FRET-Based Calcium Imaging: A Tool for High-Throughput/Content Phenotypic Drug Screening in Alzheimer Disease

Perturbed intracellular store calcium homeostasis is suggested to play a major role in the pathophysiology of Alzheimer disease (AD). A number of mechanisms have been suggested to underlie the impairment of endoplasmic reticulum calcium homeostasis associated with familial AD-linked presenilin 1 mutations (FAD-PS1). Without aiming at specifically targeting any of those pathophysiological mechanisms in particular, we rather performed a high-throughput phenotypic screen to identify compounds that can reverse the exaggerated agonist-evoked endoplasmic reticulum calcium release phenotype in HEK293 cells expressing FAD-PS1. For that purpose, we developed a fully automated high-throughput calcium imaging assay using a fluorescence resonance energy transfer-based calcium indicator at single-cell resolution. This novel robust assay offers a number of advantages compared with the conventional calcium measurement screening technologies. The assay was employed in a large-scale screen with a library of diverse compounds comprising 20,000 low-molecular-weight molecules, which resulted in the identification of 52 primary hits and 4 lead structures. In a secondary assay, several hits were found to alter the amyloid (A) production. In view of the recent failure of AD drug candidates identified by target-based approaches, such a phenotypic drug discovery paradigm may present an attractive alternative for the identification of novel AD therapeutics

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

Conformational altered p53 as an early marker of oxidative stress in Alzheimer\u27s disease

In order to study oxidative stress in peripheral cells of Alzheimer\u27s 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

Conformational Altered p53 as an Early Marker of Oxidative Stress in Alzheimer's Disease

In order to study oxidative stress in peripheral cells of Alzheimer's 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

FRET-Based Calcium Imaging: A Tool for High-Throughput/Content Phenotypic Drug Screening in Alzheimer Disease

Perturbed intracellular store calcium homeostasis is suggested to play a major role in the pathophysiology of Alzheimer disease (AD). A number of mechanisms have been suggested to underlie the impairment of endoplasmic reticulum calcium homeostasis associated with familial AD-linked presenilin 1 mutations (FAD-PS1). Without aiming at specifically targeting any of those pathophysiological mechanisms in particular, we rather performed a high-throughput phenotypic screen to identify compounds that can reverse the exaggerated agonist-evoked endoplasmic reticulum calcium release phenotype in HEK293 cells expressing FAD-PS1. For that purpose, we developed a fully automated high-throughput calcium imaging assay using a fluorescence resonance energy transfer-based calcium indicator at single-cell resolution. This novel robust assay offers a number of advantages compared with the conventional calcium measurement screening technologies. The assay was employed in a large-scale screen with a library of diverse compounds comprising 20,000 low-molecular-weight molecules, which resulted in the identification of 52 primary hits and 4 lead structures. In a secondary assay, several hits were found to alter the amyloid (A) production. In view of the recent failure of AD drug candidates identified by target-based approaches, such a phenotypic drug discovery paradigm may present an attractive alternative for the identification of novel AD therapeutics

Validation of primary hits, preliminary structure-activity-relationship (SAR) analysis and their <i>in vitro</i> cytotoxicity.

<p>(<b>a</b>) The activity of all 53 primary hits was validated in PS1-M146L/YC3.6 line. All the hits were capable of reducing the peak response of CCh-induced calcium release to <90% of DMSO-treated controls. (<b>b</b>) The structure-activity-relationship (SAR) map of the screened compounds. The symbols represent compound clusters generated by Benchware DataMiner software. The distance between the clusters correlates with the similarity between their chemical structures. The number of compounds within a cluster is illustrated by the size of the symbol. A cluster with more than 50% active compounds is represented by a star, and marked in blue if the actual number of active compounds is greater than four. The highlighted identified lead structures belong to compound classes Thiazolidine (blue), Phenothiazine (green), Imidazole (turquoise) and Benzhydrilpiperidinamine (brown). Primary hits were also active in HEK293 cells expressing (<b>e</b>) PS1-DeltaE9/YC3.6, (<b>f</b>) PS1-C92S/YC3.6, and (<b>g</b>) PS1-D385N/YC3.6, by attenuating the mutant PS1-induced amplified calcium release. (<b>c</b>) and (<b>d</b>) The hits from the primary screen were classified into 8 categories based on their efficacy in lowering the CCh-evoked calcium release. These categories are separated according to the value of normalized ER calcium response. The noted numbers in each category indicates the number of compounds belonging to that category. (<b>h</b>) Viability of HEK293 cells treated with the primary screen hits was assessed by means of MTT assay after 24 h compound treatment. Values are presented as percentage of viable cells. In (a), (e), (f) and (g), the peak response of DMSO-treated control is set to one. Each color denotes a different lead structure in (a), (b), (e), (f), (g) and (h). The data for analogous molecules belonging to the same lead structure are marked with the same color.</p

Effect of on Bepridil on APP processing.

<p>(<b>a</b>) Reduced production of Aβ38, Aβ40 and Aβ42 after 16 h Bepridil (30 µM) treatment in HEK293 cells coexpressing APPsw and PS1-M146L. Sulindac sulfide (50 µM) was used as a γ-secretase modulator control. (<b>b</b>) Increased levels of sAPPα and decreased sAPPβ secreted fragments after 16 h treatment with Bepridil in HEK293 cells coexpressing APPsw and PS1-M146L. (n.s.: non-significant; * P<0.05, ** P<0.01 and *** P<0.001).</p