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.

ESCRT-I fuels lysosomal degradation to restrict TFEB/TFE3 signaling via the Rag-mTORC1 pathway.

Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of proteins residing in endosomal membranes. Here, we show that mammalian ESCRT-I restricts the size of lysosomes and promotes degradation of proteins from lysosomal membranes, including MCOLN1, a Ca2+ channel protein. The altered lysosome morphology upon ESCRT-I depletion coincided with elevated expression of genes annotated to biogenesis of lysosomes due to prolonged activation of TFEB/TFE3 transcription factors. Lack of ESCRT-I also induced transcription of cholesterol biosynthesis genes, in response to inefficient delivery of cholesterol from endolysosomal compartments. Among factors that could possibly activate TFEB/TFE3 signaling upon ESCRT-I deficiency, we excluded lysosomal cholesterol accumulation and Ca2+-mediated dephosphorylation of TFEB/TFE3. However, we discovered that this activation occurs due to the inhibition of Rag GTPase-dependent mTORC1 pathway that specifically reduced phosphorylation of TFEB at S112. Constitutive activation of the Rag GTPase complex in cells lacking ESCRT-I restored S112 phosphorylation and prevented TFEB/TFE3 activation. Our results indicate that ESCRT-I deficiency evokes a homeostatic response to counteract lysosomal nutrient starvation, that is, improper supply of nutrients derived from lysosomal degradation.

Novel calcineurin A (PPP3CA) variant associated with epilepsy, constitutive enzyme activation and downregulation of protein expression.

PPP3CA encodes calmodulin-binding catalytic subunit of calcineurin, a ubiquitously expressed calcium/calmodulin-regulated protein phosphatase. Recently de novo PPP3CA variants were reported as a cause of disease in 12 subjects presenting with epileptic encephalopathy and dysmorphic features. We describe a boy with similar phenotype and severe early onset epileptic encephalopathy in whom a novel de novo c.1324C>T (p.(Gln442Ter)) PPP3CA variant was found by whole exome sequencing. Western blot experiments in patient's cells (EBV transformed lymphocytes and neuronal cells derived through reprogramming) indicate that despite normal mRNA abundance the protein expression level is strongly reduced both for the mutated and wild-type protein. By in vitro studies with recombinant protein expressed in E. coli we show that c.1324C>T (p.(Gln442Ter)) results in constitutive activation of the enzyme. Our results confirm the role of PPP3CA defects in pathogenesis of a distinct neurodevelopmental disorder including severe epilepsy and dysmorphism and provide further functional clues regarding the pathogenic mechanism.

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.

P53 Dysfunction in Neurodegenerative Diseases - The Cause or Effect of Pathological Changes?

Neurodegenerative diseases are a heterogeneous, mostly age-associated group of disorders characterized by progressive neuronal loss, the most prevalent being Alzheimer disease. It is anticipated that, with continuously increasing life expectancy, these diseases will pose a serious social and health problem in the near feature. Meanwhile, however, their etiology remains largely obscure even though all possible novel clues are being thoroughly examined. In this regard, a concept has been proposed that p53, as a transcription factor controlling many vital cellular pathways including apoptosis, may contribute to neuronal death common to all neurodegenerative disorders. In this work, we review the research devoted to the possible role of p53 in the pathogenesis of these diseases. We not only describe aberrant changes in p53 level/activity observed in CNS regions affected by particular diseases but, most importantly, put special attention to the complicated reciprocal regulatory ties existing between p53 and proteins commonly regarded as pathological hallmarks of these diseases, with the ultimate goal to identify the primary element of their pathogenesis.

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.

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 ß (Aß) 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.

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 Alzheimer's 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 Aß production by lowering BACE1 activity. Indeed, we also detected lowered Aß, increased sAPPα and decreased sAPPß 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.

The prion protein M129V polymorphism: longevity and cognitive impairment among Polish centenarians.

The PRNP gene encodes the cellular isoform of prion protein (PrP (c) ). The M129V polymorphism influences the risk of prion diseases and may modulate the rate of neurodegeneration with age. We present the first study of the polymorphism among Polish centenarians. In the control group (n = 165, ages 18 to 56 years) the observed M129V genotype frequencies agreed with those expected according to the Hardy-Weinberg equilibrium (MM, MV, VV): 43%, 44%, 13% (HWE p > 0.05). Among centenarians (n = 150, ages 100 to 107) both homozygotes were more common than expected and HWE was rejected: 46%, 37%, 17% (expected 42%, 46%, 13%; HWE p = 0.025). This finding is consistent with a higher mortality rate among heterozygotes. However, the observed allele and genotype frequencies did not differ significantly between the oldest-old and the young controls. The genotypic frequencies were not related to severe cognitive impairment among the centenarians.

APBB2 genetic polymorphisms are associated with severe cognitive impairment in centenarians.

APBB2 gene encodes for ß-amyloid precursor protein-binding family B member 2, (APBB2, FE65-like, FE65L1), an adaptor protein binding to the cytoplasmatic domain of ß-amyloid precursor protein (ßAPP). Over-expression of APBB2 promotes formation of ß-amyloid (Aß), the main constituent of senile plaques. Polymorphisms within APBB2 gene have been proposed as candidate risk factors for Alzheimer's disease. However, their association with longevity has never been investigated. Here we present the first attempt to analyze APBB2 polymorphisms in centenarians. We used a PCR-RFLP method to analyze two intronic nucleotide substitutions: hCV1558625 (rs17443013) and rs13133980. We found no differences in genotype or allele distribution between centenarians and young controls. After stratification of centenarians upon their cognitive performance, the APBB2 rs13133980 G allele was over-represented in centenarians with severe cognitive impairment compared to individuals without this disability. Also the hCV1558625-rs13133980 AG haplotype increased relative risk for severe cognitive impairment in centenarians. Our results support the concept of APBB2 polymorphism association with cognitive performance in the oldest age.

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.

Variation in NPC1, the gene encoding Niemann-Pick C1, a protein involved in intracellular cholesterol transport, is associated with Alzheimer disease and/or aging in the Polish population.

There is abundant evidence that cholesterol metabolism, especially as mediated by the intercellular transporter APOE, is involved in the pathogenesis of sporadic, late-onset Alzheimer disease (SLAD). Identification of other genes involved in SLAD pathogenesis has been hampered since gene association studies, whether individual or genome-wide, experience difficulty in finding appropriate controls in as much as 25% or more of normal adults will develop SLAD. Using 152 centenarians as additional controls and 120 "regular", 65-75-year-old controls, we show an association of genetic variation in NPC1 with SLAD and/or aging. In this preliminary study, we find gradients of two non-synonymous SNP's allele frequencies in NPC1 from centenarians through normal controls to SLAD in this non-stratified Polish population. An intervening intronic SNP is not in Hardy-Weinberg equilibria and differs between centenarians and controls/SLAD. Haplotypes frequencies determined by fastPHASE were somewhat different, and the predicted genotype frequencies were very different between the three groups. These findings can also be interpreted as indicating a role for NPC1 in aging, a role also suggested by NPC1's role in Dauer formation (hibernation, a longevity state) in Caenorhabditis elegans.

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.

The relationship between adiponectin levels and metabolic status in centenarian, early elderly, young and obese women.

OBJECTIVES: Adipose tissue secretes proteins which regulate energy metabolism and insulin sensitivity. Adiponectin possesses anti-diabetic, anti-atherogenic and insulin-sensitizing properties. To assess the prognostic factors in prolonged survival and the potential protective role of adiponectin in aging, we examined the release of adiponectin in relation to the metabolic status of centenarians, compared with young, early elderly and obese subjects. MATERIAL AND METHODS: The study was carried out on 122 women: 22 centenarians aged 100-102 yrs, 45 younger women aged 20-43 yrs, 19 early elderly women aged 64-67 yrs, and 36 obese women aged 26-54 yrs. Anthropometric data, clinical features and blood samples were obtained. Plasma adiponectin and insulin concentrations were measured by RIA methods. Fasting plasma glucose levels, lipid profile and creatinine concentrations were determined using routine laboratory procedures. RESULTS: In centenarians we found that adiponectin concentrations were significantly increased, compared with young, early elderly and obese women. Insulin concentrations were lower than those in young and obese subjects. HOMA-IR was significantly lower than in obese women. Positive correlations were found between adiponectin and HDL, and negative correlations between adiponectin and HOMA-IR, total cholesterol, LDL, triglycerides, blood pressure and BMI. CONCLUSION: Our results indicate that adiponectin may play a protective role that contributes to longevity.