53 isoform, named “unfolded p53”, was recognized by the use of two specific conformational anti-p53 antibodies. Immunoprecipitation experiments, performed with the monoclonal antibodies PAb1620 (that recognizes p53wt) and PAb240 (that is direct towards unfolded p53), and followed by the immunoblotting with anti-4-hydroxynonenal (HNE) and anti- 3-nitrotyrosine (3NT) antibodies, showed a preferential increase of nitrated tyrosine residues in unfolded p53 isoform comparing to p53 wt protein, in both ADmut and EOSAD. In addition, a correlation between unfolded p53 and SOD activity was further found. Thus this study suggests that ROS/RNS contributed to change of p53 tertiary structure and that unfolded p53 can be considered as an early marker of oxidative imbalance in these patients.

Polish Centenarians Programme. Multidisciplinary studies of successful ageing: aims, methods, and preliminary results.

Studies of centenarians as a model of successful ageing may help identify various environmental, social, psychological, and genetic factors supporting longevity. The scientific aims of the programme were to assess health status and environmental determinants of ageing of Polish centenarians, and to collect biological material for studying selected aspects of longevity, including genetic factors. The social aim of the project was to bring public attention to ageing of the population, as well as living conditions of elderly individuals. The intention of the authors of this paper is to present aims, scope, methods and preliminary results of the Polish Centenarians Programme, as well as to provide potential new partners for studying various aspects of longevity and ageing with the information about available materials collected during the programme. In this study, 346 subjects aged 100+ were visited, biological material was collected from 285 subjects, and 153 lymphocyte cell lines were immortalized.

Impact of SNPs on methylation readouts by Illumina Infinium HumanMethylation450 BeadChip Array: implications for comparative population studies

BackgroundInfinium HumanMethylation 450 BeadChip Arrays by Illumina (Illumina HM450K) are among the most popular CpG microarray platforms widely used in biological and medical research. Several recent studies highlighted the potentially confounding impact of the genomic variation on the results of methylation studies performed using Illumina’s Infinium methylation probes. However, the complexity of SNPs impact on the methylation level measurements (β values) has not been comprehensively described.ResultsIn our comparative study of European and Asian populations performed using Illumina HM450K, we found that the majority of Infinium probes, which differentiated two examined groups, had SNPs in their target sequence. Characteristic tri-modal or bi-modal patterns of β values distribution among individual samples were observed for CpGs with SNPs in the first and second position, respectively. To better understand how SNPs affect methylation readouts, we investigated their impact in the context of SNP position and type, and of the Illumina probe type (Infinium I or II).ConclusionsOur study clearly demonstrates that SNP variation existing in the genome, if not accounted for, may lead to false interpretation of the methylation signal differences suggested by some of the Illumina Infinium probes. In addition, it provides important practical clues for discriminating between differences due to the methylation status and to the genomic polymorphisms, based on the inspection of methylation readouts in individual samples. This approach is of special importance when Illumina Infinium assay is used for any comparative population studies, whether related to cancer, disease, ethnicity where SNP frequencies differentiate the studied groups.

Highly Pathogenic Alzheimer's Disease Presenilin 1 P117R Mutation Causes a specific Increase in p53 and p21 Protein Levels and Cell Cycle Dysregulation in Human Lymphocytes

Cell cycle (CC) reentry in neurons precedes the formation of amyloid-β (Aβ) plaques in Alzheimers disease (AD). CC alterations were also detected in lymphocytes from sporadic AD patients. In the present study, we investigated the influence of nine presenilin 1 (PS1) mutations (P117R, M139V, L153V, H163R, S170F, F177L, I213F, L226F, E318G) on CC and Aβ production in immortalized B-lymphocytes from familial AD (FAD) patients and in stably transfected human embryonic kidney cells. In both cell types, only the P117R mutation increased levels of key G1/S phase regulatory proteins, p53, and its effector p21, causing G1 phase prolongation with simultaneous S phase shortening, and lowering basal apoptosis. The CC changes were rescued by inhibition of p53, but not of γ-secretase. Moreover, the investigated PS1 mutants showed differences in the increased levels of secreted Aβ40 and Aβ42 and in Aβ42/Aβ40 ratios, but these differences did not correlate with CC patterns. Altogether, we found that both CC regulation and Aβ production differentiate PS1 mutations, and that CC PS1 activity is mediated by p53/p21 signaling but not by γ-secretase activity. The identified CC dysregulation linked with increased p53 and p21 protein levels distinguishes the highly pathogenic PS1 P117R mutation and may contribute to the specific severity of the clinical progression of FAD associated with the mutation in the PS1 117 site. These findings suggest that impairment in lymphocyte CC might play a pathogenic function in AD and are relevant to the development of new diagnostic approaches and personalized therapeutic strategies.

Mitochondrial genotype in vulvar carcinoma - cuckoo in the nest

Vulvar squamous cell carcinoma (VSCC) is a rare female genital neoplasm. Although numerous molecular changes have been reported in VSCC, biomarkers of clinical relevance are still lacking. On the other hand, there is emerging evidence on the use of mtDNA as a diagnostic tool in oncology. In order to investigate mtDNA status in VSCC patients, haplogroup distribution analysis and D-loop sequencing were performed. The results were compared with available data for the general Polish population, cancer free-centenarians as well as patients with endometrial and head and neck cancer. The obtained data were also compared with the current status of mitochondrial databases. Significant differences in haplogroup distribution between VSCC cohort, general Polish population and cancer-free centenarians cohort were found. Moreover, a correlation between the VSCC patients haplogroup and HPV status was observed. Finally, a specific pattern of mtDNA polymorphisms was found in VSCC. Our results suggest that the mitochondrial genetic background may influence the risk of VSCC occurrence as well as susceptibility to HPV infection.

The E318G substitution in PSEN1 gene is not connected with Alzheimer's disease in a large Polish cohort

Mutations in the presenilin 1 (PSEN1) gene are known to cause nearly 50% of early-onset, familial Alzheimers disease (AD) cases. To determine whether E318G mutation is related causally to AD in the Polish population E318G mutation frequency was assessed using PCR-RFLP method in a total of 659 subjects: 256 AD patients, 210 healthy, age-matched control subjects, 100 Parkinsons disease patients and 93 centenarians. When the mutation frequencies were compared to healthy controls, no significant differences between the groups were found. It could be concluded that E318G mutation is not related causally to AD in the Polish population, either as a risk factor or a disease causing mutation.

Development and implementation of a high-throughput compound screening assay for targeting disrupted ER calcium homeostasis in Alzheimer's disease.

Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimers disease (AD) pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER). Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease.