Impaired iron recycling from erythrocytes is an early hallmark of aging.

Aging affects iron homeostasis, as evidenced by tissue iron loading and anemia in the elderly. Iron needs in mammals are met primarily by iron recycling from senescent red blood cells (RBCs), a task chiefly accomplished by splenic red pulp macrophages (RPMs) via erythrophagocytosis. Given that RPMs continuously process iron, their cellular functions might be susceptible to age-dependent decline, a possibility that has been unexplored to date. Here, we found that 10- to 11-month-old female mice exhibit iron loading in RPMs, largely attributable to a drop in iron exporter ferroportin, which diminishes their erythrophagocytosis capacity and lysosomal activity. Furthermore, we identified a loss of RPMs during aging, underlain by the combination of proteotoxic stress and iron-dependent cell death resembling ferroptosis. These impairments lead to the retention of senescent hemolytic RBCs in the spleen, and the formation of undegradable iron- and heme-rich extracellular protein aggregates, likely derived from ferroptotic RPMs. We further found that feeding mice an iron-reduced diet alleviates iron accumulation in RPMs, enhances their ability to clear erythrocytes, and reduces damage. Consequently, this diet ameliorates hemolysis of splenic RBCs and reduces the burden of protein aggregates, mildly increasing serum iron availability in aging mice. Taken together, we identified RPM collapse as an early hallmark of aging and demonstrated that dietary iron reduction improves iron turnover efficacy.

Overactive BRCA1 Affects Presenilin 1 in Induced Pluripotent Stem Cell-Derived Neurons in Alzheimer's Disease.

The BRCA1 protein, one of the major players responsible for DNA damage response has recently been linked to Alzheimer's disease (AD). Using primary fibroblasts and neurons reprogrammed from induced pluripotent stem cells (iPSC) derived from familial AD (FAD) patients, we studied the role of the BRCA1 protein underlying molecular neurodegeneration. By whole-transcriptome approach, we have found wide range of disturbances in cell cycle and DNA damage response in FAD fibroblasts. This was manifested by significantly increased content of BRCA1 phosphorylated on Ser1524 and abnormal ubiquitination and subcellular distribution of presenilin 1 (PS1). Accordingly, the iPSC-derived FAD neurons showed increased content of BRCA1(Ser1524) colocalized with degraded PS1, accompanied by an enhanced immunostaining pattern of amyloid-ß. Finally, overactivation of BRCA1 was followed by an increased content of Cdc25C phosphorylated on Ser216, likely triggering cell cycle re-entry in FAD neurons. This study suggests that overactivated BRCA1 could both influence PS1 turnover leading to amyloid-ß pathology and promote cell cycle re-entry-driven cell death of postmitotic neurons in AD.

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

BACKGROUND: Infinium 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 (ß values) has not been comprehensively described. RESULTS: In 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 ß 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). CONCLUSIONS: Our 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-ß (Aß) plaques in Alzheimer's 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 Aß 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 Aß40 and Aß42 and in Aß42/Aß40 ratios, but these differences did not correlate with CC patterns. Altogether, we found that both CC regulation and Aß 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.

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.

Mitochondrial genotype and breast cancer predisposition.

Breast cancer is the most commonly diagnosed cancer in women. Despite recent advances in breast cancer research, a comprehensive set of genetic markers of increased breast cancer risk remain elusive. Recently mitochondrial DNA (mtDNA) mutations have been found in many types of cancer, including breast cancer. To investigate the possible role of mitochondrial genetics in breast cancer predisposition and biology we analyzed the D-loop sequence of cancer patients and assigned mitochondrial haplogroup using RFLP analysis. We detected a significantly greater incidence of mtDNA polymorphisms T239C, A263G and C16207T and a significant lower incidence of A73G, C150T, T16183C, T16189C, C16223T, T16362C in patients with breast cancer compared to database controls. The mitochondrial haplogroup distribution in patients with breast cancer differs from a group of cancer-free controls and the general Polish population in that haplogroup I is over-represented in individuals with cancer. These findings suggest that mitochondrial haplogroup I as well as other polymorphic variants defined by SNPs in the D-loop may be associated with an increased risk of developing breast cancer.

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.

Decreased expression and the Lys751Gln polymorphism of the XPD gene are associated with extreme longevity.

Aging is associated with progressing genomic instability. The XPD gene encodes a DNA helicase involved in nucleotide excision repair and in transcription. We analyzed the common XPD polymorphisms that were previously shown to affect protein's DNA repair efficiency and to increase the risk of developing various cancers. Analysis was performed in 149 centenarians (mean age 101.1 years old) and in 413 young subjects (mean age 27.1 years old). We showed that the distribution of the Lys751Gln genotypes differed significantly between these groups (P = 0.017). In centenarians, the homozygous genotypes AA and CC were found less frequently than in young controls (29 vs. 36%, OR = 0.71, and 14 vs. 20%, OR = 0.652, respectively). The Arg156Arg and Asp312Asn were not significantly associated with extreme longevity. Analysis of the XPD mRNA level in blood mononuclear cells of people divided into three age groups (mean ages 28.7, 65.8 and 92.7 years old) showed that extreme longevity is associated with the decrease of the mean level of the specific mRNA; the differences between young or middle-aged vs. extremely old group were significant (P < 0.0001, P < 0.0001, respectively). In addition, the methylation pattern of the XPD promoter was analyzed in 30 people divided into three age groups (29.5, 65.9, and 101.4 years old). We showed that overall methylation of the XPD promoter is a rare event; however, aging is associated with the increase of methylation level upstream of the transcription start site. In summary, we showed for the first time that both the XPD polymorphic variants and the decreased level of its expression might be associated with aging.

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 Alzheimer's 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.